HTS assay and configuration for inhibitors of the UDP-Mur formation pathway

Colorado State University, Fort Collins CO

Investigators

Abstract

DESCRIPTION (provided by applicant): Disruption of peptidoglycan (PG) synthesis is a well established mode of action of effective antibacterial and is primarily accomplished with 2-lactams and vancomycin. However, M. tuberculosis is resistant to these drugs, and inhibitors of other enzymes in the pathway of PG biosynthesis are highly desirable. In addition, new inhibitors of PG synthesis are needed for eubacteria in general, such a methicillin resistant Staphylococcus aureus and bioterrorist agents including Burkholderia pseudomallei and Yersinia pestis. Here, we target the formation of the key PG biosynthetic precursor, UDP-N-acetyl muramic acid (UDP-MurNAc). It is biosynthesized in bacteria from glucosamine-6-phosphate (GlcNH2-6P) in 5 steps by 4 different proteins [GlmM, GlmU (bifunctional), MurA, and MurB]. Although the later two enzymes have received significant attention in recent years as targets for new antibiotics, very little attention has been focused on the initial two enzymes. Furthermore, it is highly desirable to screen the entire biosynthetic pathway in one assay because it allows an economical approach to the screen-one rather than 5 screens. Also, since substrates and products move along a chemically related pathway, inhibitors could have effects on more than one enzyme. We present preliminary data successfully assaying the last 4 steps of the pathway (catalyzed by GlmU, MurA and MurB) where glucosamine-1-phosphate (GlcNH2-1P) is converted to UDP-MurNAc. The readout is the oxidation of NADPH by MurB in the final step forming UDP-MurNAc. The assay can be run in either the kinetic or end point mode. We show adequate signal to noise with all substrates below their Km values except phosphoenol pyruvate (PEP) which has a very low Km of 5<M. We propose to add GlmM and thus expand to 5 enzymatic steps beginning with GlcNH2-6P and configure this assay for roadmap screening. The hits from the NIH Roadmap screening center will be evaluated by testing for activity (MICs) against M. tuberculosis and a panel of gram positive and gram negative organisms. Concomitantly we will deconvolute which enzyme or enzymes are inhibited in a straightforward fashion using essentially the same assay system but starting at a different point in the pathway. We will also verify our results by HPLC or GC/MS analysis of the enzyme products using individual enzyme assays. We will also follow up by performing initial toxicity testing against Vero cells. Further development will focus on development of hits to drugs against tuberculosis as this is our lab's mission, although as appropriate, collaborative efforts against other pathogens will be pursued. This follow up work will utilize X-ray crystallography (all enzymes have crystal structures except GlmM and GlmM structure determination is in progress), chemical optimization of inhibitor structures, and in vivo testing. Such work is currently on going in the PI's laboratory with other potential TB drugs using collaborations with an X-ray crystallographer, medicinal chemist, and animal modeler in the context of a P01 grant. PUBLIC HEALTH RELEVANCE: The relevance of this project for public health is to develop new drugs to treat bacterial infections. The primary focus will be new tuberculosis drugs but the findings will help develop new drugs against many drug resistant bacteria plaguing the American health system and also help develop new drugs against bacterial bioterrorist agents.