title: Novel Chemical Scaffolds for Inhibition of Rifamycin-Resistant RNA Polymerase Discovered from High-Throughput Screening.
abstract: Rifampin has been a cornerstone of tuberculosis (TB) treatment since its introduction. The rise of multidrug-resistant and extensively drug-resistant TB makes the development of novel therapeutics effective against these strains an urgent need. Site-specific mutations in the target enzyme of rifampin, RNA polymerase (RNAP) comprises the majority (~97%) of rifamycin-resistant (RifR) strains of Mycobacterium tuberculosis (MTB). To identify novel inhibitors of bacterial RNAP, an in vitro plasmid-based transcription assay that uses malachite green (MG) to detect transcribed RNA containing MG aptamers was developed. This assay was optimized in a 384-well plate format and used to screen 150,000 compounds against an Escherichia coli homolog of the most clinically relevant RifRRNAP (βS531L) containing a mutation (β'V408G) that compensates for the fitness defect of this RifRmutant. Following confirmation and concentration-response studies, 10 compounds were identified with similar in vitro inhibition values across a panel of wild-type and RifRE. coli and MTB RNAPs. Four compounds identified from the screen are active against MTB in culture at concentrations below 200 µM. Initial follow-up has resulted in the elimination of one scaffold due to potential pan-assay interference.
authors.affiliation: 1 Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Nathan T
authors.initials: NT
authors.lastname: Scharf
authors.affiliation: 2 Department of Biochemistry and Molecular Biology, The Center for RNA Molecular Biology, The Pennsylvania State University, University Park, PA, USA.
authors.forename: Vadim
authors.initials: V
authors.lastname: Molodtsov
authors.affiliation: 1 Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Arrin
authors.initials: A
authors.lastname: Kontos
authors.affiliation: 2 Department of Biochemistry and Molecular Biology, The Center for RNA Molecular Biology, The Pennsylvania State University, University Park, PA, USA.
authors.forename: Katsuhiko S
authors.initials: KS
authors.lastname: Murakami
authors.affiliation: 1 Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA.
authors.forename: George A
authors.initials: GA
authors.lastname: Garcia
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D055808:Drug Discovery; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D054908:Extensively Drug-Resistant Tuberculosis / Q000188:drug therapy / Q000382:microbiology; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology; D012294:Rifamycins / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D000995:Antitubercular Agents; D052157:Aptamers, Nucleotide; D001426:Bacterial Proteins; D012294:Rifamycins; D054852:Small Molecule Libraries; C584549:malachite green RNA aptamer; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin
keywords: RNA polymerase; antibiotic resistance; high-throughput screen; rifampin; tuberculosis