ACS Bio & Med Chem Au最新文献

{"title":"Forodesine and Riboprine Exhibit Strong Anti-SARS-CoV-2 Repurposing Potential: In Silico and In Vitro Studies","authors":"Amgad M. Rabie*,&nbsp; and ,&nbsp;Mohnad Abdalla*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00039","DOIUrl":"https://doi.org/10.1021/acsbiomedchemau.2c00039","url":null,"abstract":"<p >Lately, nucleos(t)ide antivirals topped the scene as top options for the treatment of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Targeting the two broadly conserved SARS-CoV-2 enzymes, RNA-dependent RNA polymerase (RdRp) and 3′-to-5′ exoribonuclease (ExoN), together using only one shot is a very successful new tactic to stop SARS-CoV-2 multiplication irrespective of the SARS-CoV-2 variant type. Herein, the current studies investigated most nucleoside analogue (NA) libraries, searching for the ideal drug candidates expectedly able to act through this double tactic. Gradual computational filtration gave rise to six different promising NAs along with their corresponding triphosphate (TP) nucleotides. The subsequent biological assessment proved for the first time that, among the six NAs, riboprine and forodesine are able to hyperpotently inhibit the replication of the Omicron strain of SARS-CoV-2 with extremely low <i>in vitro</i> anti-RdRp, anti-ExoN, and anti-SARS-CoV-2 EC₅₀ values of about 0.18, 0.28, and 0.40 μM for riboprine and about 0.20, 0.31, and 0.65 μM for forodesine, respectively, surpassing remdesivir and molnupiravir. The significant probability that both compounds may also act as prodrugs for their final TP nucleotides <i>in vivo</i> pushed us to examine the same activities for forodesine-TP and riboprine-TP. Both nucleotides similarly displayed very promising results, respectively, which are much better than those for the two reference TP nucleotides, GS-443902 and β-<span>d</span>-<i>N</i>⁴-hydroxycytidine 5′-TP (NHC-TP). The prior <i>in silico</i> data supported these biochemical findings, suggesting that riboprine and forodesine molecules and their expected active TP metabolites strongly hit the key catalytic pockets of the SARS-CoV-2 RdRp’s and ExoN’s main active sites. In brief, the current important results of this comprehensive study revealed the interesting repurposing potentials of, mainly, the two bioactive nucleosides forodesine and riboprine and their TP nucleotides to effectively shut down the polymerase/exoribonuclease-RNA nucleotide interactions of SARS-CoV-2 and consequently treat COVID-19 infections.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 6","pages":"565–585"},"PeriodicalIF":0.0,"publicationDate":"2022-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.2c00039","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72200956","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dual-Reporter System for Real-Time Monitoring of SARS-CoV-2 Main Protease Activity in Live Cells Enables Identification of an Allosteric Inhibition Path","authors":"Yaron Bram,&nbsp;Xiaohua Duan,&nbsp;Benjamin E. Nilsson-Payant,&nbsp;Vasuretha Chandar,&nbsp;Hao Wu,&nbsp;Derek Shore,&nbsp;Alvaro Fajardo,&nbsp;Saloni Sinha,&nbsp;Nora Hassan,&nbsp;Harel Weinstein*,&nbsp;Benjamin R. TenOever*,&nbsp;Shuibing Chen* and Robert E. Schwartz*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00034","DOIUrl":"10.1021/acsbiomedchemau.2c00034","url":null,"abstract":"<p >The SARS-CoV-2 pandemic is an ongoing threat to global health, and the continuing emergence of contagious variants highlights the urgent need for additional antiviral therapy to attenuate COVID-19 disease. The SARS-CoV-2 main protease (3CL^pro) presents an attractive target for such therapy due to its high sequence conservation and key role in the viral life cycle. In this study, we designed a fluorescent–luminescent cell-based reporter for the detection and quantification of 3CL^pro intracellular activity. Employing this platform, we examined the efficiency of known protease inhibitors against 3CL^pro and further identified potent inhibitors through high-throughput chemical screening. Computational analysis confirmed a direct interaction of the lead compounds with the protease catalytic site and identified a prototype for efficient allosteric inhibition. These developments address a pressing need for a convenient sensor and specific targets for both virus detection and rapid discovery of potential inhibitors.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 6","pages":"627–641"},"PeriodicalIF":0.0,"publicationDate":"2022-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/8d/aa/bg2c00034.PMC9603010.