Novel Chemical Scaffolds for Inhibition of Rifamycin-Resistant RNA Polymerase Discovered from High-Throughput Screening

Abstract

Rifampin has been a cornerstone of tuberculosis (TB) treatment since its introduction. The rise of multidrug-resistant and extensively drug-resistant TB makes the development of novel therapeutics effective against these strains an urgent need. Site-specific mutations in the target enzyme of rifampin, RNA polymerase (RNAP) comprises the majority (~97%) of rifamycin-resistant (Rif^R) strains of Mycobacterium tuberculosis (MTB). To identify novel inhibitors of bacterial RNAP, an in vitro plasmid-based transcription assay that uses malachite green (MG) to detect transcribed RNA containing MG aptamers was developed. This assay was optimized in a 384-well plate format and used to screen 150,000 compounds against an Escherichia coli homolog of the most clinically relevant Rif^R RNAP (βS531L) containing a mutation (β'V408G) that compensates for the fitness defect of this Rif^R mutant. Following confirmation and concentration-response studies, 10 compounds were identified with similar in vitro inhibition values across a panel of wild-type and Rif^R E. coli and MTB RNAPs. Four compounds identified from the screen are active against MTB in culture at concentrations below 200 µM. Initial follow-up has resulted in the elimination of one scaffold due to potential pan-assay interference.

Keywords: RNA polymerase; antibiotic resistance; high-throughput screen; rifampin; tuberculosis.

Figure 1

Figure 1A. RNAP plasmid-based transcription assay. RNAP transcribes pMGA12 in presence of σ⁷⁰, resulting in RNA transcript containing 12 repeats of the MGA sequence. The MGAs fold into their secondary structure which can bind malachite green (MG). Aptamer-bound MG is detectable by fluorescence measurement. Figure 1B. Inhibition plot for the 150,554 compounds tested. Compound number is an arbitrary reassignment of CCG compound numbers starting at 1 for visualization purposes. Positive control values are green, negative control values are red, and sample values are black. Blue line indicates 20% inhibition.

Figure 2

Flow chart of selection criteria for high-throughput screen. Values in parentheses are the number, or percent, of compounds that did not pass that particular selection criteria. *As discussed in Data Analysis, a less stringent cutoff (45%) was used for two rows of the 384-well plates in the MG•MGA control screen.