Highly potent half-sandwich iridium and ruthenium complexes as lysosome-targeted imaging and anticancer agents.

Novel drug development for KRAS-mutated non-small cell lung cancer ............................................................................................................. ...... Shakil Md. Salman A thesis submitted for the degree of Master of Science at the University of Otago Dunedin, New Zealand October 2020 Abstract Lung cancer is the leading cause of cancer-related death among both men and women and nonsmall cell lung cancer (NSCLC) accounts for 85 to 88% of all lung cancer cases. In NSCLC, kirsten rat sarcoma (KRAS) viral oncogene homologue mutations are the third most frequent mutation. KRAS-mutant patients show a shorter overall survival as a clinically approved therapeutic agent against KRAS has yet to be developed. Therefore, novel drugs are urgently needed for these patients. The current project examined the cytotoxic effects, selectivity, and anticancer mechanism of novel synthetic compounds towards KRAS-mutated NSCLC in vitro. In this study, A549 (KRAS-mutated NSCLC cells), H522 (p53-mutated NSCLC cells), NIH3T3 (pre-neoplastic) and PNT1A (normal prostate epithelial cells) cell lines were used to screen out potent drug candidate for KRAS-mutated NSCLC. A549 cells were treated with 11 different metal-based and non-metal-based novel compounds of four different drug class (HDAC inhibitors: JAZZ-90, JAZZ-166, and JAZZ-167; Hydroxythiopyridone derivatives: M1S, M2S, M1S-Ru, and M2S-Ru; Metal-based PCA ligands and complexes: AASH-122 and JAZZ-121; Kinetically inert metal(arene) complexes of PCA: ZR-012 and ZR-014) at 0.015 to 200 µM for 72 h and cell viability was determined using the sulforhodamine B assay. Hydroxythiopyridone derivatives M1S and M2S were the smallest but most potent compounds in A549 cells with EC50 values of 0.36 and 0.32 µM, respectively. Additionally, M1S and M2S were 1.3 and 1.4 times more potent against H522 cells. Furthermore, M1S and M2S exhibited more selectivity towards these NSCLC cells and pre-neoplastic NIH3T3 cells as compared to PNT1A cells. Time-course cytotoxicity studies showed that both the drug candidates had cytostatic and cytotoxic effects at 2×EC50 concentration in A549 and H522 cells, respectively. Western blotting results indicated that the drug candidates were unable to increase the acetylation of histone 3. In A549 cells the drug candidates did not change cyclin D1 levels, after 24 h. However, 2×EC50 concentration of M1S and M2S significantly decreased cyclin D1 expression in H522 cells by 68.1 and 84.9% of control, respectively at 24 h. In both the cell lines, the drug candidates did not produce a significant effect in reducing cyclin B1 expression. Cell cycle analysis showed that the 2×EC50 concentration of M1S and M2S arrested 3.3 and 7.1% of A549 cells at the G2/M phase compared to control at 12 h. While 9.9 and 8.9% of H522 cells were arrested at the G1 phase after M1S and M2S treatment at 2×EC50 concentration. The same treatment also increased the number of sub-G1 apoptotic H522 cells. Thus, our findings provide evidence that M1S and M2S have potent anticancer activity in KRAS-mutated A549 cells and p53 mutated H522 cells, both the compounds warrant further in vitro experimentation to elucidate their specific mechanism of action(s). I Acknowledgments Foremost, I would like to praise the Almighty for his consent to accomplish the journey. Then, my heartiest gratitude and deepest respect go to my mentor, Prof. Rhonda J. Rosengren, for her continuous support, expert guidance, motivation, enthusiasm, and sharing of immense knowledge to finish my MSc thesis. Your professionalism always teaches me how to handle difficult situations so easily. My special thanks to Dr. Muhammad Hanif for providing novel compounds that are key to explore novel drugs for the KRAS-mutant NSCLC line. I am also grateful to A/Prof. John Aston, Dr. Greg Giles, and Prof. Helen Nicholson for donating experimental cell lines. My appreciation also extends to my friendly lab colleagues (Zohaib, Mhairi, Abby, Ravneel, Nensi, Jessie, Risha, Lucy, Nayla, Matt, Yi Zhen) and postgrads (Steph, Jamie, Geetanjali, Amreen, Ben, Hayley, Helen, Ellie, Caitlin, Maddie, Fergus, Monica, and Louise) for their insightful comments, motivation, and guidance. Especially, I would like to thank Zohaib for teaching me core research skills, knowledge sharing, continuous motivation, and inspiration. I would also like to recall helpful guidance from Rumpa, Arpita, and Mhairi for Western blotting trouble shootings. I am thankful to Katie, Abby, and Mhairi for teaching data analysis for flow cytometry. I also acknowledge the valuable comments from Dr. Laura Burga and Dr. Belinda Cridge during lab meetings. I am highly indebted to Fred Fastier Summer Student Scholarship and the University of Otago, Masters Scholarship; these fundings facilitated my cancer research. I appreciate the continuous support from the Dept. of Pharmacology and Toxicology, its administrative staff, and especially the HOD (Prof. Michelle Glass). I want to express my gratitude to Adib who has been supportive to me through my ups and downs since the beginning. I am also grateful to OUSA members (especially Brony and Hahna) and BDSA members (especially Sabbir, Saif, Lutfur, Saadlee, Tajul, Supti) for their guidance and motivation during my hard times. I am thankful to my nice flatmates (Ifti, Rabbi, Maruf, and Riad) for taking care of me and offering delicious cuisine. Lastly, I recall all of my previous mentors, seniors, juniors, and peers especially Dr. Ashraf, Dr. Sattya, Papri, Sakib, Shihab, Dr. Shiplu, Forhad, Morshed, Yasir, Rayhan, Leon, Muhit and Achal who always inspire me. Above all, I am extremely grateful highly indebted to my family for their unconditional love and support throughout my life. I also acknowledge my beloved wife Niloy for her motivation that synthesised energy for my hard work. II Table of contents Abstract ...................................................................................................................................... i Acknowledgments ...................................................................................................................... i Table of contents ...................................................................................................................... iii List of figures ............................................................................................................................ v List of tables ............................................................................................................................. vi List of abbreviations ............................................................................................................... vii 1. Introduction ....................................................................................................................... 1 1.1 Lung cancer ......................................................................................................................... 1 1.1.1 Epidemiology and risk factors of lung cancer ........................................................ 1 1.1.2 Histological classification of lung cancer ............................................................... 2 1.1.3 KRAS mutation and NSCLC ................................................................................ 2 1.2 Therapeutic targets of KRAS-mutated NSCLC.................................................................... 3 1.2.1 Targeting RAS and associated pathways ............................................................... 3 1.2.2 Cell cycle as a therapeutic target for KRAS-mutant NSCLC ................................ 6 1.3 Novel metal-based drugs ...................................................................................................... 8 1.4 Organometallic arene complexes ........................................................................................ 11 1.5 Different drug classes investigated in the current study ..................................................... 12 1.5.1 HDAC inhibitors ................................................................................................. 12 1.5.2 Hydroxythiopyridone derivatives ........................................................................ 18 1.5.3 Metal-based PCA ligands and complexes ............................................................ 21 1.5.4 Kinetically inert metal(arene) complexes of PCA ................................................ 22 1.6 Aims of the current study ................................................................................................... 24 1.7 Hypothesis and objectives of the project............................................................................. 25 2 Materials and methods .................................................................................................... 26 2.1 Materials ............................................................................................................................ 26 2.1.1 Cell lines .............................................................................................................. 26 2.1.2 Chemicals ............................................................................................................ 26 2.1.3 Experimental drugs ............................................................................................. 27 2.2 Methods ............................................................................................................................. 27 2.2.1 Cell maintenance ................................................................................................. 27 2.2.2 Drug preparation ................................................................................................ 27 2.2.3 Cell cytotoxicity study using the SRB assay ......................................................... 27 2.2.4 Western blotting .................................................................................................. 28 2.2.5 Cell cycle analysis by flow cytometry................................................................... 30 III 2.2.6 3 Experimental data calculation and statistical analysis ......................................... 31 Results............................................................................................................................... 33 3.1 Cytotoxicity ........................................................................................................................ 33 3.1.1 Dose-response cytotoxicity of novel compounds in A549 cells .............................. 33 3.1.2 Dose-response cytotoxicity of novel potent compounds in H522 cells ................... 36 3.1.3 Dose-response cytotoxicity of M1S and M2S on NIH3T3 cells ............................. 38 3.1.4 Dose-response cytotoxicity of M1S and M2S on PNT1A cells .............................. 39 3.2 Time course cytotoxicity assessment in A549 and H522 cells .............................................. 40 3.3 Drug-mediated changes in protein levels ............................................................................ 41 3.3.1 Acetylated histone-H3 (Acetyl H3) ...................................................................... 41 3.3.2 Cyclin D1............................................................................................................. 42 3.3.3 Cyclin B1 ............................................................................................................. 44 3.4 Cell cycle analysis ............................................................................................................... 45 3.5 Summary of results ............................................................................................................ 49 4 Discussions ....................................................................................................................... 50 4.1 Rationale for using different cell lines ................................................................................ 50 4.1.1 A549 and H522 cell lines...................................................................................... 50 4.1.2 NIH3T3 cell line .................................................................................................. 52 4.1.3 PNT1A cell line.................................................................................................... 53 4.2 Critiques of experimental design and methods ................................................................... 53 4.2.1 SAHA as a control drug ...................................................................................... 53 4.2.2 Preclinical cytotoxicity study using the SRB assay............................................... 54 4.2.3 Western blotting .................................................................................................. 55 4.2.4 Flow cytometry.................................................................................................... 56 4.3 Interpretation of results ..................................................................................................... 57 4.3.1 Dose-response cytotoxicity study ......................................................................... 57 4.3.2 Time course of cytotoxicity .................................................................................. 63 4.3.3 Drug-mediated changes in protein levels ............................................................. 64 4.3.4 Effects of M1S and M2S in cell cycle ................................................................... 67 4.4 Conclusions and future directions ...................................................................................... 69 5 References ........................................................................................................................ 70 IV List of figures Figure 1.1: Region‐specific incidence rates by sex for lung cancer in 2018. ................................... 1 Figure 1.2: Ten most common mutated genes in NSCLC. ............................................................... 2 Figure 1.3: Potential therapeutic targets for KRAS-mutated NSCLC. .......................................... 4 Figure 1.4: Non-catalytic functions of cyclin D1. .............................................................................. 8 Figure 1.5: Various arene and organometallic metal arene structures. ......................................... 12 Figure 1.6. Histone deacetylase (HDAC) mechanism of action. ..................................................... 13 Figure 1.7: Multiple HDACi-activated antitumour pathways. ....................................................... 15 Figure 1.8: Novel HDAC inhibitors examined in this study. .......................................................... 17 Figure 1.9: Cellular uptake mechanism of thiol-reactive groups. .................................................. 19 Figure 1.10: Hydroxythiopyridone derivatives used in this study. ................................................. 20 Figure 1.11: Structure of metal-based PCA ligands and complexes. ............................................. 22 Figure 1.12: Kinetically inert metal(arene) complexes of PCA examined in this study. .............. 24 Figure 3.1: Cytotoxicity of SAHA, JAZZ-90, JAZZ-166, JAZZ-167, JAZZ-121, and ASH-122 in A549 cells. ..................................................................................................................................... 34 Figure 3.2: Cytotoxicity of M1S, M2S, M1S-Ru, M2S-Ru, ZR-012, and ZR-014 in A549 cells. . 35 Figure 3.3: Cytotoxicity of SAHA, JAZZ-90, M1S, M2S, M1S-Ru, and M2S-Ru in H522 cells.. 37 Figure 3.4: Cytotoxicity of M1S and M2S in NIH3T3 cells. ........................................................... 38 Figure 3.5: Cytotoxicity of M1S and M2S in PNT1A cells. ............................................................. 39 Figure 3.6: Time course cytotoxicity assessment of M1S and M2S in A549 and H522 cells. ....... 40 Figure 3.7: Effect of M1S and M2S on acetyl H3 expression in A549 and H522 cells. ................ 42 Figure 3.8: Effect of M1S and M2S on cyclin D1 expression in A549 and H522 cells. ................. 43 Figure 3.9: Effect of M1S and M2S on cyclin B1 expression in A549 and H522 cells................... 44 Figure 3.10: Cell cycle analysis in A549 cells exposed to M1S and M2S. ...................................... 46 Figure 3.11: Cell cycle analysis in H522 cells exposed to M1S and M2S. ...................................... 47 Figure 3.12: Apoptotic cells in sub-G1 phage in H522 cells after M1S and M2S treatment. ...... 48 V List of tables Table 1.1: Classification of HDACs .................................................................................................. 14 Table 3.1: EC50 values of different classes of drug candidates in A549 cells ................................ 36 Table 3.2: EC50 values of different classes of drug candidates in H522 cells ................................ 38 Table 3.3: Selectivity index (SI) of M1S and M2S ........................................................................... 39 Table 3.4: EC50 values of different classes of drug candidates in A549, H522, NIH3T3, and PNT1A cells ................................................................................................................................. 49 Table 3.5: Effects of M1S and M2S on cell signalling proteins and cell cycle .................... 49 Table 4.1: Diversities in KRAS mutation in NSCLC patients and cell lines.................................. 51 Table 4.2: Co-mutation frequency of KRAS gene in NSCLC patients .......................................... 52 VI List of abbreviations 10a 1-Phenyltriazolylethyl-3-hydroxypyridine-2-thione 10d 1-Phenyltriazolylpentyl-3-hydroxypyridine-2-thione 1-HPT 1-Hydroxypyridine-2-thiones 3-HP 3-Hydroxypyridin-2-one 3-HPT 3-Hydroxypyridin-2-thione 7k 4-((3-((4,6-Dioxo-2-thioxotetrahydropyrimidine-5(2H)-ylidene)methyl)2-methyl-1H-indol-1-yl)methyl)benzoate) 8i 5-Methoxy-2-(6-oxo-1-o-tolyl-1,6-dihydropyridine-3-carbonyl)phenyl propyl carbonate AASH-122 [Chlorido(η6-p-cymene)(N-(4-fluorophenyl)-2-pyridinecarbothioamide) ruthenium(II)]chloride ADC Adenocarcinoma ADM Adenomas AIB1 Amplified in breast cancer 1 AKT Protein kinase B AMP Adenosine monophosphate AMPK AMP activated protein kinase ANOVA Analysis of variance AR Androgen receptor ATP Adenosine triphosphate ATPis ATP-competitive inhibitors BCA 4, 4′-Dicarboxy-2, 2′-biquinoline acid BIM Bcl-2-like protein 11 bip Biphenyl BRCA2 Breast cancer 2 gene bz Benzene C/EBPβ CCAAT-enhancer-binding proteins β C1 2-(4-Hexyloxyphenyl)-5-(4-hydroxyphenyl)pyrimidine CBP CREB-binding protein CDKs Cyclin depended kinases CGMARN Cancer Genome Atlas Research Network VII cGMP Cyclic guanosine monophosphate CKIs Cyclin-dependent kinase inhibitors clogP Calculated logP c-Met A receptor tyrosine kinase belonging to the MET Cp Pentamethylcyclopentadienato cyp Cyclopentadienyl Cur Curcumin DHA Dihydroanthracene DMP1 Dentin matrix acidic phosphoprotein 1 DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DOX Doxorubicin EC50 The concentration of a drug that gives half-maximal response EDTA Ethylenediaminetetraacetic acid EGFR Epidermal growth factor receptor EGTA Ethylene glycol tetraacetic acid ERKs Extracellular signal-regulated kinases ERα Oestrogen receptor-α FAK Focal adhesion kinase FBS Foetal bovine serum FDA Food and Drug Administration FOXM1 Forkhead box transcription factor M1 FOXO1 Forkhead box protein O1 FOXO3a Forkhead box protein O3a FSC Forward scattered GDC Genome Data Commons GDP Guanosine diphosphate GRB2 Growth factor receptor-bound protein 2 GRIP1 Glutamate receptor-interacting protein 1 GTP Guanosine triphosphate HATs Histone acetyltransferases HC11 [(η6-Tetrahydroanthracene)Ru(ethylenediamine)Cl]PF6 VIII HDACis Histone deacetylase inhibitors HDACs Histone deacetylases HIF-1α Hypoxia-induced factor-1α HSP90 Heat-shock protein 90 IC50 The concentration of an inhibitor where the response is reduced by half IKK IkB kinase Ir Iridium JAHA Jay amin hydroxamic acid JAZZ-121 [Chlorido(η5-pentamethylcyclopentadienyl)(N-(4- fluorophenyl) pyridine-2-carbothioamide)rhodium(III)]chloride JAZZ-166 [Chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-(4(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)iridium(III)] chloride JAZZ-167 [Chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-(4(pyridine-2-carbothioamido- κ2N,S)phenyl)octanediamide) rhodium(III)]chloride JAZZ-90 N1-hydroxy-N8-(4-(pyridine-2-carbothioamido)phenyl)octanediamide KRAS Kirsten rat sarcoma L1 1,2-bis(diphenylphosphino)-benzene M1S 1-Benzyl-2-methyl-3-hydroxypyridin-4-(1H)-thione M1S-Ru Chlorido[3-oxo-1-benzyl-2-methylpyridin-4(1H)-thionato-κ2O,S](pcymene)ruthenium(II)] M2S 1-Ethylbenzyl-2-methyl-3-hydroxypyridin-4-thione M2S-Ru Chlorido[3-oxo-1,ethylbenzyl-2-methylpyridin-4(1H)-thionato-κ2O,S] (cymene) ruthenium(II) MAPK Mitogen-activated protein kinase MEK Mitogen-activated protein kinase kinase MI Mdm2 inhibitor M-OTf Metal triflates mTOR Mammalian target of rapamycin MTS [5-(3-Carboxymethoxyphenyl)-2-(4,5,-dimethylthiazolyl)-3-(4sulfophenyl)tetrazolium, inner salt] MTT 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide MW Molecular weight IX MYOD1 Myoblast determination protein 1 NAD Nicotinamide adenine dinucleotide NADP+ Nicotinamide adenine dinucleotide phosphate NCI National Cancer Institute NEUROD1 Neurogenic differentiation factor 1 NFKB Nuclear factor of the κ-chain in B-cells NRU Neutral red uptake NSCLC Non-small cell lung cancer Os Osmium OTf Trifluromethylsulfonate P/CAF3 CBP-associated factor 3 PCA Pyridine-2-carbothioamide p-cym p-cymene PDEδ Phosphodiesterase 6 delta PI Propidium iodide PI3K Phosphoinositide-3-kinase PLD1 Phospholipase D1 Plecstatin-1 [Chlorido(η6-p-cymene)(N-(4-fluorophenyl)-2-pyridinecarbothioamide) ruthenium(II)]chloride PMSF Phenylmethylsulfonyl fluoride PP1 Protein phosphatase 1 PPARγ Peroxisome proliferator-activated receptor-γ PPh3 Tri-phenyl phosphine PTEN Phosphatase and tensin PTMs Post-translational modifications PVDF Polyvinylidene difluoride QTc Corrected QT interval RAC A subfamily of the Rho family of GTPases RAF Rapidly accelerated fibrosarcoma RAL Ras-like RALGDS Ral guanine nucleotide dissociation stimulator RAS Rat sarcoma X RES Resazurin reduction Rh Rhodium RHOA Ras homologue family member A RNA Ribonucleic acid ROS Reactive oxygen species RTK Receptor tyrosine kinase Ru Ruthenium SAHA Suberoylanilide hydroxamic acid SAR Structure-activity relationship SCLC Small-cell lung carcinoma SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEM Standard error of the mean SH Thiol SI Selectivity index SIRT Sirtuin SOS Son of sevenless SRB Sulforhodamine B SRC1 Steroid receptor coactivator 1 SS Disulfide SSC Side scattered STAT3 Signal transducer and activator of transcription 3 TAF1 TATA binding protein-associated factor 1 TBP2 Thioredoxin binding protein 2 TCA Trichloroacetic acid TEMED Tetramethylene diamine THA Tetrahydroanthracene TIAM1 T-cell lymphoma invasion and metastasis inducing factor 1 TKIs Tyrosine kinase inhibitors TME Tumour microenvironment Tris HCl Trizma hydrochloride Trx Thioredoxin XI VEGF Vascular endothelial growth factor VEGFR2 Vascular endothelial growth factor receptor 2 WEE1 A tyrosine kinase WST-1 4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate XTT 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]2H-tetrazolium hydroxide ZBG Zinc-binding group ZR-012 [(Triphenylphosphine)(η6-p-cymene)(N-(4-chlorophenyl)pyridine-2carbothioamide)]ruthenium(II)triflate ZR-014 [(Triphenylphosphine)(η6-biphenyl)(N-(4-chlorophenyl)pyridine-2carbothioamide)]osmium(II)triflate XII Chapter 1 Introduction 1. Introduction 1.1 Lung cancer 1.1.1 Epidemiology and risk factors of lung cancer Lung cancer has the highest mortality and morbidity globally, with 1.38 million new cases and over one million deaths in the world annually [1]. According to GLOBOCAN 2018, lung cancer is the most frequently diagnosed cancer (11.6% of the total cases) in both males and females, and causes 18.4% of the total cancer deaths globally [2]. Lung cancer is more common in Polynesians and is rare among Western Africans (Figure 1.1). Didkowska and colleagues predicted that 3 million people will die globally due to lung cancer in the year 2035 [3]. Figure 1.1: Region‐specific incidence rates by sex for lung cancer in 2018. Rates are displayed in descending order of the world (W) age‐standardised rate among men, and the highest national rates among men and women are superimposed. Source: GLOBOCAN 2018. Adapted from Bray et al. [2]. Cigarette smoking is the greatest risk factor for the progression of lung cancer, other risk factors include age, race, gender, radon exposure, occupational exposures, environmental pollution, and pre-existing lung disease. However, people without any known risk factors can also develop 1 lung cancer [4]. It has been confirmed that genetic changes make some people susceptible to lung cancer [5]. Additionally, smokers respond differently towards lung carcinogens (only 15% of smokers develop lung cancer) and acquired genetic changes, for instance, current or exsmokers more frequently gain transversion mutations (substitution of a pyrimidine to a purine nucleotide or vice versa) while never smokers commonly have transition mutations (pyrimidine to pyrimidine or purine to purine nucleotide exchange) in the KRAS kirsten rat sarcoma (KRAS) viral oncogene homologue [5-7]. 