Therapeutic Exploration of Novel Reactive-oxygen Species-Mediated Apoptotic Mechanism by Modulating Electron Transfer in Respiratory Complex III

Therapeutic Exploration of Novel Reactive-oxygen Species-Mediated Apoptotic Mechanism by Modulating Electron Transfer in Respiratory Complex III | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Therapeutic Exploration of Novel Reactive-oxygen Species-Mediated Apoptotic Mechanism by Modulating Electron Transfer in Respiratory Complex III Muhammad A. Hagras, Tomas Jager This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6357772/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Reactive oxygen species (ROS) are by-products of normal cellular aerobic metabolism, which play a crucial role in several cellular processes and contribute to the development and progression of multiple diseases such as cancer. ROS are generated naturally by various enzymes, including respiratory complexes such as respiratory complex III (a.k.a. bc 1 complex), which is a homodimer where each monomer encompasses four redox centers (Fe 2 S 2 , heme b L , heme b H, and heme c 1 ) and two native binding sites (Q o and Q i sites). In the current study, we explored a novel apoptotic mechanism by binding ET-modulating agents at the newly discovered NQ-site in the bc 1 complex, thereby controlling the fluctuation of the Phe90 residue and enhancing or reducing ET between heme b L and heme b H redox centers, which will ultimately reduce or increase the ROS production levels. We performed extensive virtual screening simulations of 1,489,806 ligands of the ZINC database against Q o , Q i , and NQ binding sites of the bc 1 complex and obtained 272 patented ligands that bind preferentially at the NQ-binding site compared to Q o and Q i binding sites. Afterward, we purchased the top 14 ligands to characterize their biological activities with respect to their ROS-regulatory and cytotoxic activities against a breast cancer MCF7 model cell line. Eventually, we discovered two lead ROS up-regulators and two lead ROS down-regulators. Additionally, we found a promising cytotoxic activity for the single treatment of the lead ROS up-regulator and even a more synergistic cytotoxic activity for the combined sequential treatment of ROS up-regulator and down-regulator. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. INTRODUCTION Reactive oxygen species (ROS) are by-products of normal cellular aerobic metabolism, which includes various compounds such as superoxide radical anion ( \(\:{\text{O}}^{{\bullet\:}-}\) ), hydroxyl radical ( \(\:{\text{O}\text{H}.}^{{\bullet\:}}\) ), and hydrogen peroxide (H 2 O 2 ). ROS play a crucial role in several cellular processes, including cellular signaling pathways and maintaining the immune system, and are implicated in various essential physiological functions such as cell cycle progression and proliferation 1 – 3 . Therefore, imbalances in ROS contribute to the development and progression of multiple diseases such as cancer 4 , musculoskeletal disorders such as rheumatoid arthritis and osteoarthritis 5 – 8 , and neurodegenerative diseases such as Alzheimer's and Parkinson's diseases 9 , 10 . ROS are generated naturally by multiple enzymes, including respiratory complexes such as respiratory complex III (a.k.a. bc 1 complex). Electrons that pass through the bc 1 complex are a primary source of mitochondrial ROS, specifically the superoxide radical ion ( \(\:{\text{O}}_{2}^{\text{⦁}-}\) ) that is produced during the oxidative phosphorylation process 11 – 13 . Disruptions in the bc 1 complex can result in ROS imbalances, and therefore, strategies for modulating the activity of the bc 1 complex could have therapeutic implications in an array of human diseases, including multiple forms of cancer, such as breast cancer. Interestingly, ROS was recently recognized as a " double-edged sword " and is found to be the underlying mechanism for most of the anticancer therapeutic methods through elevating the cellular levels of ROS above the apoptotic threshold level, thereby triggering apoptosis in cancer cells while leaving the normal cells at the under-the-threshold level of ROS 14 – 16 . The bc 1 complex is a homodimer where each monomer encompasses four redox centers (Fe 2 S 2 , heme b L , heme b H, and heme c 1 ) and two native binding sites (Q o and Q i sites). Electrons flow in the bc 1 complex in a series of protonmotive ET reactions known as Q-cycle, proposed by Mitchell 17 , 18 . Upon binding the ubiquinol (UQH 2 ) molecule at the Q o site, one electron of the bound UQH 2 molecule transfers to the [2Fe-2S] cluster of the Rieske domain, docked at the proximal docking site. Another electron transfers to heme b L , which subsequently passes it to heme b H , and finally to a bound ubiquinone (UQ) or semiquinone (SQ) molecule bound at the Q i -site 19 – 23 . Rieske domain undergoes a domain movement of ~ 22 Å to bind at the distal docking site, where [2Fe-2S] cluster passes its electron to heme c 1 , which in turn passes it to heme c of the water-soluble cytochrome c carrier 24 , 25 . The enzyme turnover takes two Q-cycles to collectively transport 4 protons to the membrane's positive side, uptake 2 protons from the negative side, reduce two cytochrome c molecules, oxidize two ubiquinol molecules, and reduce one ubiquinone molecule (Fig. 1 ) 19 , 22 , 23 . In a previous study, we calculated the atomistic details of the tunneling pathways and the corresponding ET rates between all redox pairs in the bc 1 complex. Interestingly, we discovered that the electron transfer between the heme b L and the heme b H redox centers is controlled by a key phenylalanine residue (Phe90) that primarily can assume two different conformations (a.k.a. ON/OFF conformations) 26 – 29 . We found that the Phe90 residue only exists in the ON conformation when the Q o -site is occupied. Additionally, we performed extensive MD simulations that confirmed our previous discoveries regarding the role of Phe90 residue as an ET switch or an ET gate, whose conformation influences the rate of the ET reaction between heme b L and heme b H redox pairs significantly. We also discovered a novel orphan binding site (NQ-site) in the bc 1 complex that has never been characterized before (Fig. 2 ) 28 , 30 . The NQ-binding site is deep enough to modulate the Phe90 conformation and thus modulate the ET between heme b L and heme b H redox centers. In the current study, we extend upon our previous discoveries to develop a novel apoptotic mechanism of regulating the cellular ROS levels by modulating the ET reaction between heme b L and heme b H redox centers in the bc 1 complex. The proposed ROS regulation mechanism is achieved by binding ET-modulating agents at the newly discovered NQ-site in the bc 1 complex, thereby controlling the fluctuation of the Phe90 residue and enhancing or reducing ET between heme b L and heme b H redox centers, ultimately reducing or increasing the ROS production levels. Therefore, this work provides a novel direct approach to regulate cellular ROS levels in a controlled fashion, thereby modulating cellular oxidative damage and triggering apoptosis in cancer cells while leaving the normal cells intact. In addition, our proposed mechanism is superior to the previously reported techniques 31 – 33 because it can be manipulated to upregulate or downregulate ROS levels. For example, our mechanism provides the necessary means to first " sensitize " the cancer cells by down-regulating oxidative ROS levels and thereby downregulate the antioxidant defense mechanism of cancer cells, and then subsequently elevate ROS levels above the apoptotic threshold level, thereby triggering apoptosis in cancer cells while leaving normal cells at under-the-threshold ROS levels 15 , 16 . To discover the desired ROS modulators, we performed extensive virtual screening simulations of 1,489,806 ligands of the ZINC database against Q o , Q i , and NQ binding sites of the bc 1 complex and obtained 272 patented ligands that bind preferentially at the NQ-binding site compared to Q o and Q i binding sites. Afterward, we purchased the top 14 ligands to characterize their biological activities with respect to their ROS-regulatory and cytotoxic activities against a breast cancer model cell line. Eventually, we found two lead ROS up-regulators and two lead ROS down-regulators. Additionally, we found a promising cytotoxic activity for the single treatment of the lead ROS up-regulator and even a more synergistic cytotoxic activity for the combined sequential treatment of ROS up-regulator and down-regulator. 2. MATERIALS AND METHODS 2.1 Molecular Dynamics Simulation The orientation of the bc 1 complex (PDB: 1NTZ) with respect to the membrane was computed using the Peripheral Proteins in Membranes (PPM) web server 34 . The oriented protein was inserted in the membrane using the CHARMM-GUI Membrane Builder 35 online tool using a membrane composition of Phosphatidylcholine (PC): Phosphatidylethanolamine (PE): Cardiolipin (CL) equal to 50:30:20 36 . The protein-membrane system was placed in a water box with 0.15M KCl neutralizing ions. The assembled system was energy minimized and then equilibrated using the GROMACS 37 program with CHARMM PARAM36 force field 38 , 39 under periodic boundary conditions. Afterward, molecular dynamics (MD) simulation was performed on the assembled system for 1000 ns. MD simulations for the bc 1 complex with pharmacophores bound at the NQ binding site were performed as mentioned above, starting with the bc 1 complex with the most stable docked conformation of the pharmacophores at the NQ binding site. 2.2 Virtual Screening We selected 1,489,806 compounds from the ZINC20 database 40 with a range of molecular weight of 250–550 Daltons and LogP of 0–5, satisfying the druglike properties based on Lipinski's Rule of 5. We performed the virtual screening simulations using the PyRx package 41 with the Autodock Vina 42 scoring method and at multiple stages: First, we performed virtual screening of 1,489,806 compounds against the NQ site where its residues were allowed to be flexible and using exhaustiveness equals 12 and number of modes equals 10. We then selected 10,038 compounds that bind at the NQ site more strongly than the native ligand UQH 2 . Next, we performed a virtual screening of the 10,038 compounds against the NQ site with all its residues fixed. We then selected the 4,751 compounds that bind strongly at the NQ site in the flexible mode by at least 5 kcal/mol compared to the fixed mode. Then, we performed a virtual screening of the 4,751 compounds against the Q o site with its residues being flexible. We then selected the 540 compounds that bind at the Q o higher than UQH 2 . Finally, we performed a virtual screening of the 540 compounds against the Q i site with its flexible residues, yielding 272 compounds that bind at the Q i site higher than UQH 2 . 2.3 Total Cellular ROS and Mitochondrial Superoxide Anion Measurement Assay The MCF7 breast cancer cell line (ATCC, HTB-22) was purchased from American Type Culture Collection (ATCC, Manassas, VA) and propagated in Eagle's minimum essential medium (EMEM, ATCC) supplemented with 10% FBS (Thermo Scientific), 1% L-glutamine and 1% penicillin/streptomycin (ATCC) in a humidified incubator at 37°C and 5% CO 2 . Cells were harvested and seeded into a 96-well black plate at a cell density of 25,000 cells/well and left overnight to adhere. Afterward, cells were treated with 25 µM of the 14 purchased ligands from MolPort (Riga, Latvia), positive control (i.e., doxorubicin), and negative control (i.e., DMSO) and left in the incubator for 6–8 hours. For the Q o and Q i inhibition experiments, we also incubated 25 µM of myxothiazol or antimycin A (Sigma-Aldrich), respectively. Negative controls for the inhibition experiments were composed of DMSO and either 25 µM of myxothiazol or antimycin A. Afterward, cells were washed and stained for 30 minutes at 37°C and 5% CO 2 in the dark using the CellRox Green (Invitrogen, USA) or the MitoSOX Red Assay Kit (Invitrogen, USA). Afterward, cells were washed, and total cellular ROS or mitochondrial superoxide anion levels were measured using the Synergy Biotek UV/Vis/fluorescence microplate reader with excitation/emission wavelengths of 490/525 nm or 396/610 nm, respectively. Nuclei were labeled using NucBlue Live ReadyProbes Reagent (Hoechst 33342). All experiments were performed in at least triplicate and repeated for at least three independent trials. 