The impact of biomolecule interactions on the cytotoxic effects of rhenium(I) tricarbonyl complexes.

{"full_text": " Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\n Contents lists available at ScienceDirect\n\n\n Journal of Inorganic Biochemistry\n journal homepage: www.elsevier.com/locate/jinorgbio\n\n\n\n\nThe impact of biomolecule interactions on the cytotoxic effects of rhenium\n(I) tricarbonyl complexes\nTayna\u0301 Saraiva de Lavor a, 1, Maria Henriqueta Silvestre Teixeira b, 1, Patr\u00edcia Alves de Matos a,\nRicardo Campos Lino b, Clara Maria Faria Silva b, Marcos Eduardo Gomes do Carmo c,\nMarcelo Em\u00edlio Beletti d, Antonio Otavio T. Patrocinio c, Robson Jose\u0301 de Oliveira J\u00fanior b, *,\nTayana Mazin Tsubone a, *\na\n Laborato\u0301rio Interdisciplinar de Fototerapia e Biomole\u0301culas (LIFeBio), Instituto de Qu\u00edmica (IQ), Universidade Federal de Uberla\u0302ndia (UFU), Uberla\u0302ndia, Minas Gerais,\nBrazil\nb\n Laborato\u0301rio de Citogene\u0301tica, Instituto de Biotecnologia (IBTEC), Universidade Federal de Uberla\u0302ndia (UFU), Uberla\u0302ndia, Minas Gerais, Brazil\nc\n Laboratory of Photochemistry and Materials Science, Chemistry Institute, Federal University of Uberla\u0302ndia, Uberla\u0302ndia, Minas Gerais, Brazil\nd\n Instituto de Cie\u0302ncias Biome\u0301dicas (ICBIM), Universidade Federal de Uberla\u0302ndia (UFU), Uberla\u0302ndia, Minas Gerais, Brazil\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: Rhenium complexes show great promise as anticancer drug candidates. Specifically, compounds with a Re\nAnti-cancer drugs (CO)3(NN)(py)+ core in their architecture have shown cytotoxicity equal to or greater than that of well-\nRhenium complexes established anticancer drugs based on platinum or organic molecules. This study aimed to evaluate how the\nBiomolecules\n strength of the interaction between rhenium(I) tricarbonyl complexes fac-[Re(CO)3(NN)(py)]+, NN = 1,10-\nCytotoxicity\nDrug design\n phenanthroline (phen), dipyrido[3,2-f:2\u2032,3\u2032-h]quinoxaline (dpq) or dipyrido[3,2-a:2\u20323\u2019-c]phenazine (dppz) and\nOrganometallic complexes biomolecules (protein, lipid and DNA) impacted the corresponding cytotoxic effect in cells. Results showed that\n fac-[Re(CO)3(dppz)(py)]+ has higher Log Po/w and binding constant (Kb) with biomolecules (protein, lipid and\n DNA) compared to complexes of fac-[Re(CO)3(phen)(py)]+ and fac-[Re(CO)3(dpq)(py)]+. As consequence, fac-\n [Re(CO)3(dppz)(py)]+ exhibited the highest cytotoxicity (IC50 = 8.5 \u03bcM for HeLa cells) for fac-[Re(CO)3(dppz)\n (py)]+ among the studied compounds (IC50 > 15 \u03bcM). This highest cytotoxicity of fac-[Re(CO)3(dppz)(py)]+ are\n probably related to its lipophilicity, higher permeation of the lipid bilayers of cells, and a more potent interaction\n of the dppz ligand with biomolecules (protein and DNA). Our findings open novel avenues for rational drug\n design and highlight the importance of considering the chemical structures of rhenium complexes that strongly\n interact with biomolecules (proteins, lipids, and DNA).\n\n\n\n\n1. Introduction candidates [1]. The main reason for this is the flexibility of metal\n complexes in adopting various types of chemical structures that enable\n The search for new anticancer drugs is still ongoing, as well- them to engage in effective binding interactions with target bio\u00ad\nestablished drugs containing platinum, such as cisplatin, carboplatin, molecules, thereby enhancing their effectiveness [2].\nand oxaliplatin, have demonstrated resistance and severe side effects Among transition metal complexes, rhenium (Re) organometallic\nduring treatment [1]. Thus, the primary challenge for scientists in complexes demonstrate noteworthy characteristics, such as facile syn\u00ad\nadvancing anticancer research is to achieve minimal or negligible side thesis via a one-step strategy, remarkable thermal and kinetic stability,\neffects for novel therapeutic avenues in the field. In recent decades, and excellent photochemical and photophysical properties [2]. Anti\u00ad\nseveral metal complexes have shown great potential as anticancer drug cancer applications of rhenium complexes have only recently been\n\n\n Abbreviations: Re(CO)3, rhenium(I) tricarbonyl complexes; py, pyridine; phen, 1,10-phenantroline; dpq, dipyrido[3,2-f:2\u2032,3\u2032-h]quinoxaline; dppz, dipyrido[3,2-\na:2\u20323\u2019-c]phenazine; IR, infrared; RMN, 1H Nuclear Magnetic Resonance; BSA, bovine serum albumin; DNA, deoxyribonucleic acid; DOPG, 1,2-dioleoyl-sn-glycero-3-\nphospho-(1\u2032-rac-glycerol) (sodium salt)..\n * Corresponding authors.\n E-mail addresses: oliveirajunior@ufu.br (R.J. de Oliveira J\u00fanior), tayana.tsubone@ufu.br (T.M. Tsubone).\n 1\n These authors contributed equally to the work.\n\nhttps://doi.org/10.1016/j.jinorgbio.2024.112600\nReceived 1 March 2024; Received in revised form 26 April 2024; Accepted 7 May 2024\nAvailable online 10 May 2024\n0162-0134/\u00a9 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\nexplored [3]. In particular, compounds comprising a Re(CO)3 core in 2. Experimental section\ntheir architecture have shown the possibility of overcoming the limita\u00ad\ntions of platinum-based drugs [4]. Several studies have demonstrated 2.1. Synthesis and characterization of rhenium(I) tricarbonyl complexes\nthe therapeutic potential of these compounds [5\u20139]. Knopf et al. have\nconducted research on various rhenium tricarbonyl complexes to eval\u00ad ReCl(CO)5, 1,10-phenantroline (phen), pyridine (py), and tri\u00ad\nuate their anticancer properties [6]. They discovered a drug candidate fluoromethanesulfonic acid were purchased from Aldrich (Darmstadt,\nthat demonstrates greater potency than cisplatin against HeLa cells [6]. Germany) and used without further purification. The ligands, dipyrido\nThis particular complex trigger cell death through a unique mechanism, [3,2-f:2\u2032,3\u2032-h]quinoxaline (dpq) and dipyrido[3,2-a:2\u20323\u2019-c]phenazine\ndifferent from that of cisplatin, thereby avoiding cross-resistance be\u00ad (dppz) were synthesized as described previously [12\u201314]. The fac-[Re\ntween the two compounds [6]. Further studies on in vivo biodistribution (CO)3(NN)(py)]PF6 complexes, NN = phen (1), dpq (2), and dppz (3),\nand metabolites of the complex have verified its stability and indicated were prepared according to previously described procedures [15\u201318].\nits excretion through the renal and hepatobiliary systems [6]. Product purity was confirmed by elemental analysis, infrared (IR) and\n 1\n Leonidova and colleagues have discussed the cytotoxic Re organo\u00ad H Nuclear Magnetic Resonance (NMR).\nmetallic complexes mainly focused on IC50 values and their modes of Attenuated total reflectance Fourier-transformed infrared (ATR-\ncytotoxic action [1]. Most of the reported cytotoxic effects toward FTIR) spectra were recorded in a Perkin Elmer Frontier spectrometer\nhuman cancer cell lines present remarkable outcomes associated with equipped with a diamond crystal plate, using 16 scans at a resolution of\nrhenium complexes characterized by the general formula fac-Re(CO)3, 2 cm\u2212 1. 1H NMR spectra were recorded in a Bruker Ascend 400 MHz\nexhibiting IC50 values <30 \u03bcM [1]. The toxicity of the compounds spectrometer using the residual solvent signal as internal standard.\ntypically rose with their lipophilicity, likely because of enhanced uptake Elemental analysis was carried out in a Perkin Elmer 2400 CHNS\nby cells [1]. Enslin et al. demonstrated remarkably low cytotoxicity analyzer. Electronic absorption spectra were recorded in a Shimadzu\n(IC50 = 30\u201350 nM) of Re(I) carbonyl complexes against prostate UV\u2013Vis spectrophotometer UV-1650 PC. Room temperature emission\nadenocarcinoma (PC3) [10]. The exceptional toxicity was attributed to measurements were performed in argon degassed CH3CN solutions in a\nthe compounds\u2019 localization in mitochondria and nuclei, leading to the 1.000 cm quartz cuvette. Emission quantum yields were determined\ndownregulation of mitochondrial ATP production in PC3 cells [10]. taking the fac-[ClRe(CO)3(phen)] as standard (\u03c6em = 0.018 in CH3CN at\nAlthough recent studies have shown the potential of Re tricarbonyl 298 K) [19].\ncomplexes as anticancer drugs [1,5\u201310], there remains a gap in the fac-[Re(CO)3(phen)(py)]+: IR (cm\u2212 1): \u03bdC-H(arom) w(3088), \u03bdC-H(arom)\nunderstanding of the influence of biomolecule interactions with w(2970), \u03bdC\u2261O s(2013), \u03bdC\u2261O s(1922), \u03bdC\u2261O s(1872), \u03bdC=C(arom) m(1627),\nrhenium(I) tricarbonyl complexes on their cytotoxic effects in cells. \u03bdC=C(arom) m(1601), \u03bdC=N(arom) m(1583), \u03bdC=N(arom) m(1518), \u03bdC=N(arom)\n Recently, Konkankit and colleagues showed that one of the factors m(1490), \u03bdP-F s(850), \u03bdC-H(arom) s(722), \u03bdC-H(arom) m(641), \u03bdP-F s(546), \u03bdRe-\ncontributing to the increased cytotoxic activity of rhenium(I) tricarbonyl N s(271).\ncomplexes was their lipophilic and hydrophilic nature [11]. They fac-[Re(CO)3(dpq)(py)]+: IR (cm\u2212 1): \u03bdC-H(arom) w(3093), \u03bdC-H(arom) w\ndemonstrated that by increasing the alkyl chain length of Re(CO)3, (3044), \u03bdC\u2261O s(2019), \u03bdC\u2261O s(1916), \u03bdC\u2261O s(1870), \u03bdC=C(arom) m(1608),\ncellular uptake was swifter, and cytotoxic effects manifested on a \u03bdC=C(arom) m(1577), \u03bdC=N(arom) w(1547), \u03bdC=N(arom) m(1530), \u03bdC=N(arom)\nsignificantly accelerated timescale [11]. Although this study elucidates m(1479), \u03bdP-F m(824), \u03bdC-H(arom) m(730), \u03bdC-H(arom) m(644), \u03bdP-F m(526),\nthe influence of the lipophilic/hydrophilic nature of rhenium(I) tri\u00ad \u03bdRe-N m(277).