By utilization of three-dimensional structure information of rifamycins bound to RNA polymerase (RNAP) and the human pregnane X receptor (hPXR), representative examples (2b-d) of a novel subclass of benzoxazinorifamycins have been synthesized. Relative to rifalazil (2a), these analogues generally display superior affinity toward wild-type and Rif-resistant mutants of the Mycobacterium tuberculosis RNAP but lowered antitubercular activity in cell culture under both aerobic and anaerobic conditions. Lowered affinity toward hPXR for some of the analogues is also observed, suggesting a potential for reduced Cyp450 induction activity. Mouse and human microsomal studies of analogue 2b show it to have excellent metabolic stability. Mouse pharmacokinetics in plasma and lung show accumulation of 2b but with a half-life suggesting nonoptimal pharmacokinetics. These studies demonstrate proof of principle for this subclass of rifamycins and support further expansion of structure-activity relationships (SARs) toward uncovering analogues with development potential.
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) where over 9 million people are infected globally and approximately 2 million lives are claimed annually. Rifampin (a semi‐synthetic rifamycin derivative) inhibits the MTB RNA polymerase (RNAP) and is one of the first‐line anti‐TB drugs. However rifamycin‐resistant (RifR) mutations are frequent and the individual substitutions of three β‐subunit residues of the RNAP (Asp435, His445, and Ser450) together account for 84% of MTB RifR strains found in clinical isolates. To better understand the interaction between rifamycins and wild‐type/RifR mutant MTB RNAPs, the IC50 values of known/novel rifamycins against RifR MTB RNAPs were determined. The inhibition of the different MTB RNAPs by rifamycins was investigated via dose‐response studies with an in vitro rolling circle transcription assay. Rifamycins bind tightly to the wild‐type MTB RNAP and inhibit the enzyme in the 10−9 M (nM) range. Whereas for the RifR mutants, a dramatic ~102–105 fold loss of affinity for rifamycins was observed where these different mutants were inhibited at much higher concentrations of rifamycins (10s–100s μM range). A direct binding assay is being developed to determine the equilibrium binding constant (Kd) values to further characterize the rifamycin·RNAP interactions.
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