2.4 MTS Assay MCF7 cells were harvested and seeded at a cell density of 5,000 cells/well into a 96-well white plate and left overnight to adhere. Afterward, cells were treated with different concentrations (0, 5, 10, 25, 50, and 100 µM) of pharmacophores, positive control (i.e., tamoxifen and doxorubicin), and negative control (i.e., DMSO) for 48 hours. Then, cells were washed and assayed for cytotoxicity using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (MTS) (Promega, USA), which is a colorimetric assay containing colorless tetrazolium compound that is bio-reduced by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells into a colored formazan product that can be measured at wavelength 490 nm using the Synergy Biotek plate reader. Sequential treatment was performed by incubating the overnight attached MCF7 cells with 25 µM of MH-200102 for 24 hours. Then, the cells were washed once and incubated with different concentrations of MH-101015 for another 24 hours. Then, the cells were assayed for cytotoxicity, as mentioned above. 2.5 Statistical analysis All the data and results obtained by three independent experiments are expressed as the mean fold change compared to negative control ± SD. Comparisons between groups were determined using Student's t-test. A difference with P < 0.05 was considered to be significant. 3. RESULTS AND DISCUSSION We initially constructed a system composed of the bc 1 complex (PDB: 1NTZ) embedded in a membrane with a 50:30:20 composition of PC:PE:CL, surrounded by a water box and simulated for 1000 ns under periodic boundary conditions to obtain a more realistic structure of the bc 1 complex. Afterward, we performed virtual screening of 1,489,806 compounds from the ZINC20 database at multiple stages to ensure that the obtained set of compounds has the following properties as follows: 1- Screening of 1,489,806 compounds and the native ligands (UQ and UQH 2 ) against the NQ site and selecting 10,038 pharmacophores that bind more strongly at the NQ site compared to the native ligands and hence suffer no competition upon binding at the NQ site (Fig. 3 A). 2- screening of the selected 10,038 compounds against the NQ site in both flexible mode (i.e., the residues of the NQ-site are allowed to move) and fixed mode (i.e., the residues of the NQ-site are fixed in position) and selecting 4,751 compounds that bind strongly in flexible mode by at least 5 kcal/mol compared to the fixed mode and hence would potentially induce conformational changes at the NQ site that could eventually modulate the conformation of the key phenylalanine residue (Phe90) (Fig. 3 B). 3- screening of 4,751 compounds against the Q o site (Fig. 3 C) and Q i site (Fig. 3 D) to eventually obtain 272 compounds that bind preferentially at the NQ site compared to UQ and UQH 2 but less firmly at the Q o and the Q i sites 43 . We then picked the top 14 ligands out of the obtained 272 compounds that were commercially available through Molport (Fig. 4 ). We then screened the biological activities of those 14 ligands with respect to their cytotoxic and ROS-modulating activities. We performed a cytotoxicity assay for all the purchased 14 pharmacophores with tamoxifen and doxorubicin as the positive controls and DMSO as the negative control against the MCF7 cell line. We found that the MH-200102 compound has a cytotoxic activity with IC 50 equal to 14.57 µM, lower than IC 50 of tamoxifen (~ 22.07 µM) 44 yet higher than the IC 50 of doxorubicin (~ 1 µM) 45 (Fig. 5). Additionally, MH-04504 showed a modest cytotoxic activity compared to that of MH-200102. Other compounds showed either proliferative activity or no activity at all. More interestingly, we found that sequential treatment with MH-200102 MH-01015 showed higher cytotoxic activity than the single treatment with MH-200102 (Fig. 6 ). We found that the sequential treatment with MH-200102 and then MH-01015 lowered the IC 50 to 5 µM compared to 14.57 µM with a single treatment with MH-200102 (Fig. 6 ). Our biochemical assays for the total cellular ROS levels showed that the MH-200102 exhibits an almost twofold increase in the total ROS upregulating activity compared to the ROS level of the negative control (i.e., DMSO) against the MCF7 cells, while the MH-04504 shows a modest total ROS upregulating activity (Fig. 7 ). More interestingly, we found that MH-131013 and MH-1015 show total ROS down-regulating activity compared to the ROS level of the negative control (Fig. 7 ). Additionally, we measured the mitochondrial superoxide radical anion levels where we found that the two ROS up-regulator compounds (MH-200102 and MH-04504) and the two ROS down-regulator compounds (MH-131013 and MH-1015) show a similar regulating activity on the mitochondrial superoxide anion levels proportional to their regulatory activities for the total cellular ROS levels (Fig. 8 ). To verify if the discovered ROS-regulator compounds bind at the NQ site of the bc 1 complex, we additionally measured the mitochondrial superoxide anion levels for each one of the lead-hit ROS regulators with either myxothiazole (MYX) or antimycin. MYX is a specific inhibitor for the Q o site of the bc 1 complex and hence blocks its catalytic activity 46 . For the ROS up-regulators, our results show a decrease in the mitochondrial superoxide anion levels in the MCF7 cells treated with both ligand and MYX compared to ligand only (Fig. 9 ). Those results show that MYX blocks the ROS up-modulating activity of MH-200102 and MH-04504. For the ROS down-regulators, we found that treatment with both ligand and MYX restores the mitochondrial superoxide levels to that of the negative control (i.e., DMSO and MYX). Hence, those results also show that MYX blocks any ROS down-modulating activity of MH-131013 and MH-1015. Antimycin (ANT) is a specific inhibitor for the Q i site of the bc 1 complex and hence enhances the reverse electron transfer reaction in the bc 1 complex, increasing the mitochondrial superoxide anion levels 47 , 48 . We found that treatment with ROS up-regulator and ANT has a higher superoxide anion level than negative control treatment (i.e., DMSO and ANT) (Fig. 9 ). Those results show a synergistic activity between the ROS up-regulator and ANT in elevating the mitochondrial superoxide anion levels. On the other hand, treatment with both ROS down-regulator and ANT shows a comparable mitochondrial superoxide anion level to the negative control treatment, which emphasizes that ANT blocks any ROS down-regulating activity of the ligands MH-131013 and MH-1015. To understand the molecular activities of the ROS up-regulators and down-regulators, we simulated the molecular dynamics of the bc 1 complex where one monomer has UQH 2 , UQ, and either MH-200102 or MH01015 ligands docked at the Q o , Q i , and NQ sites, respectively while leaving the other monomer ligand-free. We measured the electron transfer (ET) through-space distance between the heme b L , Phe90, and heme b H for the ligand-free monomer and for both MH-200102 or MH01015 docked monomers (Fig. 10). Our results show that the ROS up-regulator MH-200102 increases ET through-space distance to ~ 6.5 Å in close agreement with the ligand-free monomer. On the other hand, MH01015 reduces ET through-space distance to ~ 5.5 Å, as observed previously 49 . Interestingly, we found that the MH-200102 interacts unfavorably with Tyr95 residue where their aromatic rings stack sideways and hence destabilizing Tyr95, leading to a series of aromatic-aromatic interactions that eventually orient Phe90 in unfavorable conformation for ET between heme b L and heme b H (i.e., OFF conformation as previously described 26 – 30 ) (Fig. 10, right inset). On the other hand, we found that the MH01015 interacts favorably with Tyr95 through face-to-face configuration 50 , 51 that was found to eventually induce favorable conformation of Phe90 (i.e., ON conformation as previously described 26 – 30 ) (Fig. 10, left inset). 4. CONCLUSION In the current study, we managed to experimentally support our proposed ROS regulation mechanism through the binding of an ET-modulating ligand at the newly discovered NQ-site in the bc 1 complex, thereby controlling the orientation of the Phe90 residue and enhancing or reducing ET between heme b L and heme b H redox centers, ultimately reducing or increasing the ROS production levels. Therefore, this work provides a novel direct approach to regulate mitochondrial ROS levels in a controlled fashion, thereby modulating mitochondrial oxidative damage and controlling the underlying inflammatory pathogenesis that drives several critical disease processes such as cancer. To our knowledge, the proposed mechanism is the first of a kind that relies entirely on regulating ROS levels in a direct ET-modulating mechanism rather than through antioxidant activity or competitively inhibiting native ligands from binding at their respective sites 52 – 56 . We previously discovered that the electron transfer reaction between heme b L and heme b H is controlled by the conformation of a key phenylalanine residue (Phe90) that exists in two conformations, ON and OFF, that either facilitates or hinders the ET reaction, respectively. Under normal physiological conditions (Fig. 11 A), ET occurs in the bc 1 complex via the Q-cycle, where the key residue Phe90 exists in the ON conformation, promoting ET between heme b L and heme b H . In the current study, we discovered two lead hit up-regulator ligands (MH-200102 and MH-04504) that were proven to bind at a different site of the bc 1 complex than the Q o or the Q i sites. Hence, we conclude that those lead hit ROS up-regulator ligands bind at the NQ site, forcing the Phe90 residue to exist in an OFF conformation, thus inhibiting the ET rate by 2–3 orders of magnitude 29 , 30 , leading to reverse ET reaction and the subsequent observed increased mitochondrial superoxide anion and total cellular ROS levels (Fig. 11 B). Also, we found that the lead-hit ROS up-regulator (MH-200102) has a higher cytotoxic activity against MCF7 cells with IC 50 of 14.57 µM than tamoxifen (IC 50 ~ 22.07 µM). We also discovered two lead-hit ROS down-regulator ligands (MH-131013 and MH-1015) that were proven to bind at the bc 1 complex at the NQ site, stabilizing the Phe90 residue's ON conformation (Fig. 11 C), hence enhancing the ET forward reaction between heme b L and heme b H redox centers and thus leading to the observed decreased mitochondrial superoxide and cellular total ROS production levels. We found that ROS down-regulator ligands (MH-131013 and MH-1015) have proliferative activity against MCF7 cells. We also found that sequential treatment with MH-200102 MH-01015 showed higher cytotoxic activity against MCF7 with IC 50 of 5 µM. It is worth noting that other reported studies work similarly by modulating ET reactions in complex I or complex III using free fatty acids or myxothiazol, which block the binding of native ligands to their respective binding sites, such as the Q o site 52 , 53 . By contrast, our proposed mechanism depends solely on non-competing with binding the native ligands UQH 2 and UQ at the Q o and the Q i sites of the bc 1 complex, respectively. Instead, our non-competitive mechanism depends on binding the native ligands at their respective sites, thereby modulating the forward ET reaction between the bound UQH 2 at the Q o site and the bound UQ at the Q i site and regulating cellular ROS levels. Hence, our proposed