👤 Reisner E

Reactive molecules, including oxygen and nitrogen species, serve dual roles in human physiology. While they function as essential signaling molecules under normal physiological conditions, they contribute to cellular dysfunction and damage when produced in excess by normal metabolism or in response to stressors. Oxidative/nitrosative stress is a pathological state, resulting from the overproduction of reactive species exceeding the antioxidant capacity of the body, which is implicated in several chronic human diseases. Antioxidant therapies aimed at restoring redox balance and preventing oxidative/nitrosative stress have demonstrated efficacy in preclinical models. However, their clinical applications have met with inconsistent success owing to efficacy, safety, and bioavailability concerns. This summative review analyzes the role of reactive species in human pathophysiology, the mechanisms of action of antioxidant protection, and the challenges that hinder their translation into effective clinical therapies in order to evaluate potential emerging strategies such as targeted delivery systems, precision medicine, and synergistic therapeutic approaches, among others, to overcome current limitations. By integrating recent advances, this review highlights the value of targeting reactive species in the prevention and management of chronic diseases. Show less

Ewing sarcoma (EwS) cell line culture largely relies on standard techniques, which do not recapitulate physiological conditions. Here, we report on a feasible and cost-efficient EwS cell culture technique with increased physiological relevance employing an advanced medium composition, reduced fetal calf serum, and spheroidal growth. Improved reflection of the transcriptional activity related to proliferation, hypoxia, and differentiation in EwS patient tumors was detected in EwS cells grown in this refined in vitro condition. Moreover, transcriptional signatures associated with the oncogenic activity of the EwS-specific FET::ETS fusion transcription factors in the refined culture condition were shifted from proliferative toward metabolic gene signatures. The herein-presented EwS cell culture technique with increased physiological relevance provides a broadly applicable approach for enhanced in vitro modeling relevant to advancing EwS research and the validity of experimental results. Show less

The aim of the UniProt Knowledgebase (UniProtKB; https://www.uniprot.org/) is to provide users with a comprehensive, high-quality and freely accessible set of protein sequences annotated with functional information. In this publication, we describe ongoing changes to our production pipeline to limit the sequences available in UniProtKB to high-quality, non-redundant reference proteomes. We continue to manually curate the scientific literature to add the latest functional data and use machine learning techniques. We also encourage community curation to ensure key publications are not missed. We provide an update on the automatic annotation methods used by UniProtKB to predict information for unreviewed entries describing unstudied proteins. Finally, updates to the UniProt website are described, including a new tab linking protein to genomic information. In recognition of its value to the scientific community, the UniProt database has been awarded Global Core Biodata Resource status. Show less

Hypoxia, a hallmark of many solid tumors, is linked to increased cancer aggressiveness, metastasis, and resistance to conventional therapies, leading to poor patient outcomes. This challenges the efficiency of photodynamic therapy (PDT), which relies on the generation of cytotoxic reactive oxygen species (ROS) through the irradiation of a photosensitizer (PS), a process partially dependent on oxygen levels. In this work, we introduce a novel family of potent PSs based on ruthenium(II) polypyridyl complexes with 2,2'-bipyridyl ligands derived from COUPY coumarins, termed COUBPYs. Ru-COUBPY complexes exhibit outstanding in vitro cytotoxicity against CT-26 cancer cells when irradiated with light within the phototherapeutic window, achieving nanomolar potency in both normoxic and hypoxic conditions while remaining nontoxic in the dark, leading to impressive phototoxic indices (>30,000). Their ability to generate both Type I and Type II ROS underpins their exceptional PDT efficiency. The lead compound of this study, SCV49, shows a favorable in vivo pharmacokinetic profile, excellent toxicological tolerability, and potent tumor growth inhibition in mice bearing subcutaneous CT-26 tumors at doses as low as 3 mg/kg upon irradiation with deep-red light (660 nm). These results allow us to propose SCV49 as a strong candidate for further preclinical development, particularly for treating large hypoxic solid tumors. Show less

Title: Necrosis inducing tetranuclear Ru(II)-Re(I) metal complex for anticancer therapy. Abstract: Chemotherapy is one of the most widely used anticancer treatments worldwide. However, despite its clinical effectiveness, most chemotherapeutic agents are associated with severe side effects. To address this limitation, there is an urgent need for the development of novel anticancer agents. Among the promising alternatives, Ruthenium and Rhenium complexes have garnered significant attention in the scientific literature. This study proposes combining these two metal moieties into a single tetranuclear complex, bridged by a 2,2'-bipyrimidine ligand. Cytotoxicity tests revealed broad activity of the novel metal complex against multiple cancer cell lines. Mechanistic studies suggested that the complex induces cell death by necrosis. Further analyses demonstrated its ability to eradicate colon carcinoma tumor spheroids at micromolar concentrations. To the best of our knowledge, this represents the first example of a Ru(II)-Re(I) tetranuclear metal complex as an anticancer agent. Show less