\ncarbonyl complexes in modulating the rate of cell death, it is not clear fac-[Re(CO)3(dppz)(py)]+: IR (cm\u2212 1): \u03bdC-H(arom) w(3088), \u03bdC-H(arom)\nhow lipophilic characteristics can influence the combination of Re w(2923), \u03bdC\u2261O s(2032), \u03bdC\u2261O s(1936), \u03bdC\u2261O s(1908), \u03bdC=C(arom) w(1605),\ncomplex with biomolecules (i.e., proteins, lipids, and DNA) for opti\u00ad \u03bdC=C(arom) w(1578), \u03bdC=N(arom) w(1547), \u03bdC=N(arom) m(1495), \u03bdC=N(arom)\nmizing its intrinsic pharmacological properties. Expanding on previous w(1465), \u03bdP-F s(834), \u03bdC-H(arom) s(807), \u03bdC-H(arom) m(637), \u03bdP-F m(530),\nefforts, the objective of this study was to examine how the interactions of \u03bdRe-N w(271).\nthe Re complex with biomolecules affect cell death. In this context, we For fac-[Re(CO)3(phen)(py)]PF6 (1), the yield was 87%, and anal.\naimed to prepare a series of Re(CO)3 molecules with different ligand Calcd. for ReC20H13N3O3PF6: C, 35.61%; N, 6.23%; H, 1.94%, found: C,\ncharacteristics (Fig. 1) to understand the relationship between lip\u00ad 35.14%; N, 6.06%; H, 2.16%. 1H NMR (400 MHz, CD3CN) d 10.36 (dd,\nophilicity, strength of biomolecule binding, and cytotoxicity. 2H, J 1.32, 5.10 Hz), 9.59 (dd, 2H, J 1.36, 8.10 Hz), 9.02 (dd, 2H, J 1.48,\n 6.50 Hz), 8.92 (s, 2H), 8.87 (dd, 2H, J 5.12, 8.11 Hz), 8.50 (m, 1H), 7.94\n (m, 2H).\n For fac-[Re(CO)3(dpq)(py)]PF6 (2), the yield was 92%. calcd. For\n ReC25H22N5O3PF6: C, 47.91%; N, 11.18%; H, 3.54%, found: C, 47.86%;\n N, 11.05%; H, 3.36%. 1H NMR (400 MHz, CD3CN) d 10.54 (dd, 2H, J\n\n\n\n\nFig. 1. Rhenium(I) tricarbonyl complexes examined in this study are listed as follows: (1) fac-[Re(CO)3(phen)(py)]+; (2) fac-[Re(CO)3(dpq)(py)]+ and (3) fac-[Re\n(CO)3(dppz)(py)]+.\n\n 2\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\n1.24, 8.40 Hz), 10.44 (dd, 2H, J 1.48, 5.14 Hz), 9.95 (s, 2H), 9.06 (dd,\n (F0 \u2212 F)\n2H, J 1.40, 6.38 Hz), 9.01 (dd, 2H, J 5.40, 8.46 Hz), 8.50 (m, 1H), 7.96 log = logKb + nlog[Q] (4)\n F\n(m, 2H).\n For fac-[Re(CO)3(dppz)(py)]PF6 (3), the anal yield was 79%. calcd. where F0 and F are the fluorescence intensities in the absence and\nFor ReC26H15N5O3PF6: C, 40.01%; N, 8.97%; H, 1.94%; found: C, presence of the rhenium complex, respectively, and [Q] is the quencher\n39.86%; N, 8.75%; H, 1.80%. 1H NMR (400 MHz, CD3CN) d 10.66 (dd, (in this case, the rhenium complex).\n2H, J 1.36, 8.30 Hz), 10.42 (dd, 2H, J 1.32, 5.28 Hz), 9.22 (dd, 2H, J\n3.36, 6.52 Hz), 9.11 (dd, 2H, J 1.52, 6.64 Hz), 9.02 (dd, 2H, J 5.28, 8.30 2.4. Liposome-binding assay by UV\u2013Vis spectroscopy\nHz), 8.89 (dd, 2H, J 3.20, 6.68 Hz), 8.52 (m, 1H), 7.99 (m, 2H).\n For liposome preparation, lipid films of DOPG were produced by\n2.2. Partition coefficient determination of rhenium(I) tricarbonyl evaporating chloroform in argon air, followed by hydration with 2.0 mL\ncomplexes of Tris buffer (10 mM, pH 7.4) solution [25,26]. Stock DOPG lipid\n concentration was 1.5 mM. All vesicle suspensions were extruded 21\n The coefficient of partitioning of the rhenium complexes was times through polycarbonate membranes comprising pores with a\ndetermined using octanol and water in a shake-flask method according diameter of 100 nm using an Avanti Mini-Extruder [27].\nto the method described in previous studies [20,21]. Briefly, the Solutions of each rhenium complex (20 \u03bcM in Tris buffer with <1.0%\nrhenium complex (50 \u03bcM) was added to a mixture consisting of octanol v/v DMSO) were prepared. The changes in Re(I) complex (20 \u03bcM) ab\u00ad\nand water in a 50% (v/v) ratio. After intense stirring, the samples were sorption spectra were followed with successive small-volume additions\nincubated in the dark for 24 h. The organic phase was then separated of DOPG liposomes (1.5 \u03bcM). The incubation time needed to achieve\nfrom the aqueous phase, and the Re(I) complex concentration in each equilibrium was 30 min (data not shown), as determined experimen\u00ad\nphase was evaluated using UV\u2013vis spectroscopy. The coefficient of tally. Then, after each addition of an aliquot of DOPG liposomes (1.5\npartitioning in the octanol phase (log Po/w) was determined using Eq. (1) \u03bcM) and incubation of 30 min, the absorption spectra of Re(I) complex\n[22]: (20 \u03bcM) were recorded in the range 210\u2013800 nm at room temperature.\n (\n [ReComplex]octanol\n ) The binding constant (Kb) of the rhenium complex to liposomes was\nLog Po/w = Log10 (1) calculated using Eq. (5), as previously described [28,29]:\n [ReComplex]water\n A0 + Af Kb [L]\nwhere [ReComplex]octanol and [ReComplex]water represent the molar A= (5)\n 1 + kb [L]\nconcentrations of the Re(I) complex in octanol and water.\n The experiment was performed in independent duplicates, with three where A is the rhenium complex absorbance intensity recorded in the\nrepetitions in each experiment (n = 6), and the average and standard presence of the lipid at concentration [L], A0 is the rhenium complex\ndeviation of the log Po/w values obtained in each experiment were absorbance measured in the absence of the lipid, and Af is the asymptotic\ncalculated. value of the absorbance of the complete rhenium complex.\n\n2.3. Bovine serum albumin (BSA)-binding assay by fluorescence analysis 2.5. DNA-binding assay by UV\u2013Vis spectroscopy\n\n Interactions between rhenium(I) complexes and bovine serum al\u00ad A straightforward method for assessing the potential interactions\nbumin (BSA) were studied using spectroscopic techniques. The solution between DNA and a drug involves monitoring alterations in the ab\u00ad\nof BSA (3 \u03bcM) was prepared in Tris buffer (10 mM, pH 7.4). Then, sorption properties of either the drug or DNA molecules [30].\nfluorescence spectra of BSA (3 \u03bcM, in Tris buffer 10 mM, pH 7.4) were A solution of salmon sperm DNA (ss-DNA) was prepared by dis\u00ad\nrecorded in the absence or presence of the rhenium(I) tricarbonyl solving 5 mg of ss-DNA in 5 mL of Tris buffer (50 mM) containing NaCl\ncomplexes (with concentrations ranging from 0 to 16 \u03bcM in Tris buffer (5 mM) at pH 7.4 [31]. The DNA solution with a ratio of absorbance of\nwith <1.0% v/v DMSO) (\u03bbex = 280 nm, slit widths of 5.0 nm for exci\u00ad approximately 1.8 recorded at 260 and 280 nm (Abs260nm/Abs280nm)\ntation and 1.0 nm for emission) in the range 300\u2013450 nm at 35 \u25e6 C. indicates that the DNA was sufficiently free of protein and that the\n Because each rhenium complex exhibited notable absorption at the double helix structure was intact [32,33]. Thus, before starting the\nexcitation and emission wavelengths (280 and 350 nm, respectively), experiment, DNA integrity was checked by ensuring that Abs260nm/\nthe inner filter effect was adjusted using Eq. (2) [23]: Abs280nm \u2265 1.8, and ss-DNA concentration was calculated using the\n molar absorption coefficient, \u03b5260 = 6600 M\u2212 1 cm\u2212 1 [32,33]. The stock\nFcor = Fobs 10[(Aex +Aem )/2 ] (2)\n ss-DNA solution was refrigerated at 2\u201310 \u25e6 C using an ice bath during all\n Fcor and Fobs denote the corrected and observed fluorescence intensity experiments.\nvalues, respectively, whereas Aex and Aem represent the experimental Solutions of each Re(I) complex were adjusted to a fixed concen\u00ad\nabsorbance values at the excitation and emission wavelengths, tration of 20 \u03bcM in Tris buffer (50 mM) and NaCl (5 mM) containing\nrespectively. <1.0% v/v acetonitrile at a pH of 7.4. Then, after each addition of 10 \u03bcL\n The quenching constants were calculated from Stern-Volmer Eq. (3): of aliquots of ss-DNA solution, the mixture containing the Re(I) complex\nF0 was incubated with DNA for 5 min, and the absorption spectrum with\n = 1 + kq .\u03c40 [Q] = 1 + KSV .[Q] (3) varying ss-DNA concentrations (0\u201350 \u03bcM) was recorded in the range\nF\n 210\u2013800 nm at room temperature.\nwhere F0 and F are the fluorescence intensities in the absence and The binding constant (Kb) was obtained from the intercept-to-slope\npresence of the quencher, respectively, KSV is the Stern\u2013Volmer ratios of A0/(A - A0) vs. 1/[DNA] according to the following Eq. (6)\nquenching constant, kq is the bimolecular quenching constant, \u03c40 is the [30,34]:\naverage lifetime of the Trp-214 residue without the quencher (~1 \u00d7\n A0 \u03b5G 1\n10\u2212 8 s) [23] and [Q] is the concentration of the quencher. = * (6)\n A \u2212 A0 \u03b5HG \u2212 \u03b5G Kb [DNA]\n The modified Stern\u2013Volmer equation was employed to calculate the\nbinding constant (Kb) and number of binding sites (n) (Eq. (4)) [24]: where A0 is the absorbance of the complex in the absence of DNA, A is\n the absorbance in the presence of DNA at concentration [DNA], and \u03b5G\n and \u03b5HG are the absorption coefficients of the rhenium complex and the\n\n 3\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\n[ReComplex]\u2013DNA, respectively. in a 5% CO2 atmosphere at 37 \u25e6 C.