1.1.2 Histological classification of lung cancer Lung cancer is a heterogeneous disease [8] and histologically is divided into two major classes: small-cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) [9]. SCLC is more aggressive and metastatic than NSCLC, which comprises around 12–15% of total lung cancers [9-11]. NSCLC accounts for at least 85-88% of all lung cancer cases and is divided into three subtypes: large cell carcinoma (10%), squamous cell carcinoma (30%) and adenocarcinoma (50%) [9]. 1.1.3 KRAS mutation and NSCLC KRAS mutations are placed third among the ten most frequent mutations in NSCLC patients according to the cBioPortal (https://www.cbioportal.org/) database [12, 13] that compute the published data on NSCLC (Figure 1.2) [14-20]. KRAS-mutant NSCLC patients show a shorter median survival compared to KRAS wild-type NSCLC patients [21-23]. Additionally, the KRAS-mutant patients are genetically heterogeneous with a high frequency of co-occurring mutations in lung cancer-linked pathways [24]. In NSCLC, KRAS mutations predominate in lung adenocarcinomas and rarely occur in squamous-cell cancers [25]. Frequency 80 Non-small cell lung cancer 60 40 20 p LR 53 P1 K B R K AS EA P PC 1 LO R E K LN M T K 2C M T2 FA D S T T1 K 11 0 Mutated Gene Figure 1.2: Ten most common mutated genes in NSCLC. This frequency distribution was calculated based on cBioPortal data on NSCLC patients (n=1872) [26]. 2 As the response of KRAS-mutant NSCLC to chemotherapy is not satisfactory, numerous novel therapeutic strategies including direct targeting of KRAS, targeting KRAS membrane associations, the use of KRAS synthetic lethality, targeting downstream signalling pathways, and immunotherapy have been tried as an alternative to chemotherapy [25]. However, clinically approved therapies for KRAS-mutant NSCLC have yet to be developed. 1.2 Therapeutic targets of KRAS-mutated NSCLC 1.2.1 Targeting RAS and associated pathways KRAS mutations are diagnosed in around 20-40% of NSCLC patients and occur frequently, but not exclusively, in adenocarcinoma and life-long smokers [27, 28]. In NSCLC KRAS is linked to poor prognosis and drug resistance [29]. The high frequency of KRAS mutations in NSCLC identifies it as a potential therapeutic target, however, KRAS remains an undruggable target as it is difficult to downregulate mutated KRAS-induced intracellular activity [30-34]. The development of drugs that target KRAS remains a key challenge in cancer research for the following reasons. Firstly, the activation and inactivation of KRAS is depended on its GTP or GDP binding status in preference to a substrate catalytic reaction [28, 35]. Secondly, KRAS has an affinity towards GTP at the picomolar level and cancer cells have micromolar levels of GTP [28]. Finally, KRAS lacks a suitable binding pocket for small molecule inhibitors, aside from the challenging nucleoside binding pockets [28]. Considering the key challenges, Figure 1.3 illustrates the possible therapeutic targets for KRAS-mutant NSCLC. RAS proteins must associate with the cell membrane to link with guanine nucleotide exchange factors and other upstream regulators to transfer the extracellular signal to the downstream molecules. RAS proteins are produced as soluble precursors, these soluble precursors then undergo post-translational modifications (PTMs) before they interact with cell membranes. PTMs are mediated by enzymes that are an interesting target for therapy [25]. KRA-533, a novel KRAS agonist, suppressed KRAS-mutant lung cancer cells both in vitro and in vivo [28]. KRA-533 specifically interacts with the GTP/GDP binding pocket of KRAS to inhibit GTP cleavage, thereby increasing active GTP-bound KRAS which induced apoptotic and autophagic cell signalling pathways in cancer cells. cGMP phosphodiesterase 6 delta (PDEδ) subunit protein has a hydrophobic pocket that interacts with a farnesylatedhydrphobic cysteine residue located at the C terminus of RAS proteins. Deltarasin, a small molecule inhibitor, interfered with the interaction of PDEδ with RAS proteins, thereby inhibiting activation of RAS. In vitro and in vivo investigations confirmed that deltarasin induced apoptosis and autophagy in KRASmutant lung cancer cells. After binding with PDEδ, deltarasin induced apoptosis through RAS downstream signalling pathways, while it induced autophagy via the AMPK (AMP activated 3 protein kinase)-mTOR (mammalian target of rapamycin) signalling pathway. Interestingly, 3methyl adenine (an autophagy inhibitor) enhanced deltarasin-mediated apoptosis through elevation of reactive oxygen species (ROS). On the other hand, the ROS scavenger Nacetylcysteine remarkably suppressed deltarasin-mediated cell death [36]. RTK Grb2 Sos c-MET RAS RAS RAF PI3Ks RALGDS TIAM1 MEK1/2 AKT RAL RAC ERK1/2 RhoA FAK Cyclin1 mTOR IKK IkB Cyclin D1 CDK4/6 PLD1 NFKB Therapeutic target for KRAS mutated NSCLC Figure 1.3: Potential therapeutic targets for KRAS-mutated NSCLC. Direct targeting of RAS protein or its downstream effector molecules linked with cell proliferation are potential therapeutic targets for KRAS-mutant NSCLC. RTK: receptor tyrosine kinase; GRB2: growth factor receptor-bound protein 2; SOS: son of sevenless; RAS: rat sarcoma; c-Met: a receptor tyrosine kinase belonging to the MET; RAF: rapidly accelerating fibrosarcoma; MEK: mitogen-activated protein kinase kinase; ERK: extracellular signal-regulated kinases; RHOA: ras homologue family member A; FAK: focal adhesion kinase; phosphatidylinositol-3-kinase (PI3K); AKT: Protein kinase B; mTOR: mammalian target of rapamycin; IKK: IkB kinase; NFKB: nuclear factor of the κ-chain in B-cells; RALGDS: Ral guanine nucleotide dissociation stimulator; RAL: Ras-like; PLD1: phospholipase D1; TIAM1: T-cell lymphoma invasion and metastasis inducing factor 1; RAC: a subfamily of the Rho family of GTPases. Information for the figure was taken from Roman et al. [21]. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs, e.g. gefitinib and erlotinib) suppress the RAS-RAF (rapidly accelerating fibrosarcoma)-MEK (mitogen-activated protein kinase kinase)-ERK (extracellular signal-regulated kinases) signalling pathway by inhibiting the catalytic activity of EGFR. However, the TKIs fail to inhibit the signalling cascades in the presence of KRAS mutation as RAS remains downstream to EGFR [37]. Antibody-based small molecule ligands interact with mutant KRAS (at sub-μM affinity), thereby inhibit RAS-effector protein-protein interactions and endogenous RAS-linked signalling in cancer cells of human origin including lung cancer cells [38]. RAS mutations 4 (particularly KRAS) leads to hyperactivation of RAF-MEK-ERK signalling [39]. For example, KYA1797K is an antiproliferative agent that effectively suppressed KRAS-mediated tumourigenesis by inhibiting the RAS-ERK pathway in a KRASLA2 mouse model [37]. Another therapeutic strategy is to target MEK as a downstream effector of RAS [39]. Kelly et al. reported that the combination of trametinib (a MEK inhibitor) and pemetrexed (an antimetabolite used in NSCLC treatment) produced a therapeutic benefit in 50% of KRAS-mutant NSCLC patients (n=20) with a disease control rate 65% of in a phase I/Ib study. These patients experienced fatigue, nausea, and peripheral oedema due to the side effects of the combination [40]. Additionally, selumetinib, a MEK inhibitor, suppressed the growth of NSCLC cells in a dosedepended manner while the growth inhibitory activity was accelerated by co-treatment with LY2228820 (a p38 inhibitor) [41]. Genetically modified mouse models of KRASG12C lung tumours displayed significantly higher sensitivity to selumetinib in comparison to KRASG12D tumours. MEK inhibition also enhanced chemotherapeutic activity and significantly promoted progression-free survival in KRASG12C mice. However, the co-mutation of p53 rendered KRASG12C lung tumours less responsive to combination treatment with selumetinib and chemotherapy [42]. Furthermore, small interfering RNAs towards MAPK14 that encodes p38α MAPK, accelerated the growth inhibitory potential of MEK inhibitors [41]. On the other hand, preclinical studies showed that phosphatidylinositol-3-kinase (PI3K) inhibitors are inefficient in KRAS mutations since the RAS-ERK signalling cascade hijacks tumour proliferation via a compensatory mechanism [21]. Combination therapy with mTOR inhibitor AZD2014 and WEE1 tyrosine kinase inhibitor AZD1775 exhibited synergistic and selective antiproliferative activity against KRAS-mutant NSCLC cell lines, which delayed the growth of human NSCLC xenografts and regressed murine adenocarcinoma. Furthermore, mTOR inhibition potentiated WEE1 inhibition through inactivation of DNA repair, accelerating DNA damage in KRAS-mutant NSCLC, and reduced cyclin D1 [43]. In addition, forkhead box transcription factor M1 (FOXM1) is a crucial transcription factor for KRAS-ERK signalling pathway in respiratory epithelial cells. FOXM1 is also involved in oncogenic KRAS-ERK signalling in neoplastic lung epithelial cells. FOXM1 deletion suppressed the KRASG12D activated lung tumourigenesis. Therefore, it is postulated that pharmacological inhibition of FOXM1 could be beneficial for KRAS-mutant lung cancer patients [44]. KRAS mutation inactivates tumour suppressor genes INK4a/ARF/p16 leading to hyperactivation of the GTPase RHOA (ras homologue family member A) by MEK1/2 and ERK1/2 and RHOA-FAK (focal adhesion kinase) signalling cascade is involved in cell migration. Currently, defactinib, a FAK inhibitor is under clinical trial in KRAS-mutant NSCLC [21]. 5 1.2.2 Cell cycle as a therapeutic target for KRAS-mutant NSCLC Cell cycle occurs through different phases including G0/G1 (Gap phase), S (DNA synthesis phase), and G2/M (cell division) [45]. In the G1 phase, cells examine the internal and external environment to ensure conditions are favourable and complete the preparation before proceeding to the S and M phases [46]. Each stage of the cell cycle is controlled by the activity of a unique combination of cyclins and cyclin depended kinases (CDKs). The level of cyclins fluctuate throughout the cell cycle, which is primarily controlled by transcriptional activation and proteolytic degradation [47]. The net effect controls the “on” or “off” switches of CDK proteins and thereby regulates the stages of the cell cycle [48]. Additionally, the cyclin-CDK complexes often bind to cyclin-dependent kinase inhibitors (CKIs) such as p15, p18, p21, and p27, which inhibits the activity of CDKs, and control cell cycle progression [49]. The efficiency of an antiproliferative drug depends on the ability to stop the division of cancer cells by cell cycle arrest in the aforementioned phases [45]. The G1 cyclins consist of D-type cyclins such as cyclins D1, D2, and D3. G1 cyclins act in the early G1 phase of the cell cycle along with CDK4 and CDK6 [50]. G1 cyclins are relatively unstable when not bound with their catalytic partner (CDK). Cellular level of G1 cyclins remain low in the G0 phase and increase gradually upon the activity of mitogens [51]. Cyclin D1, D2, and D3 are almost indistinguishable biochemically and only cyclin D1 overexpression is prominent in cancer [52]. Compared to cyclin D2 and cyclin D3, cyclin D1 has greater functional characterisation and plays a widespread role in human cancers including lung cancer [53, 54]. Mitogens activate cyclin D1 through a complex mechanism that occurs at transcriptional and posttranscriptional levels. At the transcriptional level, growth factor-induced activation of cyclin D1 is dependent on the RAS-RAF-MEK-ERK signalling pathways [51]. Furthermore, disruption of the G1-S checkpoints leads to uncontrolled cell division and cancer progression [55]. Cyclin D1 together with CDK4/6 and cyclin E/CDK2 inactive the retinoblastoma protein through phosphorylation, thereby committing the cell to progress into DNA replication and mitosis [55, 56]. Cyclin D1 overexpression is linked to the early onset of cancer and enhance the risk of cancer progression and metastasis [56]. Cyclins A, D, and E control the transition from Gl phase to S phase, while cyclins A and B regulate the passage from the G2 phase to M phase [57]. G1 to S phase progression is not only dependent on the cyclins and CDK activity but also dependent on the down‐regulation of CDK inhibitors, such as p27 and p21 [58]. In mammalian cells, cyclin B1 and Cdk1 expression occurs in late S and G2 phases and are active at the late G2 phase. Cyclin B1 is synthesised in the cytoplasm during S phase and imported to the nucleus at the late G2 phase, and degraded during anaphase through 6 a ubiquitin-related pathway [59]. KRAS-mutant lung cancer cells are susceptible to impairment of cell cycle control, as they have a particular requirement for kinases that control key components of cell cycle transition [60]. Among the driver’s of cyclin/CDK complexes, cyclin D1/CDK4/6 and cyclin B1/Cdc2 have drawn considerable attention as cell cycle regulator as the former controls G1-S-phase transition while the latter regulates the G2-M-phase checkpoint [61]. Puyol et al. reported that there is a synthetic lethal interaction between CDK4 and KRAS oncogene in a mouse tumour model which mimics human NSCLC. Briefly, inhibition of CDK4 activity either by genetic knockdown or CDK4 selective inhibitor (e.g. PD0332991) in the mice carrying KRASG12V lung tumours halted tumour progression and confirmed the role of CDK4 in KRAS-mutant lung cancer. Therefore, targeting CDK4 could be a potent therapeutic target for KRAS-mutant NSCLC [62]. There is a positive correlation between KRAS mutation and CKD4/cyclin D levels in lung cancer patients. Therefore, ATP-competitive inhibitors (ATPis) of CDK4 could be used in the treatment of cancer patients, unfortunately, these inhibitors have limited specificity and efficacy. Targeting the interaction of CDK4 and cyclin D is an alternative to the ATPis. Abemaciclib (a competitive inhibitor of ATP) in combination with staple peptide. Staple peptide rapidly and efficiently enters into the cultured cells and inhibits CDK4 catalytic activity by colocalising with CDK4 and cyclin D1, thereby suppressed KRAS-mutant orthotopic NSCLC tumours [63]. Cyclin D1 is not only involved in cell cycle progression at the G1 checkpoint but also in the regulation of apoptosis and growth suppression by binding with several transcription factor families (Figure 1.4) [53, 64]. Multifunctional activities of cyclin D1 could influence the therapeutic response of NSCLC patients (cyclin D1 positive) [64]. KRAS-mutant NSCLC patients having low cyclin D1 expression showed a better prognosis compared to patients having high cyclin D1 expression (median overall survival 41.7 vs 3.5 months, p=0.037) [65]. Like cyclin D1, cyclin B1 is also overexpressed in KRAS mutations [66]. Cyclin B1 not only controls G2/M phase progression but is also involved in differentiation, cell growth, metastasis, and apoptosis in many cancers including lung cancer [67, 68]. The knockdown of cyclin B1 suppressed the proliferation of cancer cells in both in vitro and in vivo models [69]. Additionally, purvalanol A, a highly selective inhibitor of CDK1 (cyclin B1 partner), accelerated taxol-induced apoptosis, inhibited colony formation, and cell proliferation in NCIH1299 NSCLC cells [70]. Additionally, NSCLC patients with cyclin B1 positive tumours showed shorter survival time compared to cyclin B1 negative tumours [57]. Therefore, cyclin 7 B1 has become a novel prognostic marker and potential therapeutic target for NSCLC [61]. P27 Cyclin D1 BRCA2, RAD51 and p21 Migration PPARγ Adipogenesis Transcriptional regulation Chromatin modification Erα, SRC1, GRIP1 and AIB1 Cell cycle progression DNA damage response Nuclear hormone receptors STAT3, DMP1 BMYM and TAFII250 Cell cycle progression NEROD, C/EBPβ and MYOD AR HATs, PCAF and P300/CBP Androgen receptor –dependent cell cycle progression HDACs C/EBPβ Differentiation Tissue-specific differentiation Cell growth Figure 1.4: Non-catalytic functions of cyclin D1. Although p21 and p27 are constituents of CDK4 or CDK6 complexes, cyclin D1 binding to p21 or p27 leads to effects on migration and response to DNA damage, respectively. The DNA damage responses can be achieved via interactions with RAD51 and breast cancer 2 gene (BRCA2). Additionally, cyclin D1 regulates cell proliferation, cell growth, and differentiation by binding with nuclear hormone receptor family members (oestrogen receptor-α (ERα), androgen receptor (AR) and peroxisome proliferator-activated receptor-γ (PPARγ) and their coactivators (SRC1, GRIP1, and AIB1), BMYB and the MYB-related transcription factor DMP1, as well as the helix–loop–helix transcription factors NEUROD1, MYOD, and C/EBPβ. Furthermore, cyclin D1 binds with histone acetyltransferases (HATs) such as P/CAF3, p300/CBP, and histone deacetylases (HDACs). Generalised effects of cyclin D1 on transcription results from its binding to TAFII250 (also known as TAF1), a subunit of the basal transcriptional machinery. SRC1: steriod receptor coactivator 1; AIB1: amplified in breast cancer 1; GRIP1: glutamate receptor-interacting protein 1; NEUROD1: neurogenic differentiation factor 1; MYOD: myoblast determination protein 1; C/EBPβ: CCAATenhancer-binding proteins β; P/CAF3: CBP-associated factor 3; STAT3: signal transducer and activator of transcription 3. Information for the figure was taken from Musgrove et al. [53]. 1.3 Novel metal-based drugs Transition metal complexes are found in a diverse range of novel diagnostic and therapeutic agents, including anticancer drugs [71]. Metal-based drugs became a focus of potential cancer therapeutic agents after the discovery of platinum drugs (such as cisplatin, oxaliplatin, and carboplatin) [72, 73]. The growing interest of metal-incorporated complexes in cancer therapy is due to unique properties of metals such as charge variation, geometry, variable coordination modes, lewis acid property, redox activity and reactivity towards organic substrates [74, 75]. Interestingly, metals form positively charged ions in aqueous solution which can interact with the negatively charged biomolecules. The charge can be manipulated based on coordination molecules, leading to the generation of cationic, anionic, or neutral species [73]. Furthermore, the lewis acid properties of metal ions characterised by high electron affinity can remarkably polarise groups that are linked with them, thus promoting hydrolysis reactions [73, 74]. It has 8 been reported that single charged cations are generally more toxic, and alkyl group linked metals displayed higher toxicity compared to the aryl group [76]. Another feature of metals is their redox potential that makes them a suitable candidate to target cancer cells with disturbed redox homeostasis [77]. Additionally, organometallic complexes are “pro-drugs” that can be activated by the cellular environment (by redox reactions or ligand exchange), and the ligands may act as the active drug [75, 78]. The special features of metals become an attractive probe in metal-based drug design via ligand modification and substitution of the available chemical structures or novel structures that selectively interact with the cellular target with an improved cytotoxic and pharmacokinetic profile [74]. Platinum-based metallodrugs, cisplatin, and its analogue have been prescribed for lung cancer treatment. However, noticeable side effects and tumour resistance have limited their clinical application [79]. Many platinum-based drugs have been investigated as an alternative to cisplatin, however, due to the lack of a superior advantage platinum drugs including spiroplatin, miboplatin, sebriplatin, cycloplatam, SPI-077, BBR3464, SPI-077, aroplatin, TRK-710, iproplatin, enloplatin, zeniplatin, ormaplatin, NSC 170898 and JM 11 have been discontinued from clinical trials [74]. Therefore, rhodium (Rh), iridium (Ir), ruthenium (Ru), and osmium (Os) based compounds have been considered as an alternative to platinum-based compounds [80, 81]. Rh is one of the noble, rare, and precious transition metals of the platinum metals group [82]. Rh complexes have drawn much attention due to their synthetic versatility and a range of potential biomedical applications including antimicrobial and anticancer agents [83, 84]. Organorhodium complexes are present in various oxidation states and are reported to inhibit cell division by disrupting DNA synthesis and act as cytostatic agents before inducing cell death [83]. Many Rh compounds exhibited antiproliferative activity against Lewis lung carcinoma, lung metastatic tumours, B16 melanoma, Ehrlich ascitic tumours, oral carcinoma, P388 lymphocytic leukaemia, L1210 leukaemia, and MCa mammary carcinoma among others [85, 86]. Rh (III) complexes are the most explored organorhodium [83]. Rh (III) metalloinsertors exhibited anticancer activity at low micromolar concentration against HCT116O and HCT116N isogenic colon cancer cell lines. The potential mechanism was their binding with DNA mismatches and thereby disruption of DNA synthesis [87]. Specifically, pentamethylcyclopentadienato (Cp) arene ring containing half sandwich Rh curcumin (cur) complex [Rh(*Cp)Cl(cur)] displayed antiproliferative activity against human epithelial A549 lung cancer cell lines with an IC50 value of 31 μM [88]. Due to promising anticancer potential, many Rh complexes have entered the phase I clinical trials [86]. 