mechanism provides a non-competitive direct scheme for regulating mitochondrial ROS levels by controlling Phe90 conformation and modulating the ET reaction between heme b L and heme b H . Also, the lack of intervening enzymes and the proposed non-competitive inhibitory activity would lead to better control of the proposed mechanism to regulate the cellular ROS levels with a lower effective dose of the ET-modulating drug. Hence, our mechanism should have a lower probability of developing drug resistance and lead to fewer undesirable side effects. Additionally, our ET-modulating mechanism should mainly either downregulate or upregulate the cellular ROS levels; hence, it does not suffer from the drawback of mixed ROS down-regulation and up-regulation effects in different cell lines, as reported in other ROS-modulating studies 31 – 33 , 53 – 58 . Declarations Data availability All data generated or analyzed during this study are available within the article. Funding This work was supported by the Faculty Research Incentive Fund (FIRF) intermural grant sponsored by the University of Health Sciences and Pharmacy in St. Louis. CRediT authorship contribution statement Muhammad A. Hagras : Conceptualization, Methodology, Validation, Formal analysis, Investigation, Visualization, Writing – original draft, Data Curation, Supervision, Project administration, Resources, Funding acquisition. Tomas Jager : Methodology, Investigation, Data Curation, Writing – review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ACKNOWLEDGEMENT The authors would like to thank Prof. Jesika Faridi and Dr. Nahid Sultana at the University of the Pacific (Stockton, CA) for their initial support in establishing the cytotoxic protocol used in the current study. Funding This work was supported by the Faculty Research Incentive Fund (FIRF) intermural grant sponsored by the University of Health Sciences and Pharmacy in St. Louis. References Boonstra J, Post JA. Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene 2004; 337 : 1-13. Yang Y, Bazhin AV, Werner J, Karakhanova S. Reactive oxygen species in the immune system. International reviews of immunology 2013; 32 : 249-70. Zhang J, Wang X, Vikash V, Ye Q, Wu D, Liu Y, Dong W. ROS and ROS-mediated cellular signaling. Oxidative medicine and cellular longevity 2016; 2016 . Waris G, Ahsan H. Reactive oxygen species: role in the development of cancer and various chronic conditions. Journal of carcinogenesis 2006; 5 : 14. López-Armada MJ, Riveiro-Naveira RR, Vaamonde-García C, Valcárcel-Ares MN. Mitochondrial dysfunction and the inflammatory response. Mitochondrion 2013; 13 : 106-18. Bolduc JA, Collins JA, Loeser RF. Reactive oxygen species, aging and articular cartilage homeostasis. Free Radical Biol Med 2019; 132 : 73-82. Abbas M, Monireh M. The role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis. Iranian Journal of Allergy, Asthma and Immunology 2008: 195-202. McGarry T, Biniecka M, Veale DJ, Fearon U. Hypoxia, oxidative stress and inflammation. Free Radical Biol Med 2018; 125 : 15-24. Sorce S, Krause K-H. NOX enzymes in the central nervous system: from signaling to disease. Antioxidants & redox signaling 2009; 11 : 2481-504. Manoharan S, Guillemin GJ, Abiramasundari RS, Essa MM, Akbar M, Akbar MD. The role of reactive oxygen species in the pathogenesis of Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease: a mini review. Oxidative medicine and cellular longevity 2016; 2016 . Drose S, Brandt U. The mechanism of mitochondrial superoxide production by the cytochrome bc1 complex. J Biol Chem 2008; 283 : 21649-54. Lanciano P, Khalfaoui-Hassani B, Selamoglu N, Ghelli A, Rugolo M, Daldal F. Molecular mechanisms of superoxide production by complex III: a bacterial versus human mitochondrial comparative case study. Biochimica et Biophysica Acta (BBA)-Bioenergetics 2013; 1827 : 1332-9. Muller F. The nature and mechanism of superoxide production by the electron transport chain: its relevance to aging. Journal of the American Aging Association 2000; 23 : 227-53. Yang Y, Karakhanova S, Hartwig W, D'Haese JG, Philippov PP, Werner J, Bazhin AV. Mitochondria and mitochondrial ROS in cancer: novel targets for anticancer therapy. J Cell Physiol 2016; 231 : 2570-81. Watson J. Oxidants, antioxidants and the current incurability of metastatic cancers. Open biology 2013; 3 : 120144. Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nature reviews Drug discovery 2009; 8 : 579-91. Mitchell P. Coupling of Phosphorylation to Electron and Hydrogen Transfer by a Chemi-Osmotic type of Mechanism. Nature 1961; 191 : 144-8. Mitchell P. The protonmotive Q cycle: a general formulation. FEBS Lett 1975; 59 : 137-9. Mitchell P. Possible molecular mechanisms of the protonmotive function of cytochrome systems. J Theor Biol 1976; 62 : 327-67. Brandt U, Trumpower B. The Protonmotive Q Cycle in Mitochondria and Bacteria. Crit Rev Biochem Mol Biol 1994; 29 : 165-97. Crofts AR, Hong S, Ugulava N, Barquera B, Gennis R, Guergova-Kuras M, Berry EA. Pathways for proton release during ubihydroquinone oxidation by the bc(1) complex. Proc Natl Acad Sci USA 1999; 96 : 10021-6. Crofts AR, Shinkarev VP, Kolling DR, Hong S. The modified Q-cycle explains the apparent mismatch between the kinetics of reduction of cytochromes c1 and bH in the bc1 complex. J Biol Chem 2003; 278 : 36191-201. Osyczka A, Moser CC, Dutton PL. Fixing the Q cycle. Trends Biochem Sci 2005; 30 : 176-82. Crofts AR, Guergova-Kuras M, Huang L, Kuras R, Zhang Z, Berry EA. Mechanism of Ubiquinol Oxidation by the bc1 Complex: Role of the Iron Sulfur Protein and Its Mobility. Biochemistry 1999; 38 : 15791-806. Zhang Z, Huang L, Shulmeister VM, Chi Y-I, Kim KK, Hung L-W, Crofts AR, Berry EA, Kim S-H. Electron transfer by domain movement in cytochrome bc1. Nature 1998; 392 : 677-84. Hagras MA. Respiratory Complex III: A Bioengine with a Ligand-Triggered Electron-Tunneling Gating Mechanism. The Journal of Physical Chemistry B 2024. Hagras MA, Hayashi T, Stuchebrukhov AA. Quantum calculations of electron tunneling in respiratory complex III. The Journal of Physical Chemistry B 2015; 119 : 14637-51. Hagras M, Stuchebrukhov A. Inhibition of respiratory complex III by ligands that interact with a regulatory switch: United States Patent 11,058,645, 2021, 2021. Hagras MA, Stuchebrukhov AA. Internal switches modulating electron tunneling currents in respiratory complex III. Biochimica et Biophysica Acta (BBA)-Bioenergetics 2016; 1857 : 749-58. Hagras MA, Stuchebrukhov AA. Novel inhibitors for a novel binding site in respiratory complex III. The Journal of Physical Chemistry B 2016; 120 : 2701-8. Woo J-H, Kim Y-H, Choi Y-J, Kim D-G, Lee K-S, Bae JH, Min DS, Chang J-S, Jeong Y-J, Lee YH. Molecular mechanisms of curcumin-induced cytotoxicity: induction of apoptosis through generation of reactive oxygen species, down-regulation of Bcl-X L and IAP, the release of cytochrome c and inhibition of Akt. Carcinogenesis 2003; 24 : 1199-208. Uğuz AC, Öz A, Nazıroğlu M. Curcumin inhibits apoptosis by regulating intracellular calcium release, reactive oxygen species and mitochondrial depolarization levels in SH-SY5Y neuronal cells. Gong G, Qin Y Fau - Huang W, Huang W Fau - Zhou S, Zhou S Fau - Yang X, Yang X Fau - Li D, Li D. Rutin inhibits hydrogen peroxide-induced apoptosis through regulating reactive oxygen species mediated mitochondrial dysfunction pathway in human umbilical vein endothelial cells. Lomize MA, Pogozheva ID, Joo H, Mosberg HI, Lomize AL. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res 2012; 40 : D370-6. Jo S, Kim T, Iyer VG, Im W. CHARMM-GUI: A web-based graphical user interface for CHARMM. J Comput Chem 2008; 29 : 1859-65. Zinser E, Sperka-Gottlieb CD, Fasch EV, Kohlwein SD, Paltauf F, Daum G. Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae. J Bacteriol 1991; 173 : 2026-34. Lindahl E, Hess B, van der Spoel D. GROMACS 3.0: a package for molecular simulation and trajectory analysis. J Mol Model 2001; 7 : 306-17. Brooks BR, Brooks CL, 3rd, Mackerell AD, Jr., Nilsson L, Petrella RJ, Roux B, Won Y, Archontis G, Bartels C, Boresch S, Caflisch A, Caves L, et al. CHARMM: the biomolecular simulation program. J Comput Chem 2009; 30 : 1545-614. Best RB, Zhu X, Shim J, Lopes PE, Mittal J, Feig M, Mackerell AD, Jr. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone phi, psi and side-chain chi(1) and chi(2) dihedral angles. Journal of chemical theory and computation 2012; 8 : 3257-73. Irwin JJ, Tang KG, Young J, Dandarchuluun C, Wong BR, Khurelbaatar M, Moroz YS, Mayfield J, Sayle RA. ZINC20—A Free Ultralarge-Scale Chemical Database for Ligand Discovery. Journal of Chemical Information and Modeling 2020; 60 : 6065-73. Dallakyan S, Olson AJ. Small-molecule library screening by docking with PyRx. Methods in molecular biology (Clifton, NJ) 2015; 1263 : 243-50. Eberhardt J, Santos-Martins D, Tillack AF, Forli SJJoci, modeling. AutoDock Vina 1.2. 0: New docking methods, expanded force field, and python bindings 2021; 61 : 3891-8. Hagras MA. US Provisional Patent Application No. 63/638,833, filed on April 25, 2024. Seeger H, Huober J, Wallwiener D, Mueck AO. Inhibition of human breast cancer cell proliferation with estradiol metabolites is as effective as with tamoxifen. Hormone and metabolic research 2004; 36 : 277-80. Fang XJ, Jiang H, Zhu YQ, Zhang LY, Fan QH, Tian Y. Doxorubicin induces drug resistance and expression of the novel CD44st via NF-κB in human breast cancer MCF-7 cells. Oncol Rep 2014; 31 : 2735-42. Von Jagow G, Gribble GW, Trumpower BL. Mucidin and strobilurin A are identical and inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain at the same site as myxothiazol. Biochemistry 1986; 25 : 775-80. Quinlan CL, Gerencser AA, Treberg JR, Brand MD. The mechanism of superoxide production by the antimycin-inhibited mitochondrial Q-cycle. J Biol Chem 2011; 286 : 31361-72. Sun J, Trumpower BL. Superoxide anion generation by the cytochrome bc1 complex. Arch Biochem Biophys 2003; 419 : 198-206. Hagras MA. Respiratory Complex III: A Bioengine with a Ligand-Triggered Electron-Tunneling Gating Mechanism. The Journal of Physical Chemistry B 2024; 128 : 990-1000. Anjana R, Vaishnavi MK, Sherlin D, Kumar SP, Naveen K, Kanth PS, Sekar K. Aromatic-aromatic interactions in structures of proteins and protein-DNA complexes: a study based on orientation and distance. Bioinformation 2012; 8 : 1220. Burley SK, Petsko GA. Aromatic-Aromatic Interaction: A Mechanism of Protein Structure Stabilization. Science 1985; 229 : 23-8. Young TA, Cunningham CC, Bailey SM. Reactive oxygen species production by the mitochondrial respiratory chain in isolated rat hepatocytes and liver mitochondria: studies using myxothiazol. Arch Biochem Biophys 2002; 405 : 65-72. Schönfeld P, Wojtczak L. Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport. Biochimica et Biophysica Acta (BBA)-Bioenergetics 2007; 1767 : 1032-40. Kumar D, Lundgren Dw Fau - Moore RM, Moore Rm Fau - Silver RJ, Silver Rj Fau - Moore JJ, Moore JJ. Hydrogen peroxide induced apoptosis in amnion-derived WISH cells is not inhibited by vitamin C. Ham A, Kim B Fau - Koo U, Koo U Fau - Nam K-W, Nam Kw Fau - Lee S-J, Lee Sj Fau - Kim KH, Kim Kh Fau - Shin J, Shin J Fau - Mar W, Mar W. Spirafolide from bay leaf (Laurus nobilis) prevents dopamine-induced apoptosis by decreasing reactive oxygen species production in human neuroblastoma SH-SY5Y cells. Ahlemeyer B, Krieglstein J. Retinoic acid reduces staurosporine-induced apoptotic damage in chick embryonic neurons by suppressing reactive oxygen species production. Kang JS, Cho D Fau - Kim Y-I, Kim Yi Fau - Hahm E, Hahm E Fau - Yang Y, Yang Y Fau - Kim D, Kim D Fau - Hur D, Hur D Fau - Park H, Park H Fau - Bang S, Bang S Fau - Hwang YI, Hwang Yi Fau - Lee WJ, Lee WJ. L-ascorbic acid (vitamin C) induces the apoptosis of B16 murine melanoma cells via a caspase-8-independent pathway. Chang YF, Chi Cw Fau - Wang J-J, Wang JJ. Reactive oxygen species production is involved in quercetin-induced apoptosis in human hepatoma cells. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6357772","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":437678282,"identity":"33d2ce75-bfc7-4f52-9349-7c16d923691f","order_by":0,"name":"Muhammad A. Hagras","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYFACHjDJ2CAB4coxQBjMxGsxJl1LYgMhLbozcg9+upljJ9sg3fz4dUVNXfqG2+3PHjBUWCc24NBidiMvWTp3W7Jxg8wxM8szxw7nbrhzxtyA4Uw6Hi05BkAtzED3JJgZNrAdyN1wI4dNgrHtMD4txr9zt9UDtaR/M2z4V5ducCP9mQTjP7xazIC2ABVI5Bg/bGxjTjC4kWAmwdiAR8uZN2bWuduOG7dJ5JQxNvYdNpx5I8fcIOFYujFOLcdzjG/nbquW7ZdI3/yx4VudPB/QYQ8+1FjL4tICB2xAJAFnJxBSDgXMHxDaR8EoGAWjYBQgAAC821/lZjMSSAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Health Sciences and Pharmacy","correspondingAuthor":true,"prefix":"","firstName":"Muhammad","middleName":"A.","lastName":"Hagras","suffix":""},{"id":437678284,"identity":"86d7570d-f150-44b1-81fe-2b723b8a337e","order_by":1,"name":"Tomas Jager","email":"","orcid":"","institution":"University of Health Sciences and Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Tomas","middleName":"","lastName":"Jager","suffix":""}],"badges":[],"createdAt":"2025-04-02 05:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6357772/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6357772/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79842249,"identity":"ba298dfd-645c-4f5c-aaad-154c334cbc07","added_by":"auto","created_at":"2025-04-03 12:59:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":509786,"visible":true,"origin":"","legend":"\u003cp\u003eThe \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex showing all 4 redox centers in each monomer: Fe\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, heme \u003cem\u003ec\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e, heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e, and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e. Native ligands are shown: ubiquinol (UQH\u003csub\u003e2\u003c/sub\u003e) and ubiquinone (UQ). Distances between corresponding redox centers or ligands are displayed in Angstrom.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6357772/v1/dffaef8bb57d6773282e453f.png"},{"id":79842250,"identity":"865e9802-8e4f-4f13-ad86-13bc9f02969e","added_by":"auto","created_at":"2025-04-03 12:59:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":956138,"visible":true,"origin":"","legend":"\u003cp\u003eThe \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e homodimer complex (PDB 1NTZ) embedded in the membrane. Different binding sites are visualized, including the Q\u003csub\u003eo\u003c/sub\u003e, Q\u003csub\u003ei\u003c/sub\u003e, and NQ sites. Water molecules are displayed as red spheres. In the right inset, the NQ site entrance is displayed. In the left inset, the NQ site is shown as a solid surface with the lining Tyr95 residue displayed in red. Phe90 and Phe91 are also displayed as yellow and orange licorice residues, respectively.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6357772/v1/3bf2c6a877a1286a5eee528d.png"},{"id":79842772,"identity":"d2fb0c04-1885-4b22-933b-12a1f0571822","added_by":"auto","created_at":"2025-04-03 13:07:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":139451,"visible":true,"origin":"","legend":"\u003cp\u003eVirtual screening results of the ZINC database against the NQ, Q\u003csub\u003eo\u003c/sub\u003e, and Q\u003csub\u003ei\u003c/sub\u003e binding sites. (A) Results of screening 1,489,806 compounds and the native ligands (UQ and UQH\u003csub\u003e2\u003c/sub\u003e) against the NQ site. The dashed red line shows the binding energies of the native ligands. (B) Screening results against the NQ site in the fixed mode (upper plot) and flexible mode (middle plot). The difference in binding energies between fixed and flexible modes is shown in the lower plot. (C) Screening results against the Q\u003csub\u003eo\u003c/sub\u003e site. The dashed line indicates the binding energy of the native ligand. (D) Screening results against the Q\u003csub\u003ei\u003c/sub\u003e site. The dashed line indicates the binding energy of the native ligand.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6357772/v1/e5cbf87bea4ab5bf809eaff1.png"},{"id":79842252,"identity":"39dca466-5102-4ad1-95f1-9b7c0d91f0ca","added_by":"auto","created_at":"2025-04-03 12:59:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":231350,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structures of the purchased 14 ligands.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6357772/v1/bafdcde73b63680d75d8a83a.png"},{"id":79843784,"identity":"3a7baa27-d77f-483e-b00d-65139f13f6db","added_by":"auto","created_at":"2025-04-03 13:15:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":148958,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability results for the 14 screened ligands.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6357772/v1/6a75558a2c50c0dd7779c008.png"},{"id":79843785,"identity":"03f8a5c4-0e0a-419d-bd8b-83d6ad187e61","added_by":"auto","created_at":"2025-04-03 13:15:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":72237,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability results for the sequential treatment versus regular treatment with MH-200102 or positive controls.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6357772/v1/f53f624a3a7f42f74fd39b8e.png"},{"id":79842776,"identity":"b1afaa8c-8e59-4333-8070-c741db8f9198","added_by":"auto","created_at":"2025-04-03 13:07:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":287467,"visible":true,"origin":"","legend":"\u003cp\u003e(Top) A plot of the total ROS fold intensity of the three lead hit compounds against the negative control (i.e., DMSO). Data are shown as mean fold change ± SD. The results were evaluated using Student's t-test, and differences were considered significant at the \u003cem\u003e**P \u0026lt; 0.05\u003c/em\u003e or \u003cem\u003e***P \u0026lt; 0.005\u003c/em\u003e or ****\u003cem\u003eP\u003c/em\u003e \u003cem\u003e\u0026lt; 0.0001\u003c/em\u003e level. (Bottom) Representative fluorescent images where the green fluorescence indicates the cellular total ROS levels and the nuclei are labeled in blue fluorescence.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6357772/v1/2fa75041805286f079c3f060.png"},{"id":79842271,"identity":"3555fa28-1967-4572-956e-9cdc30a8f34a","added_by":"auto","created_at":"2025-04-03 12:59:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":310240,"visible":true,"origin":"","legend":"\u003cp\u003e(Top) A plot of the mitochondrial superoxide anion fold intensity of the lead hit compounds against the negative control (i.e., DMSO). Data are shown as mean fold change ± SD. The results were evaluated using Student's t-test, and differences were considered significant at the \u003cem\u003e*P \u0026lt; 0.05 or **P \u0026lt; 0.01\u003c/em\u003e or \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e or *\u003cem\u003e***P \u0026lt; 0.0001\u003c/em\u003e level. (Bottom) Representative fluorescent images where the red fluorescence indicates the mitochondrial superoxide anion levels and the nuclei are labeled in blue fluorescence.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6357772/v1/e7118494bd2a73593d72b70b.png"},{"id":79842266,"identity":"3a00f19b-9248-48d8-8a4d-8bc5beca5d32","added_by":"auto","created_at":"2025-04-03 12:59:27","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":327188,"visible":true,"origin":"","legend":"\u003cp\u003e(Top) A plot of the mitochondrial superoxide anion fold intensity of the four lead hit compounds against the negative control (i.e., DMSO, or DMSO, and either MYX or ANT). Data are shown as mean fold change ± SD. The results were evaluated using Student's t-test, and differences were considered significant at the \u003cem\u003e*P \u0026lt; 0.1 or **P \u0026lt; 0.01\u003c/em\u003e or \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e or *\u003cem\u003e***P \u0026lt; 0.0001\u003c/em\u003e level. (Bottom) Representative fluorescent images where the red fluorescence indicates the mitochondrial superoxide anion levels.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6357772/v1/ace8f1a63d2ae1f5aa742f4e.png"},{"id":79842777,"identity":"6b824945-3c1b-4841-a06e-4c59fd6a8a3c","added_by":"auto","created_at":"2025-04-03 13:07:27","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":406397,"visible":true,"origin":"","legend":"\u003cp\u003e(Upper plot) Electron transfer through-space distance between the heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e, Phe90, and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH \u003c/sub\u003efor the ligand-free monomer and for both MH-200102 or MH01015 docked monomers. Lower insets show the docked MH-200102 and MH-01015 conformation relative to Tyr95 (red) and the phenylamine dimer (Phe91-Phe90).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6357772/v1/96f53ade7eb246f363f8ea4f.png"},{"id":79842263,"identity":"8e19ed03-053d-4a33-a098-0721a997db6b","added_by":"auto","created_at":"2025-04-03 12:59:27","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":276346,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Normal ET state (the Q-cycle) where Phe90 exists in the ON conformation. (B) A malfunctioned ET state where Phe90 exists in the OFF conformation due to the bound ROS up-regulator (UP) at the NQ site, leading to increased ROS production. (C) Stabilized ET state where Phe90 exists in the ON conformation due to the bound ROS down-regulator drug (DOWN) at the NQ site. Red and blue arrows show the forward ET pathways for the two electrons of the UQH\u003csub\u003e2,\u003c/sub\u003e while orange arrows show the reverse ET pathway.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6357772/v1/c686a099aff50418d2e163c7.png"},{"id":81039022,"identity":"8e152b92-82b5-461e-982c-584671a14b7e","added_by":"auto","created_at":"2025-04-21 13:08:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4206075,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6357772/v1/ba113f83-a021-45d4-851a-972c2763305d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Therapeutic Exploration of Novel Reactive-oxygen Species-Mediated Apoptotic Mechanism by Modulating Electron Transfer in Respiratory Complex III","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eReactive oxygen species (ROS) are by-products of normal cellular aerobic metabolism, which includes various compounds such as superoxide radical anion (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{O}}^{{\\bullet\\:}-}\\)\u003c/span\u003e\u003c/span\u003e), hydroxyl radical (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{O}\\text{H}.