title: Identification of novel inhibitors of M. tuberculosis growth using whole cell based high-throughput screening.
abstract: Despite the urgent need for new antitubercular drugs, few are on the horizon. To combat the problem of emerging drug resistance, structurally unique chemical entities that inhibit new targets will be required. Here we describe our investigations using whole cell screening of a diverse collection of small molecules as a methodology for identifying novel inhibitors that target new pathways for Mycobacterium tuberculosis drug discovery. We find that conducting primary screens using model mycobacterial species may limit the potential for identifying new inhibitors with efficacy against M. tuberculosis. In addition, we confirm the importance of developing in vitro assay conditions that are reflective of in vivo biology for maximizing the proportion of hits from whole cell screening that are likely to have activity in vivo. Finally, we describe the identification and characterization of two novel inhibitors that target steps in M. tuberculosis cell wall biosynthesis. The first is a novel benzimidazole that targets mycobacterial membrane protein large 3 (MmpL3), a proposed transporter for cell wall mycolic acids. The second is a nitro-triazole that inhibits decaprenylphosphoryl-β-D-ribose 2'-epimerase (DprE1), an epimerase required for cell wall biosynthesis. These proteins are both among the small number of new targets that have been identified by forward chemical genetics using resistance generation coupled with genome sequencing. This suggests that methodologies currently employed for screening and target identification may lead to a bias in target discovery and that alternative methods should be explored.
authors.affiliation: The Broad Institute, 7 Cambridge Center, Cambridge, MA 02142, USA.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Stanley
authors.forename: Sarah Schmidt
authors.initials: SS
authors.lastname: Grant
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Kawate
authors.forename: Noriaki
authors.initials: N
authors.lastname: Iwase
authors.forename: Motohisa
authors.initials: M
authors.lastname: Shimizu
authors.forename: Carl
authors.initials: C
authors.lastname: Wivagg
authors.forename: Melanie
authors.initials: M
authors.lastname: Silvis
authors.forename: Edward
authors.initials: E
authors.lastname: Kazyanskaya
authors.forename: John
authors.initials: J
authors.lastname: Aquadro
authors.forename: Aaron
authors.initials: A
authors.lastname: Golas
authors.forename: Michael
authors.initials: M
authors.lastname: Fitzgerald
authors.forename: Huanqin
authors.initials: H
authors.lastname: Dai
authors.forename: Lixin
authors.initials: L
authors.lastname: Zhang
authors.forename: Deborah T
authors.initials: DT
authors.lastname: Hung
mesh_terms: D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology*; D001671:Biochemistry / Q000379:methods; D002473:Cell Wall / Q000378:metabolism; D002626:Chemistry, Pharmaceutical / Q000379:methods; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005990:Glycerol / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008956:Models, Chemical; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*
chemical_list: D000995:Antitubercular Agents; D049452:Green Fluorescent Proteins; D005990:Glycerol