Two new Ru(II) complexes, mononuclear [RuCl₂(η⁶-p-cymene)(3,4-dmph-κN)] (1) and the binuclear complex [{RuCl(η⁶-p-cymene)}₂(μ-Cl)(μ-3,4-dmph-κ²N,N')]Cl (2; 3,4-dmph = 3,4-dimethylphenylhydrazine), are synthesized and experimentally and theoretically structurally characterized utilizing ¹H and ¹³C NMR and FTIR spectroscopy, as well as DFT calculations. Degradation product of 2, thus ([{RuCl(η⁶-p-cymene)}₂(μ-Cl)(μ-3,4-dmph-κ²N,N')][RuCl₃(η⁶-p-cymene)] (2b) was characterized with SC-XRD. In the crystals of 2b, the cationic and anionic parts interact through N-H^...Cl hydrogen bridges. The spectrofluorimetric measurements proved the spontaneity of the binding processes of both complexes and HSA. Spin probing EPR measurements implied that 1 and 2 decreased the amount of bound 16-doxylstearate and implicated their potential to bind to HSA more strongly than the spin probe. The cytotoxicity assessment of both complexes against the MDA-MB-231 and MIA PaCa-2 cancer cell lines demonstrated a clear dose-dependent decrease in cell viability and no effect on healthy HS-5 cells. Determination of the malondialdehyde and protein carbonyl concentrations indicated that new complexes could offer protective antioxidant benefits in specific cancer contexts. Gel electrophoresis measurements showed the reduction in MMP9 activity and indicated the potential of 1 in limiting the cancer cells' invasion. The annexin V/PI apoptotic assay results showed that 1 and 2 exhibit different selectivity towards MIA PaCa-2 and MDA-MB-231 cancer cells. A comparative molecular docking analysis of protein binding, specifically targeting acetylcholinesterase (ACHE), matrix metalloproteinase-9 (MMP-9), and human serum albumin (HSA), demonstrated distinct binding interactions for each complex. Show less

Monofunctional platinum complexes offer a promising alternative to cisplatin in cancer chemotherapy, showing a unique mechanism of action. Their ability to induce minor helix distortions effectively inhibits DNA transcription. In our study, we synthesized and characterized three monofunctional Pt(II) complexes with the general formula [Pt(en)(L)Cl]NO3, where en = ethylenediamine, and L = pyridine (py), 2-methylpyridine (2-mepy), and 2-phenylpyridine (2-phpy). The hydrolysis rates of [Pt(en)(py)Cl]NO3 (1) and [Pt(en)(2-mepy)Cl]NO3 (2) decrease with the bulkiness of the auxiliary ligand with k(1) = 2.28 ± 0.15 × 10-4 s-1 and k(2) = 8.69 ± 0.98 × 10-5 s-1 at 298 K. The complex [Pt(en)(2-phpy)Cl]Cl (3) demonstrated distinct behavior. Upon hydrolysis, an equilibrium (Keq = 0.385 mM) between the complexes [Pt(en)(2-phpy)Cl]+ and [Pt(en)(2-phpy-H+)]+ was observed with no evidence (NMR or HR-ESI-MS) for the presence of the aquated complex [Pt(en)(2-phpy)(H2O)]2+. Despite the kinetic similarities between phenanthriplatin and (2), complexes (1) and (2) exhibit minimal activity against A549 lung cancer cell line (IC50 > 100 μΜ), whereas complex (3) exhibits notable cytotoxicity (IC50 = 41.11 ± 2.1 μΜ). In examining the DNA binding of (1) and (2) to the DNA model guanosine (guo), we validated their binding through guoN7, which led to an increased population of the C3'-endo sugar conformation, as expected. However, we observed that the rapid transition 2E (C2'-endo) ↔ 3E (C3'-endo), in the case of [Pt(en)(py)(guo)](NO3)2 ([1-guo]), slows down in the case of [Pt(en)(2-mepy)(guo)](NO3)2 ([2-guo]), resulting in separate signals for the two conformers in the 1H NMR spectra. This phenomenon arises from the steric hindrance between the methyl group of pyridine and the sugar moiety of guanosine. Notably, this hindrance is absent in [2-(9-MeG)] (9-MeG = 9-methylguanine), probably due to the absence of a bulky sugar unit in 9-MeG. In the case of (3), where the bulkiness of the substitution on the pyridine is further increased by a phenyl group, we observed a notable proximity between 9-MeGH8 and the phenyl ring of 2-phpy. Considering that only (3) exhibited good cytotoxicity against the A549 cancer cell line, it is suggested that auxiliary ligands, L, with an extended aromatic system and proper orientation in complexes of the type cis-[Pt(en)(L)Cl]NO3, may enhance the cytotoxic activity of such complexes. Show less

