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.
Method: 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
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.
Conclusion: 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.