Supplementary file 1_High-throughput screening to identify inhibitors of the MTB CARD•RNA polymerase protein-protein interaction.docx

The MTB RNA polymerase (RNAP) is a proven target for TB treatment by rifampin (RMP), which kills both active and latent MTB. However, RMP suffers from several drawbacks including: long treatment times, activation of hPXR (inducing Cyp3A4 which is problematic for HIV-TB coinfection), and resistant mutations within RNAP. Two newer treatments with novel mechanisms of action include bedaquiline and delamanid, the former has been approved by the FDA for the treatment of multiple-drug resistant tuberculosis (MDR-MTB); however, even these new drugs already have incidences of acquired resistance.

Our goal is to develop novel drug candidates for TB treatment that: target RNAP, have the potential to shorten treatment times, and have activity against RIF^R mutants. Our approach, orthogonal to the rifamycins, is to target the interaction of MTB RNAP with the required transcription factor CarD. We have previously labeled CarD with the BODIPY-FL fluorophore via engineered cysteine mutations and developed a fluorescence polarization assay to monitor its interaction with RNAP and promoter DNA.

Leveraging this target, we screened a 24 K small molecule library against CarD binding to MTB RNAP. We have now extended this screening to∼88,000 additional molecules.</p>