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium. Show less

Expert curation is essential to capture knowledge of enzyme functions from the scientific literature in FAIR open knowledgebases but cannot keep pace with the rate of new discoveries and new publications. In this work we present EnzChemRED, for Enzyme Chemistry Relation Extraction Dataset, a new training and benchmarking dataset to support the development of Natural Language Processing (NLP) methods such as (large) language models that can assist enzyme curation. EnzChemRED consists of 1,210 expert curated PubMed abstracts where enzymes and the chemical reactions they catalyze are annotated using identifiers from the protein knowledgebase UniProtKB and the chemical ontology ChEBI. We show that fine-tuning language models with EnzChemRED significantly boosts their ability to identify proteins and chemicals in text (86.30% F1 score) and to extract the chemical conversions (86.66% F1 score) and the enzymes that catalyze those conversions (83.79% F1 score). We apply our methods to abstracts at PubMed scale to create a draft map of enzyme functions in literature to guide curation efforts in UniProtKB and the reaction knowledgebase Rhea. Show less

The modular synthesis of the heteroscorpionate core is explored as a tool for the rapid development of ruthenium-based therapeutic agents. Starting with a series of structurally diverse alcohol-NN ligands, a family of heteroscorpionate-based ruthenium derivatives was synthesized, characterized, and evaluated as an alternative to platinum therapy for breast cancer therapy. In vitro, the antitumoral activity of the novel derivatives was assessed in a series of breast cancer cell lines using UNICAM-1 and cisplatin as metallodrug control. Through this approach, a bimetallic heteroscorpionate-based metallodrug (RUSCO-2) was identified as the lead compound of the series with an IC₅₀ value range as low as 3-5 μM. Notably, RUSCO-2 was found to be highly cytotoxic in TNBC cell lines, suggesting a mode of action independent of the receptor status of the cells. As a proof of concept and taking advantage of the luminescent properties of one of the complexes obtained, uptake was monitored in human breast cancer MCF7 cell lines by fluorescence lifetime imaging microscopy (FLIM) to reveal that the compound is evenly distributed in the cytoplasm and that the incorporation of the heteroscorpionate ligand protects it from aqueous processes, conversion in another entity, or the loss of the chloride group. Finally, ROS studies were conducted, lipophilicity was estimated, the chloride/water exchange was studied, and stability studies in simulated biological media were carried out to propose structure-activity relationships. Show less

Ruthenium(II) complexes containing diimine ligands have contributed to the development of agents for photoactivated chemotherapy. Several approaches have been used to obtain photolabile Ru(II) complexes. The two most explored have been the use of monodentate ligands and the incorporation of steric effects between the bidentate ligands and the Ru(II). However, the introduction of electronic effects in the ligands has been less explored. Herein, we report a systematic experimental, theoretical, and photocytotoxicity study of a novel series of Ru(II) complexes Ru1-Ru5 of general formula [Ru(phen)₂(N^∧N')]²⁺, where N^∧N' are different minimal strained ligands based on the 1-aryl-4-benzothiazolyl-1,2,3-triazole (BTAT) scaffold, being CH₃ (Ru1), F (Ru2), CF₃ (Ru3), NO₂ (Ru4), and N(CH₃)₂ (Ru5) substituents in the R4 of the phenyl ring. The complexes are stable in solution in the dark, but upon irradiation in water with blue light (λ_ex = 465 nm, 4 mW/cm²) photoejection of the ligand BTAT was observed by HPLC-MS spectrometry and UV-vis spectroscopy, with t_1/2 ranging from 4.5 to 14.15 min depending of the electronic properties of the corresponding BTAT, being Ru4 the less photolabile (the one containing the more electron withdrawing substituent, NO₂). The properties of the ground state singlet and excited state triplet of Ru1-Ru5 have been explored using density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations. A mechanism for the photoejection of the BTAT ligand from the Ru complexes, in H₂O, is proposed. Phototoxicity studies in A375 and HeLa human cancer cell lines showed that the new Ru BTAT complexes were strongly phototoxic. An enhancement of the emission intensity of HeLa cells treated with Ru5 was observed in response to increasing doses of light due to the photoejection of the BTAT ligand. These studies suggest that BTAT could serve as a photocleavable protecting group for the cytotoxic bis-aqua ruthenium warhead [Ru(phen)₂(OH₂)₂]²⁺. Show less

Five cationic ruthenium-arene complexes with the generic formula [Ru(SAc)(S₂C·NHC)(p-cymene)](PF₆) (5a-e) were prepared in almost quantitative yields using a straightforward one-pot, two-step experimental procedure starting from [RuCl₂(p-cymene)]₂, an imidazol(in)ium-2-dithiocarboxylate (NHC·CS₂) zwitterion, KSAc, and KPF₆. These half-sandwich compounds were fully characterized by various analytical techniques and the molecular structures of two of them were solved by X-ray diffraction analysis, which revealed the existence of an intramolecular chalcogen bond between the oxygen atom of the thioacetate ligand and a proximal sulfur atom of the dithiocarboxylate unit. DFT calculations showed that the C=S^…O charge transfer amounted to 2.4 kcal mol^-1. The dissolution of [Ru(SAc)(S₂C·IMes)(p-cymene)](PF₆) (5a) in moist DMSO-d₆ at room temperature did not cause the dissociation of its sulfur ligands. Instead, p-cymene was slowly released to afford the 12-electron [Ru(SAc)(S₂C·IMes)]⁺ cation that could be detected by mass spectrometry. Monitoring the solvolysis process by ¹H NMR spectroscopy showed that more than 22 days were needed to fully decompose the starting ruthenium-arene complex. Compounds 5a-e exhibited a high antiproliferative activity against human glioma Hs683 and human lung carcinoma A549 cancer cells. In particular, the IMes derivative (5a) was the most potent compound of the series, achieving toxicities similar to those displayed by marketed platinum drugs. Show less