\n A cell viability assay using the MTT method was used to assess the\n2.6. Molecular docking and ligand preparation cytotoxicity of Re (I) complexes [37,38]. Cells were grown to 70\u201380%\n confluence in a 75 cm2 flask, detached with trypsin/EDTA, seeded in 48-\n The ligands were designed using the Avogadro software version 1.1.1 well plates at a density of 3 \u00d7 104 cells/well in 300 \u03bcL of the growth\nand saved in mol2 format after structural optimization. The files were media, and incubated at 37 \u25e6 C in an atmosphere containing 5% CO2 for\nthen configured in Avogadro software using the extensions tool to obtain 24 h. Compounds were dissolved in DMSO to prepare stock solutions and\nan optimized geometry in a Universal Force Field (UFF). In AutoDock subsequently diluted in growth media (DMEM, 10% FBS, 100 \u03bcg/ml\n1.5.7, the ligands were prepared by adding polar hydrogen atoms and penicillin, and 100 \u03bcg/ml streptomycin) to maintain a DMSO concen\u00ad\nGasteiger charges, and possible water molecules were deleted. tration of <1%. Following removal of the medium, cells were incubated\n Molecular docking studies of the metallocomplexes of rhenium were with fresh medium (300 \u03bcL) containing varying concentrations (0\u2013100\ncarried out using the Auto Dock 1.5.7 software with DNA and BSA \u03bcM) of the desired rhenium complex for 24 h at 37 \u25e6 C in an atmosphere\nbiomolecules. The crystal structures of DNA (126D) with a sequence of containing 5% CO2. The growth medium containing the Re(I) complex\nCATGGCCATG at a resolution of 2 \u00c5 and BSA (3 V03) at a resolution of was removed and incubated for an additional 24 h to allow adequate\n2.7 \u00c5 were obtained from the Protein Data Bank (www.rcsb.org). Polar time for cell proliferation. Subsequently, 100 \u03bcL of MTT (0.75 mg/mL)\nhydrogen atoms and Kollman charges were added to the structure, and was added to 300 \u03bcL of DMEM comprising 10% FBS. After 3 h, the MTT/\nthe water molecules around the DNA/BSA were removed. DMEM solution was removed, and formazan crystals were dissolved in\n To study the binding state in blind docking, the DNA was analyzed in 300 \u03bcL of DMSO. The absorbance of each well was measured at 570 nm\na grid box with dimensions of 62 \u00d7 58 \u00d7 96 for the three complexes. BSA using a microplate reader. Cell viability was calculated by normalizing\nwas analyzed by considering the location of two possible binding sites in the absorbance of the treated cells to that of the untreated cells (without\nthe region of tryptophan 134 with the coordinates x = 45.989286, y = the complex; normalized to 100% cell viability). The percentage cell\n36.667143, and z = 25.500143, and tryptophan 213 with the co\u00ad viability and IC50 values shown in this study represent the average of\nordinates x = 101.169786, y = 28.225500, and z = 19.778429. two independent experiments with three replicates per concentration\n For the BSA binding study, two possible sites were analyzed in a level. For IC50 analysis (concentration that inhibits 50% of cell growth),\n70x76x78 scan box. Lamarkian genetic algorithms were used to perform GraphPad Prism 8.0 software (GraphPad Software Inc., La Jolla, Cali\u00ad\nthe ligand-receptor binding calculations. The number of genetic algo\u00ad fornia, USA) was used based on nonlinear regression, where the per\u00ad\nrithm runs was set to 100, and the number of evaluations was set to 2.5 centage of cell viability was determined as a base 10 logarithmic\nmillion [35,36] The DNA/BSA interactions were produced using Biovia function of the concentrations tested assuming a 95% confidence in\u00ad\nDiscovery Studio Visualizer version 2021 and Maestro Schro\u0308dinger terval (p < 0.05).\nsoftware, version 13.6.122. To assess the selectivity of the complexes against tumor cells, the\n selectivity index (SI) was calculated as the ratio between the concen\u00ad\n2.7. DNA cleavage studies trations needed to reduce cell viability by 50% (IC50) in non-tumor cells\n and tumor cells. The following formula was used: SI = (IC50 of the non-\n To investigate the interaction of the complexes with DNA, we com\u00ad tumor lineage)/(IC50 of the tumor lineage). An SI \u2265 2 designated the\nbined the fac-[Re(CO)3(phen)(py)]+, fac-[Re(CO)3(dpq)(py)]+, and fac- compound as more selective for tumor cell lines, indicating it kills twice\n[Re(CO)3(dppz)(py)]+ complexes with plasmid DNA from the siS\u00ad as many neoplastic cells as healthy cells. This designation underscores\nTRIKE\u2122 U6 Hairpin Cloning System (Human) \u2013 hMGFP. The isolated its potential as a promising anticancer candidate [39].\nplasmid was incorporated into a series of 11 solutions, each with a total\nvolume of 20 \u03bcL. These solutions included 30 ng/\u03bcL of the plasmid 2.9. Cell cycle analysis by flow cytometry\ntreated at the concentrations of 40 \u03bcM and 80 \u03bcM of fac-[Re(CO)3(phen)\n(py)]+ and fac-[Re(CO)3(dpq)(py)]+ and 10 \u03bcM and 20 \u03bcM of fac-[Re To explore the potential antiproliferative effects of the rhenium\n(CO)3(dppz)(py)]+ in PBS 1\u00d7 with or without dimethyl sulfoxide metallocomplexes, HeLa tumor cells (2 \u00d7 105 cells/mL) were incubated\n(DMSO - 0.05%), hydrogen peroxide (H2O2\u201315 mM), and a combination with varying concentrations of different complexes. In this study, fac-\nof both. Controls consisted of an untreated plasmid, a plasmid treated [Re(CO)3(phen)(py)]+ and fac-[Re(CO)3(dpq)(py)]+ were administered\nwith the unique site restriction enzyme Xba I, and a plasmid treated with at concentrations of 10 \u03bcM, 20 \u03bcM, and 40 \u03bcM, whereas fac-[Re\nH2O2. The reaction mixtures underwent incubation at 37 \u25e6 C for 12 h, (CO)3(dppz)(py)]+ was employed at concentrations of 5 \u03bcM, 10 \u03bcM, and\nfollowed by the addition of 3 \u03bcL of loading buffer (loader - 0.25% bro\u00ad 20 \u03bcM, with the selected concentrations aligning with their respective\nmophenol blue, 0.25% xylene cyanol, 30% glycerol, 10 mM EDTA). IC50 values. The cells were incubated for 24 h. Subsequently, the cells\nSubsequently, electrophoresis was conducted on a 0.8% agarose gel were centrifuged for 10 min at 1000 rpm, resulting in a pellet that un\u00ad\ncontaining 0.05% ethidium bromide (10 \u03bcg/mL) in 90 mM Tris-borate derwent two washes with PBS1\u00d7 and subsequent fixation in a 70%\nbuffer (pH 8.0) and 20 mM EDTA (0.5 \u00d7 TBE). Gel electrophoresis ethanol solution in PBS 1\u00d7. The fixed samples were stored at 4 \u25e6 C\nwas performed at 80 V for 3 h, and the results were examined under overnight. Subsequently, the cells were centrifuged for 10 min at 1000\nultraviolet light. Bands were quantified using the ImageJ software rpm, and the pellet was re-suspended in PBS 1\u00d7 containing 10 \u03bcg/ml\nversion 1.53 k (Java 1.8.0_172). propidium iodide and 100 \u03bcg/ml RNase. This step aims to eliminate RNA\n contamination, allowing only DNA to be stained. The cells were then\n2.8. Cytotoxicity of rhenium(I) tricarbonyl complexes incubated in the dark for 45 min at 37 \u25e6 C, after which the samples were\n subjected to analysis using the ACCURI flow cytometer (BD). The\n The HeLa cell line, derived from cervical cancer cells, was obtained analysis was conducted on the FL2 channel, and the obtained cytometric\nfrom the American Type Culture Collection (ATCC) and cultivated in data were analyzed using the FloJo software (version 10).\nDulbecco\u2019s Modified Eagle\u2019s Medium (DMEM) supplemented with 10%\nfetal bovine serum (FBS), 100 \u03bcg/ml penicillin, and 100 \u03bcg/mL strep\u00ad 2.10. Statistical analysis\ntomycin. PNT2 (human prostate epithelial cell line), B16-F10 (murine\nmelanoma cells), and NIH/3 T3 (murine fibroblasts) were cultured in Octave 7.2.0 was utilized for comparative statistical analyses. The\nRPMI-1640 medium (Gibco\u00ae, Paisley, UK) supplemented with 2 mM/L data, which were derived from a minimum of two (often three) inde\u00ad\nglutamine, 25 mM HEPES, 100 \u03bcg/mL penicillin, 100 \u03bcg/mL strepto\u00ad pendent experiments, were presented as mean values \u00b1 standard error\nmycin, and 10% fetal bovine serum (FBS). All cultures were maintained (SE). Initially, adherence to a Gaussian curve was assessed for pairwise\n\n 4\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\ncomparisons. Subsequently, depending on the nature of the data, para\u00ad Table 1\nmetric or non-parametric analyses were performed using Student\u2019s t-test Photophysical data for the studied complexes in acetonitrile at 298 K (\u03bbexc = 375\nor Holm-Sidak test for independent samples. Multiple comparisons were nm) [15].\nperformed using one-way analysis of variance (ANOVA) with Tukey or Rhenium(I) \u03bbmax (nm) (\u03b5 / 104 (L mol\u2212 1 \u03bbem \u03d5em \u03c4/\nBonferroni tests, based on variance homogeneity and the number of Complex cm\u2212 1)) (nm) \u03bcs\ngroups involved. The strength of the linear correlation was determined fac-[Re 222 (4.11), 252 (2.71), 275 545 0.020 1.6\nusing Pearson\u2019s coefficient (r). Statistical significance was set at a (CO)3(phen) (3.40), 327 (0.72), 369 (0.48)\nthreshold of P < 0.05. In the figures, significance levels concerning the (py)]+\n fac-[Re(CO)3(dpq) 258 (5.43), 286 (2.73), 370 557 0.012 0.45\ncontrol are denoted with asterisks above the bars, and multiple or\n (py)]+ (0.50)\npairwise comparisons are indicated accordingly. fac-[Re 278 (8.98), 362 (1.85), 381 554, 0.001 108\n (CO)3(dppz) (1.81) 600\n3. Results and discussion (py)]+\n\n\n3.1. Synthesis and photophysical properties\n yield and emission lifetime (Table 1). In contrast, for fac-[Re\n (CO)3(dppz)(py)]+, a structured emission band was observed, which\n The photophysics of the fac-[Re(CO)3(NN)(py)] employed in this\n +\n was attributed to radiative decay of the triplet intraligand excited state\nstudy have been extensively discussed in a previous study [15]. As\n (3ILdppz). As expected, the quantum yield was one order of magnitude\nshown in Fig. 2, the electronic absorption and emission spectra of the\n smaller, and the corresponding lifetime reached 108 \u03bcs (Table 1).\ncomplexes in acetonitrile were obtained at 298 K. The main absorption\nand physical parameters are summarized in Table 1.\n The parent complex fac-[Re(CO)3(phen)(py)]+ exhibited high- 3.2. Partition coefficient of rhenium(I) tricarbonyl complexes\nenergy fully allowed absorption bands attributed to the internal IL (\u03c0\n\u2192 \u03c0*) transitions in the phen ligand. Low-energy metal-to-ligand charge Drug absorption in an organism is mediated via passage through\ntransfer (MLCT) absorption was observed at 370 nm (Fig. 2), which was biological membranes. The lipophilicity of drug molecules strongly in\u00ad\nresponsible for the main photophysical properties of the fluid solution at fluences receptor binding, cellular uptake, and bioavailability [40].