title: A High-Throughput Assay for Developing Inhibitors of PhoP, a Virulence Factor of Mycobacterium tuberculosis.
abstract: BACKGROUND
  Tuberculosis (TB) kills over 1.5 million people per year despite the available anti-TB drugs. The long duration needed to treat TB by the current TB drugs, which target the essential cellular activities, inevitably leads to the emergence of drug-resistance. The emergence of drug-resistant TB prompts for an urgent need for new and more effective drugs.
  
  
  OBJECTIVE
  The response regulator PhoP, an essential virulence factor of Mycobacterium tuberculosis (MTB), is an attractive target for developing novel anti- TB drugs. This study aims to develop a robust high-throughput screening assay to identify PhoP inhibitors that disrupt the PhoP-DNA binding.
  
  
  METHODS
  Guided by the crystal structure of the PhoP-DNA complex, we designed and developed an assay based on Foster resonance energy transfer (FRET) by labeling Cy3 on the DNA and Cy5 on PhoP. We screened compound libraries for inhibitors that dissociated the PhoP-DNA complex by detection of the FRET signal. Hits were confirmed for their direct binding to PhoP by thermal shift assays.
  
  
  RESULTS
  From a test screening of ~6,000 bioactive compounds and approved drugs, three active compounds were identified that directly bound to PhoP and inhibited the PhoP-DNA interactions. These three PhoP inhibitors can be further developed to improve potency and are useful to study the mechanism of inhibition.
  
  
  CONCLUSIONS
  Our results demonstrated that this FRET-based PhoP-DNA binding assay is valid for additional compound library screening to identify new leads for developing novel TB drugs that target the virulence of MTB.
authors.forename: Liqin
authors.initials: L
authors.lastname: Wang
authors.forename: Miao
authors.initials: M
authors.lastname: Xu
authors.forename: Noel
authors.initials: N
authors.lastname: Southall
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
authors.affiliation: Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd. Bethesda, MD 20814, USA.. United States.
authors.forename: Shuishu
authors.initials: S
authors.lastname: Wang
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000472:pathogenicity; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; C061064:PhoP protein, Bacteria
keywords: FRET; High-throughput screening; PhoP inhibitors; TB drugs.; protein-DNA complex; tuberculosis