Inhibitors of Transcription: Antibacterial Drug Discovery

Bacterial RNA polymerase (RNAP) is a proven target for broad-spectrum antibacterial therapy.  The suitability of bacterial RNAP as a target for broad-spectrum antibacterial therapy follows from the fact that bacterial RNAP is an essential enzyme (permitting efficacy), the fact that bacterial RNAP-subunit sequences are highly conserved (providing a basis for broad-spectrum activity), and the fact that bacterial RNAP-subunit sequences are not highly conserved in human RNAPI, RNAPII, and RNAPIII (providing a basis for therapeutic selectivity).

The rifamycin antibacterial agents--rifampin, rifapentine, rifabutin, and rifamixin--bind to and inhibit bacterial RNAP. The rifamycins bind to a site on bacterial RNAP adjacent to the RNAP active center and prevent extension of RNA chains beyond a length of 2–3 nucleotides.  The rifamycins are in current clinical use in treatment of Gram-positive and Gram-negative bacterial infections.  The rifamycins are of particular importance in treatment of tuberculosis; the rifamycins are first-line antituberculosis agents and are among the only antituberculosis agents able to clear infection and prevent relapse. The clinical utility of the rifamycin antibacterial agents is threatened by the existence of bacterial strains resistant to rifamycins.  Resistance to rifamycins typically involves substitution of residues in or adjacent to the rifamycin-binding site on bacterial RNAP--i.e., substitutions that directly interfere with rifamycin binding.

In view of the public health threat posed by drug-resistant and multidrug-resistant bacterial infections, there is an urgent need for new classes of broad-spectrum antibacterial agents that (1) target bacterial RNAP (and thus have the same biochemical effects as rifamycins), but that (2) target sites within bacterial RNAP that do not overlap the rifamycin-binding site (and thus do not show cross-resistance with rifamycins).

We have identified new drug targets within the structure of bacterial RNAP.  Each of these new targets can serve as a potential binding site for compounds that inhibit bacterial RNAP and thereby kill bacteria.  Each of these new targets is present in most or all bacterial species, and thus compounds that bind to these new targets are active against a broad spectrum of bacterial species. Each of these new targets is different from targets of current antibiotics, and thus compounds that bind to these new targets are not cross-resistant with current antibiotics.  For each of these new targets, we have identified at least one lead compound that binds to the target, and we have synthesized analogs of the lead compound comprising optimized lead compounds.  Several of the lead compounds and optimized lead compounds are extremely promising, exhibiting potent in vitro antibacterial activity against a broad spectrum of bacterial pathogens--including Gram positives Staphylococcus spp. (MSSA/MRSA/VISA/VRSA), Streptococcus spp (SPN/GAS/GBS), and Enterococcus spp. (VSE/VRE); fastidious Gram-negatives Haemophilus influenzae, Moraxella catarrhalis, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae; and non‑fastidious Gram negatives Burkholderia spp. and Acinetobacter baumannii--exhibiting high oral availability and potent in vivo antibacterial efficacy in mice, and exhibiting no cross-resistance with current antibiotics.

In support of this work, we are identifying new small-molecule inhibitors of bacterial RNAP by analysis of microbial and plant natural products, by high-throughput screening, and by virtual screening.  We are also using genetic, biochemical, biophysical, and crystallographic approaches to define the mechanism of action of each known, and each newly identified, small-molecule inhibitor of bacterial RNAP, and we are using microbiological approaches to define antibacterial efficacies, resistance spectra, and spontaneous resistance frequencies of known and new small-molecule inhibitors of bacterial RNAP.

We seek to address the following objectives: to develop new classes of antituberculosis agents and broad-spectrum antibacterial agents, to develop antibacterial agents effective against pathogens resistant to current antibiotics, to develop antibacterial agents effective against pathogens of high relevance to public health, and to develop antibacterial agents effective against pathogens of high relevance to biodefense.