\nroom temperature. A typical broad emission band was observed During this process, hydrophilic or hydrophobic pathways are employed\nfollowing light excitation (Fig. 2), which was attributed to 3MLCT to transport the substance to its site of action, where it can exert its\nradiative decay, as discussed elsewhere [15]. biological effects. The octanol-water partition coefficient (log Po/w) is\n The replacement of the phen ligand by dpq and then dppz led to useful for estimating the simple relationship between hydrophilic/hy\u00ad\nredshifts in the high-energy absorption bands, owing to the extended drophobic characteristics and biological responses, such as IC50 values\nconjugation of the quinoxaline and phenazine ligands (Fig. 2). In fac-[Re [41]. Thus, the octanol/water partition coefficient (log Po/w) was\n(CO)3(dpq)(py)]+, the MLCT transition was associated with the lowest assessed to evaluate the lipophilic and hydrophilic nature of the\nenergy absorption band, whereas in fac-[Re(CO)3(dppz)(py)]+, an rhenium(I) complexes (Table 2).\noverlap of the MLCT and IL transitions was observed, leading to an in\u00ad While positive log Po/w values indicate a greater affinity of the\ncrease in the molar absorptivity coefficient (\u03b5) (Table 1). The stabiliza\u00ad compound for the organic phase, negative log Po/w values indicate an\ntion of the IL excited states in fac-[Re(CO)3(dpq)(py)]+ and fac-[Re affinity for the aqueous phase [42]. The log Po/w value obtained for fac-\n(CO)3(dppz)(py)]+ also led to considerable changes in the emission [Re(CO)3(dppz)(py)]+ was positive and larger than that of the other\nproperties. The complex with dpq maintained its 3MLCT emitter state investigated complexes (Table 2). This indicates that fac-[Re\nwith an emission maximum demonstrating a red-shift by ~12 nm (CO)3(dppz)(py)]+ exhibited greater affinity for the organic phase,\ncompared to fac-[Re(CO)3(phen)(py)]+, albeit with a smaller quantum probably because it is the most lipophilic molecule among the rhenium\n\n\n\n\nFig. 2. Absorption (A) and emission (B) spectra of studied complexes in acetonitrile at 298 K. [Re(CO)3(phen)(py)]+ (blue), [Re(CO)3(dpq)(py)]+ (green), and [Re\n(CO)3(dppz)(py)]+ (red). \u03bbexc = 375 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)\n\n 5\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\n Table 2 fluorescence intensity of the protein (Fig. 3A and Fig. S1 in the sup\u00ad\n Log Po/w of the studied rhenium(I) tricarbonyl complexes. The plemental material). Stern\u2013Volmer quenching constants and binding\n values correspond to the mean \u00b1 standard deviation (SD) from two parameters were determined (Table 3) as described in the experimental\n separate assays with three replications in each experiment (n = 6). section using Eq. (3) for fitting plots of log[(F0/F)/F] versus log\n Rhenium(I) complex Log Po/w [ReComplex] (Fig. 3B and Fig. S2 in the supplemental material).\n fac-[Re(CO)3(phen)(py)]\n +\n \u2212 0.39 \u00b1 0.08 From the fluorescence quenching results it is possible to obtain pa\u00ad\n fac-[Re(CO)3(dpq)(py)]+ \u2212 0.15 \u00b1 0.11 rameters such as Stern-Volmer constant (Ksv) and the quenching rate\n fac-[Re(CO)3(dppz)(py)]+ +0.65 \u00b1 0.21 constants (kq) (Table 3). The rhenium complexes showed high values of\n the quenching constant (Ksv > 104 M\u00a11) indicating their great efficiency\n to interact strongly with BSA. kq values were much greater than the\ncomplexes studied. The two negative Log Po/w values for the complexes\n maximum quenching rate constant of diffusion collision of 2.0 \u00d7 1010\nof fac-[Re(CO)3(phen)(py)]+ (Log Po/w = \u2212 0.39 \u00b1 0.08) and fac-[Re\n M\u2212 1 s\u2212 1 (Table 3), suggesting that complex formation between BSA and\n(CO)3(dpq)(py)]+ (Log Po/w = \u2212 0.15 \u00b1 0.11) corresponded to greater\n rhenium complexes may involve both types of quenching mechanisms,\naffinity for the aqueous phase (Table 2). These Log Po/w values are\n dynamic and static [23]. To unravel whether the interaction mechanism\ncomparable to other reports, which show a Log Po/w of \u2212 0.11 for a less\n is static or dynamic, time-resolved fluorescence studies should be per\u00ad\nhydrophobic rhenium complex (fac-[Re(phen)(CO)3(PyCH2O-dam\u00ad\n formed. However, investigation about the type of suppression mecha\u00ad\ninozide)]PF6) and Log Po/w of +0.65 for a more hydrophobic molecule\n nism is not the goal of this work. We are focused to estimate the strength\n(fac-[Re(DIP)(CO)3(PyCH2O-daminozide)]PF6) [4].\n of interaction between [ReComplex]-BSA (via Kb), instead of the\n The observed log Po/w values exhibited an ascending order: fac-[Re\n mechanism of BSA interaction.\n(CO)3(phen)(py)]+ < fac-[Re(CO)3(dpq)(py)]+ < fac-[Re(CO)3(dppz)\n The number of binding sites (n) indicated that the rhenium(I) tri\u00ad\n(py)]+ (Table 2), reflecting the hydrophobic characteristics of their li\u00ad\n carbonyl complexes bound to BSA in a 1:1 ratio (Table 3), which is in\ngands (Fig. 1). Hence, the more hydrophobic the NN ligand of the\n agreement with other reports [44,45]. The [ReComplex]-BSA Kb values\nrhenium complex, the higher the log Po/w value. This finding is in line\n increased in the following order: fac-[Re(CO)3(phen)(py)]+ < fac-[Re\nwith other reports showing that incorporating longer alkyl chains into\n (CO)3(dpq)(py)]+ < fac-[Re(CO)3(dppz)(py)]+ (Table 3). These data\nthe ligands of rhenium(I) tricarbonyl complexes also increases the log\n corroborated the results obtained for log Po/w values, which followed the\nPo/w values [11].\n same trend (Table 2). This indicates that greater hydrophobicity of the\n rhenium complex favors a stronger interaction with the protein.\n3.3. Binding constant (Kb) for the interaction of rhenium(I) complex with\nBSA\n 3.4. Binding constant (Kb) for the interaction of rhenium(I) complex with\n Serum albumin is a primary protein in the mammalian circulatory liposome\nsystem. This macromolecule plays an important physiological role in\nblood pH control and improves the solubility of hydrophobic molecules Liposomes, which are lipid bilayer vesicles frequently employed as\nin aqueous media [43]. Therefore, it is crucial to study the interactions models for cell membranes in permeability and diffusion studies, can be\nbetween the drug candidates and these proteins. BSA has frequently tailored to mimic various membrane structures by adjusting the lipid\nbeen employed as a protein model in these studies [43,44]. Despite their\nimportance, only a few studies have investigated the interactions be\u00ad Table 3\ntween rhenium complexes and BSA [43\u201345]. Therefore, the ability of the Stern\u2013Volmer constants (Ksv), the quenching rate constants (kq), binding con\u00ad\n stant (Kb) and number of binding sites (n) in rhenium(I) tricarbonyl complexes\nrhenium(I) tricarbonyl complexes to interact with BSA was investigated.\n for BSA.\n Typically, steady-state fluorescence techniques allow for the\nstraightforward determination of protein-molecule binding parameters. Rhenium (I) Ksv (\u00d7104 Kb (\u00d7103 kq (M\u2212 1 n R2\n complex M\u2212 1) M\u2212 1) s\u2212 1)\nAlbumins exhibit fluorescence emission due to the aromatic amino acid\nresidues present in the protein (e.g., tryptophan and tyrosine). When a fac-[Re(CO)3(phen) 4.26 \u00b1 0.10 3.10 \u00b1 4.26 \u00d7 0.91 0.968\nsmall molecule binds to albumin, the protein microenvironment is (py)]+ 0.07 1012\n fac-[Re(CO)3(dpq) 2.19 \u00b1 0.17 8.57 \u00b1 2.19 \u00d7 0.99 0.873\naltered, and the fluorescence intensity of the protein is affected [23]. (py)]+ 0.26 1012\nTherefore, the interaction between BSA and fac-[Re(CO)3(NN)(py)]+ fac-[Re(CO)3(dppz) 13.7 \u00b1 0.78 25.7 \u00b1 1.38 \u00d7 0.99 0.984\nwas determined by the fluorescence quenching of the protein. The suc\u00ad (py)]+ 0.24 1013\ncessive addition of rhenium complexes to BSA led to a reduction in the\n\n\n\n\nFig. 3. (A) Emission spectra of BSA (3.0 \u03bcM in Tris buffer, pH 7.4) with successive additions of [Re(CO)3(dppz)(py)]+ (ranging from 0 to 16 \u03bcM in Tris buffer with\n<1.0% v/v DMSO). Temperature: 35 \u25e6 C. \u03bbexc = 280 nm with excitation and emission slits of 5.0 nm and 1.0 nm, respectively. (B) The plot depicting log[(F0/F)/F]\nversus log[[Re(CO)3(dppz)(py)]+] in presence of BSA.\n\n 6\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\ncomposition [46]. The behavior of rhenium complexes in the presence of Table 4\ncell membranes and their lipophilic characteristics can be assayed by Binding constant (Kb) for the interaction between rhenium(I) tri\u00ad\ntheir interactions with liposomes [46]. The strength of the interaction carbonyl complexes and DOPG liposomes. Values represent the\nbetween the rhenium complexes and liposomes was assessed using average \u00b1 standard deviation of two independent experiments.\nUV\u2013Vis spectroscopy. The methodology comprises altering the intensity Rhenium(I) complex Kb (\u00d7103 M\u2212 1)\nof absorption after the incorporation of liposomes into molecules and fac-[Re(CO)3(phen)(py)]\n +\n 2.14 \u00b1 0.08\nconsiders that a chemical equilibrium occurs between the binding fac-[Re(CO)3(dpq)(py)]+ 4.71 \u00b1 0.10\nmolecule and the lipid bilayer to obtain a conventional binding isotherm fac-[Re(CO)3(dppz)(py)]+ 15.29 \u00b1 1.74\n[29]. Aliquots of liposomes (DOPG) were added to a solution of the\nrhenium complexes (Fig. 4A and Fig. S3 in the supplemental material).\n in the presence of various concentrations of DNA (Fig. S5) were sub\u00ad\nBinding constant values (Kb) shown in Table 4 were calculated by\n tracted from the absorbance of the DNA spectra alone in the absence of\nadjusting a theoretical model (Eq. (4), described in the experimental\n Re molecules (Fig. S7) to evaluate the spectral changes in the region of\nsection) derived from the experimental points on the plot of absorbance\n 250\u2013300 nm. A representative spectrum of fac-[Re(CO)3(dppz)(py)]+\nat \u03bbmax calculated as a function of DOPG concentration (Fig. 4B and\n after successive additions of DNA is shown in Fig. 5A (see data from\nFig. S4 in the supplemental material).