9 Ir is found in four oxygenation states (II, III, IV, and VI) and has excellent electrical and thermal properties [89]. In recent times, Ir based compounds have been explored extensively for medical and biological applications including anticancer therapy [90]. Ma et al. tested four half sandwich Ir (III) complexes against A549 cells, two of them (IC50 values of <4.3 μM) showed promising antiproliferative activity compared to cisplatin which exhibited an IC50 value of 21.3 μM [91]. Liu and colleagues examined the growth inhibitory activity of eight halfsandwich cyclopentadienyl Ir (III) pyridine complexes and all the compounds displayed promising antiproliferative activity against A549, A2780 ovarian, and MCF7 breast cancer cells. Among these, the 4-dimethylaminopyridine based complex exhibited the highest anticancer activity in the submicromolar range and was more potent than cisplatin [92]. Ru based complexes showed quite promising pharmacological behaviour along with an acceptable toxicological profile [93]. Ru containing new chemotherapeutic agents in cancer treatment have drawn considerable attention due to their iron mimicking properties (i.e., bind to plasma proteins including transferrin, albumin), strong interaction with biomolecules such as proteins, RNA and DNA, the presence of two oxidation states (II and III), ligand exchange kinetics, low toxicity and non-cross resistance with conventional platinum-based drugs [79, 9496]. Three ruthenium-based drugs namely NAMI-A, KP1019, and NKP-1333 (a sodium salt of KP1019) have reached clinical trials [79, 97]. NAMI-A was the first Ru compound used in clinical practice. It was proven to be effective against metastatic lung cancer. Recently, NAMIA was used in combination with gemcitabine (a deoxycytidine analogue) as a second-line chemotherapy in managing advanced NSCLC [89, 98]. Os is the densest metal and is slightly denser than Ir [99]. The three-dimensional configuration of the Os compounds (generally octahedral with 6 ligands) offers a versatile platform for the selective identification and interaction with therapeutic targets (e.g. protein, DNA). Os compounds have different oxidation states such as Os (II), Os (III), Os (IV), and even higher, diverse oxidation states allows Os to regulate biological redox in cancer cells [100]. Additionally, Os displayed antiproliferative activity in a variety of oxidative states and it possessed oxidative stress via a multi-targeted mechanism that mitigated the chance of drug resistance [101]. It has been reported that the Os complex FY26 exhibited submicromolar anticancer activity towards A549, MCF7, A2780, and HCT116 (colon cancer) cell lines [80]. Furthermore, trans-[OsIIICl2(pyrazole)4]Cl displayed antiproliferative activity in the micromolar range against A549, CH1 (ovarian cancer), and SW480 (colon cancer) cell lines while the corresponding Ru analogue was less potent against these three cell lines [102]. Buchel and colleagues reported that KP1019 (a Ru based antiproliferative drug in phase I clinical trials) 10 was inactive towards A549 cells while its Os (IV) analogue exhibited anticancer activity [103]. 1.4 Organometallic arene complexes Organometallic transition metal-arene complexes have drawn considerable attention as anticancer drug candidates. The metal-carbon bond and π-bound arene bond in metal-arene complexes offers high reactivity and kinetic stability [104]. The arene substituent shares a lipophilic surface which facilitates cellular diffusion at the lipophilic cell membrane [105]. The hydrophobicity and hydrophilicity of the metal-arene complexes can be tuned via a metal-arene system, resulting in selective uptake into cancer cells [104]. Additionally, coordination of the organic compound to a metal center offers stability and facilitates the unique chemical space that cannot be attained by the organic molecule itself. Furthermore, metal coordination facilitates the outreach of the organic compound in the intracellular region [106]. Recently, metal-arene complexes were reported to have promising anticancer activity [90, 95, 107, 108]. For instance, novel organometallic tetranuclear Ru (II) arene complexes, [Ru4(η6-pcymene)4(benzil)(Cl)6] and [Ru4(η6-p-cymene)4(oxalaldehyde)(Cl)6] possessed both anticancer and antimetastatic activity against cisplatin resistant A549 lung cancer cells [95]. In another study, the anticancer potential of six half-sandwich luminescent Ir and Ru complexes containing P^P-chelating ligands 1,2-bis(diphenylphosphino)-benzene (L1) and 1,8-bis- (diphenylphosphino)-naphthalene were examined in A549 and HeLa (human cervical cancer) cells. All the compounds showed a more potent effect compared with cisplatin in both cell lines. The most potent drug [(η5-Cpxbiph)Ir(L1)Cl]PF6 (where, Cpxbiph=1-(4-biphenylyl)-2,3,4,5tetramethyl-1,3-cyclopentadiene) was 73 times more efficient than cisplatin towards A549 cells [90]. Furthermore, some half-sandwich piano-stool Os (II) complexes displayed considerable anticancer activity in vitro without cisplatin cross-resistance [109-111]. Additionally, Peacock and coworkers tested the anticancer potential of piano stool Os (II) arene complexes with the general formula [(η6-arene)Os(N,O)Cl] against A549 and A2790 (ovarian cancer) cell lines. Three of biphenyl)Os(picolinate)Cl] these compounds, [(η6-p-cymene)Os(picolinate)Cl], [(η6and [(η6-p-cymene)Os(8-hydroxyquinolinate)Cl] showed anticancer activity comparable to the platinum-based drug carboplatin [112]. Gatti et al. synthesised two structurally similar novel half-sandwich Os (II) and two Ru (II) thiocarbazone complexes that showed antiproliferative activity against A549, A2780, A2780i cisplatinresistant ovarian, PC3 prostate and HCT116 cancer cells [107]. Three Os (II) arene complexes bearing phenylazopyridine ligands with dimethylaminyl or hydroxyl substitution on the phenyl ring and chloride iodide leaving groups displayed concentration- (submicromolar to micromolar range) and time-dependent toxicity against A549 cancer cells. These compounds 11 were also reported to have nanomolar anticancer activity towards human breast, colon, ovarian, prostate, and bladder cancer cell lines [108]. Diverse structure and ligand orientation in half-sandwich complexes are an attractive choice in novel drug development [113]. Metal-based half sandwich piano-stool type arene complexes offer a wide range of chemical modifications to the arene (such as η5-C5H5, η6-C6H6) and its substituents (R), the monodentate leaving group (X), the ligands Y and Z (can be chelating or monodentate ligands), and net charge of the metal complex (n+) (Figure 1.5). Fine-tuning of structural architecture, kinetics, and thermodynamics of the system is a sensible drug design alternative to platinum-based drugs [114-117]. The fine-tuning offers desired chemical reactivity and pharmacological properties such as cellular uptake, biomolecular interactions, distribution, cytotoxic effects, and detoxification mechanisms [117, 118]. benzene (bz) p-cymene (p-cym) biphenyl (bip) cyclopentadienyl (cyp) n+ dihydroanthracene (DHA) tetrahydroanthracene (THA) orgnometallic metal (M)-arene Figure 1.5: Various arene and organometallic metal arene structures. In the organometallic Marene complex bz is used as a representative of arene (p-cym, bip, cyp, DHA, THA). In this piano-stool type M-arene complex X is the leaving group, Y and Z are chelating or monodentate ligand. 1.5 Different drug classes investigated in the current study 1.5.1 HDAC inhibitors Epigenetics studies heritable changes in gene expression that are not exerted by the alteration of genomic code [119, 120]. Three interconnected epigenetic processes control gene expression at the chromatin level including DNA methylation, nucleosomal remodeling, and covalent modifications of histone [120]. PTMs of the N-terminal end of the histone proteins occurs through acetylation, methylation, phosphorylation, deamination, ubiquitination, sumoylation, proline isomerisation, and ADP ribosylation [121]. Histone modifications via acetylation plays a major role in epigenetic regulation of gene expression which is controlled through the balance between histone acetyltransferases (HAT) and histone deacetylases (HDAC) [122]. In many 12 cancer cells including NSCLC, the balance between HAT and HDAC is disrupted (Figure 1.6) [120, 123]. Figure 1.6. Histone deacetylase (HDAC) mechanism of action. NSCLC: non-small cell lung cancer; HAT: histone acetyltransferase [124]. Eighteen human HDAC enzymes are divided into four classes based on their homology with yeast HDACs [125]. Class I, II, and IV are Zn2+ -dependent on metalloproteins, while class III is a nicotinamide adenine dinucleotide (NAD+)-dependent enzyme (Table 1.1) [126]. Class I comprises HDACs 1, 2, 3 and 8 that are located in the nucleus; class II includes HDACs 4, 5, 7, 9, and 10, which are located both the cytoplasm and nucleus; class IV comprises HDAC 11. Unlike conventional HDACs, class III is composed of seven mammalian sirtuins (SIRT1-7) [125]. The acetylation of histones in the nucleosomes alters chromatin conformation and thereby regulates gene expression. It has been reported that the aberrantly overexpressed HDACs in different tumours leads to carcinogenesis, cancer progression, and poor therapeutic outcome. Therefore, HDAC can be a therapeutic target to reverse the genetic changes that are linked with cancer progression and drug resistance [127]. Histone deacetylase inhibitors (HDACis) based dual molecular targeted therapy could be a promising therapeutic approach for KRAS-mutant NSCLCs [128, 129]. An HDACi (belinostat) in combination with a MEK inhibitor (trametinib) exhibited synergistic activity towards RAS mutated lung cancer cells via an increase of forkhead box protein O1 (FOXO1), forkhead box 13 protein O3a (FOXO3a), bcl-2-like protein 11 (BIM), cell cycle inhibitors (p21Cip1 and p27 Kip1) and acetyl H3 level [128]. Suberoylanilide hydroxamic acid (SAHA) showed antiproliferative activity against the KRAS-mutant A549 cells, it also enhanced the efficacy of carboplatin and paclitaxel in the same cell line [130]. Table 1.1: Classification of HDACs Group Classical (Zn dependent) Class Homology to yeast Class I Rpd3 Class IIa Had1 Class II b NAD Dependent Class IV Had1 Rpd3/Had1 Class III Sir2 Name HDAC 1 HDAC 2 HDAC 3 HDAC 8 HDAC 4 HDAC 5 HDAC 7 HDAC 9 HDAC 6 HDAC 10 HDAC 11 SIRT (1-7) Subcellular location Location in body Nucleus Ubiquitous Nucleus/ cytoplasm Tissue specific Cytoplasm Tissue specific Tissue specific Tissue specific Nucleus/ cytoplasm Nucleus/ cytoplasm/ mitochondria HDAC: histone deacetylase; NAD: nicotinamide adenine dinucleotide; SIRT=sirtuin [120, 126, 131133]. HDACis are relatively novel antiproliferative agents that target zinc domain to induce apoptosis, cell death, and cell cycle arrest in cancer cells through epigenetic and non-epigenetic regulation (Figure 1.7) [125, 134]. Class I, II, and IV HDACis contain a metal-binding moiety in the protein active site, an alkyl linker which is encompassed by a hydrophobic tunnel, and a capping group that is exposed to the surroundings. The metal-binding moiety is structurally similar in HDAC class I, II, and IV and is crucial for HDACi activity [135]. HDACis are categorised based on the chemical structure into hydroxamates (e.g. SAHA), benzamides (e.g. entinostat), aliphatic acids (e.g. phenylbutyrate) and cyclic peptides (e.g. romidepsin). These HDACis are active at a wide range of concentrations, millimolar to micro and nanomolar values for butyrates, entinostat/SAHA, and trichostatin A/tetrapeptides, respectively [134]. Hydroxamic acid is a potent moiety in the field of cancer therapy. Compared to other hydroxamic acid derivatives, SAHA has drawn considerable attention as a potent anticancer agent [136]. SAHA has a broad range of HDAC inhibitory activity, specifically, it inhibits class I and class II. The transcriptional effects of SAHA could be exerted either by direct binding 14 with HDAC or indirectly acting on the transcriptional factors such as E2F-1, Smad 7, YY-1, p53, GATA-1, and Bcl-2. The non-transcriptional effects of SAHA include inhibition of angiogenesis, cell cycle arrest, apoptosis, and downregulation of immunosuppressive interleukins [138]. Following FDA approved doses the most common side effects include fatigue and gastrointestinal symptoms (e.g. diarrhoea, anorexia, and nausea) [138-140]. Thrombocytopaenia, severe anaemia, pulmonary oembolism, dehydration, and squamous cell carcinoma are the life treating side effects that require specialised care and hospitalisation [138, 140]. Additionally, corrected QT interval (QTc) prolongation has also been reported for some patients after SAHA treatment. SAHA (category D drug in pregnancy) could have a potential risk when used by pregnant women, as SAHA crosses the placenta and could harm the developing foetus [138]. SAHA is more effective against haematological malignancy than solid tumours. To overcome this therapeutic barrier SAHA is used in combination with other anticancer agents in clinical trials. However, due to the unpredictable drug-drug interaction and pharmacokinetic profile, the desired therapeutic effect could not be achieved by combination treatment [141]. HDACi • • Growth arrest • • Intrinsic pathway • • • p21 Cyclins • apoptosis HDACs Anti-apoptotic factors Pro-apoptotic factors Mitrochondrial transmembrane potential Death receptors Death receptors ligands HIF-1α function VEGF Autophagic cell death Autophgic vacuoles Mitotic cell death • • Histone acetylation Mitotic failure Senescence • • Extrinsic apoptosis pathway • • Anti-angiogenesis ROS facilitated cell death • • • ROS Trx TBP2 Polyploidy Cell cycle withdrawal HDAC-6 related effects • • • • HSP90 function PP1 activation Akt function Tubulin acetylation Figure 1.7: Multiple HDACi-activated antitumour pathways. HDAC6: histone deacetylase 6; HIF1α: hypoxia-induced factor-1α; HSP90: heat-shock protein 90; PP1: protein phosphatase 1; ROS: reactive oxygen species; TBP2: thioredoxin binding protein 2; Trx: thioredoxin; VEGF: vascular endothelial growth factor. Information for the figure was taken from Xu et al. [137]. Toxicity and efficacy of HDACis are a major concern, both of them are at least partially due to the failure of the target-specific delivery of HDACis [135]. As only a few isoform-selective 15 HDACis have been reported and the clinical toxicity of HDACis is poorly characterised. Furthermore, the sequence similarity at the active site of the isoform is another concern in the design of selective HDACis [142]. Structure-activity relationship (SAR) studies, especially on SAHA’s linker area remains relatively unexplored, even though the linker region could influence efficacy [142]. C2, C3, C6-SAHA libraries summarised that the steric effect on the SAHA carbon linker reduced inhibitory activity and influenced selectivity [142-144]. Crebinostat, a hydroxamic acid-based HDACi, differs from SAHA in the length of aliphatic linker (n) and cap moiety. Variation in the linker length (n=4-6), crebinostat (n=5) analogues showed different selectivity and inhibitory activity towards HDAC 1, 2, or 3. Specifically, as the carbon number in the linker region increased the HDAC 1, 2, or 3 inhibitory potential of the crebinostat analogue [145]. Zong et al. modified SAHA through the addition of an azido linker with the hydroxamic acid group either an ester (SAHA-eAzide, ester-linker-modified SAHA) or amide bond (SAHA-aAzide, amide-linker-modified SAHA). SAHA alone and ester-linkermodified SAHA (i.e. SAHA-eAzide) increased apoptosis and induced hyperacetylation of histone protein in comparison to control. Interestingly, SAHA-eAzide failed to improve the apoptotic actions of SAHA. On the other hand, the amide modified SAHA (i.e. SAHA-aAzide) did not promote histone hyperacetylation or apoptosis, indicating that such chemical modification inactivates SAHA [135]. From docking studies, it has been reported that the linker group connects the ZBG and cap group. The ZBG chelates the Zn2+ at the active site while the cap interacts with the active site. Modification of the cap group could be an effective strategy to regulate the HDAC inhibitory activity by controlling the affinity of the compounds towards the surface group of the HDAC enzymes [141]. Spencer and colleagues reported that the threedimensional tuning of the aryl “cap” of SAHA is a promising strategy that broadens the potential in HDACi design [146]. Modification of the cap group of the SAHA analogue showed improved HDAC inhibitory activity and selectivity [141]. Jay amin hydroxamic acid (JAHA) and homo-JAHA are metal-based analogues of SAHA that incorporate the ferrocene unit in the structure (at the cap site of SAHA). Like SAHA, the metal-based analogues exhibited broad inhibitory activity toward class I HDACs, including HDAC 8. Compared to SAHA, homoJAHA was more effective against HDAC 8 and less potent towards HDAC 6 [146]. Since the application of JAHA, several metal-based analogues, including Rh and Ir complexes, have appeared [147, 148]. Amide-based molecules have drawn the attention in drug research due to their biocompatibility and potential biological activities including anticancer, antibacterial, insecticidal, fungicidal, and herbicidal actions. An amide linkage (-CO-NH-), is a core structural unit that connects the amino acids in the protein and is pervasive in nature [149]. Pyridine-2-carbothioamide (PCA) 16 contains an amide bond in its structure. PCA could act as a bidentate chelating agent through the pyridine nitrogen and either the nitrogen or the sulfur of the carbothioamide group [150]. PCA derivatives have shown promise as antimicrobial and antiproliferative agents [151, 152]. N-(8-quinolyl)pyridine-2-carboxamide, a PCA based compound that displayed significant antiproliferative activities towards the HL60 (human leukaemia), P388 (murine leukaemia), BEL7402 (human hepatocellular carcinoma) and A549 cell lines [151]. Multitargeted antiproliferative agents developed through a combination of more than one bioactive moiety may show synergistic activity [153]. Based on this idea, PCA and hydroxamic acid (a moiety of SAHA) were combined by our collaborators to synthesise N1-hydroxy-N8-(4(pyridine-2-carbothioamido)phenyl)octanediamide (JAZZ-90), a novel hydroxamate that differs from SAHA in the length of the aliphatic linker and the cap, modified with the addition of PCA with the phenyl group of SAHA [153]. [Chlorido(η5-pentamethylcyclopentadienyl)(N1 -hydroxy-N8- (4- (pyridine-2-carbothioamido-κ2N,S) phenyl) octanediamide) iridium (III)] chloride (JAZZ-166) and [chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-(4- (pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)rhodium(III)]chloride (JAZZ-167) are the Ir and Rh complexes of JAZZ-90, respectively (Figure 1.8). JAZZ-166 and JAZZ-167 contain a cyclopentadienyl ligand (Cp, also called 1,2,3,4,5-pentamethylcyclopentadiene) linked with the metal group. Cyclopentadienyl ligands increases the hydrophobicity, which may accelerate the antitumour efficacy of the metal complexes [104]. JAZZ-90, n = 5 clogP 4.15 1,2,3,4,5-pentamethylcyclopentadiene JAZZ-166, n = 5, M = Ir JAZZ-167, n = 5, M = Rh Figure 1.8: Novel HDAC inhibitors examined in this study. clog P denotes calculated log P, clogP the value determined using Molinspiration online software tool [154]. JAZZ-90, JAZZ-166, and JAZZ-167 displayed anticancer activity towards human cancer cells including HCT116, H460 (KRAS-mutated NSCLC), SiHa (cervical cancer), and SW480. The antiproliferative activity of JAZZ-90 was comparable to SAHA [153]. Additionally, JAZZ-167 17 exhibited low toxicity in haemolysis studies and in zebrafish and this might be due to the metal centre. Furthermore, JAZZ-167 downregulates vascular endothelial growth factor receptor 2 (VEGFR2) expression, while SAHA upregulated VEGFR2 [153]. 