Liver cancer is one of the leading causes of death that motivating scientists worldwide to synthesize novel chemotherapeutics. Ru(II)-polypyridyl complexes are extensively studied for possible therapeutic and cellular applications due to their tunable coordination chemistry, structural diversity, ligand-exchange kinetics, accessible redox states, and rich photophysical or photochemical properties. Herein, we have synthesized a series of Ru(II) polypyridyl complexes [Ru^II(N^N)₂(ox)] (1-3), where ox is oxalate (C₂O₄^2-) and N^N is 1,10-phenanthroline (phen) (1), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) (2), and dipyrido[3,2,-a:2',3'-c]phenazine (dppz) (3). Oxalate (ox^2-) was opted as a bioactive dioxo ligand to prevent facile hydrolysis in aqueous media, thereby increasing the stability of the Ru(II)-polypyridyl complexes in physiological media. We thoroughly characterized all the complexes using ESI-MS, FT-IR, UV-vis, and ¹H NMR spectroscopy and other physicochemical methods. The complexes were stable under physiological conditions and under low-energy green LED light (λ_irr = 530 nm). However, the photoirradiation of complexes resulted in the efficient generation of singlet oxygen (¹O₂) as a major reactive oxygen species (ROS). The role of the extended aromatic conjugation of the N^N-donor ligands in the complexes was demonstrated by their binding propensities with CT-DNA and bovine serum albumin (BSA). Both DNA intercalation and groove binding were evidenced, while tryptophan (Trp) and tyrosine (Tyr) binding site preferences were revealed from the synchronous fluorescence spectra (SFS) of BSA. The cytotoxic profiling of the complexes performed on hepatocellular carcinoma cells (HepG2) in the dark and in the presence of green light indicated their dose-dependent cytotoxicity. The [Ru^II(N^N)₂(ox)] complexes exhibited enhanced photocytotoxicity mediated by efficient generation of cytotoxic ¹O₂ and effective interaction with DNA. All the complexes were internalized by the HepG2 liver cancer cells efficiently and localized to the cytoplasm and nucleus. The complexes exhibited potent anti-proliferative, anti-clonogenic, and anti-migratory effects on the cancer cells, suggesting their potential for therapeutic applications. Show less

As novel promising anticancer candidates, the piano-stool type complexes of ruthenium, [RuCl(η⁶-p-cymene)(N,S-L_n)]PF₆, K₁-₄, were synthesized from the reaction of the substituted benzo[b]thiophene based thiosemicarbazone ligands (L_1-4) with [{RuCl(η⁶-p-cymene)}₂(μ-Cl)₂]. All complexes were fully characterized using elemental analysis, and spectroscopic methods such as FT-IR and ¹H NMR. The molecular masses of the complexes were proved by MALDI-TOF analysis. Single crystal X-ray diffraction study was employed in the structural elucidation of complex K₁ which shows a distorted octahedral geometry around the Ru(II) ion. Furthermore, spectroscopic methods revealed that in all complexes the ligands are coordinated to the metal center in neutral thione form via N, S donors. In this study, the effect of all ligands, complexes and commercial drugs with a different concentration on the viability of OVCAR-3, A2780 and OSE cells were compared. In this comparison, the cytotoxicity of ruthenium (II) complexes on two ovarian cancer cell lines (human A2780 and human OVCAR-3) was evaluated. For this purpose, the resazurin assay was performed. Based on our studies, complex K₂ showed the highest toxicity against OVCAR-3 and A2780 cells. The cytotoxic effect of K₂ was found to be higher than that of the commercial anticancer agents Oxalpin and Carbodex, 1.8-34.7-fold for OVCAR-3 cells and 1.9-11.8-fold for A2780 cells, respectively. These results provide insight into the potential of ruthenium (II) complexes as a cytotoxic agent for the treatment of ovarian cancer, particularly for primary tumors. Show less

