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Ruthenium-containing complexes have emerged as good alternative to the currently used platinum-containing drugs for malignant tumor therapy. In this work, cytotoxic effects of recently synthesized ruthenium polypyridyl complexes [Ru(bpy)₂(CFPIP)](ClO₄)₂ (bpy = 2,2'-bipyridine, CFPIP = (E)-2-(4-fluorostyryl)-1H-imidazo[4,5-f][1,10]phenanthroline, Ru(II)-1), [Ru(phen)₂(CFPIP)](ClO₄)₂ (phen = 1,10-phenanthroline, Ru(II)-2) and [Ru(dmb)₂(CFPIP)](ClO₄)₂ (dmb = 4,4'-dimethyl-2,2'-bipyridine, Ru(II)-3) toward different tumor cells were investigated in vitro and compared with cisplatin, the most widely used chemotherapeutic drug against hepatocellular carcinoma (HepG-2). The results demonstrate that target complexes show excellent cytotoxicity against HepG-2 cells with low IC₅₀ value of 21.4 ± 1.5, 18.0 ± 2.1 and 22.3 ± 1.7 μM, respectively. It was important noting that target Ru(II) complexes exhibited better antitumor activity than cisplatin (IC50 = 28.5 ± 2.4 μM) against HepG-2 cells, and has no obvious toxicity to normal cell LO2. DNA binding results suggest that Ru(II)-1, Ru(II)-2 and Ru(II)-3 interact with CT DNA (calf thymus DNA) through intercalative mode. Complexes exerted its antitumor activity through increasing anti-migration and inducing cell cycle arrest at the S phase. In addition, the apoptosis was tested by AO (acridine orange)/EB (ethidium bromide) staining and flow cytometry. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and colocalization tests were also evaluated by ImageXpress Micro XLS system. Overall, the results show that the ruthenium polypyridyl complexes induce apoptosis in HepG-2 cells through ROS-mediated mitochondria dysfunction pathway. Show less

Adenosine deaminases that act on RNA (ADARs) are present in all animals and function to both bind double-stranded RNA (dsRNA) and catalyze the deamination of adenosine (A) to inosine (I). As inosine is a biological mimic of guanosine, deamination by ADARs changes the genetic information in the RNA sequence and is commonly referred to as RNA editing. Millions of A-to-I editing events have been reported for metazoan transcriptomes, indicating that RNA editing is a widespread mechanism used to generate molecular and phenotypic diversity. Loss of ADARs results in lethality in mice and behavioral phenotypes in worm and fly model systems. Furthermore, alterations in RNA editing occur in over 35 human pathologies, including several neurological disorders, metabolic diseases, and cancers. In this review, a basic introduction to ADAR structure and target recognition will be provided before summarizing how ADARs affect the fate of cellular RNAs and how researchers are using this knowledge to engineer ADARs for personalized medicine. In addition, we will highlight the important roles of ADARs and RNA editing in innate immunity and cancer biology. Show less

The review summarizes the advances in medicinal chemistry of tetrazole biologically active compounds and drugs over the past 15 years. Most of the considered compounds are at present actively studied, are in various stages of clinical trials, or have recently been approved for use as pharmaceuticals. Show less

Hydrogen bonding principles are at the core of supramolecular design. This overview features a discussion relating molecular structure to hydrogen bond strengths, highlighting the following electronic effects on hydrogen bonding: electronegativity, steric effects, electrostatic effects, π-conjugation, and network cooperativity. Historical developments, along with experimental and computational efforts, leading up to the birth of the hydrogen bond concept, the discovery of nonclassical hydrogen bonds (C-H…O, O-H…π, dihydrogen bonding), and the proposal of hydrogen bond design principles (e.g., secondary electrostatic interactions, resonance-assisted hydrogen bonding, and aromaticity effects) are outlined. Applications of hydrogen bond design principles are presented. Show less