1.5.2 Hydroxythiopyridone derivatives The pyridine ring is considered one of the simplest heteroaromatic structures incorporated in many natural (e.g. niacin, pyridoxine, the ubiquitous redox system NADP+/NADPH, etc.) and synthetic compounds [155]. Pyridine moieties are often used in synthetic drugs due to several special features such as water solubility, basicity, molecular size, hydrogen bond-forming ability, and stability [155, 156]. Pyridine moieties play an important role in drug discovery due to their activity as the bioisosteres of amides, amine, benzene rings, and heterocyclic rings [156]. Functionalisation with the pyridine moieties sometimes exerts biological activities of the parent compounds such as anticancer, antiviral, antimicrobial, antidepressant, antidiabetic, cardiotonic, anticonvulsant, and insecticidal [157]. Furthermore, pyridine compounds coupled with metals display high cytotoxicity and anticancer activity [158]. Pyridine ring containing water-soluble Ru (II) thiosemicarbazone complex (C22H39Cl3N10P2RuS·C2H5OH·H2O) showed anticancer activity against 41M ovarian cancer, SK-BR-3 breast cancer, HT29 colon cancer, and A549 cells with IC50 values of 0.87, 39, 186, and 47 µM, respectively [159]. Many synthetic biomolecules that have thiol-reactive moieties in the structure displayed enhanced cellular association and internalisation (Figure 1.9) [160]. For instance, modification of doxorubicin (DOX), an anticancer drug, with the thiol (SH) group improved the biological function of DOX. Briefly, DOX-SH and DOX-SS-pyridine (SS=disulfide) showed significantly higher or comparable cytotoxicity against HT-1080 fibrosarcoma, HEK293 human embryonic kidney, and CCRF-CEM human leukaemia cells compared to DOX alone, which might be due to high cellular uptake and activity in these cells [161]. Selective inhibitors against HDAC remains a challenge for researchers [162]. To explore alternatives to hydroxamic acid as the zinc-binding group (ZBG), Patil et al. tested 3hydroxypyridin-2-one (3-HP) and 3-hydroxypyridin-2-thione (3-HPT) as a novel ZBG and their potential HDACi activity [163]. Preliminary in silico molecular docking analysis using AutoDock 4.2 revealed that 3-HP and 3-HTP groups were potentially able to bind at the active site of HDAC 1, 6, and 8, suggesting the heterocyclic compounds could provide the critical ZBG in the HDACi pharmacophoric model. In vitro experiments showed that HDAC 6 and HDAC 8 were inhibited by 3-HPT with IC50 values of 681 nM and 3.7 µM, respectively, however, it was inactive towards HDAC 1 [163]. On the other hand, 3-HP was inactive in these three HDAC isoforms. The variation in HDAC inhibitory activity of 3-HP and 3-HPT might be 18 due to the thiophilicity of zinc that favours 3-HPT [163]. 1-Hydroxypyridine-2-thiones (1-HPT) is another key pharmacophore for zinc-binding that form zinc-specific five-membered complex though its oxygen and sulfur atoms. The 1-HPT analogue, 1-HPT-6-carboxylic acid, exhibited selective HDAC 6 inhibitory activity (IC50 value of 150 nM). The other two analogues of 1HPT with simple amino acids exhibited 600-fold selectivity towards eleven zinc-depended HDACs. These 1-HPT analogues also displayed growth inhibitory activity against HDAC 8 overexpressing chronic myelogenous leukaemia cells at low micromolar concentrations [162]. Figure 1.9: Cellular uptake mechanism of thiol-reactive groups. Mechanism of cellular entry of thiol-reactive groups (such as free thiols, disulfide bonds, and maleimide moiety), modified biomolecules (pentacle) upon interaction with exofacial thiols. When these biomolecules reach to the cell membrane, they internalised to free cargoes through cleavage [160]. Thiopyridones are a relatively new class of S, O chelating agents and the potential applications of these compounds are under investigation. The chemical and biological properties of thiopyridones can be fine-tuned through chemical modification of the heterocyclic backbone [164]. Interestingly S, O chelating thiomaltolato ligand containing compounds displayed promising anticancer activity with low µM range IC50 values while the corresponding maltolato 19 compounds were inactive [164]. 1-Adamantylthiopyridine derivative 2-(1-adamantylthio)-5hydroxypyridine exhibited anticancer activity against HepG2 (human liver cancer), MOLT-3 (lymphoblastic leukaemia), HuCCA-1 (human cholangiocarcinoma), and A549 cells with IC50 values of 35, 17.90, 44, and 45 µg/mL, respectively [165]. Novel 1-hydroxy-2-thiopyridine derivatives displayed cytotoxicity against HepG2 cells (IC50 value range from 1.73 to 3.84 µg/mL) and A549 cells (IC50 value range from 4.96 to 8.24 µg/mL) [166]. Additionally, low toxicity and high affinity of thipyridones towards metal ions in different oxidation states are one of the influential factors for their growing demand [164]. Ru based anticancer drugs generally have low toxicity and high selectivity towards cancer cells [167]. Ru(II)-η6-p-cymene complexes containing (O, S) hydroxythiopyridone ligands exhibited anticancer activity towards human A549, SW480, and CH1 cancer cells [168]. The arene Ru unit has amphiphilic properties as the lipophilic arene ligand is counterbalanced by the hydrophilic Ru metal center. The amphiphilic properties and the diversity of the arene ligand makes Ru arene complexes an excellent scaffold for the coupling of organic moieties for targeted chemotherapy [167]. Figure 1.10 shows the hydroxythiopyridone based novel Ru arene complexes chlorido[3-oxo-1benzyl-2-methylpyridin-4(1H)-thionato-κ2O,S](p-cymene)ruthenium(II)] (M1S-Ru) and chloride[3-oxo-1,ethylbenzyl-2-methylpyridin-4(1H)-thionato- κ2O,S](cymene)ruthenium(II) (M2S-Ru) complexes, and their respective ligands i.e. 1-benzyl-2-methyl-3-hydroxypyridin-4(1H)-thione (M1S) and 1-ethylbenzyl-2-methyl-3-hydroxypyridin-4-thione (M2S) synthesised by our collaborators. clogP 3.90 p-cymene (p-cym) M1S, n = 1 M2S, n = 2 M1S-Ru, n = 1 M2S-Ru, n = 2 Figure 1.10: Hydroxythiopyridone derivatives used in this study. clogP value determined using Molinspiration online software tool [154]. 20 1.5.3 Metal-based PCA ligands and complexes Half-sandwich Ru (II) arene or Rh (III) arene complexes can exhibit promising anticancer activity which in many cases is superior or comparable to approved anticancer drugs [153, 169]. Carbon-linked π-bound arenes or cyclopentadienyl ligands can influence cellular uptake and targeting by controlling the lipophilicity and hydrophilicity of the faces of the metal arene complexes [78]. One of the key challenges in organometallic drug development is to identify the active pharmacophore, including the activity of both the ligands and the metal ion. The structural and electronic features of the pharmacophore are essential for the identification of specific cellular targets and thereby, controlling the cellular response. The electronic and steric effects regulate both the kinetics and thermodynamics of ligand exchange and redox balance of metal ions. Hence, understanding the SAR for metal-arene and metal-cyclopentadienyl containing anticancer drug candidates is crucial for drug development [170]. Plectin is a structural protein and lung cancer stem cell biomarker. Plectin is associated with invasiveness of lung adenocarcinoma and poor survival in these patients [171, 172]. Raymond and colleagues confirmed that plectin overexpressed on the surface of KRAS-mutated H358 NSCLC cells and its knockdown reduced mobility, migration, and colony formation of these cells [172]. Previously, it has been reported that PCA based Ru arene (p-cym) complexes namely plecstatin displayed target selectivity for plectin. Plectin targeting potentially suppresses tumour invasiveness in the in vitro tumour spheroid model while oral administration (30 mg/kg) of [chlorido(η6-p-cymene)(N-(4-fluorophenyl)-2-pyridinecarbothioamide) ruthenium(II)]chloride (plecstatin-1 or AASH-122) reduced tumour growth more efficiently in the B16 melanoma model than in the CT26 colon tumour model in mice [173]. A strong interaction of plecstatins with the target depends on the hydrogen bond acceptor (-F) of plecstatins. In sodium chloride media (104 nM NaCl) AASH-122 (approximate half-life of 50 min) hydrolysed faster than its corresponding isosteric osmium analogue (no significant hydrolysis within 14 h). Both the compounds displayed identical distribution patterns and remain largely intact in vivo model after 2 h post-administration [174]. AASH-122 and its Os analogue accumulated in the liver, kidney, lung, muscle, and tumours of murine CT-26 tumourbearing mice after a single i.p. dose (15 mg/kg). Interestingly, AASH-122 penetrated deeper into the organs and tumours while its Os analogue deposited more at the edge or periphery of organs and tumours [175]. AASH-122 is structurally similar with its Rh analogue [chloride(η5pentamethylcyclopentadienyl)(N-(4-fluorophenyl)pyridine-2-carbothioamide)rhodium(III)] chloride (JAZZ-121), except the p-cym is replaced with 1,2,3,4-tetramethyl-cyclopenta-1,3diene (Figure 1.11). These two compounds were synthesised by our collaborators. 21 clogP 3.67 clogP 3.90 p-cymene (p-cym) 1,2,3,4-tetramethylcyclopenta-1,3-diene AASH-122 JAZZ-121 Figure 1.11: Structure of metal-based PCA ligands and complexes. AASH-122 and JAZZ-121 were examined in this study. clogP value determined using Molinspiration online software tool [154]. 1.5.4 Kinetically inert metal(arene) complexes of PCA Kinetically inert metal complexes are potential tools for targeting biomolecules (other than DNA), especially protein. Recent literature reported that the kinetically inter half-sandwich Ru complexes selectively inhibited protein kinases [106]. Kinases catalyse cellular reactions that transfer the phosphate group from ATP to substrates and these reactions are linked with cell division and proliferation [106, 176, 177]. Thus, kinases are one of the crucial targets for anticancer drugs [178, 179]. Antiproliferative activity of the functional compounds is dependent on the metal centers, electronic configuration, oxidative state, and hard-soft nature [180]. Os is slightly softer than Ru, thereby it has different coordination preferences to biomolecules. Another interesting property of Os compounds is the increased inertness of ligand substitution compared to Ru. Therefore, Os compounds are less prone to the hydrolysis of metal-halide bonds. Furthermore, upon hydrolysis, Os-aqua species tend to be more acidic than the corresponding analogue of the Ru-aqua species [181]. An increase in intracellular acidity could not only induce apoptosis due to acidic stress but also decrease invasion and migration [182]. The cytotoxicity, cellular uptake, and mechanism of antiproliferative activity may also be relying on the coordinated halide in the metal arene complexes [183]. The trans-effect of chloride ligands stabilises the reactive ligands at the metal center electronically. The chloride ligands, therefore, are vital as the strength of the metal-carbon bond accelerates the final rate of the metathesis reaction [170]. The majority of Ru (II) and Os (II) based arene complexes have the half-sandwich piano-stool motif, where the η6-coordinating arene such as p-cymene (p-cym) and biphenyl (bip) stabilise 22 the low oxidation state [184]. Interestingly, isostructural piano-stool complexes (RM175, Ru II bip complex, and AFAP51, Os II bip complex) that only differed in the metal center displayed significant differences in the antiproliferative activity both in vitro and in vivo [180]. AFAP51 showed higher cytotoxic potential towards malignant breast MDA-MB-231, MCF-7, and normal breast HBL-100 cells (with IC50 values of 48, 15, and 16 µM, respectively) compared to its isostructural congener Ru II compounds with IC50 values of 62, 93 and 54 µM, respectively. On the other hand, RM175 displayed anti-lung metastasis activity in the in vivo mammary cancer MCa model while AFAP51 did not [185]. In another study, N-substituted PCAs containing Ru (II) and Os (II) arene (p-cym) complexes showed antiproliferative activity against SW480, CH1, and A549 cancer cells. The smaller and lipophilic congeners were most effective in the low micromolar range [184]. It has been reported that the presence of the triphenyl phosphine (PPh3) moiety in the η6-arene (p-cym) Ru II complexes enhanced the antiproliferative activity towards the leukaemia HL-60 cell line [118]. Additionally, viscosity measurement and ethidium bromide displacement experiments confirmed that the presence of PPh3 ligand in the arene-Ru II complexes promoted DNA base pairs intercalation through intramolecular π-π interactions while with PPh3 moiety bound DNA in a covalent manner. However, the reduced protein binding capability of PPh3 ligand bearing complexes could be due to steric hindrance. These effects could have an influential consequences in the cellular uptake and drug detoxification mechanisms of these complexes [118]. Trifluromethylsulfonate (trifilate, OTf, CF3SO3-) is an outstanding leaving group and widely used in organic chemistry [186]. Trifilate is well known classical non-coordinating anion, some transition metal triflates (M-OTf) act as a cationic coordinately unsaturated species that allows easy coordination of other auxiliary ligands (L) (e.g. pyridine, water, and phosphine), resulting in removal of the triflate anion out of the coordination sphere: M-OTf + L → M(L) + (OTf)− [187]. Figure 1.12 shows the pharmacophore modified PCAs containing Ru (II) and Os (II) arene complexes [(triphenylphosphine) (η6-p-cymene) (N-(4-chlorophenyl) pyridine-2-carbothioamide)] ruthenium(II)triflate (ZR-012) and [(triphenylphosphine) (η6-biphenyl) (N-(4-chlorophenyl) pyridine-2-carbothioamide)]osmium(II)triflate (ZR-014) synthesised by our collaborators. 23 clogP 4.82 clogP 3.90 clogP 3.73 p-cymene (p-cym) Triphenylphosphine (PPh3) ZR-012 biphenyl (bip) ZR-014 Figure 1.12: Kinetically inert metal(arene) complexes of PCA examined in this study. clogP value determined using Molinspiration online software tool [154]. 1.6 Aims of the current study HDACis offer therapeutic benefits to patients with KRAS-mutant NSCLC. Development of multitargeting HDACis (JAZZ-90, JAZZ-166, and JAZZ-167) through pharmacophore modification could offer additional benefits in the treatment of KRAS-mutant NSCLC patients by minimising the side effects and improving therapeutic targeting. Additionally, hydroxythiopyridone derivatives showed promising anticancer activities and the presence of a thiol group enhanced the cellular uptake of these compounds. M1S, M2S, M1S-Ru, and M2SRu are predicted to have HDACi activities due to their pharmacophore, and their potential cytotoxic activity and underlying mechanism have yet to be investigated. It has been reported that Ru based PCA ligands and complexes target plectin, a protein overexpressed in KRASmutant NSCLC. Previously, PCA based Ru and Os complexes exhibited anticancer activities against A549 cells. AASH-122 displayed antiproliferative activities against a CT-26 tumor model in vivo and have lung tissue penetration potential. Therefore, AASH-122 and its Rh analogue JAZZ-121 could also have potential antiproliferative activities in KRAS-mutant cell lines. Another PCA based drug class (i.e. kinetically inert metal(arene) complexes of PCA) composed of pharmacophore modified novel compounds (ZR-012 and ZR-014) could have antiproliferative activity against KRAS-mutant cells. As KRAS remains an undruggable target, the current study aimed to investigate the efficacy of novel compounds in different classes towards KRAS- mutated NSCLC cells. 24 1.7 Hypothesis and objectives of the project Previous studies showed that all the drug candidates displayed antiproliferative activity towards HCT116, H460, SiHa, and SW480 cell lines in the nanomolar to micromolar range (M. Hanif, personal communication). Based on the preliminary findings it was hypothesised that these novel compounds will exhibit cytotoxic EC50 values in the nanomolar to micromolar range against A549 cells. The most potent drugs will have cytotoxic activities in other NSCLC cells and have more selectivity towards cancer cells. Additionally, the potent compounds will significantly change the expression of proteins linked with cell growth and proliferation, thereby altering cell cycle progression. The following objectives will address these hypotheses: 1) To screen the potential anticancer activity of several metal-based and non-metal-based novel compounds in the KRAS-mutated A549 NSCLC cell line. 2) To examine the antiproliferative activity of potent drug candidates in p53 mutated H522 NSCLC cells. 3) To investigate the effect of the two most potent drugs in pre-neoplastic NIH3T3 cells and normal prostate epithelial PNT1A cells. 4) To examine the potential antiproliferative mechanism mediated by the two most potent drugs, M1S and M2S, by examining their effect on cell signalling proteins (acetyl H3, cyclin D1, and B1), and cell cycle progression. 25 Chapter II Materials and Methods 2 Materials and Methods 2.1 Materials 2.1.1 Cell lines A549 and H522 cells were kindly donated by A/Prof. John Aston, Department of Pharmacology and Toxicology, University of Otago. NIH3T3 cell line was kindly donated by Dr. Greg Giles, Department of Pharmacology and Toxicology, University of Otago, and the PNT1A cell line was kindly donated by Professor Helen Nicholson, Department of Anatomy, University of Otago. All the cell lines were originally purchased from American Type Culture Collection (USA). 2.1.2 Chemicals Sulforhodamine B (SRB), ammonium persulfate, copper sulfate (CuSO4.5H20), potassium chloride (KCl), sodium pyrophosphate (Na4P2O7), sodium orthovanadate (Na3VO4), sodium fluoride (NaF), sodium hydroxide (NaOH), hydrochloric acid (HCl), trizma hydrochloride (Tris HCl), triton-X 100, methanol, ponceau red stain, tetramethylene diamine (TEMED), trizma base (Tris base), ethylenediaminetetraacetic acid (EDTA), NP-40, ethylene glycol tetraacetic acid (EGTA), dimethyl sulfoxide (DMSO), bromophenol blue, β-mercaptoethanol, complete protease inhibitor cocktail tablets, glycerol and glycine for electrophoresis were purchased from Sigma-Aldrich (USA). RPMI 1640 media and foetal bovine serum (FBS) were purchased from Sigma-Aldrich (UK) and Moregate Biotech (NZ), respectively. Trypsin, penicillinstreptomycin, phenylmethylsulfonyl fluoride (PMSF), 4, 4′-dicarboxy-2, 2′-biquinoline acid (BCA), SuperSignal West Pico Plus Chemiluminescent substrate were purchased from Thermo Fisher Scientific (USA). Potassium dihydrogen phosphate (KH2PO4) was purchased from Fisher Scientific (UK). Acetic acid (CH3COOH), sodium chloride (NaCl), and trichloroacetic acid (TCA) were purchased from Merck (Germany). Sodium dodecyl sulfate (SDS), sodium hydrogen carbonate (NaHCO3), disodium hydrogen phosphate (Na2HPO4), and ethanol (96%) was purchased from Scharlau (Spain). Molecular weight marker, acetylated histone-H3, cyclin D1, cyclin B1, β-tubulin, and β-actin primary antibodies were purchased from Cell Signalling Technology (USA). Acrylamide, bisacrylamide, polyvinylidene difluoride (PVDF) membrane, goat anti-rabbit, and goat anti-mouse secondary antibodies were purchased from Bio-Rad Laboratories (USA). FxCycle propidium iodide (PI)/RNase staining solution was purchased from Life Technologies Corporation (USA). 26 2.1.3 Experimental drugs JAZZ-90, JAZZ-166, JAZZ-167, M1S, M2S, M1S-Ru, M2S-Ru, AASH-122, JAZZ-121, ZR012, and ZR-014 were synthesised by Dr. Muhammad Hanif of the Faculty of Science, University of Auckland, NZ and he also kindly donated SAHA. 2.2 Methods 2.2.1 Cell maintenance A549, H522 lung cancer, NIH3T3 mouse embryonic fibroblast, and PNT1A normal prostate epithelial cell lines were seeded in RPMI 1640 media supplemented with 2%, 10%, 10%, and 10% FBS, respectively, and 1% penicillin-streptomycin. Cells were cultured in 75 or 175 cm2 flasks and incubated in 5% CO2 at 37°C. Cells were passaged under sterile conditions when the cells were ~85% confluent. Cell culture media was aspirated off and cells were washed with 5 mL PBS. Cells were then incubated with 1 mL of trypsin for 2-3 min in 5% CO2 at 37ºC to detach the cells from the flask. The deactivation of trypsin was conducted by the addition of 5 mL complete growth media. The entire cell suspension was transferred into a 14 mL centrifuge tube and centrifuged at 1200 rpm for 3 min at 4°C. The supernatant was discarded and the cell pellet was diluted with 10 mL of fresh growth media. The cell number was determined using a haemocytometer. 2.2.2 Drug preparation Stock solutions of JAZZ-90, M1S, M2S, and SAHA were prepared by dissolving in 100% DMSO and stored at -20°C. Since the metal-based drugs could interact with DMSO in long term storage, all the metal-based drugs including JAZZ-166, JAZZ-167, M1S-Ru, M2S-Ru, AASH-122, JAZZ-121, ZR-012, and ZR-014 were freshly prepared by dissolving in 100% DMSO. The stocks of all the drugs were diluted into RMPI-1640 to a final concentration of 0.5% DMSO. DMSO (0.5 %) was used as the vehicle control. 2.2.3 Cell cytotoxicity study using the SRB assay A549, H522, NIH3T3, and PNT1A cells were seeded in 96 well plates at 4000, 10000, 3500, and 10000 cells per well, respectively, and incubated for 24 h in 5% CO2 at 37°C. Cells were then treated with metal and non-metal-based anticancer drugs at a range of different concentrations (0.015-200 µM) while vehicle control cells were treated with DMSO (0.5%). At the end of the incubation period (72 h), the viable cell number in each well was determined by the SRB assay. Briefly, the media of each well was aspirated off and cells were fixed with 100 µL of 10% TCA. Plates were then kept at 4°C for 30 min, followed by a gentle wash with distilled water. The plates were then dried for 45 min. 100 µL of SRB (0.4% SRB in 1% acetic 27 acid) was then added to each well to stain the viable cells and plates were incubated for 30 min at room temperature. Subsequently, the unbound stain was aspirated off and cells were washed 4 times with 1% acetic acid and dried for 45 min. Finally, the SRB dye in each well was solubilised in 100 µL of 10 mM Tris base (pH 10.5) and the absorbance was read at 510 nm using a Bio-Rad Benchmark Plus microplate reader. The experiments were conducted in triplicate with three technical replicates. 2.2.4 Western blotting Preparation of protein extracts A549 (1.0 × 106 cells per dish) and H522 (2.5 × 106 cells per dish) cells were seeded in 10 cm cell culture dishes in 10 mL of complete growth media and incubated for 24 h. The cells were then treated with M1S (0.36, 0.72 µM for A549 cells and 0.28, 0.56 µM for H522 cells), M2S (0.32, 0.64 µm for A549 cells and 0.23, 0.46 µM for H522 cells) and vehicle control (0.5% DMSO) for 12 h and 24 h. The media was aspirated off and cells were washed with 5 mL of ice-cold PBS (4°C) followed by the addition of 100 µL and 150 µL of lysing buffer (50 mM Tris base, 0.5% NP-40, 0.5% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 1 mM PMSF, 10 mM sodium fluoride and complete protease inhibitor cocktail tablets) for A549 and H522 cells, respectively. The dishes were incubated for 10 min and the cells were detached with a cell scraper. The cells were transferred to a 1.5 mL heat resistant Eppendorf tube and incubated for 12 h. Cell lysates were then sonicated for 3 × 7 sec and centrifuged at 14,000 rpm for 8 min at 4°C and the supernatant was collected. Protein concentrations were determined using the BCA assay. 