Organometallic complexes of the formula [Ru(N^N)(p-cymene)Cl][X] (N^N = bidentate polypyridyl ligands, p-cymene = 1-methyl-4-(1-methylethyl)-benzene, X = counter anion), are currently studied as possible candidates for the potential treatment of cancer. Searching for new organometallic compounds with good to moderate cytotoxic activities, a series of mononuclear water-soluble ruthenium(II)-arene complexes incorporating substituted pyridine-quinoline ligands, with pending -CH₂OH, -CO₂H and -CO₂Me groups in the 4-position of quinoline ring, were synthesized, for the first time, to study their possible effect to modulate the activity of the ruthenium p-cymene complexes. These include the [Ru(η⁶-p-cymene)(pqhyme)Cl][X] (X = Cl^- (1-Cl), PF₆^- (1-PF₆), pqhyme = 4-hydroxymethyl-2-(pyridin-2-yl)quinoline), [Ru(η⁶-p-cymene)(pqca)Cl][Cl] ((2-Cl), pqca = 4-carboxy-2-(pyridin-2-yl)quinoline), and [Ru(η⁶-p-cymene)(pqcame)Cl][X] (X = Cl^- (3-Cl), PF₆^- (3-PF₆), pqcame = 4-carboxymethyl-2-(pyridin-2-yl)quinoline) complexes, respectively. Identification of the complexes was based on multinuclear NMR and ATR-IR spectroscopic methods, elemental analysis, conductivity measurements, UV-Vis spectroscopic, and ESI-HRMS techniques. The solid-state structures of 1-PF₆ and 3-PF₆ have been elucidated by single-crystal X-ray diffraction revealing a three-legged piano stool geometry. This is the first time that the in vitro cytotoxic activities of these complexes are studied. These were conducted in HEK293T (human embryonic kidney cells) and HeLa cells (cervical cancer cells) via the MTT assay. The results show poor in vitro anticancer activities for the HeLa cancer cell lines and 3-Cl proved to be the most potent (IC₅₀ > 80 μΜ). In both cell lines, the cytotoxicity of the ligand precursor pqhyme is significantly higher than that of cisplatin. Show less

In this contribution we report the synthesis, characterization and in vitro anticancer activity of novel cyclometalated 4-phenylthiazole-derived ruthenium(II) (2a-e) and osmium(II) (3a-e) complexes. Formation and sufficient purity of the complexes were unambigiously confirmed by ¹H-, ¹³C- and 2D-NMR techniques, X-ray diffractometry, HRMS and elemental analysis. The binding preferences of these cyclometalates to selected amino acids and to DNA models including G-quadruplex structures were analyzed. Additionally, their stability and behaviour in aqueous solutions was determined by UV-Vis spectroscopy. Their cellular accumulation, their ability of inducing apoptosis, as well as their interference in the cell cycle were studied in SW480 colon cancer cells. The anticancer potencies were investigated in three human cancer cell lines and revealed IC₅₀ values in the low micromolar range, in contrast to the biologically inactive ligands. Show less

Title: Insights into the anticancer photodynamic activity of Ir(III) and Ru(II) polypyridyl complexes bearing β-carboline ligands. Abstract: Ir(III) and Ru(II) polypyridyl complexes are promising photosensitizers (PSs) for photodynamic therapy (PDT) due to their outstanding photophysical properties. Herein, one series of cyclometallated Ir(III) complexes and two series of Ru(II) polypyridyl derivatives bearing three different thiazolyl-β-carboline N^N' ligands have been synthesized, aiming to evaluate the impact of the different metal fragments ([Ir(C^N)2]+ or [Ru(N^N)2]2+) and N^N' ligands on the photophysical and biological properties. All the compounds exhibit remarkable photostability under blue-light irradiation and are emissive (605 < λem < 720 nm), with the Ru(II) derivatives displaying higher photoluminescence quantum yields and longer excited state lifetimes. The Ir PSs display pKa values between 5.9 and 7.9, whereas their Ru counterparts are less acidic (pKa > 9.3). The presence of the deprotonated form in the Ir-PSs favours the generation of reactive oxygen species (ROS) since, according to theoretical calculations, it features a low-lying ligand-centered triplet excited state (T1 = 3LC) with a long lifetime. All compounds have demonstrated anticancer activity. Ir(III) complexes 1-3 exhibit the highest cytotoxicity in dark conditions, comparable to cisplatin. Their activity is notably enhanced by blue-light irradiation, resulting in nanomolar IC50 values and phototoxicity indexes (PIs) between 70 and 201 in different cancer cell lines. The Ir(III) PSs are also activated by green (with PI between 16 and 19.2) and red light in the case of complex 3 (PI = 8.5). Their antitumor efficacy is confirmed by clonogenic assays and using spheroid models. The Ir(III) complexes rapidly enter cells, accumulating in mitochondria and lysosomes. Upon photoactivation, they generate ROS, leading to mitochondrial dysfunction and lysosomal damage and ultimately cell apoptosis. Additionally, they inhibit cancer cell migration, a crucial step in metastasis. In contrast, Ru(II) complex 6 exhibits moderate mitochondrial activity. Overall, Ir(III) complexes 1-3 show potential for selective light-controlled cancer treatment, providing an alternative mechanism to chemotherapy and the ability to inhibit lethal cancer cell dissemination. Show less

