Identifying new inhibitors of bacterial RNA polymerase: screening a synthetic chemical library and microbial extracts

Rifampicin (Rif) is widely used in the treatment of Gram-positive and Gram-negative bacterial infections and is currently a first-line clinical anti-tuberculosis drug. Rif functions by binding to and inhibiting bacterial RNA polymerase (RNAP). The clinical utility of existing antibiotics, including Rif, is increasingly threatened by the emergence, and spread of drug-resistant bacterial strains. Tuberculosis resistance to Rif, in particular, poses a huge threat to the health of all mankind. There is an urgent need for new antibacterial compounds that can replace Rif. (Ho et al.,2009) (AM et al., 1959) In this work, I screened a synthetic chemical library of 98 nucleoside analogs from Janssen Pharmaceuticals for their ability to inhibit Escherichia coli RNAP, Mycobacterium tuberculosis RNAP, and bacteriophage SP6 RNAP. I identified 9 compounds that showed inhibition of one or more of the above RNAPs. Of these, 6 compounds were selective for bacterial RNAP over human RNAPs I/II/III. I also confirmed the hypothesis that the mechanism of inhibition of Janssen compounds involves competition with NTP to occupy the RNAP nucleotide addition site. In addition, I inferred the base specificity of the 9 compounds competing and the possible base identity of the analogs. In addition, I tested 984 microbial extracts with my colleague for their ability to inhibit Rif-resistant E. coli RNAP (E. coli S531L RNAP), Rif-resistant M. tuberculosis RNAP (Mtb S'531'L RNAP), and bacteriophage SP6 RNAP. I found there are 54 extract fractions which can inhibit either E. coli S531L RNAP and Mtb S'531'L RNAP. I further tested whether they could inhibit the structurally unrelated bacteriophage SP6 RNAP. I found that 32 of the extract fractions also inhibit SP6 RNAP as well as or better than they inhibit E. coli S531L RNAP and Mtb S'531'L RNAP, also found that 12 extracts strongly inhibited Rif-resistant E. coli RNAP, but not bacteriophage SP6 RNAP, indicating selectivity for bacterial RNAP. Another 10 extracts showed more moderate, selective inhibition of E. coli S531L RNAP or Mtb S'531'L RNAP.M.S.Includes bibliographical reference