Aggregation-induced phosphorescence emission (AIPE) materials based on transition metal Ir(III) complexes have significant advantages in bioimaging and photodynamic therapy (PDT) due to the long lifetime, the reduced photobleaching and the good reactive oxygen species (ROS) generation. Herein, four cationic Ir(III) complexes (Ir1-Ir4) have been synthesized and studied. Tunable phosphorescence from green to red with the excellent properties of AIPE and long lifetimes can be achieved by varying the substituents. Moreover, these phosphorescence Ir(III) complexes exhibited dual-mode PDT potential (type I and type II). Complex Ir4 showed great prospect in bioimaging and PDT with the large Stokes shift (259 nm), the long lifetime (9.85 μs) and the high ROS yield (0.73). Confocal microscopy demonstrated that Ir4 accumulated in the mitochondria selectively and possessed remarkable photostability (reduced photobleaching up to 600 s). The results indicate that Ir4 may be used in dual-mode PDT guided by mitochondria-targeted imaging. This work provides an in-depth understanding of the relationship between structure and photophysical properties and facilitates the study in PDT applications. Show less

Among the brain tumors, glioma is the most common. In general, different biochemical mechanisms, involving nicotinic acetylcholine receptors (nAChRs) and the arachidonic acid cascade are involved in oncogenesis. Although the engagement of the latter in survival and proliferation of rat C6 glioma has been shown, there are practically no data about the presence and the role of nAChRs in C6 cells. In this work we studied the effects of nAChR antagonists, marine snail α-conotoxins and snake α-cobratoxin, on the survival and proliferation of C6 glioma cells. The effects of the lipoxygenase and cyclooxygenase inhibitors either alone or together with α-conotoxins and α-cobratoxin were studied in parallel. It was found that α-conotoxins and α-cobratoxin promoted the proliferation of C6 glioma cells, while nicotine had practically no effect at concentrations below 1 µL/mL. Nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor, and baicalein, a 12-lipoxygenase inhibitor, exerted antiproliferative and cytotoxic effects on C6 cells. nAChR inhibitors weaken this effect after 24 h cultivation but produced no effects at longer times. Quantitative real-time polymerase chain reaction showed that mRNA for α4, α7, β2 and β4 subunits of nAChR were expressed in C6 glioma cells. This is the first indication for involvement of nAChRs in mechanisms of glioma cell proliferation. Show less

Title: Phosphorescent rhenium(I) complexes conjugated with artesunate: Mitochondrial targeting and apoptosis-ferroptosis dual induction. Abstract: Cell death is essential for cancer, which can be induced through multiple mechanisms. Ferroptosis, a newly emerging form of non-apoptotic cell death, involves the generation of iron-dependent reactive oxygen species (ROS). In this study, we designed and synthesized two artesunate (ART) conjugated phosphorescent rhenium(I) complexes (Re(I)-ART conjugates), [Re(N^N)(CO)3(PyCH2OART)](PF6) (Re-ART-1 and Re-ART-2) (Py = pyridine, N^N = 1,10-phenanthroline (phen, in Re-ART-1) and 4,7-diphenyl-1,10-phenanthroline (DIP, in Re-ART-2)) that can specifically locate in the mitochondria of human cervical carcinoma (HeLa). Mechanism studies show that Re-ART-1 and Re-ART-2 exhibit high cytotoxicity against cancer cells lines and can induce both apoptosis and ferroptosis in HeLa cells through mitochondrial damage, caspase cascade, glutathione (GSH) depletion, glutathione peroxidase 4 (GPX4) inactivation and lipid peroxidation accumulation. As a result, this work presents the rational design of Re(I)-ART conjugates as a promising strategy to induce both apoptosis and ferroptosis and improve therapeutic efficiency of cancer treatment. Show less

Four bipyridine-type ligands variably derivatized with two bioactive groups (taken from ethacrynic acid, flurbiprofen, biotin, and benzylpenicillin) were prepared via sequential esterification steps from commercial 2,2'-bipyridine-4,4'-dicarboxylic acid and subsequently coordinated to ruthenium(II) p-cymene and iridium(III) pentamethylcyclopentadienyl scaffolds. The resulting complexes were isolated as nitrate salts in high yields and fully characterized by analytical and spectroscopic methods. NMR and MS studies in aqueous solution and in cell culture medium highlighted a substantial stability of ligand coordination and a slow release of the bioactive fragments in the latter case. The complexes were assessed for their antiproliferative activity on four cancer cell lines, showing cytotoxicity to the low micromolar level (equipotent with cisplatin). Additional biological experiments revealed a multimodal mechanism of action of the investigated compounds, involving DNA metalation and enzyme inhibition. Synergic effects provided by specific combinations of metal and bioactive fragments were identified, pointing toward an optimal ethacrynic acid/flurbiprofen combination for both Ru(II) and Ir(III) complexes. Show less