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10440074","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Activity-Based Sensing for Chemistry-Enabled Biology: Illuminating Principles, Probes, and Prospects for Boronate Reagents for Studying Hydrogen Peroxide","authors":"Marco S. Messina*,&nbsp;Gianluca Quargnali and Christopher J. Chang*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00052","DOIUrl":"10.1021/acsbiomedchemau.2c00052","url":null,"abstract":"<p >Activity-based sensing (ABS) offers a general approach that exploits chemical reactivity as a method for selective detection and manipulation of biological analytes. Here, we illustrate the value of this chemical platform to enable new biological discovery through a case study in the design and application of ABS reagents for studying hydrogen peroxide (H₂O₂), a major type of reactive oxygen species (ROS) that regulates a diverse array of vital cellular signaling processes to sustain life. Specifically, we summarize advances in the use of activity-based boronate probes for the detection of H₂O₂ featuring high molecular selectivity over other ROS, with an emphasis on tailoring designs in chemical structure to promote new biological principles of redox signaling.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 6","pages":"548–564"},"PeriodicalIF":0.0,"publicationDate":"2022-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9782337/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9339528","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Practical Synthesis of Antimicrobial Long Linear Polyamine Succinamides","authors":"Abdulaziz H. Alkhzem,&nbsp;Shuxian Li,&nbsp;Toska Wonfor,&nbsp;Timothy J. Woodman,&nbsp;Maisem Laabei and Ian S. Blagbrough*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00033","DOIUrl":"10.1021/acsbiomedchemau.2c00033","url":null,"abstract":"<p >There are many severe bacterial infections notorious for their ability to become resistant to clinically relevant antibiotics. Indeed, antibiotic resistance is a growing threat to human health, further exacerbated by the lack of new antibiotics. We now describe the practical synthesis of a series of substituted long linear polyamines that produce rapid antibacterial activity against both Gram-positive and Gram-negative bacteria, including meticillin-resistant <i>Staphylococcus aureus</i>. These compounds also reduce biofilm formation in <i>Pseudomonas aeruginosa</i>. The most potent analogues are thermine, spermine, and 1,12-diaminododecane homo- and heterodimeric polyamine succinic acid amides. They are of the order of activity of the aminoglycoside antibiotics kanamycin and tobramycin as positive controls. Their low human cell toxicity is demonstrated in ex vivo hemolytic assays where they did not produce even 5% hemolysis of human erythrocytes. These long, linear polyamines are a new class of broad-spectrum antibacterials active against drug-resistant pathogens.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 6","pages":"607–616"},"PeriodicalIF":0.0,"publicationDate":"2022-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10125363/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9711146","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"KRAS Inhibitor that Simultaneously Inhibits Nucleotide Exchange Activity and Effector Engagement","authors":"Cynthia V. Pagba,&nbsp;Amit K. Gupta,&nbsp;Ali K. Naji,&nbsp;Dharini van der Hoeven,&nbsp;Kelly Churion,&nbsp;Xiaowen Liang,&nbsp;Jacob Jakubec,&nbsp;Magnus Hook,&nbsp;Yan Zuo,&nbsp;Marisela Martinez de Kraatz,&nbsp;Jeffrey A. Frost and Alemayehu A. Gorfe*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00045","DOIUrl":"10.1021/acsbiomedchemau.2c00045","url":null,"abstract":"<p >We describe a small molecule ligand <b>ACA-14</b> (2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid) as an initial lead for the development of direct inhibitors of KRAS, a notoriously difficult anticancer drug target. We show that the compound binds to KRAS near the switch regions with affinities in the low micromolar range and exerts different effects on KRAS interactions with binding partners. Specifically, <b>ACA-14</b> impedes the interaction of KRAS with its effector Raf and reduces both intrinsic and SOS-mediated nucleotide exchange rates. Likely as a result of these effects, <b>ACA-14</b> inhibits signal transduction through the MAPK pathway in cells expressing mutant KRAS and inhibits the growth of pancreatic and colon cancer cells harboring mutant KRAS. We thus propose compound <b>ACA-14</b> as a useful initial lead for the development of broad-acting inhibitors that target multiple KRAS mutants and simultaneously deplete the fraction of GTP-loaded KRAS while abrogating the effector-binding ability of the already GTP-loaded fraction.