| dc.description.abstract | Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the leading cause of infectious death worldwide. In particular, the rise in incidence of drug resistant Mtb strains has made it imperative to develop new drugs targeting proteins that are essential to the bacteria. The utilization of genetic and biochemical techniques has led to the discovery of compounds targeting unexploited, essential Mtb targets. One such compound, the amidinourea 8918, was identified via a phenotypic screening campaign against mycobacteria. Mechanism of action studies revealed that 8918 kills Mtb via binding the active site of PptT, the type II phosphopantetheinyl transferase, resulting in partial non-competitive inhibition of the enzyme. PptT catalyzes the Mg2+ dependent transfer of the phosphopantetheine arm of Coenzyme A onto client carrier proteins, converting them from inactive apo to active holo form. Therefore, the extensive quenching of PptT’s activity attenuates Mtb’s lipid biosynthesis and virulence factor production, leading to cell death.
Furthermore, the activity of 8918 was augmented by a protein encoded just upstream of PptT, PptH. Biochemical studies revealed that the newly discovery enzyme was a phosphopantetheinyl hydrolase, possessing activity antagonistic with respect to PptT, while complementation studies confirmed that its activity contributed to 8918’s bactericidality. Structural studies indicated that the 8918-associated resistance mutations were indeed loss of function mutations which fell into one of two categories: mutations which impacted metal chelation residues and residues which introduced other structurally destabilizing mutations. Overall, these findings revealed a new mechanism of antimicrobial resistance: loss of function of an enzyme that opposes the activity of the drug target.
Another such compound, the isoindole analog LG-1-29, was identified via a phenotypic screen against mycobacteria. Subsequent resistant mutant selection suggested that the cytidine triphosphate synthetase PyrG was likely to be a target of the compound. PyrG catalyzes the ATP-dependent animation of UTP to CTP, and is therefore essential to the de-novo synthesis of CTP. While on-target studies suggested that PyrG was the target of LG-1-29, the compound failed to inhibit the recombinant enzyme. Taken together, these studies suggested that LG-1-29 may be a pro-drug, which upon chemical modification is able to inhibit PyrG. | |