}^{{\\bullet\\:}}\\)\u003c/span\u003e\u003c/span\u003e), and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). ROS play a crucial role in several cellular processes, including cellular signaling pathways and maintaining the immune system, and are implicated in various essential physiological functions such as cell cycle progression and proliferation\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, imbalances in ROS contribute to the development and progression of multiple diseases such as cancer\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, musculoskeletal disorders such as rheumatoid arthritis and osteoarthritis\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and neurodegenerative diseases such as Alzheimer's and Parkinson's diseases\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eROS are generated naturally by multiple enzymes, including respiratory complexes such as respiratory complex III (a.k.a. \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex). Electrons that pass through the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex are a primary source of mitochondrial ROS, specifically the superoxide radical ion (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{O}}_{2}^{\\text{⦁}-}\\)\u003c/span\u003e\u003c/span\u003e) that is produced during the oxidative phosphorylation process\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Disruptions in the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex can result in ROS imbalances, and therefore, strategies for modulating the activity of the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex could have therapeutic implications in an array of human diseases, including multiple forms of cancer, such as breast cancer. Interestingly, ROS was recently recognized as a \"\u003cem\u003edouble-edged sword\u003c/em\u003e\" and is found to be the underlying mechanism for most of the anticancer therapeutic methods through elevating the cellular levels of ROS above the apoptotic threshold level, thereby triggering apoptosis in cancer cells while leaving the normal cells at the under-the-threshold level of ROS \u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex is a homodimer where each monomer encompasses four redox centers (Fe\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e, heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH,\u003c/sub\u003e and heme \u003cem\u003ec\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e) and two native binding sites (Q\u003csub\u003eo\u003c/sub\u003e and Q\u003csub\u003ei\u003c/sub\u003e sites). Electrons flow in the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex in a series of protonmotive ET reactions known as Q-cycle, proposed by Mitchell\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Upon binding the ubiquinol (UQH\u003csub\u003e2\u003c/sub\u003e) molecule at the Q\u003csub\u003eo\u003c/sub\u003e site, one electron of the bound UQH\u003csub\u003e2\u003c/sub\u003e molecule transfers to the [2Fe-2S] cluster of the Rieske domain, docked at the proximal docking site. Another electron transfers to heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e, which subsequently passes it to heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e, and finally to a bound ubiquinone (UQ) or semiquinone (SQ) molecule bound at the Q\u003csub\u003ei\u003c/sub\u003e-site \u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Rieske domain undergoes a domain movement of ~\u0026thinsp;22 \u0026Aring; to bind at the distal docking site, where [2Fe-2S] cluster passes its electron to heme \u003cem\u003ec\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e, which in turn passes it to heme \u003cem\u003ec\u003c/em\u003e of the water-soluble cytochrome \u003cem\u003ec\u003c/em\u003e carrier \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The enzyme turnover takes two Q-cycles to collectively transport 4 protons to the membrane's positive side, uptake 2 protons from the negative side, reduce two cytochrome \u003cem\u003ec\u003c/em\u003e molecules, oxidize two ubiquinol molecules, and reduce one ubiquinone molecule (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn a previous study, we calculated the atomistic details of the tunneling pathways and the corresponding ET rates between all redox pairs in the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e complex. Interestingly, we discovered that the electron transfer between the heme b\u003csub\u003e\u003cem\u003eL\u003c/em\u003e\u003c/sub\u003e and the heme b\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e redox centers is controlled by a key phenylalanine residue (Phe90) that primarily can assume two different conformations (a.k.a. ON/OFF conformations)\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We found that the Phe90 residue only exists in the ON conformation when the Q\u003csub\u003eo\u003c/sub\u003e-site is occupied. Additionally, we performed extensive MD simulations that confirmed our previous discoveries regarding the role of Phe90 residue as an ET switch or an ET gate, whose conformation influences the rate of the ET reaction between heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e redox pairs significantly. We also discovered \u003cem\u003ea novel orphan binding site (NQ-site) in the bc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e \u003cem\u003ecomplex\u003c/em\u003e that has never been characterized before (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The NQ-binding site is deep enough to modulate the Phe90 conformation and thus modulate the ET between heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e redox centers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the current study, we extend upon our previous discoveries to develop a novel apoptotic mechanism of regulating the cellular ROS levels by modulating the ET reaction between heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e redox centers in the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex. The proposed ROS regulation mechanism is achieved by binding ET-modulating agents at the newly discovered NQ-site in the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex, thereby controlling the fluctuation of the Phe90 residue and enhancing or reducing ET between heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e redox centers, ultimately reducing or increasing the ROS production levels. Therefore, this work provides a novel \u003cem\u003edirect\u003c/em\u003e approach to regulate cellular ROS levels in a controlled fashion, thereby modulating cellular oxidative damage and triggering apoptosis in cancer cells while leaving the normal cells intact. In addition, our proposed mechanism is superior to the previously reported techniques\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e because it can be manipulated to upregulate or downregulate ROS levels. For example, our mechanism provides the necessary means to first \"\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003esensitize\u003c/span\u003e\" the cancer cells by down-regulating oxidative ROS levels and thereby downregulate the antioxidant defense mechanism of cancer cells, and then subsequently \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eelevate\u003c/span\u003e ROS levels above the apoptotic threshold level, thereby triggering apoptosis in cancer cells while leaving normal cells at under-the-threshold ROS levels\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo discover the desired ROS modulators, we performed extensive virtual screening simulations of 1,489,806 ligands of the ZINC database against Q\u003csub\u003eo\u003c/sub\u003e, Q\u003csub\u003ei\u003c/sub\u003e, and NQ binding sites of the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex and obtained 272 patented ligands that bind preferentially at the NQ-binding site compared to Q\u003csub\u003eo\u003c/sub\u003e and Q\u003csub\u003ei\u003c/sub\u003e binding sites. Afterward, we purchased the top 14 ligands to characterize their biological activities with respect to their ROS-regulatory and cytotoxic activities against a breast cancer model cell line. Eventually, we found two lead ROS up-regulators and two lead ROS down-regulators. Additionally, we found a promising cytotoxic activity for the single treatment of the lead ROS up-regulator and even a more synergistic cytotoxic activity for the combined sequential treatment of ROS up-regulator and down-regulator.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Molecular Dynamics Simulation\u003c/h2\u003e \u003cp\u003eThe orientation of the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex (PDB: 1NTZ) with respect to the membrane was computed using the Peripheral Proteins in Membranes (PPM) web server\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The oriented protein was inserted in the membrane using the CHARMM-GUI Membrane Builder\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e online tool using a membrane composition of Phosphatidylcholine (PC): Phosphatidylethanolamine (PE): Cardiolipin (CL) equal to 50:30:20 \u003csup\u003e36\u003c/sup\u003e. The protein-membrane system was placed in a water box with 0.15M KCl neutralizing ions. The assembled system was energy minimized and then equilibrated using the GROMACS\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e program with CHARMM PARAM36 force field\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e under periodic boundary conditions. Afterward, molecular dynamics (MD) simulation was performed on the assembled system for 1000 ns. MD simulations for the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex with pharmacophores bound at the NQ binding site were performed as mentioned above, starting with the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex with the most stable docked conformation of the pharmacophores at the NQ binding site.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Virtual Screening\u003c/h2\u003e \u003cp\u003eWe selected 1,489,806 compounds from the ZINC20 database\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e with a range of molecular weight of 250\u0026ndash;550 Daltons and LogP of 0\u0026ndash;5, satisfying the druglike properties based on Lipinski's Rule of 5. We performed the virtual screening simulations using the PyRx package\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e with the Autodock Vina \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003escoring method and at multiple stages: First, we performed virtual screening of 1,489,806 compounds against the NQ site where its residues were allowed to be flexible and using exhaustiveness equals 12 and number of modes equals 10. We then selected 10,038 compounds that bind at the NQ site more strongly than the native ligand UQH\u003csub\u003e2\u003c/sub\u003e. Next, we performed a virtual screening of the 10,038 compounds against the NQ site with all its residues fixed. We then selected the 4,751 compounds that bind strongly at the NQ site in the flexible mode by at least 5 kcal/mol compared to the fixed mode. Then, we performed a virtual screening of the 4,751 compounds against the Q\u003csub\u003eo\u003c/sub\u003e site with its residues being flexible. We then selected the 540 compounds that bind at the Q\u003csub\u003eo\u003c/sub\u003e higher than UQH\u003csub\u003e2\u003c/sub\u003e. Finally, we performed a virtual screening of the 540 compounds against the Q\u003csub\u003ei\u003c/sub\u003e site with its flexible residues, yielding 272 compounds that bind at the Q\u003csub\u003ei\u003c/sub\u003e site higher than UQH\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Total Cellular ROS and Mitochondrial Superoxide Anion Measurement Assay\u003c/h2\u003e \u003cp\u003eThe MCF7 breast cancer cell line (ATCC, HTB-22) was purchased from American Type Culture Collection (ATCC, Manassas, VA) and propagated in Eagle's minimum essential medium (EMEM, ATCC) supplemented with 10% FBS (Thermo Scientific), 1% L-glutamine and 1% penicillin/streptomycin (ATCC) in a humidified incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were harvested and seeded into a 96-well black plate at a cell density of 25,000 cells/well and left overnight to adhere. Afterward, cells were treated with 25 \u0026micro;M of the 14 purchased ligands from MolPort (Riga, Latvia), positive control (i.e., doxorubicin), and negative control (i.e., DMSO) and left in the incubator for 6\u0026ndash;8 hours. For the Q\u003csub\u003eo\u003c/sub\u003e and Q\u003csub\u003ei\u003c/sub\u003e inhibition experiments, we also incubated 25 \u0026micro;M of myxothiazol or antimycin A (Sigma-Aldrich), respectively. Negative controls for the inhibition experiments were composed of DMSO and either 25 \u0026micro;M of myxothiazol or antimycin A. Afterward, cells were washed and stained for 30 minutes at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in the dark using the CellRox Green (Invitrogen, USA) or the MitoSOX Red Assay Kit (Invitrogen, USA). Afterward, cells were washed, and total cellular ROS or mitochondrial superoxide anion levels were measured using the Synergy Biotek UV/Vis/fluorescence microplate reader with excitation/emission wavelengths of 490/525 nm or 396/610 nm, respectively. Nuclei were labeled using NucBlue Live ReadyProbes Reagent (Hoechst 33342). All experiments were performed in at least triplicate and repeated for at least three independent trials.