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 6","pages":"617–626"},"PeriodicalIF":0.0,"publicationDate":"2022-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/4b/89/bg2c00045.PMC10125367.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9413756","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Adhesive Virulence Factors of Staphylococcus aureus Resist Digestion by Coagulation Proteases Thrombin and Plasmin","authors":"Fanny Risser,&nbsp;Joanan López-Morales and Michael A. Nash*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00042","DOIUrl":"https://doi.org/10.1021/acsbiomedchemau.2c00042","url":null,"abstract":"<p ><i>Staphylococcus aureus</i> (<i>S. aureus</i>) is an invasive and life-threatening pathogen that has undergone extensive coevolution with its mammalian hosts. Its molecular adaptations include elaborate mechanisms for immune escape and hijacking of the coagulation and fibrinolytic pathways. These capabilities are enacted by virulence factors including microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and the plasminogen-activating enzyme staphylokinase (SAK). Despite the ability of <i>S. aureus</i> to modulate coagulation, until now the sensitivity of <i>S. aureus</i> virulence factors to digestion by proteases of the coagulation system was unknown. Here, we used protein engineering, biophysical assays, and mass spectrometry to study the susceptibility of <i>S. aureus</i> MSCRAMMs to proteolytic digestion by human thrombin, plasmin, and plasmin/SAK complexes. We found that MSCRAMMs were highly resistant to proteolysis, and that SAK binding to plasmin enhanced this resistance. We mapped thrombin, plasmin, and plasmin/SAK cleavage sites of nine MSCRAMMs and performed biophysical, bioinformatic, and stability analysis to understand structural and sequence features common to protease-susceptible sites. Overall, our study offers comprehensive digestion patterns of <i>S. aureus</i> MSCRAMMs by thrombin, plasmin, and plasmin/SAK complexes and paves the way for new studies into this resistance and virulence mechanism.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 6","pages":"586–599"},"PeriodicalIF":0.0,"publicationDate":"2022-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.2c00042","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72202053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Shedding Light on the Elephant in a Dark Room in the Discovery of New Medicine: Highlighting Molecular Pharmaceutics within ACS Bio & Med Chem Au","authors":"Afsaneh Lavasanifar*,&nbsp; and ,&nbsp;Lynne S. Taylor,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00047","DOIUrl":"https://doi.org/10.1021/acsbiomedchemau.2c00047","url":null,"abstract":"T parable of the “Blind men and an elephant” is a famous story in many cultures around the world. The story, which originated in the ancient Indian subcontinent, was retold by Rumi, the 13th century Persian poet, in a poem named “The elephant in the dark room”. In the 19th century, the American poet John Godfrey Saxe made his own poem based on the story (The poems of John Godfrey Saxe/The Blind Men and the Elephant). The story tells us about six blind men, who have not seen an elephant before, approaching one in a dark room. They try to learn what the elephant is like by touching it. Each man feels a different part of the elephant’s body (its side, tusk, trunk etc.) and describes it based on their experience, which of course is different from the description of others and far from reality or the big picture. This tale is a great metaphor for the limitations of isolated scientific observation in the development of real and accurate knowledge and/or the translation of discoveries into impactful solutions for real-life problems. Reflecting on the lessons learned from this tale, as applied to the scientific world, reminds us of the need for a multidisciplinary and collaborative approach for producing impactful research, and this is particularly true for the drug development process. A medicine is much more than a molecule and its development requires contributions from different scientific disciplines including, but not limited to, medicinal and analytical chemists, biologists, and formulation scientists. The key to life-changing