3 µL of protein lysate of each sample was added to a 96-well plate in triplicate followed by the addition of 17 µL of Mili Q water. BSA with a range of concentrations of 0-500 µg/µL was used to obtain the standard curve. 200 µL of BCA (BCA and 4% CuSO4.5H2O in a 50:1 ratio) was then added and the plate was then incubated for 45 min at 37°C. Absorbance was determined using a Bio-Rad Benchmark Plus microplate reader at 560 nm. A standard curve was used to determine the protein concentration of the samples. The normalisation of each sample was diluted with the required volumes of lysing buffer. Finally, a 4X sample buffer (62.5 mM tris HCl pH 6.8, 1% SDS, 10% glycerol, 0.005% bromophenol blue, 355 mM β-mercaptoethanol) was added to all samples at 1:3 (4X sample buffer: sample) ratio and the samples were heated at 95°C for 5 min and cooled on ice. Samples were then stored at −20°C until further analysis. 28 Gel electrophoresis Each gel consisted of a resolving gel and stacking gel (0.43 mL acrylamide/bisacrylamide solution (29:1), 0.63 mL upper Tris buffer, 25 µL 10% APS, 1.45 µL TEMED). There were two types of resolving gel used, one was 10% gel (2.5 mL acrylamide/bisacrylamide solution (29:1), 1.88 mL lower Tris buffer, 250 µL 50% glycerol, 37.5 µL 10% APS, 2.83 mL dH2O, 5 µL TEMED) and the another was 15% gel (3.75 mL acrylamide/bisacrylamide solution (29:1), 1.88 mL lower Tris buffer, 300 µL 50% glycerol, 37.5 µL 10% APS, 1.55 mL dH2O, 5 µL TEMED). 15% gel was used for cyclin D1, acetyl H3, and β-tubulin, while 10% gel was used for cyclin B1 and β-actin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was used to separate proteins based on molecular weight (MW). Samples containing 25 µg and 20 µg of protein extracted from A549 and H522 cell lysis, respectively, were loaded on each well of the upper gel along with an MW marker to identify the location of the protein of interest. Gels were initially run in SDS buffer (25mM Tris-base, pH 8.3, 0.192 M glycine, 0.1% (w/v) SDS) at a voltage of 70V and after the passing of upper gel, the voltage was maintained at 100V until the dye front had reached to the bottom of the resolving gel. Transferring to PVDF membrane At the end of electrophoresis the upper gel was removed and resolving gel was transferred into transfer buffer to remove excess SDS. A sandwich of equilibrated fibre pad, blotting paper, resolving gel and an activated PVDF membrane was made in the cassettes. To transfer the protein from gel to the activated PVDF membrane transfer buffer (25mM tris-base, pH 8.3, 0.192 M glycine, and 10% methanol) was used. To avoid the overheating of the apparatus, ice packs stored at −80°C were placed inside the apparatus. The proteins were transferred to PVDF membrane at 100 V for 1.5 h using a Bio-Rad wet transfer system. Blocking and antibody incubation The membrane was blocked using 2% non-fat milk powder for 30 min to prevent the nonspecific binding of antibodies. The membrane was then washed 2 times (5 min each) with TBS (0.025 mM Tris-base, 0.1 M NaCl, pH 7.4) to remove the blocking solution and then incubated overnight with the primary antibody at 4°C. At the end of the incubation period, the membrane was washed 6 times with TBST (0.05% Tween 20, 0.025 mM Tris-base, 0.1 M NaCl, pH 7.4) for 5 min each and then treated with a secondary antibody containing horseradish peroxidase enzyme for 1 h. After that the membrane was further washed 6 times with TBST, the membrane was then taken to the darkroom. 29 X-ray film development Chemilunescent solutions were added and X-ray film was exposed to the membrane, following this step the films were developed. Films were analysed using the Bio-Rad GS710 scanner and protein density was determined as a ratio of β-actin or β-tubulin. The experiments were conducted in triplicate, technical replicates were not included. As the same blot was used to screen the expression of different proteins, after the successful development of the X-ray film the blot was washed 6 times with TBST before additional primary antibody treatment. 2.2.5 Cell cycle analysis by flow cytometry Cell fixing A549 (1.0 × 106 cells per dish) and H522 (3.0 × 105 cells per well) cells were seeded in 10 cm cell culture dishes and 6-well plates with 10 mL and 2 mL of complete media and incubated for 24 h, respectively. The cells were then treated with M1S (0.36, and 0.72 µM for A549 cells; 0.28, and 0.56 µM for H522 cells), M2S (0.32, and 0.64 µM for A549 cells; 0.23, and 0.46 µM for H522 cells) using 0.5 % DMSO as the vehicle control for 6 h and 12 h. After treatment, the A549 and H522 cells were washed twice with 2 mL and 400 µL of cold isotonic PBS (137 mM NaCl, 2.7 mM KCL, 4.3 mM Na2HPO4, 1.47 mM KH2PO4; pH 7.4 maintained at 4⁰C), respectively. Then 1 mL (A549 cells) and 500 µL (H522 cells) of pre-warmed trypsin were added to each well followed by incubation at 37⁰C for less than 5 min. Plates were immediately placed on ice and the cell suspension was transferred into a fresh 15 mL tube. The tubes were centrifuged at 2000 rpm for 5 min at 4⁰C. The supernatant was removed from each tube and the cell pellet was resuspended in 300 µL isotonic PBS (4⁰C). The above centrifugation step and supernatant removing steps were repeated once. The pellet was then fixed by adding 600 µL cold 70% ethanol and vortexing gently. The tubes were stored at 4⁰C until analysis. Propidium iodide staining The tubes containing the cell suspensions were centrifuged at 2000 rpm for 5 min at 4⁰C. After that the supernatant was removed, the cells were washed two times with 500 µL cold PBS. The cell pellet was then resuspended in 250 and 200 µL of cold FxCycle PI/RNase (0.1% sodium citrate, 0.1% triton-X, 1 mg/mL PI) staining solution for A549 and H522 cells, respectively and transferred to a FAC tube followed by incubation in the dark for 30 min at room temperature. The samples were analysed via flow cytometry using a Beckman Coulter Gallios flow cytometer (USA) and data were analysed by Kaluza software. Technical replicates were not included in this experiment. 30 2.2.6 Experimental data calculation and statistical analysis To calculate the EC50 (the concentration of drug required to decrease the cell number by 50% of vehicle control) concentration, the cell number was analysed by nonlinear regression using Prism-GraphPad 8 software (USA) [188, 189]. The following formula was used to determine the EC50 value [190]. EC50 = Minimum cell viability (%) + (Maximum cell viability (%) - Minimum cell viability (%)) (( ) 1 + 10^ Log EC50 – Drug Concentration) × Hill Slope In the published literature the terminology IC50 (the concentration of drug required for 50% inhibition) and EC50 are used interchangeably for representing cytotoxicity data [91, 185, 188, 189, 191]. Yadav and colleagues presented the cytotoxicity results using the term EC50 and inhibitory activity using the term IC50 [188]. It appeared more logical to present the cytotoxic effect of a drug using the terminology EC50, as it measures the effect of a drug on cell number, not their inhibitory effects underlying this activity. Therefore, the cytotoxicity data of the current study is presented using EC50. However, in the introduction and discussion sections, the terminology used in each publication is what has been stated. The selectivity index (SI) of M1S and M2S was calculated according to the following formula [192]. EC50 of drug in normal cell line SI= EC50 of drug in cancer cell line Where PNTA1 was used as a normal cell line Western blotting results were obtained by scanning the developed blots using a Bio-Rad GS710 calibrated imaging densitometer (USA) and quantified using Quantity One software from Bio-Rad (USA). The scanner was set to scan blue x-ray film at 42.3 × 42.3 resolution. Using the volume rectangle tool in the Quantity One software, boxes of the same size were used to calculate the density of the protein as well as the corresponding background surrounding the protein band. The volume analysis report was set to determine the density of the bands. The average background density values were subtracted from the protein-band density. The value was then divided by the corresponding band of housekeeping protein i.e. either β-tubulin or βactin as a loading control. Statistical analysis was performed using Prism-GraphPad 8 software (USA) and the values presented as mean ± standard error of the mean (SEM). A one-way analysis of variance 31 (ANOVA) coupled with a Bonferroni post-hoc test was performed to determine the shift of a novel drug’s potency in comparison to SAHA. While data from the time-course cytotoxicity assay, Western blotting, and cell cycle analysis were analysed using a two-way ANOVA (parameters of time and treatment) coupled with a Bonferroni post-hoc test. Statistical significance was set at p<0.05. 32 Chapter III Results 3 Results 3.1 Cytotoxicity 3.1.1 Dose-response cytotoxicity of novel compounds in A549 cells To select the most potent drug with cytotoxicity toward KRAS-mutant NSCLC, eight metalbased and three non-metal-based synthetic compounds of four different classes were screened in A549 lung cancer cells using the SRB assay. The EC50 values for all the drugs are shown in Table 3.1. The non-metal-based HDACi JAZZ-90 displayed potent cytotoxicity compared with the control drug SAHA with EC50 values of 0.76 and 1.01 µM, respectively. On the other hand, the metal-based HDACis JAZZ-166, and JAZZ-167 were 3.2 and 4.2 times less cytotoxic than SAHA (Figure 3.1). Interestingly, of the metal-based PCA ligands and complexes, AASH showed considerable potency with an EC50 value of 2.02 µM, while JAZZ -121 was unable to reach 50 percent viability over the range of concentrations tested (0.15 to 200 µM) (Figure 3.1). Additionally, as shown in Figure 3.2, non-metal-based hydroxythiopyridone derivatives, M1S, and M2S were 4.8 and 5.2-fold more potent, respectively, compared with their metal-based complexes M1S-Ru and M2S-Ru. Furthermore, kinetically inert metal(arene) complexes of PCA, ZR-012, and ZR-014 showed almost equal potency to that of M1S-Ru and M2S-Ru (Figure 3.2). Overall, all three non-metal-based synthetic compounds M2S, M1S, and JAZZ90 and metal-based derivatives ZR-012, ZR-014, M1S-Ru, and M2S-Ru displayed cytotoxic potential comparable to SAHA, while AASH-122, JAZZ-166 and JAZZ-167 exhibited cytotoxic activity that was significantly weaker compared to SAHA (Table 3.1). The three most potent drugs M2S, M1S, and JAZZ-90 were chosen for further study. Additionally, as the metal-based derivatives ZR-012, ZR-014, M1S-Ru, and M2S-Ru showed similar potency, M1S-Ru and M2S-Ru were selected for further examination because they contained the same moieties contained within M1S and M2S, which elicited submicromolar EC50 values. 33 B) SAHA Cell number (% of control) Cell number (% of control) A) 100 EC5 0 = 1.01 µM 50 0 10 -7 10 -6 10 -5 10 -4 JAZZ-90 100 50 0 10 -8 Log concentration (M) D) JAZZ-166 Cell number (% of control) Cell number (% of control) C) 100 EC5 0 = 3.17 µM 50 0 10 -7 10 -6 10 -5 10 -4 50 0 Cell number (% of control) Cell number (% of control) EC5 0 = Not determined 10 -6 10 -5 10 -4 Log concentration (M) 100 50 EC5 0 = 2.02 µM 0 10 -6 10 -5.5 Log concentration (M) 10 -3 10 -3 AASH-122 F) 0 10 -7 EC5 0 = 4.22 µM 10 -7 10 -6 10 -5 10 -4 Log concentration (M) 100 10 -3 100 10 -3 JAZZ-121 50 10 -7 10 -6 10 -5 10 -4 Log concentration (M) JAZZ-167 Log concentration (M) E) EC5 0 = 0.76 µM 10 -5 Figure 3.1: Cytotoxicity of SAHA, JAZZ-90, JAZZ-166, JAZZ-167, JAZZ-121, and ASH-122 in A549 cells. A549 cells were seeded in 96-well plates at 4 × 103 cells per well and incubated for 24 h at 37ºC. The cells were then treated with SAHA (A), JAZZ-90 (B), JAZZ-166 (C), JAZZ-167 (D), JAZZ121 (E), and AASH-122 (F) (0.015 to 200 µM) for 72 h. Vehicle control cells were treated with DMSO (0.5%). At the end of the treatment period, the cell number was determined by the SRB assay. The points represent the mean ± SEM (n=3). EC50 values were obtained using non-linear regression using Prism software. 34 M1S EC5 0 = 0.36 µM 0 10 -7.5 10 -7 10 -6.5 10 -6 10 -5.5 Log concentration (M) EC5 0 = 1.73 µM 0 10 -6.5 10 -6 10 -5.5 Log concentration (M) EC5 0 = 1.64 µM 10 -6 10 -5.5 Log concentration (M) M2S-Ru 100 EC5 0 = 1.66 µM 50 0 10 -5 100 50 EC5 0 = 1.53 µM 0 10 -6.5 10 -5 10 -6 10 -5.5 Log concentration (M) ZR-014 F) 0 10 -6.5 10 -7 10 -6.5 10 -6 10 -5.5 Log concentration (M) 10 -6.5 100 50 0 10 -5 ZR-012 E) EC5 0 = 0.32 µM 50 D) 100 50 100 10 -7.5 Cell number (% of control) Cell number (% of control) 10 -5 M1S-Ru C) Cell number (% of control) Cell number (% of control) 100 50 M2S B) Cell number (% of control) Cell number (% of control) A) 10 -6 10 -5.5 Log concentration (M) 10 -5 Figure 3.2: Cytotoxicity of M1S, M2S, M1S-Ru, M2S-Ru, ZR-012, and ZR-014 in A549 cells. A549 cells were seeded in 96-well plates at 4 × 103 cells per well and incubated for 24 h at 37ºC. The cells were then treated with M1S (A), M2S (B), M1S-Ru (C), M2S-Ru (D), ZR-012 (E), and ZR-014 (F) (0.025 to 80 µM ) for 72 h. Vehicle control cells were treated with DMSO (0.5%). At the end of the treatment period, the cell number was determined by the SRB assay. The points represent the mean ± SEM (n=3). EC50 values were obtained using non-linear regression using Prism software. 35 Table 3.1: EC50 values of different classes of drug candidates in A549 cells Class Name Molecular (Mol.) formula I SAHA JAZZ-90 II III IV EC50 (µM) C14H20N2O3 Mol. weight (g/mol) 234.32 1.01 95% confidence interval 0.92 to 1.10 C20H24N4O3S 400.49 0.76 0.59 to 0.97 * 2.29 to 4.30 JAZZ-166 C30H38Cl2IrN4O3S 797.83 3.17 JAZZ-167 C30H38Cl2RhN4O3S 708.52 4.22* 3.69 to 4.99 JAZZ-121 C22H24Cl2FN2RhS 541.31 AASH-122 C22H23Cl2FN2RuS 538.47 Not determined** 2.02* Not determined 1.95 to 2.09 M1S C13H13NOS 231.31 0.36 0.34 to 0.38 M2S C14H15NOS 245.34 0.32 0.29 to 0.34 M1S-Ru C23H26NOSClRu 501.05 1.73 1.69 to 1.77 M2S-Ru C24H28NOSClRu 515.07 1.66 1.56 to 1.77 ZR-012 C41H37ClF3N2O3RuPS2 894.37 1.64 1.58 to 1.70 ZR-014 C43H33ClF3N2O3OsPS2 1003.52 1.53 1.50 to 1.57 I: HDACis; II: Metal-based PCA ligands and complexes; III: Hydroxythiopyridone derivatives; IV: Kinetically inert metal(arene) complexes of PCA. * Significantly increased compared to SAHA, p<0.01. Data were analysed with one-way ANOVA coupled with a Bonferroni post-hoc test. **Cytotoxicity did not reach 50%. 3.1.2 Dose-response cytotoxicity of novel potent compounds in H522 cells To examine the consistency of JAZZ-90, M1S, M2S, M1S-Ru, and M2S-Ru potency towards other NSCLC cells, their activity was examined in H522 cells. SAHA was nearly 4.5 times more potent in H522 cells compared with JAZZ-90 (Table 3.2) but the potency of JAZZ-90 in H522 cells was reduced by 2-fold when compared to A549 cells (Figure 3.1 and 3.3). On the other hand, M1S and M2S displayed more cytotoxicity in comparison with their metal-based counterparts M1S-Ru and M2S-Ru (Table 3.2). Notably, in comparison with A549 cells, the potency of M1S and M2S increased nearly 1.3 and 1.4 times against H522 cells, while the cytotoxic potential of MIS-Ru and M2S-Ru decreased by almost 3.8 and 4.5-fold, respectively (Figure 3.2 and 3.3). In summary, M1S and M2S were the most potent compounds against H522 cells and cytotoxicity was comparable with the control drug SAHA, which was similarly seen in A549 cells. The antiproliferative activity of the other three potent drugs JAZZ-90, M1S-Ru, and M2SRu (in A549) shifted significantly in comparison to SAHA in H522 cells (Table 3.1, 3.2). Since M1S and M2S exhibited submicromolar EC50 values in multiple cell lines, they were selected for further examination. 36 SAHA B) Cell number (% of control) Cell number (% of control) A) 100 EC5 0 = 0.35 µM 50 0 10 -8 10 -7 10 -6 10 -5 JAZZ-90 100 EC5 0 = 1.55 µM 50 0 10 -4 10 -7 10 -6 10 -5 Log concentration (M) Log concentration (M) M1S EC5 0 = 0.28 µM 0 10 -7.5 10 -7 10 -6.5 10 -6 Log concentration (M) EC5 0 = 6.66 µM 50 0 10 -5.5 10 -5 Log concentration (M) EC5 0 = 0.23 µM 50 0 100 EC5 0 = 7.42 µM 50 0 10 -6 10 -4.5 10 -7 10 -6.5 10 -6 Log concentration (M) M2S-Ru F) 100 10 -6 100 10 -7.5 10 -5.5 M1S-Ru E) Cell number (% of control) Cell number (% of control) 100 50 M2S D) Cell number (% of control) Cell number (% of control) C) 10 -4 10 -5.5 10 -5 Log concentration (M) 10 -4.5 Figure 3.3: Cytotoxicity of SAHA, JAZZ-90, M1S, M2S, M1S-Ru, and M2S-Ru in H522 cells. H522 cells were seeded in 96-well plates at 10 × 103 cells per well and incubated for 24 h at 37ºC. The cells were then treated with SAHA (A), JAZZ-90 (B), M1S (C), M2S(D), M1S-Ru (E), and M2S-Ru (F) (0.025 to 20 µM) for 72 h. Vehicle control cells were treated with DMSO (0.5%). At the end of the treatment period, the cell number was determined by the SRB assay. The points represent the mean ± SEM (n=3). EC50 values were obtained using non-linear regression using Prism software. 37 Table 3.2: EC50 values of different classes of drug candidates in H522 cells Class Name EC50 (µM) HDACis SAHA 0.35 95% confidence interval 0.31 to 0.39 JAZZ-90 1.55* 1.37 to 1.77 M1S 0.28 0.26 to 0.29 M2S 0.23 0.22 to 0.23 M1S-Ru 6.66 * 6.16 to 7.21 M2S-Ru 7.42* 7.09 to 7.77 Hydroxythiopyridone derivatives * Significantly increased compared to SAHA, p<0.01. Data were analysed with one-way ANOVA coupled with a Bonferroni post-hoc test. 3.1.3 Dose-response cytotoxicity of M1S and M2S on NIH3T3 cells To investigate the potency of M1S and M2S towards cell lines with a different origin, both the compounds were examined in pre-neoplastic NIH3T3 cells (mouse embryonic fibroblast cell line) (Figure 3.4). Similar to the results from A549 and H522 cells, M1S (EC50 value of 0.44 µM, 95% confidence interval, 0.43 to 0.47 µM) and M2S (EC50 value of 0.34 µM, 95% confidence interval, 0.33 to 0.36 µM) showed consistent cytotoxic potential towards NIH3T3 cells. The potency of M2S in NIH3T3 cells is almost identical to A549 cells while in comparison with A549 cells the cytotoxicity of M1S decreased by 1.2-fold. On the other hand, the potency of M1S and M2S decreased by 1.6 and 1.5-fold in NIH3T3 cell compared with H522 cells. Cell number (% of control) Cell number (% of control) 100 50 EC5 0 = 0.44 µM 0 10 -6.5 10 -6 Log concentration (M) M2S B) M1S A) 10 -5.5 100 50 EC5 0 = 0.34 µM 0 10 -7 10 -6.5 10 -6 Log concentration (M) 10 -5.5 Figure 3.4: Cytotoxicity of M1S and M2S in NIH3T3 cells. NIH3T3 cells were seeded in 96-well plates at 3.5 × 103 cells per well and incubated for 24 h at 37ºC. The cells were then treated with M1S (A) and M2S (B) (0.075 to 2) µM for 72 h. Vehicle control cells were treated with DMSO (0.5%). At the end of the treatment period, the cell number was determined by the SRB assay. The points represent the mean ± SEM (n=3). EC50 values were obtained using non-linear regression using Prism software. 38 3.1.4 Dose-response cytotoxicity of M1S and M2S on PNT1A cells To screen the selectivity of M1S and M2S against non-cancerous cells, the potency of the compounds was tested on PNT1A cells (normal prostate epithelial cells) (Figure 3.5). In comparison with the neoplastic (A549 and H522 cells) and pre-neoplastic cells (NIH3T3 cells), the potency of M1S (EC50 value of 1.29 µM, 95% confidence interval, 1.19 to 1.39 µM) and M2S (EC50 value of 1.12 µM, 95% confidence interval, 1.08 to 1.16 µM) decreased (Figure 3.2, 3.3, 3.4, and 3.5). The highest selectivity of M1S and M2S was observed in H522 cells, with a SI value of 4.47 and 4.87, respectively. While the SI value decreased to 3.55 and 3.5 in A549 cells. The drugs showed the lowest selectivity for NIH3T3 cells as the SI value further decreased to 2.91 and 3.29 for M1S and M2S, respectively (Table 3.3). M1S 100 EC5 0 = 1.29 µM 50 0 10 -6.5 10 -6 M2S B) Cell number (% of control) Cell number (% of control) A) 10 -5.5 100 0 10 -7 10 -5 EC5 0 = 1.12 µM 50 Log concentration (M) 10 -6.5 10 -6 10 -5.5 Log concentration (M) 10 -5 Figure 3.5: Cytotoxicity of M1S and M2S in PNT1A cells. PNT1A cells were seeded in 96-well plates at 10 × 103 cells per well and incubated for 24 h at 37ºC. The cells were then treated with M1S (A) and M2S (B) (0.15 to 5 µM) for 72 h. Vehicle control cells were treated with DMSO (0.5%). At the end of the treatment period, the cell number was determined by the SRB assay. The points represent the mean ± SEM (n=3). EC50 values were obtained using non-linear regression using Prism software. Table 3.3: Selectivity index (SI) of M1S and M2S Drug EC50 values in normal cell line M1S PNT1A (1.29 µM) M2S PNT1A (1.12 µM) EC50 values in neoplastic or pre-neoplastic cell lines SI A549 (0.36 µM) 3.55 H522 (0.28 µM) 4.46 NIH3T3 (0.44 µM) 2.91 A549 (0.32 µM) 3.5 H522 (0.23 µM) 4.87 NIH3T3 (0.34 µM) 3.29 The values of SI were determined using PNT1A as a normal cell line. 39 3.2 Time course cytotoxicity assessment in A549 and H522 cells To determine the time-dependent cytotoxicity of M1S and M2S, A549 and H522 cells were treated at 2×EC50 concentrations for 12, 24, 36, 48, 60, and 72 h (Figure 3.6 A and B). M1S and M2S produced a similar cytostatic pattern in A549 cells (Figure 3.6 A). A statistically significant decrease in cell number was observed following 12 and 24 h of M2S and M1S treatment, respectively, and cell number was relatively stable throughout the remainder of the treatment period compared to control (Figure 3.6 A). Though the cytotoxic profile of both the A) A549 50000 Control M1S (2×EC5 0) 40000 Cell number Cell number M2S M1S 50000 30000 20000 10000 * * * * * Control 40000 M2S (2×EC50) 30000 20000 10000 * 0 * * * * * 0 0 20 40 60 80 0 20 40 60 Time (hour) Time (hour) M1S M2S 80 B) H522 40000 Control M1S (2×EC5 0) 30000 Cell number Cell number 40000 20000 10000 * * * * * * 20 20000 10000 * * * * * * * 0 0 0 Control M2S (2×EC5 0) 30000 40 60 0 80 20 40 60 80 Time (hour) Time (hour) Figure 3.6: Time course cytotoxicity assessment of M1S and M2S in A549 and H522 cells. A549 cells and H522 were seeded in 96-well plates at 4 × 103 and 10 × 103 cells per well respectively and incubated for 24 h at 37ºC. A549 cells were then treated with 0.72 µM of M1S and 0.64 µM of M2S (A) while H522 cells were treated with 0.56 µM of M1S and 0.46 µM of M2S (B) for 12-72 h. Vehicle control cells were treated with DMSO 0.5%. At the end of the treatment period, the cell number was determined by the SRB assay. The points represent the mean ± SEM (n=3). Data were analysed using a two-way ANOVA coupled with a Bonferroni post-hoc test. *Significantly different from control, p<0.01. No significant differences were calculated between each treatment point from 12 to 72 h. 40 compounds towards H522 cells (Figure 3.6 B) was different in comparison to A549 cells (Figure 3.6 A), the two drugs showed almost identical initial cytotoxicity against H522 cells. Specifically, M1S and M2S showed a significant (nearly 2 and 2.9-fold, respectively) decrease in cell number at 12 h, and this effect was maintained for the rest of the treatment time. The cytotoxicity of M1S and M2S was different in the two NSCLC cell lines. To investigate the potential mechanism that drives the time and cell line dependent variation in cytotoxicity, Western blotting was performed to determine the expression of acetyl H3, cyclin D1, and cyclin B1. 