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 MTS Assay\u003c/h2\u003e \u003cp\u003eMCF7 cells were harvested and seeded at a cell density of 5,000 cells/well into a 96-well white plate and left overnight to adhere. Afterward, cells were treated with different concentrations (0, 5, 10, 25, 50, and 100 \u0026micro;M) of pharmacophores, positive control (i.e., tamoxifen and doxorubicin), and negative control (i.e., DMSO) for 48 hours. Then, cells were washed and assayed for cytotoxicity using CellTiter 96\u0026reg; AQueous One Solution Cell Proliferation Assay kit (MTS) (Promega, USA), which is a colorimetric assay containing colorless tetrazolium compound that is bio-reduced by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells into a colored formazan product that can be measured at wavelength 490 nm using the Synergy Biotek plate reader. Sequential treatment was performed by incubating the overnight attached MCF7 cells with 25 \u0026micro;M of MH-200102 for 24 hours. Then, the cells were washed once and incubated with different concentrations of MH-101015 for another 24 hours. Then, the cells were assayed for cytotoxicity, as mentioned above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the data and results obtained by three independent experiments are expressed as the mean fold change compared to negative control\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Comparisons between groups were determined using Student's t-test. A difference with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cp\u003eWe initially constructed a system composed of the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex (PDB: 1NTZ) embedded in a membrane with a 50:30:20 composition of PC:PE:CL, surrounded by a water box and simulated for 1000 ns under periodic boundary conditions to obtain a more realistic structure of the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex. Afterward, we performed virtual screening of 1,489,806 compounds from the ZINC20 database at multiple stages to ensure that the obtained set of compounds has the following properties as follows: 1- Screening of 1,489,806 compounds and the native ligands (UQ and UQH\u003csub\u003e2\u003c/sub\u003e) against the NQ site and selecting 10,038 pharmacophores that bind more strongly at the NQ site compared to the native ligands and hence suffer no competition upon binding at the NQ site (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). 2- screening of the selected 10,038 compounds against the NQ site in both flexible mode (i.e., the residues of the NQ-site are allowed to move) and fixed mode (i.e., the residues of the NQ-site are fixed in position) and selecting 4,751 compounds that bind strongly in flexible mode by at least 5 kcal/mol compared to the fixed mode and hence would potentially induce conformational changes at the NQ site that could eventually modulate the conformation of the key phenylalanine residue (Phe90) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). 3- screening of 4,751 compounds against the Q\u003csub\u003eo\u003c/sub\u003e site (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC) and Q\u003csub\u003ei\u003c/sub\u003e site (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD) to eventually obtain 272 compounds that bind preferentially at the NQ site compared to UQ and UQH\u003csub\u003e2\u003c/sub\u003e but less firmly at the Q\u003csub\u003eo\u003c/sub\u003e and the Q\u003csub\u003ei\u003c/sub\u003e sites\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWe then picked the top 14 ligands out of the obtained 272 compounds that were commercially available through Molport (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). We then screened the biological activities of those 14 ligands with respect to their cytotoxic and ROS-modulating activities. We performed a cytotoxicity assay for all the purchased 14 pharmacophores with tamoxifen and doxorubicin as the positive controls and DMSO as the negative control against the MCF7 cell line. We found that the MH-200102 compound has a cytotoxic activity with IC\u003csub\u003e50\u003c/sub\u003e equal to 14.57 \u0026micro;M, lower than IC\u003csub\u003e50\u003c/sub\u003e of tamoxifen (~\u0026thinsp;22.07 \u0026micro;M)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e yet higher than the IC\u003csub\u003e50\u003c/sub\u003e of doxorubicin (~\u0026thinsp;1 \u0026micro;M)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e (Fig. 5). Additionally, MH-04504 showed a modest cytotoxic activity compared to that of MH-200102. Other compounds showed either proliferative activity or no activity at all.\u003c/p\u003e\n\u003cp\u003eMore interestingly, we found that sequential treatment with MH-200102 \u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABYAAAATCAYAAACUef2IAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAAFiUAABYlAUlSJPAAAACNSURBVDhP1dPBCcQwDERRaTuwu1QprsQux67o72VhQYFEMskhA7oY8S4eKYA8kI9/uCuX8FpLaq2iqocZY/j1fwhkzkkpBRE5TO/drwMQgtnAwzBJPAWTwNMwF3hrDQDl12NV9f+6HTOTR2CJ9Hg374LNLHZ5PpFWpOEzdLvHUZQMnEGJwlmUCLyDAnwBrzECb8Sk4u8AAAAASUVORK5CYII=\" width=\"22\" height=\"19\"\u003e MH-01015 showed higher cytotoxic activity than the single treatment with MH-200102 (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). We found that the sequential treatment with MH-200102 and then MH-01015 lowered the IC\u003csub\u003e50\u003c/sub\u003e to 5 \u0026micro;M compared to 14.57 \u0026micro;M with a single treatment with MH-200102 (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eOur biochemical assays for the total cellular ROS levels showed that the MH-200102 exhibits an almost twofold increase in the total ROS upregulating activity compared to the ROS level of the negative control (i.e., DMSO) against the MCF7 cells, while the MH-04504 shows a modest total ROS upregulating activity (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). More interestingly, we found that MH-131013 and MH-1015 show total ROS down-regulating activity compared to the ROS level of the negative control (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). Additionally, we measured the mitochondrial superoxide radical anion levels where we found that the two ROS up-regulator compounds (MH-200102 and MH-04504) and the two ROS down-regulator compounds (MH-131013 and MH-1015) show a similar regulating activity on the mitochondrial superoxide anion levels proportional to their regulatory activities for the total cellular ROS levels (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eTo verify if the discovered ROS-regulator compounds bind at the NQ site of the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex, we additionally measured the mitochondrial superoxide anion levels for each one of the lead-hit ROS regulators with either myxothiazole (MYX) or antimycin. MYX is a specific inhibitor for the Q\u003csub\u003eo\u003c/sub\u003e site of the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex and hence blocks its catalytic activity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. For the ROS up-regulators, our results show a decrease in the mitochondrial superoxide anion levels in the MCF7 cells treated with both ligand and MYX compared to ligand only (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). Those results show that MYX blocks the ROS up-modulating activity of MH-200102 and MH-04504. For the ROS down-regulators, we found that treatment with both ligand and MYX restores the mitochondrial superoxide levels to that of the negative control (i.e., DMSO and MYX). Hence, those results also show that MYX blocks any ROS down-modulating activity of MH-131013 and MH-1015.\u003c/p\u003e\n\u003cp\u003eAntimycin (ANT) is a specific inhibitor for the Q\u003csub\u003ei\u003c/sub\u003e site of the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex and hence enhances the reverse electron transfer reaction in the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex, increasing the mitochondrial superoxide anion levels\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. We found that treatment with ROS up-regulator and ANT has a higher superoxide anion level than negative control treatment (i.e., DMSO and ANT) (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). Those results show a synergistic activity between the ROS up-regulator and ANT in elevating the mitochondrial superoxide anion levels. On the other hand, treatment with both ROS down-regulator and ANT shows a comparable mitochondrial superoxide anion level to the negative control treatment, which emphasizes that ANT blocks any ROS down-regulating activity of the ligands MH-131013 and MH-1015.\u003c/p\u003e\n\u003cp\u003eTo understand the molecular activities of the ROS up-regulators and down-regulators, we simulated the molecular dynamics of the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex where one monomer has UQH\u003csub\u003e2\u003c/sub\u003e, UQ, and either MH-200102 or MH01015 ligands docked at the Q\u003csub\u003eo\u003c/sub\u003e, Q\u003csub\u003ei\u003c/sub\u003e, and NQ sites, respectively while leaving the other monomer ligand-free. We measured the electron transfer (ET) through-space distance between the heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e, Phe90, and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e for the ligand-free monomer and for both MH-200102 or MH01015 docked monomers (Fig. 10). Our results show that the ROS up-regulator MH-200102 increases ET through-space distance to ~\u0026thinsp;6.5 \u0026Aring; in close agreement with the ligand-free monomer. On the other hand, MH01015 reduces ET through-space distance to ~\u0026thinsp;5.5 \u0026Aring;, as observed previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Interestingly, we found that the MH-200102 interacts unfavorably with Tyr95 residue where their aromatic rings stack sideways and hence destabilizing Tyr95, leading to a series of aromatic-aromatic interactions that eventually orient Phe90 in unfavorable conformation for ET between heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e (i.e., OFF conformation as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e) (Fig.\u0026nbsp;10, right inset). On the other hand, we found that the MH01015 interacts favorably with Tyr95 through face-to-face configuration\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e that was found to eventually induce favorable conformation of Phe90 (i.e., ON conformation as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e) (Fig. 10, left inset).