new discoveries in drug development is in the communication and exchange of ideas between scientific teams from these different disciplines. Recognizing the need for a fully open access, multidisciplinary scientific communication platform, particularly for scientists working in drug development, has led the American Chemical Society to launch ACS Bio & Med Chem Au. The journal, which is one of the nine gold (Au) open access journals, has a broad scope and showcases research from biological, medicinal, and pharmaceutical sciences to nurture communication and information sharing between scientists from different but related disciplines, and this is hugely useful for drug development research. ACS Bio & Med Chem Au particularly recognizes the importance of the molecular and mechanistic understanding of drug formulations as well as translational research in areas of pharmaceutical chemistry, existing and emerging drug delivery systems, biological performance of formulations, and other multidisciplinary research projects, especially those in the field of pharmaceutical development which are within the scope of Molecular Pharmaceutics. In this context, the journal welcomes submission of Articles, Letters, Perspectives, and Reviews with a focus on understanding the physicochemical properties of drugs and drug formulations (including small molecules, proteins, and genes) affecting their in vitro/in vivo function, the development of ","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 4","pages":"313–315"},"PeriodicalIF":0.0,"publicationDate":"2022-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsbiomedchemau.2c00047","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72198245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modeling the Effect of Cooperativity in Ternary Complex Formation and Targeted Protein Degradation Mediated by Heterobifunctional Degraders","authors":"D. Park, J. Izaguirre, R. Coffey, Hu Xu","doi":"10.1101/2022.03.22.485399","DOIUrl":"https://doi.org/10.1101/2022.03.22.485399","url":null,"abstract":"Chemically induced proximity between certain endogenous enzymes and a protein of interest (POI) inside cells may cause post-translational modifications to the POI with biological consequences and potential therapeutic effects. Heterobifunctional (HBF) molecules that bind with one functional part to a target POI and with the other to an E3 ligase induce the formation of a target-HBF-E3 ternary complex, which can lead to ubiquitination and proteasomal degradation of the POI. Targeted protein degra-dation (TPD) by HBFs offers a promising approach to modulating disease-associated proteins, especially those that are intractable using other therapeutic approaches, such as enzymatic inhibition. The three-way interactions among the HBF, the target POI, and the ligase—including the protein-protein interaction (PPI) between the POI and the ligase—contribute to the stability of the ternary complex, manifested as positive or negative binding cooperativity in its formation. How such cooperativity affects HBF-mediated degradation is an open question. In this work, we develop a pharmaco-dynamic model that describes the kinetics of the key reactions in the TPD process, and we use this model to investigate the role of cooperativity in the ternary complex formation and in the target POI degradation. Our model predicts that, under certain conditions, increasing cooperativity may diminish degradation, implying an optimal range of cooperativity values for efficient degradation. We also develop a statistical inference model for determining cooperativity in intracellular ternary complex formation from cellular assay data, and demonstrate it by quantifying the change in cooperativity due to site-directed mutagenesis at the POI-ligase interface of the SMARCA2-ACBI1-VHL ternary complex. Our pharmacodynamic model provides a quantitative framework to dissect the complex HBF-mediated TPD process and may inform the rational design of effective HBF degraders.","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"3 1","pages":"74 - 86"},"PeriodicalIF":0.0,"publicationDate":"2022-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46056866","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Plant Cysteine Oxidase Oxygen-Sensing Function Is Conserved in Early Land Plants and Algae","authors":"Leah J. Taylor-Kearney,&nbsp;Samuel Madden,&nbsp;Jack Wilson,&nbsp;William K. Myers,&nbsp;Dona M. Gunawardana,&nbsp;Elisabete Pires,&nbsp;Philip Holdship,&nbsp;Anthony Tumber,&nbsp;Rosalind E. M. Rickaby and