In vitro Characterization of Wild-type and Rifamycin-resistant Mycobacterium tuberculosis RNA polymerases.

Abstract

Rifampin (a semi-synthetic rifamycin derivative) is one of the first-line anti-tuberculosis drugs. Rifampin inhibits transcription by binding to the beta-subunit of the RNA polymerase (RNAP). The majority of the clinically-relevant Mycobacterium tuberculosis (MTB) rifamycin-resistant (RifR) mutations result from amino acid substitutions of one of the following three beta residues: Asp435, His445, and Ser450 in a highly conserved 27 amino acid region of the RNAP beta-subunit. The core subunits of wild-type and RifR mutants (Asp435Val, His445Tyr, Ser450Leu) of MTB RNAP were overexpressed in and purified from E. coli. The rifamycins were all found to bind tightly (IC50) to the wild-type MTB and E. coli RNAPs, whereas dramatic (~10^2-10^5 fold) losses of affinity for rifamycins were observed for the RifR mutants from both bacteria. Additional studies with efflux pump-deficient E. coli (EC2880) confirm that the differential sensitivity of MTB and E. coli to rifamycin antibiotic activity is due to rifamycin efflux from E. coli, rather than any differences in the target RNAPs. The activities of C-8 modified rifamycins are consistent with X-ray crystal structures that show Ser450 acting as a hydrogen bond donor to the C-8 hydroxyl of rifamycins and that rifamycin resistance in the Ser450 mutants is likely due to loss of this hydrogen bond and loss of affinity. A series of novel benzoxazinorifamycin analogues displayed superior affinity toward wild-type and RifR mutants of the MTB RNAP than rifampin and rifalazil (RifR mutants, but not WT), but the IC50 values were still in the 10^-6 M range with the RifR MTB RNAPs. Rifampin exhibits significant drug-drug interactions via potent induction of cytochrome P450 3A4 (CYP3A4). Selected commercially available rifamycins and our synthetic analogues were screened in the human pregnane X receptor (hPXR) activation assay to determine their extents of hPXR activation as an indicator of potential for CYP3A4 induction. One of our analogues exhibited very low (similar to rifalazil) activation of hPXR, while the others did show significant activation and some cytotoxicity. The results of these studies have provided encouraging evidence that rifamycins with improved activity against RifR MTB RNAP and lower drug-drug interaction liabilities can be developed.