\u003c/p\u003e"},{"header":"4. CONCLUSION","content":"\u003cp\u003eIn the current study, we managed to experimentally support our proposed ROS regulation mechanism through the binding of an ET-modulating ligand at the newly discovered NQ-site in the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex, thereby controlling the orientation of the Phe90 residue and enhancing or reducing ET between heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e redox centers, ultimately reducing or increasing the ROS production levels. Therefore, this work provides a novel \u003cem\u003edirect\u003c/em\u003e approach to regulate mitochondrial ROS levels in a controlled fashion, thereby modulating mitochondrial oxidative damage and controlling the underlying inflammatory pathogenesis that drives several critical disease processes such as cancer. To our knowledge, the proposed mechanism is the first of a kind that relies entirely on regulating ROS levels in a direct ET-modulating mechanism rather than through antioxidant activity or competitively inhibiting native ligands from binding at their respective sites\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWe previously discovered that the electron transfer reaction between heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e is controlled by the conformation of a key phenylalanine residue (Phe90) that exists in two conformations, ON and OFF, that either facilitates or hinders the ET reaction, respectively. Under normal physiological conditions (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eA), ET occurs in the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex via the Q-cycle, where the key residue Phe90 exists in the ON conformation, promoting ET between heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e. In the current study, we discovered two lead hit up-regulator ligands (MH-200102 and MH-04504) that were proven to bind at a different site of the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex than the Q\u003csub\u003eo\u003c/sub\u003e or the Q\u003csub\u003ei\u003c/sub\u003e sites. Hence, we conclude that those lead hit ROS up-regulator ligands bind at the NQ site, forcing the Phe90 residue to exist in an OFF conformation, thus inhibiting the ET rate by 2\u0026ndash;3 orders of magnitude\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, leading to reverse ET reaction and the subsequent observed increased mitochondrial superoxide anion and total cellular ROS levels (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eB). Also, we found that the lead-hit ROS up-regulator (MH-200102) has a higher cytotoxic activity against MCF7 cells with IC\u003csub\u003e50\u003c/sub\u003e of 14.57 \u0026micro;M than tamoxifen (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;22.07 \u0026micro;M). We also discovered two lead-hit ROS down-regulator ligands (MH-131013 and MH-1015) that were proven to bind at the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex at the NQ site, stabilizing the Phe90 residue\u0026apos;s ON conformation (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eC), hence enhancing the ET forward reaction between heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e redox centers and thus leading to the observed decreased mitochondrial superoxide and cellular total ROS production levels. We found that ROS down-regulator ligands (MH-131013 and MH-1015) have proliferative activity against MCF7 cells. We also found that sequential treatment with MH-200102 \u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAABYAAAATCAYAAACUef2IAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAAFiUAABYlAUlSJPAAAACNSURBVDhP1dPBCcQwDERRaTuwu1QprsQux67o72VhQYFEMskhA7oY8S4eKYA8kI9/uCuX8FpLaq2iqocZY/j1fwhkzkkpBRE5TO/drwMQgtnAwzBJPAWTwNMwF3hrDQDl12NV9f+6HTOTR2CJ9Hg374LNLHZ5PpFWpOEzdLvHUZQMnEGJwlmUCLyDAnwBrzECb8Sk4u8AAAAASUVORK5CYII=\" width=\"22\" height=\"19\"\u003e MH-01015 showed higher cytotoxic activity against MCF7 with IC\u003csub\u003e50\u003c/sub\u003e of 5 \u0026micro;M.\u003c/p\u003e\n\u003cp\u003eIt is worth noting that other reported studies work similarly by modulating ET reactions in complex I or complex III using free fatty acids or myxothiazol, which block the binding of native ligands to their respective binding sites, such as the Q\u003csub\u003eo\u003c/sub\u003e site\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. By contrast, our proposed mechanism depends solely on \u003cem\u003enon-competing\u003c/em\u003e with binding the native ligands UQH\u003csub\u003e2\u003c/sub\u003e and UQ at the Q\u003csub\u003eo\u003c/sub\u003e and the Q\u003csub\u003ei\u003c/sub\u003e sites of the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex, respectively. Instead, our non-competitive mechanism depends on binding the native ligands at their respective sites, thereby modulating the forward ET reaction between the bound UQH\u003csub\u003e2\u003c/sub\u003e at the Q\u003csub\u003eo\u003c/sub\u003e site and the bound UQ at the Q\u003csub\u003ei\u003c/sub\u003e site and regulating cellular ROS levels. Hence, our proposed mechanism provides a non-competitive direct scheme for regulating mitochondrial ROS levels by controlling Phe90 conformation and modulating the ET reaction between heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eAlso, the lack of intervening enzymes and the proposed non-competitive inhibitory activity would lead to better control of the proposed mechanism to regulate the cellular ROS levels with a lower effective dose of the ET-modulating drug. Hence, our mechanism should have a lower probability of developing drug resistance and lead to fewer undesirable side effects. Additionally, our ET-modulating mechanism should mainly either downregulate or upregulate the cellular ROS levels; hence, it does not suffer from the drawback of mixed ROS down-regulation and up-regulation effects in different cell lines, as reported in other ROS-modulating studies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are available within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Faculty Research Incentive Fund (FIRF) intermural grant sponsored by the University of Health Sciences and Pharmacy in St. Louis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMuhammad A. Hagras\u003c/strong\u003e: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Visualization, Writing – original draft, Data Curation, Supervision, Project administration, Resources, Funding acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTomas Jager\u003c/strong\u003e: Methodology, Investigation, Data Curation, Writing – review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Prof. Jesika Faridi and Dr. Nahid Sultana at the University of the Pacific (Stockton, CA) for their initial support in establishing the cytotoxic protocol used in the current study.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Faculty Research Incentive Fund (FIRF) intermural grant sponsored by the University of Health Sciences and Pharmacy in St. Louis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBoonstra J, Post JA. Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. \u003cem\u003eGene\u003c/em\u003e 2004;\u003cstrong\u003e337\u003c/strong\u003e: 1-13.\u003c/li\u003e\n\u003cli\u003eYang Y, Bazhin AV, Werner J, Karakhanova S. Reactive oxygen species in the immune system. \u003cem\u003eInternational reviews of immunology\u003c/em\u003e 2013;\u003cstrong\u003e32\u003c/strong\u003e: 249-70.\u003c/li\u003e\n\u003cli\u003eZhang J, Wang X, Vikash V, Ye Q, Wu D, Liu Y, Dong W. ROS and ROS-mediated cellular signaling. \u003cem\u003eOxidative medicine and cellular longevity\u003c/em\u003e 2016;\u003cstrong\u003e2016\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eWaris G, Ahsan H. Reactive oxygen species: role in the development of cancer and various chronic conditions. \u003cem\u003eJournal of carcinogenesis\u003c/em\u003e 2006;\u003cstrong\u003e5\u003c/strong\u003e: 14.\u003c/li\u003e\n\u003cli\u003eL\u0026oacute;pez-Armada MJ, Riveiro-Naveira RR, Vaamonde-Garc\u0026iacute;a C, Valc\u0026aacute;rcel-Ares MN. Mitochondrial dysfunction and the inflammatory response. \u003cem\u003eMitochondrion\u003c/em\u003e 2013;\u003cstrong\u003e13\u003c/strong\u003e: 106-18.\u003c/li\u003e\n\u003cli\u003eBolduc JA, Collins JA, Loeser RF. Reactive oxygen species, aging and articular cartilage homeostasis. \u003cem\u003eFree Radical Biol Med\u003c/em\u003e 2019;\u003cstrong\u003e132\u003c/strong\u003e: 73-82.\u003c/li\u003e\n\u003cli\u003eAbbas M, Monireh M. The role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis. \u003cem\u003eIranian Journal of Allergy, Asthma and Immunology\u003c/em\u003e 2008: 195-202.\u003c/li\u003e\n\u003cli\u003eMcGarry T, Biniecka M, Veale DJ, Fearon U. Hypoxia, oxidative stress and inflammation. \u003cem\u003eFree Radical Biol Med\u003c/em\u003e 2018;\u003cstrong\u003e125\u003c/strong\u003e: 15-24.\u003c/li\u003e\n\u003cli\u003eSorce S, Krause K-H. NOX enzymes in the central nervous system: from signaling to disease. \u003cem\u003eAntioxidants \u0026amp; redox signaling\u003c/em\u003e 2009;\u003cstrong\u003e11\u003c/strong\u003e: 2481-504.\u003c/li\u003e\n\u003cli\u003eManoharan S, Guillemin GJ, Abiramasundari RS, Essa MM, Akbar M, Akbar MD. The role of reactive oxygen species in the pathogenesis of Alzheimer\u0026rsquo;s disease, Parkinson\u0026rsquo;s disease, and Huntington\u0026rsquo;s disease: a mini review. \u003cem\u003eOxidative medicine and cellular longevity\u003c/em\u003e 2016;\u003cstrong\u003e2016\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eDrose S, Brandt U. The mechanism of mitochondrial superoxide production by the cytochrome bc1 complex. \u003cem\u003eJ Biol Chem\u003c/em\u003e 2008;\u003cstrong\u003e283\u003c/strong\u003e: 21649-54.\u003c/li\u003e\n\u003cli\u003eLanciano P, Khalfaoui-Hassani B, Selamoglu N, Ghelli A, Rugolo M, Daldal F. Molecular mechanisms of superoxide production by complex III: a bacterial versus human mitochondrial comparative case study. \u003cem\u003eBiochimica et Biophysica Acta (BBA)-Bioenergetics\u003c/em\u003e 2013;\u003cstrong\u003e1827\u003c/strong\u003e: 1332-9.\u003c/li\u003e\n\u003cli\u003eMuller F. The nature and mechanism of superoxide production by the electron transport chain: its relevance to aging. \u003cem\u003eJournal of the American Aging Association\u003c/em\u003e 2000;\u003cstrong\u003e23\u003c/strong\u003e: 227-53.\u003c/li\u003e\n\u003cli\u003eYang Y, Karakhanova S, Hartwig W, D\u0026apos;Haese JG, Philippov PP, Werner J, Bazhin AV. Mitochondria and mitochondrial ROS in cancer: novel targets for anticancer therapy. \u003cem\u003eJ Cell Physiol\u003c/em\u003e 2016;\u003cstrong\u003e231\u003c/strong\u003e: 2570-81.\u003c/li\u003e\n\u003cli\u003eWatson J. Oxidants, antioxidants and the current incurability of metastatic cancers. \u003cem\u003eOpen biology\u003c/em\u003e 2013;\u003cstrong\u003e3\u003c/strong\u003e: 120144.\u003c/li\u003e\n\u003cli\u003eTrachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? \u003cem\u003eNature reviews Drug discovery\u003c/em\u003e 2009;\u003cstrong\u003e8\u003c/strong\u003e: 579-91.\u003c/li\u003e\n\u003cli\u003eMitchell P. Coupling of Phosphorylation to Electron and Hydrogen Transfer by a Chemi-Osmotic type of Mechanism. \u003cem\u003eNature\u003c/em\u003e 1961;\u003cstrong\u003e191\u003c/strong\u003e: 144-8.\u003c/li\u003e\n\u003cli\u003eMitchell P. The protonmotive Q cycle: a general formulation. \u003cem\u003eFEBS Lett\u003c/em\u003e 1975;\u003cstrong\u003e59\u003c/strong\u003e: 137-9.\u003c/li\u003e\n\u003cli\u003eMitchell P. Possible molecular mechanisms of the protonmotive function of cytochrome systems. \u003cem\u003eJ Theor Biol\u003c/em\u003e 1976;\u003cstrong\u003e62\u003c/strong\u003e: 327-67.\u003c/li\u003e\n\u003cli\u003eBrandt U, Trumpower B. The Protonmotive Q Cycle in Mitochondria and Bacteria. \u003cem\u003eCrit Rev Biochem Mol Biol\u003c/em\u003e 1994;\u003cstrong\u003e29\u003c/strong\u003e: 165-97.\u003c/li\u003e\n\u003cli\u003eCrofts AR, Hong S, Ugulava N, Barquera B, Gennis R, Guergova-Kuras M, Berry EA. Pathways for proton release during ubihydroquinone oxidation by the bc(1) complex. \u003cem\u003eProc Natl Acad Sci USA\u003c/em\u003e 1999;\u003cstrong\u003e96\u003c/strong\u003e: 10021-6.\u003c/li\u003e\n\u003cli\u003eCrofts AR, Shinkarev VP, Kolling DR, Hong S. The modified Q-cycle explains the apparent mismatch between the kinetics of reduction of cytochromes c1 and bH in the bc1 complex. \u003cem\u003eJ Biol Chem\u003c/em\u003e 2003;\u003cstrong\u003e278\u003c/strong\u003e: 36191-201.\u003c/li\u003e\n\u003cli\u003eOsyczka A, Moser CC, Dutton PL. Fixing the Q cycle. \u003cem\u003eTrends Biochem Sci\u003c/em\u003e 2005;\u003cstrong\u003e30\u003c/strong\u003e: 176-82.\u003c/li\u003e\n\u003cli\u003eCrofts AR, Guergova-Kuras M, Huang L, Kuras R, Zhang Z, Berry EA. Mechanism of Ubiquinol Oxidation by the bc1 Complex:\u0026thinsp; Role of the Iron Sulfur Protein and Its Mobility. \u003cem\u003eBiochemistry\u003c/em\u003e 1999;\u003cstrong\u003e38\u003c/strong\u003e: 15791-806.\u003c/li\u003e\n\u003cli\u003eZhang Z, Huang L, Shulmeister VM, Chi Y-I, Kim KK, Hung L-W, Crofts AR, Berry EA, Kim S-H. Electron transfer by domain movement in cytochrome bc1. \u003cem\u003eNature\u003c/em\u003e 1998;\u003cstrong\u003e392\u003c/strong\u003e: 677-84.