Emily Flashman*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00032","DOIUrl":"10.1021/acsbiomedchemau.2c00032","url":null,"abstract":"<p >All aerobic organisms require O₂ for survival. When their O₂ is limited (hypoxia), a response is required to reduce demand and/or improve supply. A hypoxic response mechanism has been identified in flowering plants: the stability of certain proteins with N-terminal cysteine residues is regulated in an O₂-dependent manner by the Cys/Arg branch of the N-degron pathway. These include the Group VII ethylene response factors (ERF-VIIs), which can initiate adaptive responses to hypoxia. Oxidation of their N-terminal cysteine residues is catalyzed by plant cysteine oxidases (PCOs), destabilizing these proteins in normoxia; PCO inactivity in hypoxia results in their stabilization. Biochemically, the PCOs are sensitive to O₂ availability and can therefore act as plant O₂ sensors. It is not known whether oxygen-sensing mechanisms exist in other phyla from the plant kingdom. Known PCO targets are only conserved in flowering plants, however PCO-like sequences appear to be conserved in all plant species. We sought to determine whether PCO-like enzymes from the liverwort, <i>Marchantia polymorpha</i> (MpPCO), and the freshwater algae, <i>Klebsormidium nitens</i> (KnPCO), have a similar function as PCO enzymes from <i>Arabidopsis thaliana</i>. We report that MpPCO and KnPCO show O₂-sensitive N-terminal cysteine dioxygenase activity toward known AtPCO ERF-VII substrates as well as a putative endogenous substrate, MpERF-like, which was identified by homology to the <i>Arabidopsis</i> ERF-VIIs transcription factors. This work confirms functional and O₂-dependent PCOs from Bryophyta and Charophyta, indicating the potential for PCO-mediated O₂-sensing pathways in these organisms and suggesting PCO O₂-sensing function could be important throughout the plant kingdom.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 5","pages":"521–528"},"PeriodicalIF":0.0,"publicationDate":"2022-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9585510/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40586705","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantification of Engagement of Microtubules by Small Molecules in Living Cells by Flow Cytometry","authors":"Angelo E. Andres,&nbsp;Andres Mariano,&nbsp;Digamber Rane and Blake R. Peterson*,&nbsp;","doi":"10.1021/acsbiomedchemau.2c00031","DOIUrl":"10.1021/acsbiomedchemau.2c00031","url":null,"abstract":"<p >Drugs such as paclitaxel (Taxol) that bind microtubules are widely used for the treatment of cancer. Measurements of the affinity and selectivity of these compounds for their targets are largely based on studies of purified proteins, and only a few quantitative methods for the analysis of interactions of small molecules with microtubules in living cells have been reported. We describe here a novel method for rapidly quantifying the affinities of compounds that bind polymerized tubulin in living HeLa cells. This method uses the fluorescent molecular probe Pacific Blue-GABA-Taxol in conjunction with verapamil to block cellular efflux. Under physiologically relevant conditions of 37 °C, this combination allowed quantification of equilibrium saturation binding of this probe to cellular microtubules (<i>K</i>_d = 1.7 μM) using flow cytometry. Competitive binding of the microtubule stabilizers paclitaxel (cellular <i>K</i>_i = 22 nM), docetaxel (cellular <i>K</i>_i = 16 nM), cabazitaxel (cellular <i>K</i>_i = 6 nM), and ixabepilone (cellular <i>K</i>_i = 10 nM) revealed intracellular affinities for microtubules that closely matched previously reported biochemical affinities. By including a cooperativity factor (α) for curve fitting of allosteric modulators, this probe also allowed quantification of binding (<i>K</i>_b) of the microtubule destabilizers colchicine (<i>K</i>_b = 80 nM, α = 0.08), vinblastine (<i>K</i>_b = 7 nM, α = 0.18), and maytansine (<i>K</i>_b = 3 nM, α = 0.21). Screening of this assay against 1008 NCI diversity compounds identified NSC 93427 as a novel microtubule destabilizer (<i>K</i>_b = 485 nM, α = 0.02), illustrating the potential of this approach for drug discovery.</p>","PeriodicalId":29802,"journal":{"name":"ACS Bio & Med Chem Au","volume":"2 5","pages":"529–537"},"PeriodicalIF":0.0,"publicationDate":"2022-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/38/80/bg2c00031.PMC9585582.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40586704","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}