\n other rhenium complexes in Fig. S8 in the supplemental material). The\n The absorbance intensity of the three rhenium complexes increased,\n value of Kb was obtained as the ratio of the slope to the intercept ob\u00ad\neven at a fixed concentration (20 \u03bcM), when aliquots of liposomes\n tained from the plot of A0/A-A0 versus 1/[DNA] (Fig. 5B and Fig. S9 in\n(DOPG) were added (Fig. 4A and Fiugre S3 in the supplemental mate\u00ad\n the supplemental material) and is summarized in Table 5Table.\nrial). The observed [ReComplex]-liposomes Kb values exhibited an\n The absorption spectrum of the Re complexes without interference\nascending order: fac-[Re(CO)3(phen)(py)]+ < fac-[Re(CO)3(dpq)(py)]+\n from the characteristics of DNA absorption (Fig. 5A and Fig. S8) showed\n< fac-[Re(CO)3(dppz)(py)]+ (Table 4). The ascending order of these Kb\n alterations in the absorbance intensity in the region spanning from 250\nvalues aligns with the data for log Po/w (Table 2) and Kb values for\n to 300 nm attributed to an increase in the concentration of DNA, which\nproteins (Table 3), suggesting that the hydrophobic nature of fac-[Re\n was verified for all studies performed in triplicate. A hypochromic effect\n(CO)3(dppz)(py)]+ favors the interaction of the rhenium complex with\n (reduced absorbance) was observed with the fac-[Re(CO)3(phen)(py)]+\nbiomolecules.\n and fac-[Re(CO)3(dppz)(py)]+ complexes (Fig. S8A and Fig. 5A), sug\u00ad\n gesting intercalating-type interactions due to \u03c0-\u03c0-type stacking between\n3.5. Binding constant (Kb) for the interaction of rhenium (I) complex with ligands in the complex with the nitrogenous bases of DNA [30]. In\nDNA contrast, the complex fac-[Re(CO)3(dpq)(py)]+ showed a hyperchromic\n effect (Fig. S8B), in contrast with previous studies. The observed\n Many drugs currently in clinical use or in advanced clinical trials hyperchromism suggests that fac-[Re(CO)3(dpq)(py)]+-DNA in\u00ad\npharmacologically target DNA. Certain molecules can bind to DNA, teractions primarily involve electrostatic interactions or interactions\nthereby inhibiting cell replication, which is a critical process in cell with the DNA groove attributed to the destabilization of the double helix\ngrowth and division [47]. Metal-based anticancer complexes primarily [48]. To conclusively support that fac-[Re(CO)3(phen)(py)]+ and fac-\ntarget DNA by binding to the N-7 position of guanine residues [30]. [Re(CO)3(dppz)(py)]+ complexes display an intercalative DNA binding\nStudying the interactions of drug candidates with DNA is important for interaction and fac-[Re(CO)3(dpq)(py)]+ primarily involve interactions\nthe rational design of new drugs. UV\u2013Vis spectroscopy is employed to that destabilize the DNA double helix, additional experiments still\nstudy drug-DNA interactions, given that the intercalative binding of a would be needed, such as viscosity, thermal denaturation and/or cir\u00ad\ncomplex with DNA can result in a hypochromic and/or bathochromic cular dichroism [8,49\u201353]. Nevertheless, our research primarily focused\nshift in the absorption spectrum [30]. To substantiate the potential to assess the strength of the interaction between [ReComplex]-DNA (via\nbinding of each rhenium complex to DNA, the binding constant (Kb) was Kb) rather than the mechanism of DNA interaction.\ncomputed by tracking changes in absorbance as the concentration of The strength of [ReComplex]-DNA interaction (Kb) exhibited the\nDNA increased (Fig. S5 in the supplemental material). The DNA following ascending order: fac-[Re(CO)3(phen)(py)]+ < fac-[Re\nbiomolecule exhibited an absorption band (260 nm) in the same region (CO)3(dpq)(py)]+ < fac-[Re(CO)3(dppz)(py)]+ (Table 5). The Kb values\nas the IL bands of the Re complexes (250\u2013300 nm); Fig. S6. To enable were comparable to those of other metal complexes that bind to DNA\ndata analysis in the 250\u2013300 nm region, the UV\u2013Vis absorption spec\u00ad reported in literature [8,30,54,55]. Again, fac-[Re(CO)3(dppz)(py)]+\ntrum of DNA alone (without Re complexes) was recorded under the same showed the highest Kb value with DNA, corroborating previous data\nconditions used in the assay in the presence of complexes (Fig. S7 in the showing the highest Kb value with BSA and the highest log Po/w among\nsupplemental material). Thus, the absorbance spectra of the compounds the studied compounds. This emphasizes the importance of the\n\n\n\n\nFig. 4. (A) Absorption spectra of fac-[Re(CO)3(dppz)(py)]+ (20 \u03bcM) with successive addition of DOPG liposomes (1.5 \u03bcM) in Tris buffer, pH 7.4 at room temperature.\n(B) Plot of fac-[Re(CO)3(dppz)(py)]+ absorbance at \u03bbmaximum versus [DOPG] used for the calculation of Kb values.\n\n 7\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\n\n\nFig. 5. (A) Absorption spectra of fac-[Re(CO)3(dppz)(py)]+ (20 \u03bcM) discounting the absorption spectra of ss-DNA at different concentrations in Tris buffer (50 mM)\nand NaCl (5 mM) at pH 7.4 and room temperature. (B) Plot of A0/A-A0 versus 1/[DNA] obtained to calculate Kb value of fac-[Re(CO)3(dppz)(py)]+.\n\n\n complexes with BSA in chain A near the tryptophan 134 site. Conversely,\n Table 5 Fig. 6B, D, and F illustrate their interactions in chain B, which is close to\n Binding constant (Kb) of the interaction between rhenium(I) tri\u00ad\n the tryptophan 213 site.\n carbonyl complexes and DNA. Values represent the mean \u00b1 stan\u00ad\n As depicted in Fig. 6A, interactions were observed between the\n dard error of three independent experiments (n = 3).\n pyridyl group of fac-[Re(CO)3(phen)(py)]+ and proline 117, which were\n Rhenium(I) complex Kb (\u00d7103 M\u2212 1)\n mediated via \u03c0-alkyl hydrophobic bonds in chain A of BSA. Furthermore,\n fac-[Re(CO)3(phen)(py)] +\n 1.35 \u00b1 0.52 the phenanthroline ligand of fac-[Re(CO)3(phen)(py)]+ engaged in\n fac-[Re(CO)3(dpq)(py)]+ 2.01 \u00b1 0.77 \u03c0-alkyl hydrophobic interactions with isoleucine 141, leucine 115,\n fac-[Re(CO)3(dppz)(py)]+ 7.63 \u00b1 0.44\n arginine 185, and tyrosine 137 (Fig. 6A). Moving to the B chain of BSA\n near the tryptophan 213 site, the pyridyl group of fac-[Re(CO)3(phen)\nhydrophobicity of the dppz ligand, which favors enhanced interaction (py)]+ interacted with leucine 326, whereas phenanthroline ligand\nwith the hydrophobic regions of biomolecules (proteins, lipids, and demonstrated \u03c0-alkyl hydrophobic interactions with leucine 346 and\nDNA). 480, valine 481 and alanine 212 (Fig. 6B). Moreover, one of the carbonyl\n ligands of fac-[Re(CO)3(phen)(py)]+ formed hydrogen bonds with\n arginine 208 (Fig. 6B).\n3.6. Molecular docking\n In chain A of BSA with fac-[Re(CO)3(dpq)(py)]+ (Fig. 6C), the pyr\u00ad\n idyl group established connections with interleukin 181, tyrosine 137,\n As in vitro analyses revealed that the complexes can interact with\n and arginine 185. Simultaneously, the dipyrido[3,2-f:2\u2032,3\u2032-h]quinoxa\u00ad\nimportant cellular biomolecules, in silico simulations were performed\n line (dpq) ligand interacted with proline 117, leucine 115, lysine 114,\nusing bioinformatics tools. This study aimed to enhance our under\u00ad\n and arginine 185. Additionally, a hydrogen bond with the nitrogen of\nstanding of the potential chemical bonds that could form between Re\n leucine 115 was identified (Fig. 6C). Shifting to the B chain of BSA, in\u00ad\ncomplexes and BSA proteins or DNA.\n teractions between the pyridyl ring and aspartate 450 involving anion-\u03c0\n interactions, as well as those with tryptophan 213 involving hydro\u00ad\n3.7. In silico molecular interactions of fac-[Re(CO)3(phen)(py)]+, fac- phobic \u03c0-alkyl interactions (Fig. 6D) are depicted. In contrast, the dpq\n[Re(CO)3(dpq)(py)]+, and fac-[Re(CO)3(dppz)(py)]+ with BSA ligand of fac-[Re(CO)3(dpq)(py)]+ engaged with arginine 217 via\n cation-\u03c0 interactions, lysine 221 via cation-\u03c0 interactions, and glutamine\n Molecular docking revealed interactions between the three Re 291 via hydrophobic \u03c0-alkyl interactions (Fig. 6D). Hydrogen bonds\ncomplexes and BSA macromolecules at the examined activation sites were detected between the dpq group and alanine at positions 290, 294,\nexhibiting low binding energies indicative of favorable binding. The and 342 (Fig. 6D).\ndata in Table 6 demonstrate an inverse relationship between the hy\u00ad In fac-[Re(CO)3(dppz)(py)]+, the pyridyl ring interacted with inter\u00ad\ndrophobicity of the Re complexes and the binding energy, indicating a leukin 181 and tyrosine 160 (Fig. 6E). Additionally, the dipyrido[3,2-\nmore favorable binding affinity with an increase in the the hydropho\u00ad a:2\u20323\u2019-c]phenazine (dppz) group of fac-[Re(CO)3(dppz)(py)]+ formed\nbicity. This is consistent with the experimental findings for Kb with BSA, connections with tryptophan 134, leucine 115 and 122, tyrosine 137,\nsupporting the observation that a more hydrophobic Re complex cor\u00ad interleukin 141, lysine 136, and proline 117 in the A chain of BSA\nresponds to a higher Kb (Table 3). (Fig. 6E). Furthermore, hydrogen bonds were observed between the two\n Fig. 6A, C, and E depict the interactions of fac-[Re(CO)3(NN)(py)]+ of their C\u2013\u2013O groups and arginine 185 (Fig. 6E). In the B chain of BSA\n (Fig. 6F), hydrophobic \u03c0-alkyl interactions between the pyridyl group\nTable 6 and valine 481, alanine 209, and 212, were detected. In contrast, the\nThe energy of the interactions between rhenium complexes and the BSA mole\u00ad dppz ligand extended toward arginine 208 and 212 via hydrophobic\ncule at tryptophan sites 134 (chain A) and 213 (chain B). \u03c0-cation, \u03c0-sigma interactions, and \u03c0-alkyl interactions (Fig. 6F). Hy\u00ad\n Rhenium(I) Binding energy Tryptophan Binding energy Tryptophan drophobic \u03c0-alkyl interactions were also observed with alanine 209,\n complex 134 (kcal/mol) 213 (kcal/mol) glycine 327, alanine 349, leucine 346, lysine 350, and valine 481.\n fac-[Re Additionally, hydrogen bonds were observed between the C\u2013 \u2013O groups,\n (CO)3(phen) \u2212 6,72 \u2212 6,70 leucine 480, and serine 479 (Fig. 6F).