\u003c/li\u003e\n\u003cli\u003eHagras MA. Respiratory Complex III: A Bioengine with a Ligand-Triggered Electron-Tunneling Gating Mechanism. \u003cem\u003eThe Journal of Physical Chemistry B\u003c/em\u003e 2024.\u003c/li\u003e\n\u003cli\u003eHagras MA, Hayashi T, Stuchebrukhov AA. Quantum calculations of electron tunneling in respiratory complex III. \u003cem\u003eThe Journal of Physical Chemistry B\u003c/em\u003e 2015;\u003cstrong\u003e119\u003c/strong\u003e: 14637-51.\u003c/li\u003e\n\u003cli\u003eHagras M, Stuchebrukhov A. Inhibition of respiratory complex III by ligands that interact with a regulatory switch: United States Patent 11,058,645, 2021, 2021.\u003c/li\u003e\n\u003cli\u003eHagras MA, Stuchebrukhov AA. Internal switches modulating electron tunneling currents in respiratory complex III. \u003cem\u003eBiochimica et Biophysica Acta (BBA)-Bioenergetics\u003c/em\u003e 2016;\u003cstrong\u003e1857\u003c/strong\u003e: 749-58.\u003c/li\u003e\n\u003cli\u003eHagras MA, Stuchebrukhov AA. Novel inhibitors for a novel binding site in respiratory complex III. \u003cem\u003eThe Journal of Physical Chemistry B\u003c/em\u003e 2016;\u003cstrong\u003e120\u003c/strong\u003e: 2701-8.\u003c/li\u003e\n\u003cli\u003eWoo J-H, Kim Y-H, Choi Y-J, Kim D-G, Lee K-S, Bae JH, Min DS, Chang J-S, Jeong Y-J, Lee YH. Molecular mechanisms of curcumin-induced cytotoxicity: induction of apoptosis through generation of reactive oxygen species, down-regulation of Bcl-X L and IAP, the release of cytochrome c and inhibition of Akt. \u003cem\u003eCarcinogenesis\u003c/em\u003e 2003;\u003cstrong\u003e24\u003c/strong\u003e: 1199-208.\u003c/li\u003e\n\u003cli\u003eUğuz AC, \u0026Ouml;z A, Nazıroğlu M. Curcumin inhibits apoptosis by regulating intracellular calcium release, reactive oxygen species and mitochondrial depolarization levels in SH-SY5Y neuronal cells.\u003c/li\u003e\n\u003cli\u003eGong G, Qin Y Fau - Huang W, Huang W Fau - Zhou S, Zhou S Fau - Yang X, Yang X Fau - Li D, Li D. Rutin inhibits hydrogen peroxide-induced apoptosis through regulating reactive oxygen species mediated mitochondrial dysfunction pathway in human umbilical vein endothelial cells.\u003c/li\u003e\n\u003cli\u003eLomize MA, Pogozheva ID, Joo H, Mosberg HI, Lomize AL. OPM database and PPM web server: resources for positioning of proteins in membranes. \u003cem\u003eNucleic Acids Res\u003c/em\u003e 2012;\u003cstrong\u003e40\u003c/strong\u003e: D370-6.\u003c/li\u003e\n\u003cli\u003eJo S, Kim T, Iyer VG, Im W. CHARMM-GUI: A web-based graphical user interface for CHARMM. \u003cem\u003eJ Comput Chem\u003c/em\u003e 2008;\u003cstrong\u003e29\u003c/strong\u003e: 1859-65.\u003c/li\u003e\n\u003cli\u003eZinser E, Sperka-Gottlieb CD, Fasch EV, Kohlwein SD, Paltauf F, Daum G. Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae. \u003cem\u003eJ Bacteriol\u003c/em\u003e 1991;\u003cstrong\u003e173\u003c/strong\u003e: 2026-34.\u003c/li\u003e\n\u003cli\u003eLindahl E, Hess B, van der Spoel D. GROMACS 3.0: a package for molecular simulation and trajectory analysis. \u003cem\u003eJ Mol Model\u003c/em\u003e 2001;\u003cstrong\u003e7\u003c/strong\u003e: 306-17.\u003c/li\u003e\n\u003cli\u003eBrooks BR, Brooks CL, 3rd, Mackerell AD, Jr., Nilsson L, Petrella RJ, Roux B, Won Y, Archontis G, Bartels C, Boresch S, Caflisch A, Caves L, et al. CHARMM: the biomolecular simulation program. \u003cem\u003eJ Comput Chem\u003c/em\u003e 2009;\u003cstrong\u003e30\u003c/strong\u003e: 1545-614.\u003c/li\u003e\n\u003cli\u003eBest RB, Zhu X, Shim J, Lopes PE, Mittal J, Feig M, Mackerell AD, Jr. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone phi, psi and side-chain chi(1) and chi(2) dihedral angles. \u003cem\u003eJournal of chemical theory and computation\u003c/em\u003e 2012;\u003cstrong\u003e8\u003c/strong\u003e: 3257-73.\u003c/li\u003e\n\u003cli\u003eIrwin JJ, Tang KG, Young J, Dandarchuluun C, Wong BR, Khurelbaatar M, Moroz YS, Mayfield J, Sayle RA. ZINC20\u0026mdash;A Free Ultralarge-Scale Chemical Database for Ligand Discovery. \u003cem\u003eJournal of Chemical Information and Modeling\u003c/em\u003e 2020;\u003cstrong\u003e60\u003c/strong\u003e: 6065-73.\u003c/li\u003e\n\u003cli\u003eDallakyan S, Olson AJ. Small-molecule library screening by docking with PyRx. \u003cem\u003eMethods in molecular biology (Clifton, NJ)\u003c/em\u003e 2015;\u003cstrong\u003e1263\u003c/strong\u003e: 243-50.\u003c/li\u003e\n\u003cli\u003eEberhardt J, Santos-Martins D, Tillack AF, Forli SJJoci, modeling. AutoDock Vina 1.2. 0: New docking methods, expanded force field, and python bindings 2021;\u003cstrong\u003e61\u003c/strong\u003e: 3891-8.\u003c/li\u003e\n\u003cli\u003eHagras MA. US Provisional Patent Application No. 63/638,833, filed on April 25, 2024.\u003c/li\u003e\n\u003cli\u003eSeeger H, Huober J, Wallwiener D, Mueck AO. Inhibition of human breast cancer cell proliferation with estradiol metabolites is as effective as with tamoxifen. \u003cem\u003eHormone and metabolic research\u003c/em\u003e 2004;\u003cstrong\u003e36\u003c/strong\u003e: 277-80.\u003c/li\u003e\n\u003cli\u003eFang XJ, Jiang H, Zhu YQ, Zhang LY, Fan QH, Tian Y. Doxorubicin induces drug resistance and expression of the novel CD44st via NF-\u0026kappa;B in human breast cancer MCF-7 cells. \u003cem\u003eOncol Rep\u003c/em\u003e 2014;\u003cstrong\u003e31\u003c/strong\u003e: 2735-42.\u003c/li\u003e\n\u003cli\u003eVon Jagow G, Gribble GW, Trumpower BL. Mucidin and strobilurin A are identical and inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain at the same site as myxothiazol. \u003cem\u003eBiochemistry\u003c/em\u003e 1986;\u003cstrong\u003e25\u003c/strong\u003e: 775-80.\u003c/li\u003e\n\u003cli\u003eQuinlan CL, Gerencser AA, Treberg JR, Brand MD. The mechanism of superoxide production by the antimycin-inhibited mitochondrial Q-cycle. \u003cem\u003eJ Biol Chem\u003c/em\u003e 2011;\u003cstrong\u003e286\u003c/strong\u003e: 31361-72.\u003c/li\u003e\n\u003cli\u003eSun J, Trumpower BL. Superoxide anion generation by the cytochrome bc1 complex. \u003cem\u003eArch Biochem Biophys\u003c/em\u003e 2003;\u003cstrong\u003e419\u003c/strong\u003e: 198-206.\u003c/li\u003e\n\u003cli\u003eHagras MA. Respiratory Complex III: A Bioengine with a Ligand-Triggered Electron-Tunneling Gating Mechanism. \u003cem\u003eThe Journal of Physical Chemistry B\u003c/em\u003e 2024;\u003cstrong\u003e128\u003c/strong\u003e: 990-1000.\u003c/li\u003e\n\u003cli\u003eAnjana R, Vaishnavi MK, Sherlin D, Kumar SP, Naveen K, Kanth PS, Sekar K. Aromatic-aromatic interactions in structures of proteins and protein-DNA complexes: a study based on orientation and distance. \u003cem\u003eBioinformation\u003c/em\u003e 2012;\u003cstrong\u003e8\u003c/strong\u003e: 1220.\u003c/li\u003e\n\u003cli\u003eBurley SK, Petsko GA. Aromatic-Aromatic Interaction: A Mechanism of Protein Structure Stabilization. \u003cem\u003eScience\u003c/em\u003e 1985;\u003cstrong\u003e229\u003c/strong\u003e: 23-8.\u003c/li\u003e\n\u003cli\u003eYoung TA, Cunningham CC, Bailey SM. Reactive oxygen species production by the mitochondrial respiratory chain in isolated rat hepatocytes and liver mitochondria: studies using myxothiazol. \u003cem\u003eArch Biochem Biophys\u003c/em\u003e 2002;\u003cstrong\u003e405\u003c/strong\u003e: 65-72.\u003c/li\u003e\n\u003cli\u003eSch\u0026ouml;nfeld P, Wojtczak L. Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport. \u003cem\u003eBiochimica et Biophysica Acta (BBA)-Bioenergetics\u003c/em\u003e 2007;\u003cstrong\u003e1767\u003c/strong\u003e: 1032-40.\u003c/li\u003e\n\u003cli\u003eKumar D, Lundgren Dw Fau - Moore RM, Moore Rm Fau - Silver RJ, Silver Rj Fau - Moore JJ, Moore JJ. Hydrogen peroxide induced apoptosis in amnion-derived WISH cells is not inhibited by vitamin C.\u003c/li\u003e\n\u003cli\u003eHam A, Kim B Fau - Koo U, Koo U Fau - Nam K-W, Nam Kw Fau - Lee S-J, Lee Sj Fau - Kim KH, Kim Kh Fau - Shin J, Shin J Fau - Mar W, Mar W. Spirafolide from bay leaf (Laurus nobilis) prevents dopamine-induced apoptosis by decreasing reactive oxygen species production in human neuroblastoma SH-SY5Y cells.\u003c/li\u003e\n\u003cli\u003eAhlemeyer B, Krieglstein J. Retinoic acid reduces staurosporine-induced apoptotic damage in chick embryonic neurons by suppressing reactive oxygen species production.\u003c/li\u003e\n\u003cli\u003eKang JS, Cho D Fau - Kim Y-I, Kim Yi Fau - Hahm E, Hahm E Fau - Yang Y, Yang Y Fau - Kim D, Kim D Fau - Hur D, Hur D Fau - Park H, Park H Fau - Bang S, Bang S Fau - Hwang YI, Hwang Yi Fau - Lee WJ, Lee WJ. L-ascorbic acid (vitamin C) induces the apoptosis of B16 murine melanoma cells via a caspase-8-independent pathway.\u003c/li\u003e\n\u003cli\u003eChang YF, Chi Cw Fau - Wang J-J, Wang JJ. Reactive oxygen species production is involved in quercetin-induced apoptosis in human hepatoma cells.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6357772/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6357772/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eReactive oxygen species (ROS) are by-products of normal cellular aerobic metabolism, which play a crucial role in several cellular processes and contribute to the development and progression of multiple diseases such as cancer. ROS are generated naturally by various enzymes, including respiratory complexes such as respiratory complex III (a.k.a. \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex), which is a homodimer where each monomer encompasses four redox centers (Fe\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e, heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH,\u003c/sub\u003e and heme \u003cem\u003ec\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e) and two native binding sites (Q\u003csub\u003eo\u003c/sub\u003e and Q\u003csub\u003ei\u003c/sub\u003e sites). In the current study, we explored a novel apoptotic mechanism by binding ET-modulating agents at the newly discovered NQ-site in the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex, thereby controlling the fluctuation of the Phe90 residue and enhancing or reducing ET between heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eL\u003c/sub\u003e and heme \u003cem\u003eb\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e redox centers, which will ultimately reduce or increase the ROS production levels. We performed extensive virtual screening simulations of 1,489,806 ligands of the ZINC database against Q\u003csub\u003eo\u003c/sub\u003e, Q\u003csub\u003ei\u003c/sub\u003e, and NQ binding sites of the \u003cem\u003ebc\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e complex and obtained 272 patented ligands that bind preferentially at the NQ-binding site compared to Q\u003csub\u003eo\u003c/sub\u003e and Q\u003csub\u003ei\u003c/sub\u003e binding sites. Afterward, we purchased the top 14 ligands to characterize their biological activities with respect to their ROS-regulatory and cytotoxic activities against a breast cancer MCF7 model cell line. Eventually, we discovered two lead ROS up-regulators and two lead ROS down-regulators. Additionally, we found a promising cytotoxic activity for the single treatment of the lead ROS up-regulator and even a more synergistic cytotoxic activity for the combined sequential treatment of ROS up-regulator and down-regulator.\u003c/p\u003e","manuscriptTitle":"Therapeutic Exploration of Novel Reactive-oxygen Species-Mediated Apoptotic Mechanism by Modulating Electron Transfer in Respiratory Complex III","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-03 12:59:22","doi":"10.21203/rs.3.rs-6357772/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"073360cb-c2c0-4c63-a2a5-2b7ed848011d","owner":[],"postedDate":"April 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-21T13:08:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-03 12:59:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6357772","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6357772","identity":"rs-6357772","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}