Abstract
Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) accounts for 3.7% of new cases of TB annually worldwide and is a major threat to global public health. Due to the prevalence of the MDR-TB and extensively drug resistant tuberculosis (XDR-TB) cases, there is an urgent need for new drugs with novel mechanisms of action. CarD, a global transcription regulator in MTB, binds RNAP and activates transcription by stabilizing the transcription initiation open-promoter complex (RPo). CarD is required for MTB viability and it has highly conserved homologues in many eubacteria. A fluorescence polarization (FP) assay which monitors the association of MTB RNAP, native rRNA promoter DNA and CarD has been developed. Overall, our objective is to identify and characterize small molecule inhibitors which block the CarD/RNAP interaction and to understand the mechanisms by which CarD interacts with the molecules. We expect that the development of a new and improved anti-TB compound with a novel mechanism of action will relieve the burden of resistance. This CarD FP assay is amenable to HTS and is an enabling tool for future novel therapeutic discovery.
Highlights
Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) accounts for 3.7% of new cases of TB annually worldwide and is a major threat to global public health
CarD consists of two subdomains, an N-terminal domain (1–53) which interacts with M. tuberculosis (MTB) RNAP at the β1β2-lobes of the β-subunit, known as the “protrusion” and a C-terminal domain (64–162), which is separated from the N-terminal domain by a 10-amino acid linker
It has been shown that CarD can activate and repress transcription from different p romoters10. We11 and others have found that activation occurs when CarD stabilizes the RNAP open promoter complex (RPo) of promoters that have inherently short RPo lifetimes to facilitate transcription initiation
Summary
Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) accounts for 3.7% of new cases of TB annually worldwide and is a major threat to global public health. CarD, a global transcription regulator in MTB, binds RNAP and activates transcription by stabilizing the transcription initiation open-promoter complex (RPo). We expect that the development of a new and improved anti-TB compound with a novel mechanism of action will relieve the burden of resistance This CarD FP assay is amenable to HTS and is an enabling tool for future novel therapeutic discovery. In MTB, CarD is a global regulator that modulates transcription by stabilizing the RNAP open promoter complex (RPo)[6,7]. CarD’s function as a global transcriptional regulator that is required for MTB survival makes it an attractive and novel potential therapeutic target. A high throughput screen (HTS) comprising 23,320 small molecules was performed Hits from this screen were characterized in both biochemical and biophysical assays for validation and to probe their mechanism(s) of action.
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