3.3 Drug-mediated changes in protein levels 3.3.1 Acetylated histone-H3 (Acetyl H3) While the hydroxythiopyridone derivatives are not strong candidates for HDAC inhibition, this was confirmed by examining for changes in acetyl H3 following treatment with M1S and M2S in A549 and H522 cells. The expression of acetyl H3 was analysed using Western blotting. No significant difference in the expression of acetyl H3 was observed after the treatment of either M1S or M2S at the EC50 or 2×EC50 concentrations (Figure 3.7). 41 A) A549 cells Treatment Time (h) DMSO 12 24 M1S (EC50) M1S (2×EC50) M2S (EC50) M2S (2×EC50) 12 24 12 24 12 24 12 24 Acetyl H3 β-Tubulin H522 cells Treatment Time (h) DMSO 12 24 M1S (EC50) M1S (2×EC50) M2S (EC50) M2S (2×EC50) 12 24 12 24 12 24 12 24 Acetyl H3 β-Tubulin A549 1.5 (Acetyl H3/β-Tubulin) (Acetyl H3/β-Tubulin) B) 1.0 0.5 0.0 12 h H522 1.5 DMSO 1.0 M1S (EC5 0) M1S (2×EC5 0) 0.5 M2S (EC5 0) M2S (2×EC5 0) 0.0 12 h 24 h 24 h Time (h) Time (h) Figure 3.7: Effect of M1S and M2S on acetyl H3 expression in A549 and H522 cells. A549 (1 × 106 cells per dish) and H522 (2.5 × 106 cells per dish) cells were seeded in 10 cm cell culture dishes and left to attach for 24 h at 37ºC. A549 cells were then treated with 0.36, 0.72 µM of M1S and 0.32, 0.64 µM of M2S while H522 cells were treated with 0.28, 0.56 µM of M1S and 0.23 0.46 µM of M2S for 12-24 h. Vehicle control cells were treated with DMSO 0.5%. Cell lysates were subjected to Western blotting with a specific antibody against acetyl H3. A) Blots shown are representative of n=3. B) Scanning densitometry of the ratio of acetyl H3 to β-tubulin. Data were analysed with a two-way ANOVA coupled with a Bonferroni post-hoc test. None were significantly different. 3.3.2 Cyclin D1 To determine the potential mechanism by which M1S and M2S inhibit cell proliferation since the drugs were not HDACis, the expression of cyclin D1 protein (a G1 progression regulator) was examined in A549 and H522 cells (Figure 3.8). In A549 cells, M1S and M2S did not significantly change the cyclin D1 levels compared to control. Similarly in H522 cells treatment for 12 and 24 h with M1S or M2S at the EC50 concentration also failed to alter the cyclin D1/β- 42 tubulin ratio. However, doubling the M1S concentration to 2×EC50 reduced the cyclin D1 expression by 59.8% of control at 12 h while the same concentration significantly decreased the expression by 68.1% of control at 24 h. Furthermore, increasing the concentration of M2S to 2×EC50 significantly decreased the cyclin D1/β-tubulin ratio by 69.7 and 84.9% of control at 12 and 24 h, respectively. A) A549 cells Treatment Time (h) DMSO 12 24 M1S (EC50) M1S (2×EC50) M2S (EC50) M2S (2×EC50) 12 24 12 24 12 24 12 24 Cyclin D1 β-Tubulin H522 cells Treatment Time (h) DMSO 12 24 M1S (EC50) M1S (2×EC50) M2S (EC50) M2S (2×EC50) 12 24 12 24 12 24 12 24 Cyclin D1 β-Tubulin A549 (Cyclin D1/β-Tubulin) (Cyclin D1/β-Tubulin) B) 1.0 0.5 0.0 12 h H522 1.0 DMSO M1S (EC5 0) 0.5 M1S (2EC5 0) * * M2S (EC5 0) M2S (2EC5 0) 0.0 12 h 24 h * 24 h Time (h) Time (h) Figure 3.8: Effect of M1S and M2S on cyclin D1 expression in A549 and H522 cells. A549 (1 × 106 cells per dish) and H522 (2.5 × 106 cells per dish) cells were seeded in 10 cm cell culture dishes and left to attach for 24 h at 37ºC. A549 cells were then treated with 0.36, 0.72 µM of M1S and 0.32, 0.64 µM of M2S while H522 cells were treated with 0.28, 0.56 µM of M1S and 0.23 0.46 µM of M2S for 12-24 h. Vehicle control cells were treated with DMSO 0.5%. Cell lysates were subjected to Western blotting with a specific antibody against cyclin D1. A) Blots shown are representative of n=3. B) Scanning densitometry of the ratio of cyclin D1 to β-tubulin. Data were analysed with a two-way ANOVA coupled with a Bonferroni post-hoc test. *Significantly different from control, p<0.01. 43 3.3.3 Cyclin B1 To confirm the cell line and time-dependent effect on protein that regulate the cell cycle, the expression of cyclin B1, a G2/M progression regulator was also investigated. No significant changes in cyclin B1/β-actin ratio was seen when A549 and H522 cells were treated with either 1X or 2X the EC50 concentration of M1S or M2S for 12 or 24 h when compared to vehicle control (Figure 3.9). A) A549 cells Treatment Time (h) DMSO 12 M1S (EC50) M1S (2×EC50) M2S (EC50) M2S (2×EC50) 24 12 24 12 24 12 24 12 24 Cyclin B1 β-Actin H522 cells Treatment Time (h) DMSO 12 M1S (EC50) M1S (2×EC50) M2S (EC50) M2S (2×EC50) 24 12 24 12 24 12 24 12 24 Cyclin B1 β-Actin B) (Cyclin B1/β-Actin) (Cyclin B1/β-Actin) A549 1.5 1.0 0.5 0.0 12 h H522 1.5 DMSO M1S (EC5 0) 1.0 M1S (2×EC5 0) 0.5 M2S (EC5 0) M2S (2×EC5 0) 0.0 12 h 24 h Time (h) 24 h Time (h) Figure 3.9: Effect of M1S and M2S on cyclin B1 expression in A549 and H522 cells. A549 (1 × 106 cells per dish) and H522 (2.5 × 106 cells per dish) cells were seeded in 10 cm cell culture dishes and left to attach for 24 h at 37ºC. A549 cells were then treated with 0.36, 0.72 µM of M1S and 0.32, 0.64 µM of M2S while H522 cells were treated with 0.28, 0.56 µM of M1S and 0.23 0.46 µM of M2S for 12-24 h. Vehicle control cells were treated with DMSO 0.5%. Cell lysates were subjected to Western blotting with a specific antibody against cyclin B1. A) Blots shown are representative of n=3. B) Scanning densitometry of the ratio of cyclin B1 to β-actin. Data were analysed with a two-way ANOVA coupled with a Bonferroni post-hoc test. None were significantly different. 44 In the time course assay, M1S and M2S showed a cytostatic and cytotoxic pattern in A549 and H522 cells, respectively. Additionally, a significant difference in cell number was seen in A549 cells after 12 h of M2S treatment at 2×EC50 concentration, while the same concentration of M1S and M2S significantly decreased cell number in H522 cells (Figure 3.6). However, M1S and M2S failed to produce any significant changes in acetyl H3, cyclin D1, and cyclin B1 expression in A549 cells as shown by Western blotting after 12 h (Figure 3.7, 3.8 and 3.9). Similar to A549 cells, after 12 h the compounds were unable to significantly change acetyl H3 and cyclin B1 levels at 2×EC50 concentration in H522 cells (Figure 3.7 and 3.9). Interestingly, both the compounds showed a concentration-dependent (EC50 and 2×EC50) trend in the reduction of the cell cycle protein cyclin D1 in both A549 and H522 cells after 12 h. However, within this period only M2S significantly decreased cyclin D1 level at 2×EC50 in H522 cells (Figure 3.8). Therefore, cell cycle analysis at 12 h and one earlier time point, 6 h were conducted to examine how these compounds drive the cell cycle changes and generate their cytostatic or cytotoxic effects evident at 12 h. 3.4 Cell cycle analysis To investigate the effects of M1S and M2S on cell cycle progression of A549 and H522 cells, cell cycle analysis was performed using flow cytometry. M1S and M2S did not produce any significant effect in the cell cycle progression of A549 cells compared to control (Figure 3.10). In H522 cells significance of M1S and M2S treatments could not be determined from two biological repeats. It appeared as though the compounds exhibited a concentrationdependent trend of arresting H522 cells in the G1 phase at 12 h (Figure 3.11). However, triplicate experiments in H522 cells will be required to confirm this effect. In the sub-G1 phase, a small peak far left of the G1 peak was observed in H522 cells (Figure 3.11 A), which is an indicator of apoptotic cells. The significance of the apoptotic cells in M1S and M2S groups could not be confirmed from duplicate experiments. However, it appeared as though a higher proportion of apoptotic cells might be present in the treatment groups compared to control at 6 and 12 h (Figure 3.12). Triplicate experiments in H522 cells will also give a more precise conclusion regarding whether or not the apoptotic cells appeared in the sub-G1 phase. 45 A549 A) FL3-A::PI M2S (2×EC50) M1S (2×EC50) Histogram Histogram Histogram DMSO FL3-A::PI FL3-A::PI Propotion of cell in each phase (%) B) A549 G2/M 100 S G1 50 0 ) ) ) ) ) ) ) ) S O C 50 C 5 0 C 5 0 C 5 0 S O C 5 0 C 5 0 C 5 0 C 5 0 M E M D ( 2×E S (E 2×E D S (E 2×E S (E 2×E S ( ( ( ( 2 1 2 1 M 1S M 2S M 1S M 2S M M M M 6h 12 h Figure 3.10: Cell cycle analysis in A549 cells exposed to M1S and M2S. A549 (1 × 106 cells per dish) cells were seeded in 10 cm cell culture dishes and left to attach for 24 h at 37ºC. Cells were then treated with 0.36, 0.72 µM of M1S, and 0.32, 0.64 µM of M2S for 6-12 h. Vehicle control cells were treated with DMSO 0.5%. A) Representative histograms of control and treatment. B) Bars indicate the mean proportion of cells in different cell cycle phases (% of total) ± SEM (n=3). Data were analysed with a two-way ANOVA coupled with a Bonferroni post-hoc test. None were significantly different. 46 A) H522 Histogram Histogram Histogram FL3-A::PI M2S (2×EC50) M1S (2×EC50) DMSO FL3-A::PI FL3-A::PI Propotion of cell in each phase (%) B) H522 100 Sub G1 G1 50 S G2/M 0 ) ) ) ) ) ) ) ) SO C50 C 50 C 50 C 50 SO C 50 C 50 C 50 C 50 M E M D ( 2×E S (E 2×E D S (E 2×E S (E 2×E S ( ( ( ( 2 1 2 1 M 1S M 2S M 1S M 2S M M M M 6h 12 h Figure 3.11: Cell cycle analysis in H522 cells exposed to M1S and M2S. H522 (3.0 × 105 cells per well) cells were seeded in 6-well plates and left to attach for 24 h at 37ºC. Cells were treated with 0.28, 0.56 µM of M1S and 0.23 0.46 µM for 6-12 h. Vehicle control cells were treated with DMSO 0.5%. A) Representative histograms of control and treatment. B) Bars indicate the mean proportion of cells in different cell cycle phases (% of total) ± SEM (n=2). Data could not be statistically analysed from n=2. 47 Apoptotic cells (% of sub G1 cells) H522 100 80 DSMO M1S (EC5 0) 60 M1S (2×EC5 0) 40 M2S (EC5 0) M2S (2×EC5 0) 20 0 6h 12 h Time (h) Figure 3.12: Apoptotic cells in sub-G1 phage in H522 cells after M1S and M2S treatment. H522 (3.0 × 105 cells per well) cells were seeded in 6-well plates and left to attach for 24 h at 37ºC. Cells were treated with 0.28, 0.56 µM of M1S and 0.23 0.46 µM for 6-12 h. Vehicle control cells were treated with DMSO 0.5%. Bars indicate the mean ± SEM (n=2). Data could not be statistically analysed from n=2. 48 3.5 Summary of results Table 3.4: EC50 values of different classes of drug candidates in A549, H522, NIH3T3, and PNT1A cells Class Hydroxythiopyridone derivatives HDACis Metal-based PCA ligands and complexes Kinetically inert metal(arene) complexes of PCA Drug Candidate name Mol. weight (g/mol) EC50 (µM) A549 H522 NIH3T3 PNT1A M1S M2S 231.31 245.34 0.36 0.32 0.28 0.23 M1S-Ru M2S-Ru 501.05 515.07 1.73 1.66 6.66 7.42 SAHA JAZZ-90 234.32 400.49 1.01 0.76 0.35 1.55 JAZZ-166 JAZZ-167 797.83 708.52 3.17 4.22 JAZZ-121 541.31 AASH-122 538.47 Not determined 2.02 ZR-012 ZR-014 894.37 1003.52 1.64 1.53 0.44 0.34 1.29 1.12 Time course cytotoxicity of M1S and M2S A549: M1S and M2S produced a similar cytostatic pattern in A549 cells, approximately initial seeding number (4000 cells) was maintained from 12-72 h. H522: M1S and M2S decreased ~2 and 2.9-fold of initial cells (10,000 cells) respectively at 12 h, and this effect was maintained for 24-72 h. Table 3.5: Effects of M1S and M2S on cell signalling proteins and cell cycle Cell lines Protein Expression (at 12 and 24 h) Acetyl H3 A549 H522 Cyclin B1 Cyclin D1 Cell Cycle (at 6 and 12 h) G1, S and G2 Phases Non-significant at EC50 to 2×EC50 concentration of M1S and M2S. Non-significant at EC50 M1S: Significant changes at to 2×EC50 concentration 2×EC50 after 12 and 24 h. of M1S and M2S. M2S: Significant changes at 2×EC50 after 24 h. 49 M1S and M2S appeared to arrest at G1 phase at 2×EC50. Statistical analysis could not performed from n=2. Chapter IV Discussions 4 Discussions The current project was designed to study the cytotoxic effects and anticancer mechanisms of 11 novel metal-based and non-metal-based compounds against NSCLC cells in vitro. Previous investigations in the lung, cervical, and colorectal cancer cell lines have shown that these novel compounds have antiproliferative activity in the nanomolar to micromolar range. Therefore, it was hypothesised that potent cytotoxicity would also be observed in NSCLC cell lines. Based on the cytotoxicity screening, the two most potent compounds, M1S and M2S, were chosen to further investigate their effects on cell signalling proteins by Western blotting, and cell cycle progression by flow cytometry. To assure the reliability of the current results, it is important to understand the limitations of the protocols in order to interpret the experimental results. 4.1 Rationale for using different cell lines 4.1.1 A549 and H522 cell lines KRAS mutations are heterogeneous and different genotypes could be linked with specific clinical outcomes [25]. Table 4.1 shows the diversities in KRAS mutation and KRAS-mutated NSCLC cancer cell lines. The A549 adenocarcinoma cell line has been widely studied since its discovery in 1976 [193] and has a higher expression phospho-AKT (protein kinase B) [194]. Additionally, A549 cells are resistant to MEK inhibitors [194]. Consecutive activation of KRAS promotes continuous stimulation of downstream signalling pathways that result in tumourigenesis, including activation of RAF-MEK-ERK and PI3K-AKT-mTOR signalling cascades [36]. Furthermore, co-occurring genetic alterations could alter other signalling pathways and thereby affect biological behaviours, clinical outcomes, and therapeutic responses in KRAS-mutant lung cancer [195]. Table 4.2 depicts co-mutation frequency of KRAS gene in NSCLC patients. In the KRAS-mutant NSCLC patients, p53 (guardian of the genome) is one of the most frequent co-mutations [196, 197]. H522, a solid tumour-derived, adenocarcinoma cell line, has a mutation at exon-6 of the p53 gene [198-200]. p53 protein functions as a central hub that receives and transmits multiple signals. It plays a vital role in tumour suppression notably by promoting apoptosis, autophagy process, growth arrest, and senescence, as well as by inhibiting angiogenesis [197, 201]. Furthermore, p53 enhances the sensitivity of cancer cells to chemoradiation and thereby, prolonging the survival. Thus, p53 becomes one of the most popular targets for mechanism-based anticancer drug discovery [197, 202]. Lastly, H522 and A549 cell lines have been commonly used to study the antimetastatic activity of drugs [203-205]. 50 51 KRASAmp. KRAS Mutated gene 30.2% 48.57% 9.33% 6.12% ~15% N= 129 (NSCLC) N= 179 (ADC) N=230 (ADC) N= 183 (ADC) N= 385 (NSCLC) 27.26% N= 532 (ADM & ADC) 20% 36.81% N= 163 (ADC) N= 129 (NSCLC) 26.78% 32.61% N=230 (ADC) N= 183 (ADC) Frequency Sample et al. Wagner [218] et al. Imielinksi et al. [6] CGMARN [6] Nadal et al. [217] Svaton [216] Dang et al. [215] NCI GDC data portal [214] Ding et al. [6] Imielinksi et al. [6] CGMARN [6] Source Codon 61 Codon 13 Codon 12 Frequently mutated codon in KRAS Q61K Q61H G13C G13D G12R G2S G12D G12A G12V G12C Mutation type Representative cell Lines Calu-6 [207] H460 [207] H1355, H1734 [207] H1944, H647 [208] H157 [206, 207] A549 [206] SK-LU-1, T3M-10 [208] H2009 [206] SW 900, LCLC-97TM1, H2444, H441, H727, RERF-LC-Ad2, COR-L23 [9, 207, 208, 211-213] Calu-1, LU-65, LU-99, H1385, H1792, H2122, H23, H358, SW 1573, IA-LM, HOP-62, H2030, H2212 [206-210] Table 4.1: Diversities in KRAS mutation in NSCLC patients and cell lines NSCLC: Non-small cell lung cancer; Amp.: Amplification; ADC: Adenocarcinoma; ADM: Adenomas; CGMARN: Cancer Genome Atlas Research Network; NCI: National Cancer Institute; GDC: Genome Data Commons. Mutation of KRAS gene in NSCLC patients Table 4.2: Co-mutation frequency of KRAS gene in NSCLC patients Mutation Type Mutated Genes Co-mutation of KRAS gene in NSCLC patients KRAS-TP53 Comutation of 2 genes KRAS-STK1 KRAS-EGFR KRAS-BRAF KRAS-ALK KRAS-ROS1 KRAS-EGFR -BRAF Comutation of 3 gene KRAS-TP53STK1 Frequency Source 30.67% N=230 (ADC) 38.78% N= 183 (ADC) 35% N= 163 (ADC) 31% N= 75 (NSCLC) 26.67% N=230 (ADC) 14.29% N= 183 (ADC) 16.67% N= 163 (ADC) 8% N= 75 (NSCLC) 11% N= 75 (NSCLC) 1.4% N= 350 (NSCLC) 0.3% N= 350 (NSCLC) 0.3% N= 350 (NSCLC) 0.3% N= 350 (NSCLC) 0.3% N= 350 (NSCLC) 1.33% N=230 (ADC) 6.12% N= 183 (ADC) 5% N= 163 (ADC) CGMARN Imielinksi et al. Ref. [6] Ding et al. Lei et al. [196] CGMARN Imielinksi et al. [6] Ding et al. Lei et al. [196] Lei et al. Dang et al. Dang et al. Dang et al. [215] Dang et al. Dang et al. CGMARN Imielinksi et al. [6] Ding et al. NSCLC: Non-small cell lung cancer; ADC: Adenocarcinoma, ADM: Adenomas; CGMARN: Cancer Genome Atlas Research; Ref.: References. 4.1.2 NIH3T3 cell line NIH3T3 is an immortalised mouse embryonic fibroblast cell line used to promote survival and self-renewal of tissue progenitor cells [219, 220]. This cell line is also preferred for transfection experiments [220]. NIH3T3 is non-tumourigenic in athymic nude mice however, subcutaneous injection of NIH3T3 cells with basement membrane protein (matrigel) forms locally invasive and highly vascularised tumours. Additionally, intravenous administration of tumour-derived 52 NIH3T3 cells generates multiple colonies on the lung surface while the parental NIH3T3 cells are non-metastatic [221]. Thus, the NIH3T3 cell line is considered to be preneoplastic [222]. Fibroblasts are one of the key cellular components for tumours and reside in highly complex multicellular environments in the lung [223, 224] Fibroblast, endothelial and immune cells coexist along with transformed cells in the tumour microenvironment (TME) [225]. The TME is directly involved in tumour initiation, progression, drug resistance, and metastasis. Current therapeutic strategies focus only on the rapid-growing tumour “seeds” ignoring the tumour “soil” i.e. TEM [226]. A recent study showed that the NSCLC cell lines (A549 and H1299) cocultured with two different fibroblast cells increased mitochondrial function in the tumour cells [225]. Mitochondria influence multiple cellular mechanisms that support tumour progression via the proliferation of transformed cells, adaptation under an unfavourable microenvironment, and finally the migration to distant anatomical sites [227]. Therefore, the antiproliferative activity of M1S and M2S was examined in NIH3T3 cells as a representation of fibroblast cells. 4.1.3 PNT1A cell line PNT1A is a normal, immortalised non-transformed prostate epithelial cell line [228, 229]. PNT1A cells are positive for phosphatase and tensin (PTEN) homologue and cells harbour wild type p53 [230]. PNT1A is a reliable model for understanding the cellular processes, for instance, the proliferation of prostatic epithelium in response to androgen and growth factors [231]. It is noted that to immortalise the PNT1A cells, cells were transfected with a plasmid containing the SV40 genome with a defective replication origin and express large T protein [232]. Therefore, PNT1A cells are representative of a normal cell line having the aforementioned limitation. Pulmonary epithelial cells play a crucial role in the maintenance of lung tissue homeostasis [233]. Specialised epithelial cells located in a different part of the lung tissue function in host defense, gas exchange, xenobiotic metabolism, chemoattractant, maintenance of structural integrity, expression of adhesion receptors, lipid mediators for communication with neighbouring cells and matrix attachment [233-235]. Interruption of the formation of special epithelial cells negatively impacts on lung morphology [235]. Therefore, the cytotoxic potential of M1S and M2S were tested on PNT1A cells as a representation of normal epithelial cells. 4.2 Critiques of experimental design and methods 4.2.1 SAHA as a control drug SAHA is an FDA approved HDACi that showed antiproliferative activity toward NSCLC cells including A549 and H522 [236-239]. As the cytotoxic potency of SAHA against A549 cells 53 and H522 cells has been reported using the SRB assay [236-238], it was selected as an appropriate control drug with which to compare the various novel drugs. 