Mitochondrial damage will hinder the energy production of cells and produce excessive ROS (reactive oxygen species), resulting in cell death through autophagy or apoptosis. In this paper, four cyclometalated iridium(III) complexes (Ir1: [Ir(piq)₂L]PF₆; Ir2: [Ir(bzq)₂L]PF₆; Ir3: [Ir(dfppy)₂L]PF₆; Ir4: [Ir(thpy)₂L]PF₆; piq = 1-phenylisoquinoline; bzq = benzo[h]quinoline; dfppy = 2-(2,4-difluorophenyl)pyridine;thpy = 2-(2-thienyl)pyridine; L = 1,10-phenanthroline-5-amine) were synthesized and characterized. Cytotoxicity tests show that these complexes have excellent cytotoxicity to cancer cells, and mechanism studies indicatethat these complexes can specifically target mitochondria. Complexes Ir1 and Ir2 can damage the function of mitochondria, subsequently increasing intracellular levels of ROS, decreasing MMP (mitochondrial membrane potential), and interfering with ATP energy production, which leads to autophagy and apoptosis. Furthermore, autophagy induced by Ir1 and Ir2 can promote cell death in coordination with apoptosis. Surprisingly, these four complexes also showed moderate antibacterial activity to S. aureusand P. aeruginosa. Show less

A series of bis[3-ethyl-4-aryl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(i) complexes was synthesized and evaluated for the anti-cancer properties in sensitive and resistant ovarian carcinoma and leukemia cell lines. TLDR: The lack of effects against non-cancerous lung fibroblast SV-80 cells indicated a high selectivity towards tumor cells and thioredoxin reductase is not the main target of these complexes, because its inhibition pattern did not correlate with their biological activity. Show less

In mammalian cells, the bulky DNA adducts caused by ultraviolet radiation are mainly repaired via the nucleotide excision repair (NER) pathway; some defects in this pathway lead to a genetic disorder known as xeroderma pigmentosum (XP). Ribosomal protein S3 (rpS3), a constituent of the 40S ribosomal subunit, is a multi-functional protein with various extra-ribosomal functions, including a role in the cellular stress response and DNA repair-related activities. We report that rpS3 associates with transcription factor IIH (TFIIH) via an interaction with the xeroderma pigmentosum complementation group D (XPD) protein and complements its function in the NER pathway. For optimal repair of UV-induced duplex DNA lesions, the strong helicase activity of the TFIIH complex is required for unwinding damaged DNA around the lesion. Here, we show that XP-D cells overexpressing rpS3 showed markedly increased resistance to UV radiation through XPD and rpS3 interaction. Additionally, the knockdown of rpS3 caused reduced NER efficiency in HeLa cells and the overexpression of rpS3 partially restored helicase activity of the TFIIH complex of XP-D cells in vitro. We also present data suggesting that rpS3 is involved in post-excision processing in NER, assisting TFIIH in expediting the repair process by increasing its turnover rate when DNA is damaged. We propose that rpS3 is an accessory protein of the NER pathway and its recruitment to the repair machinery augments repair efficiency upon UV damage by enhancing XPD helicase function and increasing its turnover rate. Electronic supplementary material The online version of this article (10.1007/s00018-020-03754-x) contains supplementary material, which is available to authorized users. Show less

