Steffek, Micah (2016) Evaluation of PPAT Phosphopantetheine Site Inhibitors and Identification of Small Molecule Binders at the PPAT ATP site. Johns Hopkins Independent Research Proposal.

Abstract

In this study I propose to develop an SPR binding assay for inhibitors of phosphopantetheine adenylyltransferase (PPAT) and will use this technique to attempt to identify small molecules that bind to the ATP site of PPAT. Phosphopantetheine adenylyltransferase is an essential enzyme that catalyzes the penultimate step in the biosynthetic pathway of coenzyme A. Coenzyme A (CoA) plays an essential role in the production of fatty acids and generation of acyl chain carrying proteins in bacteria. This feature makes PPAT an attractive target for development of a novel antibiotic and the low sequence homology with the human PPAT should allow for a high degree of selectivity. PPAT utilizes both ATP and 4’-phosphopantetheine as substrates and yields dephospho-CoA. Small molecule inhibitors have been identified that specifically target E. coli PPAT but bind exclusively in the phosphopantetheine site and not the ATP site as determined by x-ray crystallography. There has been a limited amount of characterization and in this study the inhibitors will be characterized by SPR to generate the K¬D and kinetic rates (kon and koff). These inhibitors are able kill wild type E. coli but the potency is limited by the E. coli’s ability to effectively pump the inhibitor out of the periplasm. Resistance studies with phosphopantetheine site inhibitors and resistant mutations were found. These mutations were in the phosphopantetheine site and demonstrate that the bacterium can tolerate mutations in PPAT while maintaining its natural catalytic activity. The resistance mutations significantly reduce the efficacy of the inhibitors in whole cell killing assays and further limit their effectiveness. In this study the resistant mutation will be cloned into PPAT and purified for binding studies. The mutants will be characterized by determining the binding kinetics of the natural substrates and the phosphopantetheine site inhibitors to determine the effect of these mutations at the enzyme level. All potent published and internal inhibitors of PPAT bind exclusively to the phosphopantetheine site of both gram positive and negative bacteria. A small screen will be run using the SPR against PPAT with a blocked phosphopantetheine site to find novel binders to the ATP site. The library will be generated through chemical fingerprinting tools from known ATP site inhibitors of PPAT and related ATP sites. Hits will be characterized for their binding affinity and if they are competitive with ATP. Validated hits in the ATP site may provide rationale for compounds that can bridge both the phosphopantetheine and ATP sites 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/28244