4.2.2 Preclinical cytotoxicity study using the SRB assay To develop a new therapeutic agent accurate and reliable data from in vitro cytotoxicity experiments is vitally important as these results are critical to choosing a lead drug candidate [240]. In cancer drug discovery studies, several in vitro assays are used to screen cell viability and these are based on a variety of parameters. These in vitro assays have their pros and cons, hence it is important to select the most reliable ones based on the literature [240, 241]. Additionally, the cost, experimental equipment, and method of detection should be taken into account before selecting an in vitro technique [241]. The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay has become a popular method for determination of drug cytotoxicity and cell viability [242]. Several tetrazolium-based assays, such as the sodium salt of 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H5-tetrazolio]-1,3-benzene disulfonate (WST-1), [5-(3-carboxymethoxyphenyl)-2-(4,5,- dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium, inner salt] (MTS), and 2,3-bis(2-methoxy-4nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assays have yet to replace the well-established MTT assay [240, 243-245]. However, research has proven that the MTT assay is inconsistent and nonspecific due to dependency on cellular metabolic status including the number of viable mitochondria, mitochondrial activity, cellular metabolic and energy perturbations, variation in oxidoreductases activity, exo-/endocytosis and intracellular trafficking leading to both false-positive or false-negative results [242, 246-248]. Previously, resveratrol, verapamil, ursolic acid, rottlerin, imatinib, genistein nanoparticles, nano-TiO2, some polypeptides and plant extracts (of Hypericum adenotrichum, Salvia kronenburgii, and Pelargonium quercetorum) were reported to interfere with MTT reduction and thereby also produce inconsistent data between the MTT assay and other assays [246-248]. Furthermore, resazurin reduction (RES), neutral red uptake (NRU), and SRB data gave a smaller variability across the linear range while larger variation was produced by the MTT assay. The results indicate that these assays would be more reliable in the detection of small changes in cell number than the MTT assay. Among these three techniques, results from the SRB assay have generated the most reproducible data as indicated by the coefficient of determination [240]. The SRB assay is a quick and sensitive colourimetric technique for the determination of drugmediated cytotoxicity in both suspension and attached cells. This assay is described by Skehan and colleagues for the development of a large-scale in vitro anticancer discovery program at 54 the National Institute of Cancer (NCI) [249]. SRB is a bright pink aminoxanthene dye that binds to protein basic amino acid residues of TCA-fixed cells, which is related to the viable cell number. An advantage of this technique compared to others is that cell quantification is based on protein content that does not interfere with test compounds, thereby producing high reproducibility [249, 250]. Additionally, fewer steps are required to optimise the assay protocol compared with MTT [251]. Finally, this assay has higher sensitivity, better linearity, a stable endpoint, is independent of intermediary metabolism, and is less sensitivity to environmental fluctuations [249, 250]. The drawbacks of this assay include low sensitivity with nonadherent cells, as well as the need to add TCA to fix the cells [240]. In comparison with MTT, SRB stains recently lysed cells without staining the cell debris [251]. Overall, considering the test parameters, the cytotoxicity of our novel metal-based and non-metal-based synthetic compounds were screened by the SRB assay. 4.2.3 Western blotting Western blotting, also called immunoblotting, is one of the most widely used and broadly useful techniques in cancer research to understand the cell signalling mechanism of proteins that are linked with cancer treatment [252, 253]. The techniques use SDS-PAGE to separate complex protein mixtures present in a sample. The separated proteins are then transferred onto PVDF or nitrocellulose membrane, where they bind to antibodies specific for target proteins [254]. The effectiveness of the protein transfer process is dependent on the type of gel (percentage of acrylamide, bisacrylamide: acrylamide ratio) and transfer membrane. Each membrane has a capacity limit for protein binding (e.g. PVDF ~ 170 µg/cm2, 80-250 µg/cm2), so it is wise to consider how much protein lysate to run per well on the gel [255]. The membrane must be blocked after the transfer process to avoid high background and non-specific binding of the antibody. A 1 h incubation with 3-5% non-fat dry milk, goat serum or BSA solution is usually recommended [256]. After that, the membrane is incubated with a primary antibody to the target protein. The primary antibody is detected by an enzyme tagged secondary antibody. The secondary antibody catalyses an enzymatic reaction with the substrate to produce light, which can be detected by X-ray film or digital imager [254, 256]. Enhanced chemiluminescence has become the most popular method for detection in Western blotting due to its high sensitivity [255]. It is important to note that the data generated with Western blotting is typically considered semi-quantitative. Western blotting measures a relative comparison of protein levels, but not an absolute measure of quantity. Though the protocol of Western blotting is simple, many experimental errors can arise, leading to undesirable results. The unexpected results can be grouped into five categories: (i) unusual or undesirable bands, (ii) faint bands or 55 weak signal, (iii) no band, (iv) uneven or patchy spots on the blot and (v) high background on the blot [257]. The advantage of immunoblotting is the ability to identify target proteins as low as 1 ng because of the high-resolution capacity of gel electrophoresis and specificity and strong sensitivity of the immunoassay [254]. The molecular mass of the identified protein can be determined by comparing with standards of known molecular weight. Additionally, the selective nature of the specific antibody promotes the determination of a target protein in a complex mixture having >100,000 different proteins [256]. Isoforms and post-translationally modified protein with the same molecular weight can be identified using 2D electrophoresis (2DE Western). Western blotting can also be used as an effective early diagnostic tool [256, 258]. Like all other experimental techniques, Western blotting has its limitations [256]. The major limitation of this method is dependent on the primary antibody against the target protein and many protein targets cannot be determined due to the unavailability of specific antibodies [259]. Another main drawback is that many antibodies are non-specific [256]. Approximately, 50% of commercially available antibodies are of low quality and should not be used for Western blotting [259]. Furthermore, each antibody requires independent optimisation and protocol modification. Larger proteins (>140 kDa) may not be transferred from the gel to the membrane and smaller proteins (<10 kDa) may not be retained by the membrane. Other limitations include the inability of the primary antibody to detect the denatured immobilised antigen, quick decay of the detection signal, and high background [256]. However, these limitations are mainly due to sub-standard antibodies and sub-standard protocols that can be overcome with quality antibodies and proper experimental techniques [256, 259]. 4.2.4 Flow cytometry Flow cytometry is a technique that offers rapid multi-parametric analysis of various properties of single cells in solution. A flow cytometer machine utilises lasers as light sources to generate both scattered and fluorescent light signals [260, 261]. The fluorescence can be used for qualitative and quantitative determination of various cellular properties including relative size, internal complexity, DNA, RNA, or protein content [261]. Flow cytometry is a powerful tool that has a wide range of applications in the biomedical field including cancer biology [260]. Flow cytometry can be used to examine cell cycle kinetics and progression based on the DNA content of individual cells. To quantify the DNA content cells are stained with a fluorescent die that interacts with the DNA [262]. PI is a widely used DNA binding die with red fluorescence. As PI stains double-stranded nucleic acids, to avoid RNA interference PI is used in combination 56 with RNAase for the accurate determination of cell cycle phases (i.e. G1, S, G2/M phases) [262, 263]. Ethanol preserves PI stained cells for up to 3 days without significant interference with the cell-cycle distribution [264]. Additionally, ethanol fixation leaks out the highly fragmented DNA of the apoptotic cells during their dehydration and staining, while fixation with other solvents, for instance, formaldehyde preserves the fragmented DNA inside the cells [265]. Ethanol fixation thereby provides an extra advantage in the detection of sub-G1 apoptotic cells. Furthermore, 70% ethanol fixed cells can be stored up to 4 weeks [262]. It should be noted that ethanol-based fixation often produces considerable aggregation of cells. Dropwise addition of ethanol along with vortexing is used to minimise the aggregation. Advancement in analysis has improved flow cytometry as the total fluorescence of individual cells can be achieved via sophisticated gating from a cell population. The fluctuations in protein and gene expression could be due to variations in cell volume. To reduce cell variability, flow cytometry relies on “gating” (i.e. use of sequential gates in the FSC-A/SSC-A and FSC-H/FSCW dot blots to discard undesired cells that located outside of the selected gates) thereby reducing variability in the cellular optical properties [266]. Cell volume and granularity are primary confounding characteristics that are linked with the forward scattered (FSC) light and side scattered (SSC) light measurements, respectively. For instance, larger cells may show an apparent increased fluorescence. To minimise the cell size and granularity effects on the fluorescence intensity, the phenotypic variability based regression model can be used [267]. A limitation of flow cytometry is that it can generate “false-positive” heterogeneity, most of the results are associated with cytotoxicity, apoptosis, and cellular differentiation [266, 268]. The false-positive results could be due to sample preparation artifacts and staining resulting from antibody cross-reactivity and lack of morphological information. Additionally, the flow cytometric study of adherent cells has the main disadvantage that cells have to be detached into a single cell suspension before analysis [266]. 4.3 Interpretation of results 4.3.1 Dose-response cytotoxicity study To determine the most potent drug for KRAS-mutant NSCLC eight metal-based and three nonmetal-based compounds were screened in A549 cells. Potency is defined as the activity of a drug in terms of the amount or concentration of the drug required to produce the desired effect [269]. Potency has an influential role in in vitro drug testing and selection [270]. The lower the EC50 value, the lower the concentration required to kill 50% of cells, therefore, a higher potency [271]. Compounds with lower EC50 values are predicted to be more effective in in vivo with minimum side effects, as the effective dose will be small (assuming toxicity is proportional to 57 dose) [270]. The first large scale drug screen was performed in the NCI60 panel of cell lines using the SRB assay after 48 h of continuous incubation [272]. Another publicly available database (http://cancer.sanger.ac.uk/cosmic) reported that the anticancer potency of >100 drugs against ~1000 cells relied on 72 h continuous incubation with the drug [272]. Several other groups examined the potency of drugs after 72 h [273-275]. Furthermore, Gulden et al. reported that at least 72 h of incubation make the results more comparable to other in vitro assays and extrapolation to in vivo experiments more meaningful than shorter exposure times [276]. To achieve reliable and reproducible potency, all the metal-based and non-metal-based drug candidates were exposed to the experimental cell lines for 72 h. M2S, M1S, and JAZZ-90 showed the most potent cytotoxicity towards A549 cells with EC50 values <0.8 µM. On the other hand, the metal compound ZR-014, ZR-012, M2S-Ru, and M1S-Ru produced EC50 values of 1.5-1.8 µM. While the EC50 value of the control drug, SAHA, was 1 µM in A549 cells. Liu and colleagues determined the IC50 value of SAHA (2.6 µM) in A549 cells, using the SRB assay after 72 h [238]. Depending on the seeding cell number, cell viability assay, cell culture media, and experiment time, other groups have reported EC50 value of SAHA between 0.69 to 5.34 µM in A549 cells [277-279]. The antiproliferative activity of JAZZ-90, M1S, M2S, M1SRu, M2S-Ru, Z4-012, and ZR-014 was comparable to SAHA while the potency of AASH-121, JAZZ-166, and JAZZ-167 were significantly lower compared to SAHA. In terms of EC50 values, M2S, M1S, and JAZZ-90 were the most potent drug candidates and were selected for further experiments. Though M1S-Ru, M2S-Ru, ZR-012, and ZR-014 were equally potent, M1S-Ru and M2S-Ru were selected for the next experiments along with three most potent drugs considering that M1S-Ru and M2S-Ru contain the cytotoxic moieties of M1S and M2S. SAR is crucial in understanding various aspects of drug discovery as it provides information regarding structural modifications to optimise drug physicochemical properties and biological activities that include potency improvement, toxicity reduction, and ensuring sufficient bioavailability [280]. Anticancer activity of non-metal and metal-based derivatives varied in A549 cells, the variation might be due to the structural differences. JAZZ-166 (EC50 value of 3.17 µM) and JAZZ-167 (EC50 value of 4.22 µM) are the Ir and Rh arene complex of JAZZ-90 (EC50 value of 0.76 µM), where the metal arene moiety is connected with the capping group. Hanif and colleagues also observed that JAZZ-90 was more potent compared to JAZZ-166 and JAZZ-167 in multiple cancer cell lines including HCT116, H460, SiHa, and SW480 [153]. Generally, the capping group is a hydrophobic structure that interacts with the rim amino acids [281]. The surface recognition cap group offers unique interaction with biological targets including overexpressed receptors that could influence the selective accumulation of HDACis 58 [282]. Modification of the cap group affects the in vitro HDAC inhibition and antiproliferative activity [283]. Based on previous studies and current results JAZZ-166 and JAZZ-167 were less potent than JAZZ-90 and this might be due to the steric hindrance of the bulky capping group. The compounds AASH-122 (EC50 value of 2.02 µM) and JAZZ-121 (EC50 not achieved) have a similar structure (PCA in the pharmacophore), except the metal and arene ligand, the pcym (clogP value of 3.90) of AASH-122 (Ru based) was replaced with 1,2,3,4-tetramethylcyclopenta-1,3-diene (clogP value of 3.67) in JAZZ-121 (Rh based). Guichard and colleagues observed that [(η6-tetrahydroanthracene)Ru(ethylenediamine)Cl]PF6 (HC11) exhibited promising anticancer activity against A549 and H520 NSCLC cell lines with IC50 values of 0.5 and 0.53 μM, respectively. The alteration of arene ligand (THA, clogP value of 3.43) of HC11 with bip (clog P value of 3.73) in RM175 remarkably decreased the antiproliferative activity towards A549 and H520 cells with IC50 values of 3 μM and 3.5 μM, repectively [96, 154]. Like AASH-122 and JAZZ-121, ZR-012 (Ru based) and ZR-014 (Os based) contain PCA in the structure with different metal coordination. ZR-012 and ZR-014 contained PPh3 instead of chlorine atom with the metal in AASH-12, were equally potent with EC50 value of <1.64 μM, and showed slightly higher potency compared to AASH-121. Previous findings and our results indicated that metal complexes lacking the PPh3 showed poor anticancer activity, suggesting the importance of PPh3 to enhance the antiproliferative activity of metal complexes [118]. Cancer cells vary remarkably between individuals. Furthermore, some properties of in situ tumour cells are lost during the processing and immortalisation in culture. To overcome these limitations, scientists investigated the drug’s effect in multiple cell lines with genetic and phenotypic variation; expecting that consistent results could be more generalisable and have a high predictive value [270]. A549 cells have a pebble-like shape and are connected with the neighbouring cells with close cell-cell adhesion [284, 285]. On the other hand, H522 cells grow as adherent, single cells, and loosely attached clusters [200, 286]. A549 cells are growing faster (approximate doubling time 22 h) [287] than H522 cells (doubling time 38.2 h) [288]. Slower growing cells respond differently to drugs than faster-growing cells and the variation may be driven by the cell cycle [289, 290]. Additionally, the seeding density and cultural conditions also contribute to the alteration in drug sensitivity [289]. The EC50 of JAZZ-90, M1S-Ru, and M2S-Ru in H522 cells were all higher compared to A549 cells. Additionally, the EC50 values of these drug candidates in H522 cells were significantly different from the control drug SAHA. While M1S and M2S showed consistent cytotoxicity toward H522 cells with EC50 values of ~0.2 µM; this value was comparable with SAHA. Based on SRB assay results, Penthala et al. reported that the thiobarbiturate acid analogue 4-((3-((4,6-dioxo-2-thioxotetrahydropyrimidine5(2H)-ylidene)methyl)-2-methyl-1H-indol-1-yl)methyl)benzoate) 59 (7k) displayed similar cytotoxic activity in H522 cells with an IC50 value of 0.25 µM after 48 h. However, the IC50 value of the same compound rose to 1.36 µM in A549 cells [291]. The N-hyroxypyridone derivative militarinones E also exhibited 7k like antiproliferative activity in A549 cells with an IC50 value of 1.59 µM [292]. Similar to M1S and M2S, the control drug of the present study (SAHA) displayed more cytotoxicity in H522 cells compared to A549 cells with EC50 values of 0.35 and 1µM, respectively. SAHA induced more cytotoxic effect in mutant, rather than p53 null or wild-type human cancer cells [293]. However, Karelia and coworkers reported similar IC50 values (0.87 and 0.83 µM) for SAHA in A549 and H522 cells using the SRB assay after 24 h [236]. However, using the CellTiter 96 AQueous assay, it was reported that A549 cells were more sensitive to SAHA compared with H522 cells with IC50 values of 1.5 and 2.1 µM, respectively [279]. The variation in the IC50 of SAHA in the A549 and H522 might be due to different assay types, growth media, and seeding density [236, 279, 294]. The main targets of anticancer drugs are intracellular and, therefore, the drugs must pass the cell membrane and, finally, the nuclear membrane to drive pharmacological activity [295]. Lipinski’s “Rule of 5” is considered as the most prominent framework in predicting bioavailability and cell membrane permeability in the field of small-molecule drug development. One of the five rules is that compounds that have a molecular weight over 500 are less permeable to the lipid membrane, however, all the compounds do not follow this rule [296]. It has been previously reported that lung cells are naturally permeable to all the smallmolecule drugs, therapeutic peptides, and proteins [297]. It was hypothesised that the size of M1S and M2S might be a factor that determines their potency. M1S and M2S were smaller (in terms of molecular weight) and more potent among the tested compounds in both the NSCLC cell lines (A549 and H522). M1S-Ru and M2S-Ru are the Ru complex of M1S and M2S, respectively. While JAZZ-166 and JAZZ-167 are the Ir and Rh complex of JAZZ-90, respectively. The molecular weight of JAZZ-90, M1S, and M2S is lower than the metal-based counterparts of the compounds. As mentioned earlier, JAZZ-90 was more potent than JAZZ166 and JAZZ-167 in A549 cells while M1S and M2S were more potent than their corresponding metal-based compounds in both A549 and H522 cell lines. Additionally, the presence of a free sulfur group in the drug candidate structure might be another factor that modulates a drug’s activity as this group influences cellular uptake. It has been reported that the presence of thiol groups in the drug structure may facilitate the interaction with the cell surface via disulfide exchange reaction at the plasma membrane and reactive cell surface thiol groups may promote absorptive endocytosis [160]. The breakdown of the disulfide bonds in a macromolecule having disulfide bridge (-S-S-) occurs at the cell membrane after endocytosis, thus exofacial thiols can act as a natural system for the entry of extracellular compounds inside 60 the cells [160]. JAZZ-90, M1S, and M2S have an available sulfur group while the metal-based complexes of these compounds the sulfur group interacts with metal. In A549 cells, all the drug candidates (JAZZ-90, M1S, and M2S) with the free sulfur group were more potent than their metal-based counterparts, which might be due to higher cellular uptake. El-Sayed and colleagues reported that [4-(3-(4- fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-2-(2- mercaptophenylamino)-6-(naphthalen-1-yl)-nicotinonitrile] displayed promising antiproliferative activity towards HepG2 and HeLa cells with IC50 values of 12.2 and 19.4 µM, respectively. This compound contains an NH group and a free SH group, which may interact with DNA bases either via thia/aza Michael addition or through the formation of hydrogen bonds, thereby causing DNA damage [298]. Furthermore, experimental drug candidates were dissolved in DMSO, this might be unfavourable for the metal compounds. Hall and colleagues reported that DMSO interacts with platinum drugs (e.g. cisplatin) containing monodentate ligands, thereby causing ligand displacement and structural changes. These changes were found to reduce the antiproliferative activity of monodentate ligand-based platinum drugs. However, bidentate ligand-containing platinum drugs (e.g. oxaliplatin) were found to be more active in DMSO compared to aqueous solvents [299]. In the current study, all the experimental drugs contained one mono and one bidentate ligand. Hall and colleagues showed the activity of the carboplatin and [PtCl2(en)] (having both mono and bidentate ligand) decreased remarkably against DLD-1 colorectal cancer and KB-3-1 cervical carcinoma cell lines when DMSO was used as a solvent compared to saline [299]. However, the opposite effect of the solvents was observed towards cisplatin-resistant KB-CP.5 cells (cisplatin-resistant sub-line of KB-3-1). Therefore, testing the cytotoxic activity of the experimental novel metal compounds in other solvents could confirm the potential effect of DMSO. Fibroblast cells are one of the key components of the TME [225] and both the drug candidates (M1S and M2S) displayed cytotoxic potential against the NIH3T3 cell line, a fibroblast cell line with intact p53 function [300]. Based on the cytotoxicity screening of 1,408 compounds in multiple cell lines, it was summarised that NIH3T3 was one of the most sensitive cell lines to compound induced cytotoxicity [301]. M2S showed a similar cytotoxic effect in A549 cells, while the potency of M1S decreased by 1.2-fold compared to A549 cells. Using the MTT assay it was reported that 48 h post-treatment of Au(2-thiopyridine) 2- (diphenylphosphino)ethylamine-2-carbonylthiophene (a gold (I) complex) caused higher antiproliferative activity against NIH3T3 compared to A549 cells as evident from the IC50 values, 2.1 and 24.5 µM, respectively. Under the same experimental condition, cisplatin showed more cytotoxic activity towards A549 cells compared to NIH3T3 cells with IC50 values 61 of 64 and 145 µM [302]. Conventional chemotherapeutic agents are non-selective and can damage normal tissues, which results in undesirable and severe side effects [303, 304]. Higher cytotoxicity of antiproliferative agents towards normal cell line is one of the preliminary concerns in drug screening [305]. The development of a new therapeutic agent that actively or passively targets cancerous cells, can not only improve therapeutic outcomes but also minimise drug-induced toxicity [303, 304]. A SI greater than one indicated that the drugs were less toxic to normal tissue cells compared with cancer cells, therefore, compounds with high SI value were assumed to be safer for therapy [306]. In the current study, M1S and M2S showed a similar SI, as both the drugs exhibited the highest selectivity against H522 cells (SI value of 4.46 to 4.87), intermediate selectivity in A549 cells (SI value of 3.50 to 3.55) and lowest selectivity towards NIH3T3 cells (SI value of 2.91 to 3.29). It has been reported that (E)‐resveratrol 3‐O‐rutinoside (SI value of 11.3) displayed better selectivity towards A549 cells compared to vinblastine (a drug used in NSCLC treatment) (SI value of 1); SI in A549 cells was calculated using PNT2 (normal human prostate cell line) as the normal cell [306, 307]. Novel coumarin-based benzopyranone derivatives exhibited selective anticancer activity (SI value of 1.01 to 4.08) in A549 cells; the SI was calculated using LL49 lung cell line as normal cells [305]. The SI value of artemisinin in A549 cells was 2.40 which is comparable with the selectivity of M1S and M2S in A549 cells. However, the artemisinin SI value was calculated using HBE (normal human bronchial epithelial cell line normal cell line) as the normal cell line [308]. Interestingly, M1S and M2S displayed more toxicity and selectivity towards p53 mutated H522 cells compared with p53 intact cells (A549 and NIH3T3 cells). The clinically used agent, taxol, was reported to be more effective against p53 mutated cells, but toxic towards normal cells as well [309]. The hydroxythiopyridone derivatives, M1S and M2S contain a pyridine and benzene ring in their chemical structure. Novel 2 pyridone derivatives displayed anticancer activity against six human cancer cell lines including A549 lung cancer cells. 