Three phosphorescent iridium(III) complexes consisting bis-diphosphine ligands were prepared and characterized by single-crystal XRD, CHN analysis, spectroscopic techniques, cyclic voltammetry, and DFT. The synthesized complexes were the three monomeric [Ir(ppy)₂(L₁)Cl] (1), [Ir(ppy)₂(L₂)]Cl (2) and [Ir(ppy)₂(L₃)]Cl (3) where L₁ = bis-(diphenylphosphino)methane (dppm), L₂ = bis-(diphenylphosphino)propane (dppp) and L₃ = bis-(diphenylphosphino)benzene (dppbe). Complexes 1-3 gave an absorption band between 240 to 380 nm in both CH₂Cl₂ and DMSO, which is assigned as a charge transfer transition based on theoretical calculation. They showed a blue-green emission at 460-520 nm in DMSO with an absolute quantum efficiency of 0.013-0.046 at room temperature. The selective photo-induced electron transfer (PET) by Fe³⁺ in DMSO, was studied to obey the Rehm-Weller principle. The 1:1 binding soichiometry between 1-3 and Fe³⁺ was established by Job's plot. The binding constants (K_a) were determined using the Benesi-Hildebrand plot. All the complexes are extremely more potent than cisplatin for in vitro antiproliferative activity towards the human breast cancer cells, HCC1937, MCF-7, and MDA-MB-231. The values of IC₅₀ were in the range of 0.077-0.485 μM, and 1 exhibited the most effective IC₅₀ against MDA-MB-231 cell line, the triple-negative breast cancer cell. Their lipophilicities (log P) were also examined to explain the penetration ability of the studied complexes towards cell barriers, and transport to the molecular target. Show less

One approach to reduce the side effects of chemotherapy in cancer treatment is photodynamic therapy (PDT), which allows spatiotemporal control of the cytotoxicity. We have used the strategy of coordinating π-expansive ligands to increase the excited state lifetimes of Ir(III) half-sandwich complexes in order to facilitate the generation of ¹O₂. We have obtained derivatives of formulas [Cp*Ir(C^∧N)Cl] and [Cp*Ir(C^∧N)L]BF₄ with different degrees of π-expansion in the C^∧N ligands. Complexes with the more π-expansive ligand are very effective photosensitizers with phototoxic indexes PI > 2000. Furthermore, PI values of 63 were achieved with red light. Time-dependent density functional theory (TD-DFT) calculations nicely explain the effect of the π-expansion. The complexes produce reactive oxygen species (ROS) at the cellular level, causing mitochondrial membrane depolarization, cleavage of DNA, nicotinamide adenine dinucleotide (NADH) oxidation, as well as lysosomal damage. Consequently, cell death by apoptosis and secondary necrosis is activated. Thus, we describe the first class of half-sandwich iridium cyclometalated complexes active in PDT. Show less

Title: Neutral rhenium(I) tricarbonyl complexes with sulfur-donor ligands: anti-proliferative activity and cellular localization. Abstract: Rhenium(I) tricarbonyl complexes are widely studied for their cell imaging properties and anti-cancer and anti-microbial activities, but the complexes with S-donor ligands remain relatively unexplored. A series of six fac-[Re(NN)(CO)3(SR)] complexes, where (NN) is 2,2'-bipyridyl (bipy) or 1,10-phenanthroline (phen), and RSH is a series of thiocarboxylic acid methyl esters, have been synthesized and characterized. Cellular uptake and anti-proliferative activities of these complexes in human breast cancer cell lines (MDA-MB-231 and MCF-7) were generally lower than those of the previously described fac-[Re(NN)(CO)3(OH2)]+ complexes; however, one of the complexes, fac-[Re(CO)3(phen)(SC(Ph)CH2C(O)OMe)] (3b), was active (IC50 ∼ 10 μM at 72 h treatment) in thiol-depleted MDA-MB-231 cells. Moreover, unlike fac-[Re(CO)3(phen)(OH2)]+, this complex did not lose activity in the presence of extracellular glutathione. Taken together these properties show promise for further development of 3b and its analogues as potential anti-cancer drugs for co-treatment with thiol-depleting agents. Conversely, the stable and non-toxic complex, fac-[Re(bipy)(CO)3(SC(Me)C(O)OMe)] (1a), predominantly localized in the lysosomes of MDA-MB-231 cells, as shown by live cell confocal microscopy (λex = 405 nm, λem = 470-570 nm). It is strongly localized in a subset of lysosomes (25 μM Re, 4 h treatment), as shown by co-localization with a Lysotracker dye. Longer treatment times with 1a (25 μM Re for 48 h) resulted in partial migration of the probe into the mitochondria, as shown by co-localization with a Mitotracker dye. These properties make complex 1a an attractive target for further development as an organelle probe for multimodal imaging, including phosphorescence, carbonyl tag for vibrational spectroscopy, and Re tag for X-ray fluorescence microscopy. Show less

Anticancer drugs are at the frontline of cancer therapy. However, innate resistance to these drugs occurs in one-third to one-half of patients, exposing them to the side effects of these drugs with no meaningful benefit. To identify the genes and pathways that confer resistance to such therapies, we performed a genome-wide screen in haploid human embryonic stem cells (hESCs). These cells possess the advantage of having only one copy of each gene, harbour a normal karyotype, and lack any underlying point mutations. We initially show a close correlation between the potency of anticancer drugs in cancer cell lines to those in hESCs. We then exposed a genome-wide loss-of-function library of mutations in all protein-coding genes to 10 selected anticancer drugs, which represent five different mechanisms of drug therapies. The genetic screening enabled us to identify genes and pathways which can confer resistance to these drugs, demonstrating several common pathways. We validated a few of the resistance-conferring genes, demonstrating a significant shift in the effective drug concentrations to indicate a drug-specific effect to these genes. Strikingly, the p53 signalling pathway seems to induce resistance to a large array of anticancer drugs. The data shows dramatic effects of loss of p53 on resistance to many but not all drugs, calling for clinical evaluation of mutations in this gene prior to anticancer therapy. Show less