\n (py)]+ Molecular docking analysis is a valuable tool for examining in\u00ad\n fac-[Re(CO)3(dpq)\n \u2212 7,13 \u2212 6,51 teractions between metal complexes and biological molecules, such as\n (py)]+\n fac-[Re(CO)3(dppz) BSA, which plays an essential role in the transportation of ligands in the\n \u2212 8,97 \u2212 8,70\n (py)]+ body [56].\n\n\n 8\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\n\n\nFig. 6. Interaction between rhenium complexes with BSA protein (3 V03). (A) and (B) Interaction of fac-[Re(CO)3(phen)(py)]+ with the site near tryptophan 134 and\ntryptophan 213. (C) and (D) Interaction of fac-[Re(CO)3(dpq)(py)]+ with the site near tryptophan 134 and tryptophan 213. (E) and (F) Interaction of fac-[Re\n(CO)3(dppz)(py)]+ with the site near tryptophan 134 and tryptophan 213.\n\n\n Our findings align with those of a previous study [44]. In their in the ability to bind to the same amino acid residues in subdomain IIA.\nsilico analysis, they investigated the interaction between four rhenium\ncomplexes, specifically of the fac-[Re(CO)3(\u03b1-diimine){4-C11py}]\n 3.8. In silico molecular interactions of fac-[Re(CO)3(phen)(py)]+, fac-\nCF3SO3 type, with BSA. Their results revealed the presence of binding\n [Re(CO)3(dpq)(py)]+ and fac-[Re(CO)3(dppz)(py)]+ with DNA\nsites within the hydrophobic cavities in subdomains IIA and IIIA, cor\u00ad\nresponding to sites I and II, respectively [44]. Furthermore, the identi\u00ad\n Molecular docking revealed that all three complexes interacted with\nfication of two amino acid residues, Trp-134 and Trp213, within BSA,\n the DNA macromolecule, displaying low binding energies indicative of\nboth situated in subdomain IIA, demonstrated their involvement in the\n favorable binding. As shown in Table 7, an increase in the hydropho\u00ad\ninteraction between the complex and the protein [44]. Our research\n bicity of the Re complexes correlated with a decrease in the binding\ncorroborates these findings, providing additional evidence for the\n energy, signifying a more favorable binding affinity. This corresponds to\nobserved interactions between the Re complex and protein.\n our previously experimental results of Kb corresponding to interaction\n It is important to note the existence of a hydrophobic cavity of\n with DNA, reinforcing the observation that a Re complex with increased\nconsiderable size in subdomain IIA, which can bind to various drugs.\n hydrophobicity correlates with a higher Kb value (Table 5).\nInterestingly, the Re complexes investigated in this study demonstrated\n The three Re complexes studied showed an affinity for the minor\n\n 9\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\nTable 7 groove in their most stable conformation. The fac-[Re(CO)3(phen)(py)]+\nEnergy associated with the interactions between rhenium complexes and the complex engaged with double-stranded DNA via adenine 8 from chain A\nDNA molecule and number of hydrogen bonds involved in mediating the and guanine 14 from chain B, forming hydrogen bonds (represented as\ninteractions. green spheres in Fig. 7A).\n Rhenium(I) complex Binding energy (Kcal/mol) Hydrogen bonds The fac-[Re(CO)3(dpq)(py)]+ complex interacted with a single\n fac-[Re(CO)3(phen)(py)]\n +\n \u2212 6,78 2 strand of DNA by forming hydrogen bonds with adenine 8 from chain A,\n fac-[Re(CO)3(dpq)(py)]+ \u2212 6,97 1 as shown by the green spheres in Fig. 7B. In addition, fac-[Re(CO)3(dpq)\n fac-[Re(CO)3(dppz)(py)]+ \u2212 7,83 1 (py)]+ demonstrated the ability to interact with DNA through interca\u00ad\n lation in a less stable position.\n Similarly, the fac-[Re(CO)3(dppz)(py)]+ complex exclusively\n\n\n\n\nFig. 7. Interaction between (A) fac-[Re(CO)3(phen)(py)]+, (B) fac-[Re(CO)3(dpq)(py)]+, and (C) fac-[Re(CO)3(dppz)(py)]+ and DNA and corresponding magnifi\u00ad\ncation results.\n\n 10\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\nengaged with a single strand of DNA, establishing hydrogen bonds with non-oxidative conditions, an increase in the percentage of single-strand\nguanine 4 from chain A, as depicted by the green spheres in the breaks (Form II with 83.47%), a decrease in the percentage of intact\nmagnification shown in Fig. 7C. Similar to the previous complexes, it supercoiled form (Form I with 5.68%), and the appearance of double-\nalso exhibited other intermolecular interactions, such as van der Waals strand breaks (Form III with 10.86%) were observed.\nforces and induced dipole interactions. When plasmids maintained under non-oxidative conditions were\n Varma et al. synthesized rhenium complexes with heterocyclic mo\u00ad treated with DMSO, a scavenger or neutralizer of ROS (columns 5 and 6\nlecular ligands and heteronuclear rhenium(I) complexes [57,58]. The in the gel), a decrease in the percentage of DNA in Form II (60.12%) and\ninteractions of these complexes with DNA were investigated via various an increase in Form I (23.24%) were observed. Under pro-oxidative\nspectroanalytical and electrophoretic techniques [57,58]. The results conditions represented by the introduction of hydrogen peroxide into\nsuggested that all the examined complexes bonded with the grooves of the system, extensive plasmid degradation was observed, resulting in a\nDNA and induced strand breaks [57,58]. This corroborates the molec\u00ad smeared DNA pattern under all conditions, rendering observation of\nular docking data of the three rhenium complexes reported in this study, specific bands (columns 8, 9, 10, and 11) impossible.\nindicating their affinity for the minor groove of DNA. Ghosh et al. noted The fac-[Re(CO)3(dpq)(py)]+ complex also induced structural al\u00ad\nin their molecular docking studies that rhenium(II) dinitrosyl and terations in the plasmids (Fig. 8B). Untreated plasmid DNA (column 2),\nmononitrosyl complexes can bind to DNA, displaying a preference for when subjected to agarose gel electrophoresis, primarily depicted intact\norientation toward the minor groove of this biomolecule [59]. supercoiled form (form I at 84.69%), circular form with single-strand\n breaks (form II at 12.27%), and a linear form resulting from double-\n strand breaks with a small percentage (form III). When treated with\n3.9. DNA cleavage studies\n fac-[Re(CO)3(dpq)(py)]+, the band profile of the gel was altered, indi\u00ad\n cating the induction of single-strand breaks (columns 3, 4, 5, and 6 on\n The interaction between rhenium complexes and DNA is crucial for\n the gel).\nidentifying target structures of antitumor agents that rely on DNA\n In samples treated under non-oxidizing conditions, an increase in the\ncleavage. Despite the strength of binding in cases involving motifs such\n percentage of single-strand breaks (form II) and a sharp decrease in the\nas dppz, DNA cleavage abilities are also influenced by other substituents\n percentage of the intact supercoiled form (form I) were observed. Under\npresent in the complexes [54]. As both UV\u2013Vis spectroscopy results and\n pro-oxidizing conditions, represented by the introduction of hydrogen\nin silico analyses confirmed the DNA interaction capacity, we investi\u00ad\n peroxide into the system (as with the previous complex), intense plasmid\ngated the potential of these metal complexes to induce DNA breaks using\n degradation was observed, forming a smear of DNA under all conditions.\nplasmids as a model.\n This rendered observation of specific bands impossible (columns 8, 9,\n 10, and 11).\n3.10. Interaction between fac-[Re(CO)3(phen)(py)]+, fac-[Re Similar to the two preceding complexes, fac-[Re(CO)3(dppz)(py)]+\n(CO)3(dpq)(py)]+, and fac-[Re(CO)3(dppz)(py)]+ and plasmid DNA induced structural alterations in the plasmids (Fig. 8C). The untreated\n plasmid DNA (lane 2 in the gel) predominantly exhibited a supercoiled\n The complex fac-[Re(CO)3(phen)(py)]+ was capable of inducing intact form (Form I, 62.6%), circular form with single-strand breaks\nstructural alterations in plasmids at different concentrations (Fig. 8A). (Form II, 30.9%), and a small percentage in the linear form resulting\nWhen subjected to agarose gel electrophoresis, the untreated plasmid from double-strand breaks (Form III). Upon treatment with fac-[Re\nDNA (column 2 in the gel) predominantly exhibited a supercoiled intact (CO)3(dppz)(py)]+, the band profile of the gel was altered, indicating\nform (Form I, 83.07%) and circular form with single-strand breaks the induction of single-strand breaks (lanes 3, 4, 5, and 6).\n(Form II, 16.93%), whereas a linear form resulting from double-strand In samples treated under non-oxidizing conditions, there was an in\u00ad\nbreaks was absent (Form III). Upon treatment with [Re(CO)3(phen) crease in the percentage of single-stranded breaks (Form II), a significant\n(py)]+, the band pattern on the gel was altered, indicating the induction decrease in the percentage of the intact supercoiled form (Form I), and a\nof both single- and double-strand breaks (columns 3 and 4 in the gel). slight increase in band density corresponding to double-stranded breaks\nWhen treated with a concentration of 80 \u03bcM (column 3 in the gel) under\n\n\n\n\nFig. 8. Agarose gel electrophoresis images of plasmid degradation and band patterns found after treatment with rhenium complexes. (A) fac-[Re(CO)3(phen)(py)]+,\n(B) fac-[Re(CO)3(dpq)(py)]+, and (C) fac-[Re(CO)3(dppz)(py)]+. 1: Marker; 2: Control without treatment; 3: Re complexes (40 or 10 \u03bcM); 4: Re complexes (80 or 20\n\u03bcM); 5: Re complexes (40 or 10 \u03bcM) + DMSO; 6: Re complexes (80 or 20 \u03bcM) + DMSO (0.05%); 7: Xba I digested; 8: Re complexes (40 or 10 \u03bcM) + H2O2 (15 mM); 9:\nRe complexes (80 or 20 \u03bcM) + H2O2 (15 mM); 10: Re complexes (40 or 10 \u03bcM) + H2O2 (15 mM) + DMSO (0.05%); 11: Re complexes (80 or 20 \u03bcM) + H2O2 (15 mM)\n+ DMSO (0.05%); 12: H2O2 (15 mM). Data are expressed as the mean \u00b1 SE of three assays. Statistical analysis by two-way ANOVA and multiple comparisons by\nBonferroni test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant compared to the untreated control.