Antibiotic Discovery of Inhibitors of Transcription Factors-RNA Polymerase Protein-Protein Interactions in Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) is a worldwide infectious disease, primarily affecting the lungs, caused by a single infectious agent, Mycobacterium tuberculosis (Mtb). It is estimated that 1.3 million people died from TB in 2022, making TB the second leading infectious killer after COVID-19. Multidrug-resistant TB (MDR-TB) continues to pose a significant public health challenge. A more severe form, called extensively drug-resistant TB (XDR TB), is a rare type of TB disease that is resistant to nearly all medicines, necessitating prolonged treatment regimens that are often associated with higher unfavorable outcomes.

The rise and spread of drug-resistant TB are mainly associated with mismanagement of treatments and lack of novel therapeutics with alternative mechanisms of action. The MTB RNA polymerase (RNAP) is the well-established therapeutic target of rifampin (RMP), an ansamycin antibiotic effective against several types of bacterial infections, including both active and latent Mtb. However, the utility of RMP is compromised due to drawbacks such as long treatment times, metabolizing enzyme induction and resistant mutations within RNAP. RMP resistance has presented challenges for its efficacy, making it a is a major therapeutic challenge. Resistance to RMP primarily occurs through point mutations of its binding pocket in the resistance determining region (RRDR) in the β-subunit of RNAP. These mutations dramatically impair the binding of RMP to RNAP and diminish drug efficacy.

Drug discovery for TB has been revitalized in recent years due to the need for more effective and safer treatment options. Our ultimate goal is to develop novel drug candidates for TB treatment, targeting the Mtb RNAP, that could potentially have activity against MDR-TB strains. Our approach is to block the interaction of Mtb RNAP with the required transcription factors known as CarD and NusG. Transcription factors are RNAP-binding proteins known to regulate the Mtb transcription process and contribute to the Mtb’s ability to resist host defenses. CarD is a global transcription regulator that operates during the initiation step of transcription. NusG is known to promote elongation processes in transcription. Both factors are required for MTB viability and are potential targets for novel antibiotic development.

A Fluorescence Polarization (FP) assay was previously developed by labeling CarD with a green fluorophore (BODIPY-FL) and used to perform a 24K small molecule High-Throughput Screen (HTS). This FP assay monitors the association of Mtb RNAP, native rRNA promoter DNA and labeled CarD. In this work, we utilized this FP assay to screen several distinct compound libraries for inhibitors of the CarD-RNAP interaction. Preliminary hits have been identified from the screens and target identification follow up experiments have been performed. Building on the knowledge gained from the development of the CarD FP assay, we also labeled NusG with a fluorophore to enable future compound library screenings. NusG was labeled at three positions and used to develop a low throughput electrophoretic mobility shift assay (EMSA). While labeled NusG binding to RNAP was confirmed by EMSA, FP assay did not yield sufficient sensitivity for HTS. Therefore, new strategies are being explored to optimize and validate the NusG FP assay for future use.