Ferroptosis is evolving as a highly promising approach to combat difficult-to-treat tumour entities including therapy-refractory and dedifferentiating cancers1-3. Recently, ferroptosis suppressor protein-1 (FSP1), along with extramitochondrial ubiquinone or exogenous vitamin K and NAD(P)H/H+ as an electron donor, has been identified as the second ferroptosis-suppressing system, which efficiently prevents lipid peroxidation independently of the cyst(e)ine-glutathione (GSH)-glutathione peroxidase 4 (GPX4) axis4-6. To develop FSP1 inhibitors as next-generation therapeutic ferroptosis inducers, here we performed a small molecule library screen and identified the compound class of 3-phenylquinazolinones (represented by icFSP1) as potent FSP1 inhibitors. We show that icFSP1, unlike iFSP1, the first described on-target FSP1 inhibitor5, does not competitively inhibit FSP1 enzyme activity, but instead triggers subcellular relocalization of FSP1 from the membrane and FSP1 condensation before ferroptosis induction, in synergism with GPX4 inhibition. icFSP1-induced FSP1 condensates show droplet-like properties consistent with phase separation, an emerging and widespread mechanism to modulate biological activity7. N-terminal myristoylation, distinct amino acid residues and intrinsically disordered, low-complexity regions in FSP1 were identified to be essential for FSP1-dependent phase separation in cells and in vitro. We further demonstrate that icFSP1 impairs tumour growth and induces FSP1 condensates in tumours in vivo. Hence, our results suggest that icFSP1 exhibits a unique mechanism of action and synergizes with ferroptosis-inducing agents to potentiate the ferroptotic cell death response, thus providing a rationale for targeting FSP1-dependent phase separation as an efficient anti-cancer therapy. Show less

Tumor onset and its progression are strictly linked to its metabolic rewiring on the basis of the Warburg effect. In this context, fumarate emerged as a putative oncometabolite mediating cancer progression. Fumarate accumulation is usually driven by fumarate hydratase (FH) loss of function, the enzyme responsible for the reversible conversion of fumarate into malate. Fumarate accumulation acts as a double edge sword: on one hand it takes part in the metabolic rewiring of cancer cells, while on the other it also plays a crucial role in chromatin architecture reorganization. The latter is achieved by competing with a-ketoglutarate-dependent enzymes, eventually altering the cellular methylome profile, which in turn leads to its transcriptome modeling. Furthermore, in recent years, it has emerged that FH has an ability to recruit DNA double strand breaks. The accumulation of fumarate into damaged sites might also determine the DNA repair pathway in charge for the seizure of the lesion, eventually affecting the mutational state of the cells. In this work, we aimed to review the current knowledge on the role of fumarate as an oncometabolite orchestrating the cellular epigenetic landscape and DNA repair machinery. Show less

Colorectal cancer is among the most common cancers worldwide and a frequent cause of cancer related deaths. Oxaliplatin is the first line chemotherapeutics for treatment, but the development of resistance leads to recurrence of oxaliplatin insensitive tumors. To understand possible mechanisms of drug tolerance we developed oxaliplatin resistant derivatives (OR-LoVo) of the established LoVo cell line originally isolated from a metastatic colon adenocarcinoma. We compared the microRNA (miRNA) expression profile of the cell pair and found expression of miR-29a-3p significantly increased in OR-LoVo cells compared to parent cells. In addition, miR-29a-3p was significantly elevated in tumor tissue when compared to matched surrounding tissue in human, suggesting potential clinical importance. Ectopic miR-29-a-3p expression induced chemoresistance in a number of different cancer cell lines as well as colorectal tumors in mice. We further demonstrated that miR-29-a-3p downregulates expression of the ubiquitin ligase component FEM1B and that reduction of Fem1b levels is sufficient to confer oxaliplatin resistance. FEM1B targets the glioma associated oncogene Gli1 for degradation, suggesting that increased Gli1 levels could contribute to oxaliplatin tolerance. Accordingly, knockdown of GLI1 reverted chemoresistance of OR-LoVo cells. Mechanistically, resistant cells experienced significantly lower DNA damage upon oxaliplatin treatment, which can be partially explained by reduced oxaliplatin uptake and enhanced repair. These results suggest that miR-29-a-3p overexpression induces oxaliplatin resistance through misregulation of Fem1B and Gli1 levels. TCGA analyses provides strong evidence that the reported findings regarding induced drug tolerance by the miR-29a/Fem1B axis is clinically relevant. The reported findings can help to predict oxaliplatin sensitivity and resistance of colorectal tumors. Show less