\n\n 11\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\n(Form III). The ability for these complexes to cleave plasmid DNA agent favors Fenton-type reactions, where oxidation of the metal com\u00ad\nwithout oxidants or not hydrolytically is unusual and highly unexpected, plex occurs, leading to the formation of more accessible and reactive\nas rhenium compounds are inert and are unlikely to act as Lewis acid oxygen species such as hydroxyl radicals (OH\u2022), which attack DNA\ncatalysts for this type of cleavage. molecules. The same mechanism is probably responsible for the breaks\n The molecular docking data of this work show that rhenium com\u00ad induced by fac-[Re(CO)3(phen)(py)]+, fac-[Re(CO)3(dpq)(py)]+, and\nplexes interact with DNA base. The study performed by Knopf et al. fac-[Re(CO)3(dppz)(py)]+. In the presence of DMSO, the intensity of\n(2017) [6] reveals that rhenium complexes can bind covalently to bands resulting from breaks decreased in some treatments, supporting\nguanine DNA or RNA nucleobase. The exact mechanism of interaction this hypothesis. This agent acts as a free radical scavenger by neutral\u00ad\nwith DNA has not been fully elucidated; however, the study performed izing the reactive species generated.\nby Konovalov et al. in 2020 [60] suggests that ligands composed of The present study sheds light on the complex interaction mecha\u00ad\nbenzene rings serve as a driving force for the interaction of rhenium nisms between rhenium complexes and DNA, revealing intriguing in\u00ad\ncomplexes with DNA nucleotide bases by inducing a large number of sights into their potential biological applications. Future studies should\nhydrophobic interactions, rich in \u03c0 bonding, including \u03c0-\u03c0 stacking, focus on elucidating the precise mechanisms of interaction, exploring\nwhich enhances DNA intercalation. This is corroborated by the pre\u00ad the effects of different ligands on DNA binding affinity, and investigating\nsented result of molecular docking, which shows that the more benzene the potential therapeutic applications of Rhenium complexes in various\nrings present, the higher the affinity to the DNA molecule, as demon\u00ad disease contexts. By continuing to deepen our understanding of these\nstrated by the studied complexes. Another study conducted by Varna interactions, we can unlock new opportunities for the development of\net al. in 2020 [57] demonstrates that Rhenium I complexes also have the novel treatments and therapies. Ultimately, collaborative efforts across\nability to interact and cleave S. pombe genomic DNA, depending on the multidisciplinary research teams will be instrumental in advancing our\nnature of their ligands. This interaction mechanism requires further knowledge and harnessing the full potential of rhenium complexes in\nstudies, as rhenium compounds are inert and do not exhibit this type of biomedicine.\ncatalytic activity in non-oxidizing conditions.\n Similar to the two previous complexes, under pro-oxidizing condi\u00ad\ntions, represented by the introduction of hydrogen peroxide into the 3.11. Cytotoxicity of rhenium(I) tricarbonyl complexes\nsystem, intense plasmid degradation was observed, forming a DNA\nsmear under all conditions (lanes 8, 9, 10, and 11 in the gel). When we Given the reported anticancer activity of rhenium(I) tricarbonyl\nobserved the band pattern induced by fac-[Re(CO)3(dppz)(py)]+ treat\u00ad complexes [1,2,62] and considering that the inherent cytotoxicity of the\nment under this pro-oxidizing condition, notwithstanding the pro\u00ad complexes is contingent on the chemical nature of the molecules ligands\nnounced degradation of DNA, discernible intact bands persisted, which [63], we evaluated the in vitro cytotoxicity of fac-[Re(CO)3(phen)(py)]+,\ncould be attributed to the lower concentration employed during plasmid fac-[Re(CO)3(dpq)(py)]+, and fac- [Re(CO)3(dppz)(py)]+ in HeLa,\ntreatment. These concentrations were intricately tied to the IC50 values PNT2, B16F10, and NIH-3 T3 cells by the MTT assay. The half-maximal\nspecified for each metal complex, with fac-[Re(CO)3(dppz)(py)]+ inhibitory concentration (IC50) of each compound was determined\nshowing a notably lower IC50 concentration than the other complexes. (Table 8) from a dose-response curve of cell viability versus log(con\u00ad\n A consistent pattern was observed in all gel electrophoresis experi\u00ad centration) (Fig. S10 in the supplemental material).\nments. Column 7 consistently served as a positive control for double- IC50 data indicates that the cytotoxic activity of the examined Re(I)\nstrand breaks, indicating that the plasmid DNA underwent treatment complexes increased with the expansion of the \u03c0-system of the ligands in\nwith the single-site restriction enzyme XbaI. Column 1 consistently all cell lines: ICRe(dppz)\n 50 < ICRe(dpq)\n 50 < ICRe(phen)\n 50 (Table 8). This explains\nrepresented the molecular weight markers, providing a reference for the how the chemical structure of the ligands influenced the overall cyto\u00ad\nsizing of DNA fragments. Finally, column 12 consistently represented toxicity of the studied rhenium complexes, as shown in other studies\nthe plasmid that remained untreated with the complex but was exposed [4,11,63,64].\nto pro-oxidizing conditions. This uniformity in the experimental setup The fac-[Re(CO)3(phen)(py)]+ complex showed an IC50 of 32.57 \u03bcM\nand control columns across all gels ensured a standardized comparison against HeLa cells. However, its IC50 exceeded the highest tested dose of\nof the results and facilitated an accurate interpretation of the observed 100 \u03bcM for all the other cell lines tested, rendering it impractical to\noutcomes in our study. calculate its SI. The fac-[Re(CO)3(dpq)(py)]+ complex exhibited an IC50\n All rhenium complexes evaluated in this study exhibited the capacity of 26.67 \u03bcM for the HeLa strain, with an SI of 1.57, whereas for the\nto induce the cleavage of plasmid DNA in oxidative conditions, thereby B16F10 strain, the IC50 was 43.61 \u03bcM with an SI of 1.44 (Table 8). The\nsubstantiating the observations made by previous researchers. Utilizing fac-[Re(CO)3(dppz)(py)]+ complex showed an IC50 of 8.48 \u03bcM for HeLa\nagarose gel electrophoresis analyses, Varma et al. demonstrated the with a SI of 1.91, whereas for the B16F10 strain, the IC50 was 11.51 \u03bcM,\nability of heteronuclear rhenium(I) complexes to induce DNA cleavage and the SI was 1.62 (Table 8).\nin Saccharomyces cerevisiae [58].\n Zeleniuk et al. exhibited nucleolytic activity in pUC18 plasmids Table 8\ntreated with dirhenium(III) complexes [61]. Similar to the complexes IC50 of rhenium complexes in different cell lines and their respective selectivity\n indices (SI). Values correspond to the average \u00b1 standard error of two separate\nassessed in this study, increased activity was observed in the presence of\n assays (n = 6).\nhydrogen peroxide, concluding that DNA is a potential target in living\ncells. The authors indicated that the mechanism underlying DNA Rhenium(I) IC50 (\u03bcM) SI IC50 (\u03bcM) SI\n complex\ncleavage in dirhenium(III) complexes is multifaceted, emphasizing the HeLa PNT2 B16F10 NIH/3\nimportance of considering the effects of electron donation or withdrawal T3\nfrom ligands, as well as the influence of the catalytic activity of the metal fac-[Re 32.57 >100 ND* >100 >100 ND*\ncore. These results align with our findings showing that the effect of the (CO)3(phen) \u00b1 0.06\nrhenium complexes varied, likely because of the different ligands pre\u00ad (py)]+\n fac- [Re 26.67 41.77 1.57 43.61 \u00b1 62.66 1.44\nsent in each complex.\n (CO)3(dpq) \u00b1 0.05 \u00b1 4.5 3.26 \u00b1 2.3\n Ghosh et al. assessed the effects of rhenium(II) dinitrosyl and mon\u00ad (py)]+\nonitrosyl complexes on the SC plasmid (pUC19) [59]. Similar to the fac- [Re 8.48 \u00b1 16.23 1.91 11.51 \u00b1 18.63 1.62\nfindings of this study, they observed effective nucleolytic activity in the (CO)3(dppz) 0.03 \u00b1 0.97 0.61 \u00b1 0.86\npresence of H2O2, cleaving DNA into a circular form with single-strand (py)]+\n\nbreaks. According to the authors, the presence of H2O2 as an oxidizing *\n ND = Not Defined.\n\n 12\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\n The fac-[Re(CO)3(dppz)(py)]+ complex showed significantly higher Fig. 9B.\ncytotoxicity toward all cell lines than the other two Re(I) complexes In the analyses of the fac-[Re(CO)3(dppz)(py)]+ complex, an increase\n(Table 8). Lower IC50 value of fac-[Re(CO)3(dppz)(py)]+ (IC50 = 8.48 in the number of cells in the G0/G1 phase was observed from 49.4% in\n\u03bcM, Table 8) with respect to the HeLa cell line was comparable to that of the control group to 57.5% in cells subjected to treatment with a con\u00ad\na similar compound reported in literature, which also comprised the centration of 10 \u03bcM (Fig. 9C). Additionally, under experimental condi\u00ad\ndppz ligand: fac-[Re(CO)3(dppz)(nHO)]+ (IC50 = 10 \u03bcM) [63]. Notably, tions where concentrations were fixed at 5 \u03bcM and 20 \u03bcM, the\nthe IC50 value of cisplatin was approximately 25 \u03bcM against the same percentages were calculated at 53.5% and 46.3%, respectively.