Identification and Evaluation of Bacterial RNA Polymerase Inhibitors Using a Novel Plasmid-based Transcription Assay

Tuberculosis (TB) is a global health problem caused by Mycobacterium tuberculosis with 8-10 million new cases each year according to the World Health Organization. The current treatment includes a 6-9 month treatment with four different drugs. Due to the long treatment time and lack of adherence to the treatment regimen, there is a rise in the number of multi-drug resistant strains of Mycobacterium tuberculosis (MDR-TB). Rifampin, a long-time staple in TB treatment, is an effective drug against TB which acts by inhibiting the bacterial enzyme RNA polymerase (RNAP). However, there are problems of resistance to rifampin due to mutations in the gene encoding for RNAP and rifampin is a very effective inducer of CYP450s. It is of interest to develop potent inhibitors against bacterial RNAP that are effective against both the wild-type M. tuberculosis RNAP and common mutant RNAPs and do not induce CYP450s. Nucleic acid aptamers are very useful oligonucleotides that bind specifically to a target molecule based on the nucleotide sequence. An in vitro plasmid-based RNAP transcription assay was developed and adapted to high-throughput screening, in which a malachite green aptamer (MGA) is used as the detection method for RNA transcription. Compounds identified were evaluated for activity against a panel of 10 bacterial RNAPs (E. coli and MTB - WT and RifR RNAPs), and active scaffolds were further studied resulting in identification of a cyanopyrimidine scaffold for bacterial RNAP inhibition. Previous studies have shown benzoxazinorifamycins to have improved activity compared to rifampin in vitro against RifR RNAP enzymes. Additionally, benzoxazinorifamycins have decreased induction of the human pregnane X receptor (hPXR), which leads to expression of CYP3A4 and drug-drug interactions for patients taking rifampin concurrently with HIV medications. Novel benzoxazinorifamycins were evaluated for in vitro activity against WT and RifR bacterial RNAPs (E. coli and MTB) using the plasmid-based transcription assay. We hypothesize that identification of rifamycins that do not activate hPXR and are still active against M. tuberculosis RNAP and M. tuberculosis in culture will allow for the development of anti-TB drugs that can be taken concurrently with HIV medications, as TB-HIV coinfection is a global problem.PhDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140941/1/scharfn_1.pd