5-methoxy-2-(6-oxo-1-o-tolyl-1,6dihydropyridine-3-carbonyl)phenyl propyl carbonate (8i), a 2 pyridine derivative showed cytotoxicity in A549 cells with an IC50 value of 6.13 µM. The addition of benzyl and ethyl group to 8i, increased its cytotoxicity by 5-fold [310]. Both M1S and M2S have a benzyl group in their structure. Additionally, M1S and M2S have a structural moiety similar to 3-HPT. 3HPT displayed no cytotoxic potential in DU-145 (androgen-independent prostate cancer), LNCaP (androgen-dependent prostate cancer), and Jurkat (T-cell leukaemia) cells. Interestingly, 3-HPT hydroxyoxypyridin-2-thione, derivatives 1-(4-dimethylamino-(1,1'-biphenylmethyl))-3- 1-(2-cyano-(1,1'-biphenylmethyl))-3-hydroxyoxypyridin-2- 62 thione and 1-(2-methyl-(1,1'-biphenylmethyl))-3-hydroxyoxypyridin-2-thione showed cytotoxicity against all three cell lines, but these compounds were less potent compared to SAHA [163]. M2S showed slightly higher cytotoxic potential than M1S in neoplastic, pre-neoplastic, and normal prostate epithelial cells. M2S has an identical chemical structure as M1S except one additional carbon attached to the benzyl group. Lipophilicity and cytotoxicity tends to increase with the increase of carbon chain (up to a certain number). This might due to a favourable balance between lipophilicity and hydrophobicity [311, 312]. Lipophilicity is one of the crucial parameters that determine the cellular uptake of small molecules including drugs [160], it also aligned with drug potency and pharmacokinetics [313]. The octanol/water partition coefficient, logP, predicts the lipophilicity of compounds, where a high logP value corresponds to higher lipophilicity [314]. logP is also a determining factor in the anticancer activity of some compounds, for instance, crown ether compounds showed anticancer activity with an optimum logP value of 5.5 [312]. The logP value for M1S and M2S were calculated using Molinspiration online software tool [154] and the values obtained were 2.02 and 2.23 for M1S and M2S, respectively. The slightly higher logP value of M2S compared to M1S might facilitate its cellular uptake, and thereby, enhance its cytotoxic potential. 4.3.2 Time course of cytotoxicity Most of the cell-based assays are performed at a standard time point of 48 h to 72 h posttreatment to investigate the potential effect of the drug. Due to this extended time point, the effect of the drug at the early treatment period could not be determined. However, a time-course is necessary to examine whether the drug is cytostatic, cytotoxic or it simply arrests cell division [242]. Additionally, it has been reported that cytotoxicity shifts with time [253, 315, 316]. The time course study observed the anticancer potential of M1S and M2S towards A549 and H522 cells after 0, 12, 24, 36, 48, 60, and 72 h of treatment at 2×EC50 concentrations. Both drug candidates showed an almost identical cytostatic pattern in A549 cells. A statistically significant difference in cell number was observed from 12 h and 24 h of M2S and M1S treatment, respectively. No significant difference in cell number was seen between each time point of M1S and M2S treatment. Time-dependent activity of M1S and M2S towards A549 cells indicated that these compounds could have blocked cell division, without inducing cell death [317, 318]. Therefore, M1S and M2S appeared to be cytostatic at the concentrations examined in A549 cells [319]. Interestingly, the two drug candidates displayed cytotoxicity at 12 h in H522 cells. Compared with control, the initial cell population showed an approximately 2 and 2.9-fold decrease after M1S and M2S treatment, followed by a static cell number up until 63 72 h. 4.3.3 Drug-mediated changes in protein levels Diverse cell signalling proteins are involved in the cytostatic and cytotoxic activity of a drug [320]. Furthermore, cytostatic and cytotoxic activity of a drug relies on the applied drug concentration, frequency of drug treatment, the action of a drug on cell cycle phases, and cellular context [319]. To investigate the potential mechanism underlying the cytostatic and cytotoxic activity towards different cell lines, acetyl H3, cyclin D1, and cyclin B1 expression was tested. Acetyl H3 HDACis attenuate acetylation events and block several cancer‐related signalling pathways [321]. HDACis either alone or in combination with other cytotoxic agents showed promising activity in the treatment of NSCLC [321, 322]. While the structure of M1S and M2S do not support the idea that they would be potent HDACis, this was confirmed by Western blotting analysis of acetyl H3 expression. No significant differences in acetyl H3 expression were produced when A549 and H522 cells were treated with EC50 and 2×EC50 of either M1S or M2S. These results indicate that the pharmacophore of M1S and M2S was not able to interact with the active site of HDACs. Therefore, no changes in the acetylation of H3 were observed in response to drug candidates. A previous study reported that the channel to the HDAC 1 active site is small as compared to HDAC 6 whose hydrophobic channel entrance seems large enough to interact with 3-HPT [323], a compound that possesses a similar pharmacophore to the M1S and M2S zinc-binding domain. Sodji et al. observed that five –CH2 methylene groups were optimal between the hyroxythiopyridone (act as Zn2+ chelator) and triazole (act as capping group) for inhibition of HDAC 6 and HDAC 8. 1-phenyltriazolylethyl-3-hydroxypyridine-2thione (10a) (two –CH2 methylene linker) was reported to have IC50 value of >10 µM for HDAC 6 and HDAC 10, which decreased to 911 nM and 917 nM in 1-phenyltriazolylpentyl-3hydroxypyridine-2-thione (10d) (five –CH2 methylene linker) [323]. Increased potency towards HDAC 6 inhibition was reported to occur due to a stable π-stacking interaction between the phenyl ring of 10d and Phe680 at the entrance to the active site of HDAC 6, resulting in improved HDACi binding and activity. On the other hand, enhanced potency against HDAC 8 was reported to occur as a result of the capping group incorporation into an inaccessible hydrophobic pocket close to the active site, which was inaccessible for 10a due to the shorter methylene spacers [323]. M1S and M2S contains one –CH2 and two –CH2 methylene groups between the capping group and the zinc-binding domain, respectively. Previously, M1S and M2S like compounds with shorter methylene groups were reported to have HDACi activity. 64 For example, 3-HPT based compounds with one aryl group connected via single –CH2 methylene linker, 1-benzyl-3-hydroxypyridin-2-thione, showed HDAC 6 and HDAC 8 inhibitory activity with IC50 values of 457 and 1272 nM, respectively. While 1-(1,1'biphenylmethyl)-3-hydroxypyridin-2-thione, synthesised by the addition of two phenyl groups with 3-HPT, produced decreased HDACi activity towards HDAC 6 and HDAC 8, where the IC50 value increased to 847 and 4283 nM, respectively [163]. These results indicated that the pharmacophore of M1S and M2S lacks HDAC selectivity. Furthermore, drug docking modelling studies in our lab showed that M1S did not fit the active site of HDACs (Z. Rana, personal communication). Cyclin D1 D- type cyclins (cyclin D1, D2, D3) activated via mitogenic growth factors (notably RAS signalling pathways), stimulate the quiescent cells (G0 phase) to enter into the G1 phase of the cell cycle [324]. Cyclin D1 is a regulatory subunit of the CDKs including CDK4/6, which controls cell proliferation and development via its transcriptional regulatory activity [325]. Overexpression of cyclin D1 results in dysregulation of CDK activity, uncontrolled cell division under restricted mitogen signalling, bypass of cell cycle checkpoints, and ultimately oncogenic consequences [326]. These consequences include angiogenesis, through the regulation of VEGF expression, DNA damage response, and centrosome depletion [53]. Furthermore, an alternation of cyclin D1 expression could significantly affect the cellular responses to drug treatment [59]. Cyclin D1 overexpression is common in NSCLC (~5% - 76%) which leads to a loss of the G0/G1 to S checkpoint [53, 326]. Stage I and II NSCLC patients (n=98) with cyclin D1 positive tumours had shorter survival than those with cyclin D1 negative tumours (5-year survival of 48% vs 74%, respectively; p=0.006 determined through the log-rank test). A higher percentage of NSCLC patients with poor differentiation of the tumour, visceral pleural invasion, chronic pulmonary obstructive disease have positive cyclin D1 expression than other lung tumours [327]. KRAS-driven NSCLC is particularly dependent on the CKD4 and is sensitive to the cyclin D1-degrading combination bexarotene (repress cyclin D1 through proteasomal degradation) and erlotinib (selectively and reversibly inhibits the tyrosine kinase activity of EGFR), although the response of KRAS-mutated cancer to the erlotinib is poor [53, 328, 329]. Pyrimidine and pyridine based derivatives have diverse selectivity and inhibitory potential for CDK/cyclin [330, 331]. Benzyl group containing purine based compounds, olomoucine (6(benzylamino)-2-[(2-hydroxyethyl)amino]-9-methylpurine), roscovitine (6-(benzylamino)- 65 2(R)-[[1-(hydroxymethyl)propyl]amino]-9-isopropylpurine) and R-roscovitine (2-(R)-(1ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine) were more potent against CDK1 and CDK2 than CDK4 [330, 332-334]. Interestingly, the R- roscovitine (IC50 value of 14.2 µM) displayed more specificity towards CKD4/cyclin D1 compared to olomoucine and roscovitine with IC50 values >1000 and >100 µM, respectively [332, 333]. Furthermore, Rroscovitine exhibited cytotoxic activity against A549 and H460 cell lines with IC50 values of 15.9 and 13.1 µM, respectively [333]. Similar to purine based compounds, pyrimidine ring containing compounds also displayed variation in terms of selectivity towards CDKs [330, 331]. Recently, the pyrido[2,3-d]pyrimidin-7-one has become an effective pharmacophore in inhibiting CDKs. An in vitro study showed that modification of C-2 position of pyrido[2,3d]pyrimidin-7-ones with a 2-aminopyridine side-chain enhanced the selectivity for CDK4/6. 8cyclopentyl-2-(pyridin-2-ylamino)-8H-pyrido[2,3-d]-pyrimidine-7-one, synthesised through modification of pyrido[2,3-d]pyrimidin-7-ones with a 2-aminopyridine side chain at C-2, showed more selectively towards CDK4/D than CDK2/A with IC50 values of 0.14 and 5.01 µM, respectively [331]. The C-2 modification with aniline exhibited more selectivity in the inhibition of CDK2/A than CDK4/D, where the IC50 decreased to 0.01 and 0.21 µM, respectively. However, aniline modification with aminopyridine-containing side chains enhanced the efficacy and selectivity towards CDK4/D compared to the CDK2/A with IC50 values of 0.002 and 0.04 µM, respectively [331]. It should be noted that both M1S and M2S contain the benzyl group and the pyridine core. Previously it was reported that 3-hydroxy-4pyridone ring containing non-protein amino acid mimosine (β-[N-(3-hydroxy-4-oxypyridyl)]α-aminopropionic acid), decreased the cyclin D1 activity, inhibited cyclin D1-associated kinase activity, increased p21 mRNA and protein level and upregulated p27 expression [335, 336]. Western blotting analysis of cyclin D1 protein expression demonstrated that when A549 and H522 were treated with either M1S or M2S at their respective EC50 and 2×EC50 concentration, a trend of concentration-dependent decrease of cyclin D1 expression was observed at 12 h and 24 h. In A549 cells, no statistically significant difference in any of the treatment groups was observed. It should be noted that data variation in biological replicates was too large to detect a difference. On the other hand, in H522 cells, the EC50 concentration of both the compounds failed to produce a significant effect in the reduction of cyclin D1 level. The 2×EC50 concentration of M1S significantly downregulated cyclin D1 expression level by 68.1% of control at 24 h. While the 2×EC50 concentration of M2S significantly reduced the cyclin D1/βtubulin ratio by 69.7% and 84.9% of control at 12 and 24 h, respectively. A decrease of cyclin D1 activity could block the G1 phase of the cell cycle [337]. These results demonstrate that 66 M1S and M2S may provide a therapeutic advantage to the NSCLC patients by inhibiting cyclin D1 expression. However, further experiments are required to determine the cell line-specific effects. Cyclin B1 Cyclin B1 in combination with CDK1 plays a vital role in the regulation of the G2/M phase transition in the cell cycle. The expression of cyclin B1 starts to increase in the G2 phase and peaks in the M phase [312, 338]. Cyclin B1/CDK1 complexes assist chromosome condensation, nuclear envelope breakdown, and mitotic spindle assembly [338]. Besides its role in the cell cycle, the regulatory function of cyclin B1 is also involved in stem cell self-renewal, DNA damage repair, epigenetic regulation, and mediation of pro-apoptotic signalling in response to mitotic arrest [324, 339]. Cyclin B1 is overexpressed in a considerable number of NSCLC patients, whose survival time is shorter than patients with low levels of cyclin B1 [61]. Additionally, CCNB1, a gene that encodes cyclin B1, plays a vital role in the development and progression of NSCLC [340]. As the deregulation of cyclin B1 expression leads to neoplastic transformation, suppression of cyclin B1 activity could be a potential target for antiproliferative therapy [341]. Cells with an elevated cyclin B1/CDK1 activity could favour mitosis while cells with a suppressed cyclin B1/ CDK1 activity could be arrested in the G2 phase [342]. In the current study, no significant changes in cyclin B1 level between any of the treatment groups and control were observed in A549 and H522 cells after 12 h and 24 h of M1S or M2S treatment. Previously, it was reported that 12 h post-treatment of A549 cells with 2-(4hexyloxyphenyl)-5-(4-hydroxyphenyl)pyrimidine (C1) cyclin B1 increased while the phosphorylation of cdc2 was decreased. These results indicated that C1 activated cdc2/cyclin B1 kinase and induced mitotic arrest rather than G2 arrest [312]. On the other hand, in H522 cells both M1S and M2S exhibited a concentration-dependent decrease of cyclin B1/β-actin ratio at 24 h. Based on the in vitro NSCLC model using H522 and H460 cells it was reported that a decrease of cyclin B1 encoding CCNB1gene expression suppressed the proliferation, migration, and invasion of NSCLC cells [340]. 4.3.4 Effects of M1S and M2S in cell cycle Cell cycle arrest is a halting point in the cell cycle, where cells are no longer involved in the processes of duplication and division [343]. Anticancer agents may act at multiple steps during the cell cycle progression, however, G1/S or G2/M arrest occurs most frequently [344]. Cell proliferation can be effectively restricted if the G1/S checkpoint is arrested, as this is the limiting phase of the cell cycle [345]. 67 In the current study, no significant changes in the cell cycle distribution was observed in A549 cells after 6 and 12 h post-treatment of M1S and M2S at either EC50 or 2×EC50 concentration, while the statistical significance was not calculated for H522 cells as only two biological repeats were conducted. Interestingly, A549 cells were distributed more at the G2/M phase following treatment, however, a concentration-dependent effect was not seen. At 12 h, the 2×EC50 concentration of M1S and M2S increased 3.3 and 7.1% of cells in the G2/M phase compared to control. Wakasaya and colleagues reported that 12 µM (2×IC50 ) of 2-(4-butoxyphenyl)-5(4-hydroxyphenyl)pyrimidine significantly arrested A549 cells at the G2/M phase after 12 h, however, the effect disappeared at 24 h [346]. Cell cycle arrest is linked with the activity of the different cyclin/CDK complexes that control the successive phases of the cell cycle [347]. The Western blotting results of the current study showed a concentration-dependent trend of decreasing cyclin D1 level at 12 h in A549 cells. It has been reported that KRAS mediated proliferation converges on the CDK4/6 cyclin-D complex, which regulates G1 and S phase transition [348]. Huang et al. showed that cyclin D1-suppressing siRNA caused G1 arrest in rapidly proliferating A549 cells [349]. Furthermore, Ras-induced cyclin D1 expression at the G2 phase in rapidly proliferating cells and the levels of cyclin D1 in the G2 phase were sufficient to switch post-mitotic cells into their next S phase [350]. The results of the current study indicate that M1S and M2S could have a G2/M phase arresting activity and a higher concentration of the drug candidates might produce significant changes in the cell cycle. p53 mutation in cancer cells caused defective G1 checkpoint activity [351]. In H522 cells, it appeared that M1S and M2S produced a weak effect in arresting cells at the G1 phase at 12 h, where the 2×EC50 concentration of M1S and M2S increased 9.9 and 8.9% cell proportion at the G1 phase, respectively. However, the experiments were not performed in triplicate and should be considered preliminary. It has been reported that the Mdm2 inhibitor (MI) disrupts the interaction between Mdm2 and p53 and 10 to 30 μM of MI-43 induced G2 arrest (from 5% to 20%) in A549 cells, which was 5.9 to 17.6-fold higher than the IC50 concentration. Furthermore, the calculated IC50 value was 21 μM in H522 cells. On the other hand, 10 μM (approximately 0.5×IC50) of MI-43 failed to induce G1 arrest in H522 cells, but increasing the concentration to 30 μM caused 18.6% of arrest at the G1 phase [352]. In the present study, a minor sub-G1 peak that was to the far left of the G1 peak was observed both in the treatment and control groups of H522 cells. This minor peak is an indicator of apoptosis [353, 354]. While the results were considered preliminary, it appeared as though M1S and M2S treatment raised the percentage of apoptotic cells. This effect may be related to the cytotoxic response of the drug candidates towards H522 cells. In the A549 cells, no sub-G1 68 peak was observed. Therefore, the apoptotic populations could not be determined from the cell cycle analysis. A previous study showed that untreated A549 cells have a small sub-G1 population compared to untreated H522 cells after 72 h [355]. Zhu et al. observed that glycyrrhetinic acid, a natural compound, induced G0/G1 phase arrest in a time and concentration-dependent manner, without causing apoptosis [49]. 4.4 Conclusions and future directions Among the 11 screened novel compounds of four different drug classes, the two smallest hydroxythiopyridone derivatives, M1S and M2S, exhibited the most potent anticancer activity in both A549 and H522 cells. The drug candidates were also cytotoxic towards pre-neoplastic NIH3T3 cells. The SI value indicated that the compounds were more selective towards cancer cells than pre-neoplastic cells. In the time course cytotoxicity studies M1S and M2S (at 2×EC50 concentration) exhibited almost identical cytostatic and cytotoxic activity in A549 and H522 cells, respectively. Western blotting results showed that the pharmacophore of M1S and M2S was not active or potent in inhibiting HDACs. The 2×EC50 concentration of M1S or M2S significantly downregulated cyclin D1 expression only in H522 cells, suggesting that the drug candidates induced either G1/S or G2/M phase arrest. However, the same concentration was unable to produce a significant effect in changing the expression of cyclin B1 and cell cycle progression in both A549 and H522 cells. Therefore, clear conclusions could not be drawn. To better understand the effects of the compounds on cell cycle progression, a triplicate experiment is needed in H522 cells. Additionally, to confirm the anticancer activity and selectivity of M1S and M2S in KRAS-mutated NSCLC cells further investigations using multiple KRAS-mutated NSCLC and wild type non-cancerous lung cell lines are recommended. Since treatment with M1S and M2S resulted in changes in A549 and H522 cell viability, the potential mechanism underlying this activity should be confirmed. Time-course and concentration-response experiments may give a clear picture of the effects of the drug candidates on cell cycle progression and other cell signalling proteins. Expression of KRAS and KRAS associated upstream and downstream signalling proteins in both KRAS-mutated and wild type cell lines should be tested to investigate the effects of M1S and M2S on KRAS mutations. Flow cytometry analysis after annexin V/PI staining would confirm the early apoptotic and late apoptotic or necrotic effect of M1S or M2S. 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