In this report, we synthesized three new iridium(III) complexes: [Ir(piq)₂(apip)]PF₆ (Ir1, piq = 1-phenylisoquinoline, apip = 2-aminophenyl-1H-imidazo[4,5-f][1,10]phenanthroline), [Ir(piq)₂(maip)]PF₆ (Ir2, maip = 3-aminophenyl-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(piq)₂(paip)]PF₆ (Ir3, paip = 4-aminophenyl-1H-imidazo[4,5-f][1,10]phenanthroline). The DNA binding was investigated. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to detect the cytotoxic activity of Ir1, Ir2 and Ir3, the complexes show highly active against B16 cells with IC₅₀ values of 0.3 ± 0.2 μM, 3.7 ± 0.2 μM and 4.6 ± 1.1 μM, respectively. Subsequently, cellular uptake suggested that the cytotoxicity of the complexes is attributed to their differences in cellular uptake levels. In addition, complexes Ir1, Ir2 and Ir3 induce cell cycle arrest at the G0/G1 phase and regulate the cell cycle mediators such as cyclin D1, CDK6 (cyclin-dependent kinase 6), CDK4 and p21, leading to the inhibition of B16 cells proliferation. The autophagy was investigated by monodansylcadaverine (MDC) staining. The complexes can promote the change from LC3-I to LC3-II, up-regulate levels of Beclin-1 and down-regulate expression of p62. The complexes induced apoptosis by regulating the expression levels of related indicators such as PARP (poly ADP-ribose polymerase), PI3K (phosphoinositide-3 kinase), AKT (protein kinase B), Caspase, Bcl-2 (B-cell lymphoma-2), Bad (Bcl2 associated death promoter), Bax (Bcl2-associated X) and Cyto C (cytochrome C). Additionally, Ir1 exerted significant antitumor activity in the suppression of malignant melanoma proliferation in vivo. As indicated in the above results, these complexes were highly effective for malignant melanoma treatment through the intrinsic pathway and provided much insight into anticancer drugs for tumor therapy. Show less

AbstractA library of eleven cationic gold(III) complexes of the general formula [(C C)Au(N N)]+ when C C is either biphenyl or 4,4’‐ditertbutyldiphenyl and N N is a bipyridine, phenanthroline or dipyridylamine derivative have been synthesized and characterized. Contrasting effects on the viability of the triple negative breast cancer cells MDA‐MB‐231 was observed from a preliminary screening. The antiproliferative activity of the seven most active complexes were further assayed on a larger panel of human cancer cells as well as on non‐cancerous cells for comparison. Two complexes stood out for being either highly active or highly selective. Eventually, reactivity studies with biologically meaningful amino acids, glutathione, higher order DNA structures and thioredoxin reductase (TrxR) revealed a markedly different behavior from that of the well‐known coordinatively isomeric [(C N C)Au(NHC)]+ structure. This makes the [(C C)Au(N N)]+ complexes a new class of organogold compounds with an original mode of action. TLDR: Reactivity studies with biologically meaningful amino acids, glutathione, higher order DNA structures and thioredoxin reductase (TrxR) revealed a markedly different behavior from that of the well-known coordinatively isomeric [(C^N^C)Au(NHC)] + structure, making the cationic gold(III) complexes a new class of organogold compounds with an original mode of action. Show less

The new ligand BBIP (BBIP = 2-(7-bromo-2H-benzo[d]imidazole-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) with its iridium(III) complexes: [Ir(ppy)₂(BBIP)](PF₆) (ppy = 2-phenylpyridine, Ir1), [Ir(bzq)₂(BBIP)](PF₆) (bzq = benzo[h]quinolone, Ir2) and [Ir(piq)₂(BBIP)](PF₆) (piq = 1-phenylisoquinoline, Ir3) were synthesized and characterized by elemental analysis, High Resolution Mass Spectrometer (HRMS), ¹H NMR and ¹³C{1H} NMR. The cytotoxicity of the complexes against A549, HepG2, SGC-7901, BEL-7402, HeLa and normal LO2 was evaluated through 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) method. The results show that Ir1 exhibits high cytotoxic activity against A549 cells with a low IC₅₀ value of 4.9 ± 0.5 μM. A series of biological activities such as cell cycle arrest, endoplasmic reticulum localization assay, apoptosis, western blotting, cellular uptake determination and in vivo antitumor activity were investigated. The assays implied that the complexes inhibit cancer cell migration through blocking mitotic progress. Cell cycle distribution stated that the complexes depress cell growth at G0/G1 phase. Additionally, the complexes acted on the endoplasmic reticulum and induce apoptosis through endoplasmic reticulum stress pathway. Especially, the western blotting showed that the complexes activated Bcl-2 (B-cell lymphoma-2) family and decreased PI3K (phosphoinositide-3 kinase) and AKT (protein kinase B), up-regulated the expression of mTOR (mammalian target of rapamycin) and p-mTOR (phosphorylated mammalian target of rapamycin). Therefore, the complexes induce apoptosis through activating PI3K-AKT-mTOR pathway. Antitumor in vivo demonstrated that Ir1 can effectively prevent the tumor growth with an inhibitory rate of 48.89%. Show less

