Structure-based design of novel benzoxazinorifamycins with potent binding affinity to wild-type and rifampin-resistant mutant Mycobacterium tuberculosis RNA polymerases.

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.

Synthesis and structure-activity relationships of novel substituted 8-amino, 8-thio, and 1,8-pyrazole congeners of antitubercular rifamycin S and rifampin.

A series of rifamycin S and rifampin analogues incorporating substituted 8-amino, 8-thio, and 1,8-pyrazole substituents has been synthesized. The compounds were made by activation of the C-8 phenol as a sulfonate ester, followed by displacement with selected nitrogen and sulfur nucleophiles. The analogues were screened in assays to quantify their antitubercular activity under both aerobic and anaerobic conditions, and for inhibition of wild-type Mycobacterium tuberculosis (MTB) RNAP and rifamycin-resistant MTB RNAP (S450L) via an in vitro rolling circle transcription assay. Additionally, the MIC(90) values were determined for these analogues against Escherichia coli strains. Although none of the analogues displayed superior enzymatic or microbiological activity to their parent scaffolds, the results are consistent with the Rif C-8 hydroxyl acting as a hydrogen bond acceptor with S450 and that Rif resistance in the S450L mutant is due to loss of this hydrogen bond. Representative analogues were also evaluated in the human pregnane X receptor (PXR) activation assay.

Rifamycin inhibition of WT and Rif-resistant Mycobacterium tuberculosis and Escherichia coli RNA polymerases in vitro.

Mycobacterium tuberculosis (MTB) infects over 9 million people globally and claims approximately 2 million lives annually. Rifampin (Rif) is one of the first-line anti-tuberculosis drugs that inhibits transcription by binding to the ß subunit (encoded by the rpoB gene) of the prokaryotic RNA polymerase (RNAP). A highly conserved 81 base pair core region among the ß subunit of prokaryotes harbors most of the point mutations leading to rifamycin-resistant (RifR) mutations, where the majority of the clinically relevant MTB RifR mutations result from amino acid substitutions of one of the following three amino acids: ßAsp435, ßHis445, and ßSer450 (MTB numbering). In this study, to determine the direct effect of rifamycins on the MTB RNAP, co-overexpression vectors were constructed to co-express the core subunits of wild-type and RifR mutants of MTB RNAP. The three aforementioned amino acids were each mutated to the most prevalent substitution found in the MTB clinical isolates (Asp435Val, His445Tyr, Ser450Leu) in the rpoB gene via site-directed mutagenesis. After purification via two-step column chromatography, the in vitro activity of the wild-type and RifR mutant MTB RNAPs was assessed via rolling circle transcription assay. The apparent IC(50) values for three key rifamycins (rifampin (Rif), rifabutin (Rbn), and rifaximin (Rfx)) were determined and these results indicate that the mutant RNAPs demonstrate approximately 10(3)-fold or greater loss of affinities for rifamycins relative to wild-type MTB RNAP. Along with the MTB RNAPs, rifamycin inhibition of the Escherichia coli RNAP counterparts was also assessed. Previously, it has been reported that Gram-positive bacteria (particularly mycobacteria) are more sensitive to rifamycins than Gram-negative bacteria. Under our experimental conditions, the rifamycin IC(50)s for wild-type and RifR mutants of MTB and E. coli RNAPs (wild-type and corresponding mutants) were very similar; therefore, the difference in sensitivity toward rifamycins does not reside in the RNAP itself. The correlation between the sensitivity of rifamycins and permeability into cells was evaluated using the wild-type E. coli strains (TG2 and DH5α) and a mutant E. coli strain with efflux pump defects (EC2880, tolC(-)/imp(-)). The MICs were drastically lower in the EC2880 strain, consistent with previous reports that the differential sensitivity of MTB and E. coli to rifamycins is not related to the RNAP, but rather has to do with efflux pumps in E. coli. Future work will focus on the elucidation of the molecular interaction of these MTB RifR mutants with rifamycins to provide insight to the design of novel rifamycins.