title: Target mechanism-based whole-cell screening identifies bortezomib as an inhibitor of caseinolytic protease in mycobacteria.
abstract: A novel type of antibacterial screening method, a target mechanism-based whole-cell screening method, was developed to combine the advantages of target mechanism- and whole-cell-based approaches. A mycobacterial reporter strain with a synthetic phenotype for caseinolytic protease (ClpP1P2) activity was engineered, allowing the detection of inhibitors of this enzyme inside intact bacilli. A high-throughput screening method identified bortezomib, a human 26S proteasome drug, as a potent inhibitor of ClpP1P2 activity and bacterial growth. A battery of secondary assays was employed to demonstrate that bortezomib indeed exerts its antimicrobial activity via inhibition of ClpP1P2: Down- or upmodulation of the intracellular protease level resulted in hyper- or hyposensitivity of the bacteria, the drug showed specific potentiation of translation error-inducing aminoglycosides, ClpP1P2-specific substrate WhiB1 accumulated upon exposure, and growth inhibition potencies of bortezomib derivatives correlated with ClpP1P2 inhibition potencies. Furthermore, molecular modeling showed that the drug can bind to the catalytic sites of ClpP1P2. This work demonstrates the feasibility of target mechanism-based whole-cell screening, provides chemical validation of ClpP1P2 as a target, and identifies a drug in clinical use as a new lead compound for tuberculosis therapy.
  
  
  OBJECTIVE
  During the last decade, antibacterial drug discovery relied on biochemical assays, rather than whole-cell approaches, to identify molecules that interact with purified target proteins derived by genomics. This approach failed to deliver antibacterial compounds with whole-cell activity, either because of cell permeability issues that medicinal chemistry cannot easily fix or because genomic data of essentiality insufficiently predicted the vulnerability of the target identified. As a consequence, the field largely moved back to a whole-cell approach whose main limitation is its black-box nature, i.e., that it requires trial-and-error chemistry because the cellular target is unknown. We developed a novel type of antibacterial screening method, target mechanism-based whole-cell screening, to combine the advantages of both approaches. We engineered a mycobacterial reporter strain with a synthetic phenotype allowing us to identify inhibitors of the caseinolytic protease (ClpP1P2) inside the cell. This approach identified bortezomib, an anticancer drug, as a specific inhibitor of ClpP1P2. We further confirmed the specific "on-target" activity of bortezomib by independent approaches including, but not limited to, genetic manipulation of the target level (over- and underexpressing strains) and by establishing a dynamic structure-activity relationship between ClpP1P2 and growth inhibition. Identifying an "on-target" compound is critical to optimize the efficacy of the compound without compromising its specificity. This work demonstrates the feasibility of target mechanism-based whole-cell screening methods, validates ClpP1P2 as a druggable target, and delivers a lead compound for tuberculosis therapy.
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Moreira
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.
authors.forename: Grace J Y
authors.initials: GJ
authors.lastname: Ngan
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.
authors.forename: Jian Liang
authors.initials: JL
authors.lastname: Low
authors.affiliation: Experimental Therapeutics Center, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Anders
authors.initials: A
authors.lastname: Poulsen
authors.affiliation: Experimental Therapeutics Center, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Brian C S
authors.initials: BC
authors.lastname: Chia
authors.affiliation: Experimental Therapeutics Center, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Melgious J Y
authors.initials: MJ
authors.lastname: Ang
authors.affiliation: Experimental Therapeutics Center, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Amelia
authors.initials: A
authors.lastname: Yap
authors.affiliation: Experimental Therapeutics Center, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Justina
authors.initials: J
authors.lastname: Fulwood
authors.affiliation: Experimental Therapeutics Center, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Umayal
authors.initials: U
authors.lastname: Lakshmanan
authors.affiliation: Experimental Therapeutics Center, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Jolander
authors.initials: J
authors.lastname: Lim
authors.affiliation: Experimental Therapeutics Center, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Audrey Y T
authors.initials: AY
authors.lastname: Khoo
authors.affiliation: Experimental Therapeutics Center, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Horst
authors.initials: H
authors.lastname: Flotow
authors.affiliation: Experimental Therapeutics Center, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Hill
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA.
authors.forename: Ravikiran M
authors.initials: RM
authors.lastname: Raju
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Rubin
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore thomas_dick@nuhs.edu.sg.
authors.forename: Thomas
authors.initials: T
authors.lastname: Dick
mesh_terms: D000995:Antitubercular Agents / Q000302:isolation & purification* / Q000494:pharmacology; D000069286:Bortezomib / Q000302:isolation & purification* / Q000494:pharmacology; D020134:Catalytic Domain; D004353:Drug Evaluation, Preclinical / Q000379:methods; D058492:Drug Repositioning; D057166:High-Throughput Screening Assays; D056004:Molecular Dynamics Simulation; D009161:Mycobacterium / Q000187:drug effects* / Q000201:enzymology*; D011480:Protease Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000995:Antitubercular Agents; D011480:Protease Inhibitors; D000069286:Bortezomib; D012697:Serine Endopeptidases