Developing novel therapeutics often follows three steps: target identification, design of strategies to suppress target activity and drug development to implement the strategies. In this review, we recount the evidence identifying the basic leucine zipper transcription factors ATF5, CEBPB, and CEBPD as targets for brain and other malignancies. We describe strategies that exploit the structures of the three factors to create inhibitory dominant-negative (DN) mutant forms that selectively suppress growth and survival of cancer cells. We then discuss and compare four peptides (CP-DN-ATF5, Dpep, Bpep and ST101) in which DN sequences are joined with cell-penetrating domains to create drugs that pass through tissue barriers and into cells. The peptide drugs show both efficacy and safety in suppressing growth and in the survival of brain and other cancers in vivo, and ST101 is currently in clinical trials for solid tumors, including GBM. We further consider known mechanisms by which the peptides act and how these have been exploited in rationally designed combination therapies. We additionally discuss lacunae in our knowledge about the peptides that merit further research. Finally, we suggest both short- and long-term directions for creating new generations of drugs targeting ATF5, CEBPB, CEBPD, and other transcription factors for treating brain and other malignancies. Show less

An increasing number of novel Ru(II) polypyridyl complexes have been successfully applied as photosensitizers (PSs) for photodynamic therapy (PDT). Despite recent advances in optimized PSs with refined photophysical properties, the lack of tumoral selectivity is often a major hurdle for their clinical development. Here, classical maleimide and versatile NHS-activated acrylamide strategies were employed to site-selectively conjugate a promising Ru(II) polypyridyl complex to the N-terminally Cys-modified Bombesin (BBN) targeting unit. Surprisingly, the decreased cell uptake of these novel Ru-BBN conjugates in cancer cells did not hamper the high phototoxic activity of the Ru-containing bioconjugates and even decreased the toxicity of the constructs in the absence of light irradiation. Overall, although deceiving in terms of selectivity, our new bioconjugates could still be useful for advanced cancer treatment due to their nontoxicity in the dark. Show less

Arene-ruthenium(II) complexes with carbothioamidopyrazoles at the C-2 and C-5 positions have been recognized as chemotherapeutic agent alternatives to cisplatin and its oxaliplatin analogs. The aim of this study was to continue research on the biological aspect of arene-ruthenium(II) complexes and their anticancer activity. The present paper includes an additional 12 new tumor cells, analyzed by MTT, and employs a series of extended bioassays to better understand their potential mechanism of antitumor activity. The following tests were conducted: membrane permeability studies, intramolecular reactive oxygen and nitrogen species (ROS/RNS) assays, mitochondrial potential changes, DNA analysis by comet assay using the electrophoresis method, measurement of cleaved PARP protein levels, and determination of apoptotic and necrotic cell fractions by fluorescence microscopy. Additionally, the article presents lipophilicity studies based on RP-TLC and molecular docking studies. We hope that the presented data will prove useful in practical treatment, especially for patients with cancer. Show less

The toxicity of and resistance to platinum complexes as cisplatin, oxaliplatin or carboplatin calls for the replacement of these therapeutic agents in clinical settings. We have previously identified a set of half sandwich-type osmium, ruthenium and iridium complexes with bidentate glycosyl heterocyclic ligands exerting specific cytostatic activity on cancer cells but not on non-transformed primary cells. The apolar nature of the complexes, conferred by large, apolar benzoyl protective groups on the hydroxyl groups of the carbohydrate moiety, was the main molecular feature to induce cytostasis. We exchanged the benzoyl protective groups to straight chain alkanoyl groups with varying length (3 to 7 carbon units) that increased the IC₅₀ value as compared to the benzoyl-protected complexes and rendered the complexes toxic. These results suggest a need for aromatic groups in the molecule. The pyridine moiety of the bidentate ligand was exchanged for a quinoline group to enlarge the apolar surface of the molecule. This modification decreased the IC₅₀ value of the complexes. The complexes containing [(η⁶-p-cymene)Ru(II)], [(η⁶-p-cymene)Os(II)] or [(η⁵-Cp*)Ir(III)] were biologically active unlike the complex containing [(η⁵-Cp*)Rh(III)]. The complexes with cytostatic activity were active on ovarian cancer (A2780, ID8), pancreatic adenocarcinoma (Capan2), sarcoma (Saos) and lymphoma cell lines (L428), but not on primary dermal fibroblasts and their activity was dependent on reactive oxygen species production. Importantly, these complexes were cytostatic on cisplatin-resistant A2780 ovarian cancer cells with similar IC₅₀ values as on cisplatin-sensitive A2780 cells. In addition, the quinoline-containing Ru and Os complexes and the short chain alkanoyl-modified complexes (C3 and C4) proved to be bacteriostatic in multiresistant Gram-positive Enterococcus and Staphylococcus aureus isolates. Hereby, we identified a set of complexes with submicromolar to low micromolar inhibitory constants against a wide range of cancer cells, including platinum resistant cells and against multiresistant Gram-positive bacteria. Show less