\ncell line [4,65]. Furthermore, it is notable that the compound exhibited a consistent\n Pete et al [66] reported the IC50 values of rhenium complexes against trend of reduction in cells in the S phase of the cell cycle, coupled with a\nHeLa described by various authors, which ranged from 36 to 371 \u03bcM. gradual increase in the proportion of cells in the G2 phase with the in\u00ad\nThese data corroborate the range of IC50 values found in the present crease in the concentration of the complex; approximately 40% of cells\nstudy, where the three analyzed Re complexes showed lower IC50 values were found in the G2 phase upon treatment with 20 \u03bcM. This dose-\nthan those described for the other complexes, demonstrating greater dependent relationship highlights the direct influence of the com\u00ad\ncytotoxicity compared to the complexes described in the literature [66]. pound on the cell cycle stages. Consistent with the results observed for\n The SI is crucial for understanding the selectivity of compounds to\u00ad previous compounds, fac-[Re(CO)3(dppz)(py)]+ also stands out for its\nward tumor cells versus normal cells [67]. The SI values obtained for all ability to inhibit cell proliferation. This inhibitory effect was supported\nrhenium complexes were < 2, (the SI for HeLa cells was 1.91), indicating by the observation of cell trapping in the G0/G1 and G2 phases, as\nthat targeted delivery or bioisosterism strategies should be used to depicted in Fig. 9C.\nimprove the selectivity of the complex. According to the classification In the context of the intrinsic cellular division process, checkpoint\ndescribed by Bauer et al., the Re complexes examined in this study can verification mechanisms, when activated in response to DNA damage,\nbe considered moderately toxic to the HeLa strain, since all IC50 values impede half of the progression of the cell cycle. This translates into the\nwere lower than 50 \u03bcM [68]. trapping of cells in specific phases, such as G0/G1, as demonstrated by\n A control assay was performed to evaluate the toxicity of the isolated Machado et al. in 2021 with a copper complex, or even in the G2 phase\nligand and compare it with its corresponding complex. Results indicated [69], as evidenced by Simpson et al. in 2017 while studying tricarbonyl\nthat the cytotoxicity of the fac-[Re(CO)3(dppz)(py)]+ complex was rhenium complexes [70]. These observations are consistent with the\nsignificantly greater than that of its dppz ligand (Fig. S11 in the sup\u00ad results of the present study. When the cell cycle is blocked during these\nplemental material), confirming the cytotoxicity of the fac-[Re phases, two possible pathways emerge. The first pathway involves the\n(CO)3(dppz)(py)]+ complex, rather than solely the dppz ligand. There\u00ad repair of cellular damage, whereas the second can trigger the process of\nfore, fac-[Re(CO)3(dppz)(py)]+ may represent a promising an antitumor programmed cell death, known as apoptosis, a phenomenon observed in\ndrug candidate. cells treated with the three complexes used in this study.\n\n3.12. Anti-proliferative activity in HeLa: effects on the cell cycle 4. Conclusions\n\n To assess the impact of fac-[Re(CO)3(phen)(py)]+, fac-[Re In vitro and in silico interaction studies of rhenium complexes with\n(CO)3(dpq)(py)]+, and fac-[Re(CO)3(dppz)(py)]+) on cell proliferation, biomolecules (proteins, DNA, and lipids) revealed that greater hydro\u00ad\nthe HeLa cell line was treated with the complexes for 24 h, and the phobicity of the ligands in the complexes can favor interactions with\ndistribution of cells in each phase of the cell cycle was evaluated by flow biomolecules and consequently increase cytotoxicity in cells. The Re(I)\ncytometry after staining the cell nuclei with propidium iodide. An in\u00ad complex containing the dppz ligand exhibited the highest cytotoxicity\ncrease in the number of cells in the G0/G1 and/or G2 phases was among the studied compounds, probably because of its lipophilicity and\nobserved after 24 h of treatment with all the studied rhenium complexes. stronger biomolecular interactions. These interactions induced cell-\n Regarding the fac-[Re(CO)3(phen)(py)]+ complex, at a treatment cycle arrest, culminating in programmed cell death. Our findings\nconcentration of 20 \u03bcM, approximately 57.9% of cells were in the G0/G1 emphasize the importance of considering these characteristics in\nphase (Fig. 9A). This result was in contrast with the control group, which rational drug design.\nrecorded a proportion of 49.4%. Additionally, in groups treated with\nconcentrations of 10 \u03bcM and 40 \u03bcM, the percentages were approxi\u00ad Funding\nmately 55.2% and 59.5%, respectively. Simultaneously, the compound\nreduced the number of cells in the S phase of the cell cycle while This work was supported by Funda\u00e7a\u0303o de Amparo a\u0300 Pesquisa de\ndemonstrating a proportional increase in the number of cells in the G2 Minas Gerais/FAPEMIG (APQ-00704-21 and APQ-01087-21) and Con\u00ad\nphase. This phenomenon manifested in a dose-dependent manner, selho Nacional de Desenvolvimento Cient\u00edfico e Tecnolo\u0301gico/CNPq\nhighlighting the role of cell cycle regulation. The complex demonstrated (407282/2023\u20138). Tayana M. Tsubone expresses gratitude to the\na remarkable inhibitory effect on cell proliferation by trapping cells in L\u2019Ore\u0301al Brazil-UNESCO-ABC Award \u201cFor Women in Science\u201d 2023, in\nthe G0/G1 and G2 phases (Fig. 8A). chemistry field. This study was financed in part by the Coordena\u00e7a\u0303o de\n Concerning the complex fac-[Re(CO)3(dpq)(py)]+, a significant in\u00ad Aperfei\u00e7oamento de Pessoal de N\u00edvel Superior/CAPES - Finance Code\ncrease in the percentage of cells in the G0/G1 phase was observed, 001 and CAPES scholarship grant 88887.828352/2023\u201300.\nincreasing from 49.4% in the control group to approximately 74.8% in\nthe group subjected to treatment with 20 \u03bcM (Fig. 9B). Additionally, CRediT authorship contribution statement\nunder experimental conditions with concentrations of 10 \u03bcM and 40 \u03bcM,\nvalues of approximately 60.3% and 59.7% were recorded, respectively. Tayna\u0301 Saraiva de Lavor: Methodology, Investigation, Formal\nNotably, the compound consistently reduced the number of cells in the S analysis. Maria Henriqueta Silvestre Teixeira: Methodology, Investi\u00ad\nphase of the cell cycle, coupled with a gradual increase in the proportion gation, Formal analysis. Patr\u00edcia Alves de Matos: Investigation, Formal\nof cells in the G2 phase recorded with an increase in the concentration. analysis. Ricardo Campos Lino: Investigation, Formal analysis. Clara\nAs previously observed, this phenomenon followed a dose-dependent Maria Faria Silva: Investigation, Formal analysis. Marcos Eduardo\nrelationship, reinforcing the hypothesis of cell cycle modulation by Gomes do Carmo: Investigation, Formal analysis. Marcelo Em\u00edlio\nthis complex. Similar to the previously reported complex, fac-[Re Beletti: Resources. Antonio Otavio T. Patrocinio: Validation, Re\u00ad\n(CO)3(dpq)(py)]+ inhibited cell proliferation. This inhibition was man\u00ad sources, Formal analysis. Robson Jose\u0301 de Oliveira J\u00fanior: Writing \u2013\nifested by the trapping of cells in the G0/G1 and G2 phases, as shown in review & editing, Writing \u2013 original draft, Validation, Supervision,\n\n 13\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\n\n\nFig. 9. Representative histograms of the cell cycle phases of HeLa tumorigenic cells treated with different concentrations of rhenium complexes. (A) fac-[Re\n(CO)3(phen)(py)]+, (B) fac-[Re(CO)3(dpq)(py)]+ and (C) fac-[Re(CO)3(dppz)(py)]+. For the bar graph, the data represent the mean \u00b1 standard error. *p < 0.05. **p\n< 0.01, ***p < 0.001, and ****p < 0.0001 (compared to the negative control by one-way ANOVA followed by Bonferroni post-test).\n\n\n\n\n 14\n\fT.S. de Lavor et al. Journal of Inorganic Biochemistry 257 (2024) 112600\n\n\nResources, Formal analysis. Tayana Mazin Tsubone: Writing \u2013 review geometry on the properties of d6 polypyridinic transition metal complexes, Chem.\n Phys. 326 (2006) 54\u201370, https://doi.org/10.1016/j.chemphys.2006.01.040.\n& editing, Writing \u2013 original draft, Visualization, Validation, Supervi\u00ad\n [15] C.L. Ramos, F.S. Prado, M.E.G. Carmo, G. Farias, B. Souza, A.E.H. MacHado, A.O.\nsion, Resources, Project administration, Methodology, Funding acqui\u00ad T. Patrocinio, Temperature dependent emission properties of ReI tricarbonyl\nsition, Formal analysis, Conceptualization. complexes with dipyrido-quinoxaline and phenazine ligands, J. Braz. Chem. Soc.\n 33 (2022) 425\u2013436, https://doi.org/10.21577/0103-5053.20210161.\n [16] S.F. Sousa, R.N. Sampaio, N.M. Barbosa Neto, A.E.H. Machado, A.O.T. Patrocinio,\n The photophysics of fac-[re(CO)3(NN)(bpa)]+ complexes: a theoretical/\nDeclaration of competing interest\n experimental study, Photochem. Photobiol. Sci. 13 (2014) 1213\u20131224, https://doi.\n org/10.1039/c4pp00074a.\n The authors declare that they have no known competing financial [17] F.S. Prado, S.F. Sousa, A.E.H. Machado, A.O.T. Patrocinio, Influence of the\n protonatable site in the photo-induced proton-coupled electron transfer between\ninterests or personal relationships that could have appeared to influence\n rhenium (I) Polypyridyl complexes and hydroquinone, J. Braz. Chem. Soc. 28\nthe work reported in this paper. (2017) 1741\u20131751, https://doi.org/10.21577/0103-5053.20170022.\n [18] L.A. Faustino, A.E. Hora Machado, A.O.T. Patrocinio, Photochemistry of fac-[Re\nData availability (CO)3(dcbH2)(trans-stpy)]+: new insights on the isomerization mechanism of\n coordinated stilbene-like ligands, Inorg. Chem. 57 (2018) 2933\u20132941, https://doi.\n org/10.1021/acs.inorgchem.8b00093.\n Data will be made available on request. [19] A.O.T. Patrocinio, M.K. Brennaman, T.J. Meyer, N.Y. Murakami Iha, Excited-state\n dynamics in fac-[re(CO)3(Me4phen)(L)]+, J. Phys. Chem. A 114 (2010)\n 12129\u201312137, https://doi.org/10.1021/jp104692w.\nAcknowledgments [20] B.M. Rodrigues, H.F.V. Victo\u0301ria, G. Leite, K. Krambrock, O.A. Chaves, D.F. de\n Oliveira, R.Q. de Garcia, L. De Boni, L.A.S. Costa, B.A. Iglesias, Photophysical,\n photobiological, and biomolecule-binding properties of new tri-cationic meso-tri\n The authors acknowledge the National Institute of Science and\n (2-thienyl)corroles with Pt(II) and Pd(II) polypyridyl derivatives, J. 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