Rational design and synthesis of non-competitive transcription inhibitors targeting a conserved RNA polymerase-σ ^A...

RNA polymerase (RNAP) inhibitors that block RNA chain elongation or disrupt DNA unwinding have long served as frontline therapeutic agents. However, a major limitation of competitive inhibitors like rifampicin is the rapid emergence of resistance due to point mutations at the RNAP active site. Transcription initiation, the first step in mRNA synthesis, depends on the interaction between RNAP and a sigma (σ) factor. We targeted the conserved region of the RNAP-σ ^A interaction interface to inhibit transcription initiation. Mycobacterium tuberculosis σ ^A was used as a template for inhibitor design due to the extensive experimental structural data on Mycobacterial RNAP in the apo and holo forms, with a promoter DNA fragment, as well as complexes with rifampicin and Nα-aroyl-N-aryl-phenylalaninamide inhibitors. Sequence comparison and subsequent mutational analysis validated Asp319, Glu322, and Gln323 in M. tuberculosis σ ^A as critical residues for interaction with the RNAP β' subunit. The interface encompassing this polypeptide segment served as a target for inhibitor design. Structure-based virtual screening (SBVS) of a Ugi reaction derived library (URDL) led to the identification of hydroxamate 2a that could bind to M. tuberculosis σ ^A (K _D 2.8 µM). Systematic exploration of SAR via 22 analogues led to non-hydroxamate 2u as a superior binder (K _D 0.9 µM). Both 2a and 2u also displayed almost complete inhibition of transcription at 1 mM. In a whole-cell bactericidal assay, 2u exhibited clear bactericidal activity against Staphylococcus aureus (MIC 50 µM) and M. tuberculosis (MIC 250 µM). Overall, this study showcases the rational design of non-competitive inhibitors of M. tuberculosis transcription through a systematic application of in silico screening and multicomponent reaction chemistry.