Abstract:

Antibiotic resistance has become a serious threat to global human health, with emergence of drug-resistant strains of Mycobacterium tuberculosis of particular concern. Rifampicin has been the frontline antitubercular therapeutic for the past thirty years. Rifampicin binds bacterial RNA polymerise, thereby blocking transcription. Specific point mutations in the rifampicin-binding pocket of the enzyme render the drug unable to bind its target. A new inhibitor, rifalazil, has recently been shown to possess enhanced antibacterial activity against several rifampicin-resistant clinical isolates of TB. Rifalazil is structurally identical to rifampicin with the exception of the identity and positioning of a short side chain.

In Chapter Two, the first example invoking a direct comparison of the in vitro activities of rifampicin and rifalazil against a panel of mutant RNA polymerise enzymes is presented. Rifalazil is shown to inhibit transcription in both wild-type and rifampicin-resistant RNA polymerises, revealing a direct correlation between antibacterial activity and transcription inhibition with this new drug. These results clearly indicate that the enhanced activity of rifalazil relative to rifampicin is due to specific inhibition of transcription rather than pharmacokinetic and/or cell permeability effects.

In Chapter Three, the role of the side chain of rifalazil is examined more closely. Drug-based chemical probes, in which the crucial aliphatic ansa bridges of rifamycin S and rifalazil have been removed, are evaluated with an in vitro transcription inhibition assay. The rifalazil-based probe is shown to possess significantly greater activity against both wild-type and mutant RNA polymerises, thereby directly implicating the rifalazil side chain in drug activity.

In Chapter Four, rifalazil is shown to bind mutant polymerises, while rifampicin does not. Additionally, a new transcription assay provides evidence that rifalazil inhibits a different step in the process of transcription. Finally, a unique strategy is employed as steps are taken toward the goal of obtaining a crystallographic co-complex of rifalazil and RNA polymerise.

This thesis represents a successful implementation of a strategy developed in the Kahne group for dissecting the mechanism of action of antibiotics. Through the synthesis and evaluation of drug-derived chemical probes, significant insight is gained into the mechanism of action of rifalazil.