Steffek, Micah (2016) Characterization of PPAT Resistance Mutants and Identification of Small Molecule Binders at the PPAT ATP site. Independent Research Project Johns Hopkins University.

Abstract

Phosphopantetheine adenylyltransferase (PPAT) is an essential bacterial enzyme in the CoA biosynthetic pathway that catalyzes the reaction between ATP and 4’-phosphopantetheine to form 3’-dephospho-CoA. Inhibiting PPAT through blocking substrate binding and shutting down the CoA pathway is an attractive target because CoA is an essential metabolite and bacteria cannot salvage exogenous CoA. There are two different substrate binding sites on PPAT, ATP and phosphopantetheine, with differing ligandability. The phosphopantetheine site is a highly ligandable and potent inhibitors have been found to this site but resistance is a problem. . In this study, four point mutants were isolated from E. coli that was resistant to potent phosphopantetheine site inhibitors. These mutants were characterized in vitro for their ability to bind to two different series of PPAT inhibitors. All of the point mutations were effective at reducing the affinity of phosphopantetheine site inhibitors from 10 to 14000 fold. A structure of an inhibitor bound to PPAT allow for structural analysis of the inhibitor binding site and the location of the mutations. This analysis provides a design rationale for future medicinal chemistry efforts to avoid these mutagenic hotspots. Due to the presence of resistance mutations in the phosphopantetheine site; a screen was done to find ATP site binders. Competition methods using SPR were developed to characterize the PPAT inhibitors and assign the binding sites. The ATP site has been resistant to small molecule binders in previous hit finding strategies so to increase the odds of finding compounds in the ATP site a second site screening technique was employed by blocking the phosphopantetheine site. The screen was done using a small focused library and identified several compounds that could bind to inhibitor bound PPAT. After competition studies, three selective ATP site compounds were identified. These ATP site specific compounds represent new chemical starting points that could be used to design potent inhibitors of PPAT.

Item Type:	Article
Date Deposited:	12 May 2016 23:45
Last Modified:	12 May 2016 23:45
URI:	https://oak.novartis.com/id/eprint/29338