High-throughput screening to identify inhibitors of the MTB CARD•RNA polymerase protein-protein interaction

In 2023, tuberculosis (TB) once again became the world's deadliest infectious disease, overtaking COVID-19 as the leading cause of death from a single infectious agent, Mycobacterium tuberculosis (MTB). The World Health Organization (WHO) reports that the COVID-19 pandemic has reversed years of progress in providing essential TB services and reducing TB disease burden. In 2025, TB was responsible for an estimated 1.23 million deaths globally (95% uncertainty interval: 1.13-1.33 million), and there were 400 K cases of rifamycin resistant/multidrug resistant TB (RR, RIFR, MDR). While treatment success for drug susceptible TB remains high (88%), outcomes for MDR/RR-TB are lower but improving, reaching 71% globally. Clearly, there is a critical unmet need for novel antibiotics against MTB, exacerbated now due to COVID-19. The limited success in pursuing novel targets underscores the urgent need to revisit and reevaluate the mechanisms of action behind existing and once effective drugs, a strategy that may hold untapped potential in the fight against growing antibiotic-resistant TB. 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.