AbstractThree [1,3‐diethyl‐4‐(p‐methoxyphenyl)‐5‐(3,4,5‐trimethoxyphenyl)imidazol‐2‐ylidene](L)gold(I) complexes, 4 a (L=Cl), 5 a (L=PPh3), and 6 a (L=same N‐heterocyclic carbene (NHC)), and their fluorescent [4‐(anthracen‐9‐yl)‐1,3‐diethyl‐5‐phenylimidazol‐2‐ylidene](L)gold(I) analogues, 4 b, 5 b, and 6 b, respectively, were studied for their localisation and effects in cancer cells. Despite their identical NHC ligands, the last three accumulated in different compartments of melanoma cells, namely, the nucleus (4 b), mitochondria (5 b), or lysosomes (6 b). Ligand L was also more decisive for the site of accumulation than the NHC ligand because the couples 4 a/4 b, 5 a/5 b, and 6 a/6 b, carrying different NHC ligands, afforded similar results in cytotoxicity tests, and tests on targets typically found at their sites of accumulation, such as DNA in nuclei, reactive oxygen species and thioredoxin reductase in mitochondria, and lysosomal membranes. Regardless of the site of accumulation, cancer cell apoptosis was eventually induced. The concept of guiding a bioactive complex fragment to a particular subcellular target by secondary ligand L could reduce unwanted side effects. TLDR: The concept of guiding a bioactive complex fragment to a particular subcellular target by secondary ligand L could reduce unwanted side effects. Show less

Werner's Complex, as a cationic coordination complex (CCC), has hitherto unappreciated biological properties derived from its binding affinity to highly anionic biomolecules such as glycosaminoglycans (GAGs) and nucleic acids. Competitive inhibitor and spectroscopic assays confirm the high affinity to GAGs heparin, heparan sulfate (HS), and its pentasaccharide mimetic Fondaparinux (FPX). Functional consequences of this affinity include inhibition of FPX cleavage by bacterial heparinase and mammalian heparanase enzymes with inhibition of cellular invasion and migration. Werner's Complex is a very efficient condensing agent for DNA and tRNA. In proof-of-principle for translational implications, it is demonstrated to display antiviral activity against human cytomegalovirus (HCMV) at micromolar concentrations with promising selectivity. Exploitation of non-covalent hydrogen-bonding and electrostatic interactions has motivated the unprecedented discovery of these properties, opening new avenues of research for this iconic compound. Show less

Iridium(III) complexes have the potential to serve as novel therapeutic drugs for treating tumor. In this work, three new complexes [Ir(ppy)₂(cdppz)](PF₆) (1) (ppy = 2-phenylpyridine, cdppz = 11-chlorodipyrido[3,2-a,2',3'-c]phenazine), [Ir(bzq)₂(cdppz)](PF₆) (2) (bzq = benzo[h]quinolone) and [Ir(piq)₂(cdppz)](PF₆) (3) (piq = 1-phenylisoquinoline) were prepared as well as characterized. MTT (3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide) assay revealed that the complex 2 exerted potent cytotoxicity against to various cancer cells lines and particularly for SGC-7901 cells. Meanwhile, the complexes could suppress cell colonies formation and migration ability. Apoptosis assays of AO/EB staining as well as flow cytometry revealed that the synthesized complexes may cause apoptosis of SGC-7901 cells. Moreover, the decline of mitochondrial membrane potential (MMP), elevation of intracellular reactive oxygen species (ROS) levels and release of cytochrome c demonstrated the complexes could cause apoptosis mainly through the mitochondrial death pathway and arrest cell at G0/G1 phase. Additionally, the complexes have significant influence on the expression of proteins which is interrelated to cell apoptosis. In summary, our studies provided fundamental information regarding the further study of the possible anticancer mechanisms of iridium (III) complexes. Show less

Ruthenium complexes are developed as substitutes for platinum complexes to be used in the chemotherapy of hematological and gynecological malignancies, such as ovarian cancer. We synthesized and screened 14 ruthenium half-sandwich complexes with bidentate monosaccharide ligands in ovarian cancer cell models. Four complexes were cytostatic, but not cytotoxic on A2780 and ID8 cells. The IC₅₀ values were in the low micromolar range (the best being 0.87 µM) and were similar to or lower than those of the clinically available platinum complexes. The active complexes were cytostatic in cell models of glioblastoma, breast cancer, and pancreatic adenocarcinoma, while they were not cytostatic on non-transformed human skin fibroblasts. The bioactive ruthenium complexes showed cooperative binding to yet unidentified cellular target(s), and their activity was dependent on reactive oxygen species production. Large hydrophobic protective groups on the hydroxyl groups of the sugar moiety were needed for biological activity. The cytostatic activity of the ruthenium complexes was dependent on reactive species production. Rucaparib, a PARP inhibitor, potentiated the effects of ruthenium complexes. Show less

The studies of iridium (III) complexes as potent anticancer reagents have attracted great attention. Here, a new iridium (III) complex [Ir(bzq)₂(PYIP)](PF₆) (Ir1, bzq = benzo[h]quinoline, PYIP = 2-(pyren-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) was synthesized and its liposomes (Ir1Lipo) was prepared. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to detect the cytotoxic activity of Ir1 and Ir1Lipo on HepG2, SGC-7901, BEL-7402, HeLa, B16, A549 and normal NIH3T3 cells. The complex Ir1 displays no obvious inhibitory effect on the growth of BEL-7402 cells, while the Ir1Lipo shows significant cytotoxic activity on BEL-7402 cells (IC₅₀ = 2.6 ± 0.03 μM). In further studies, Ir1Lipo induced apoptosis by the mitochondrial pathways, such as increasing intracellular reactive oxygen species (ROS) content and intracellular Ca²⁺ level, decreasing the mitochondrial membrane potential (MMP). In addition, after incubation with Ir1Lipo, the colony formation of BEL-7402 cells was significantly inhibited. Moreover, flow cytometry was used to detect the impact of Ir1Lipo on cell cycle distribution, and western blot was used to detect the expression of caspases and Bcl-2 (B-cell lymphoma-2) family proteins. Furthermore, Ir1Lipo exhibited significant antitumor activity in vivo with an inhibitory rate of 65.8%. These results indicated that Ir1Lipo induces apoptosis in BEL-7402 cells through intrinsic mitochondrial pathway. Show less

Abstract It is known that Triton X-100 (TX) reversibly inhibits activity of cytochrome c oxidase (CcO). The mechanism of inhibition is analyzed in this work. The action of TX is not directed to the reaction of CcO with cytochrome c, does not cause transition of the enzyme to the “slow” form, and is not associated with monomerization of the enzyme complex. TX completely suppresses oxygen reduction by CcO, but inhibition is prevented and partially reversed by dodecyl-β–D-maltoside (DDM), a detergent used to maintain CcO in solution. A 1/1 stoichiometry competition is shown between DDM and TX for binding to CcO, with Ki = 0.3 mM and affinity of DDM for the enzyme of 1.2 mM. TX interaction with the oxidized enzyme induces spectral response with maximum at 421 nm and [TX]1/2 = 0.28 mM, presumably associated with heme a3. When CcO interacts with excess of H2O2 TX affects equilibrium of the oxygen intermediates of the catalytic center accelerating the FI-607 → FII-580 transition, inhibits generation of O2 by the enzyme, and, to a lesser extent, suppresses the catalase partial activity. The observed effects can be explained by inhibition of the conversion of the intermediate FII-580 to the free oxidized state during the catalytic cycle. TX suppresses intraprotein electron transfer between hemes a and a3 during enzyme turnover. Partial peroxidase activity of CcO remains relatively resistant to TX under conditions that block oxidase reaction effectively. These features indicate an impairment of the K proton channel conductivity. We suggest that TX interacts with CcO at the Bile Acid Binding Site (BABS) that is located on the subunit I at the K-channel mouth and contacts with amphipathic regulators of CcO [Buhrow et al. (2013) Biochemistry, 52, 6995-7006]. Apparently, TX mimics the physiological ligand of BABS, whereas the DDM molecule mimics an endogenous phospholipid bound at the edge of BABS that controls effective affinity for the ligand. Show less