Mechanism and activity of beta-lactam resistant enzymes in E. faecium and E. faecalis

Enterococci (e.g., E. faecalis and E. faecium) cause severe and often fatal nosocomial and community-acquired infections. Therapy of enterococcal infections is frequently compromised by their decreased susceptibility (increased resistance) to many classes of antibiotics, especially β-lactams. This resistance is overwhelmingly attributable to the expression of low-affinity penicillin-binding proteins PBP4 (E. faecalis) and PBP5 (E. faecium), both of which are members of a family of low-affinity PBPs. In the clinical setting, especially E. faecium strains show widespread high-level penicillin resistance due to amino acid substitutions. In this renewal grant, we are building on our significant novel insights gained over the last 6 years and coupling it with extensive new structural and functional preliminary data to define the molecular basis of stem peptide/peptidoglycan recruitment and translate our discoveries into novel antibiotic therapeutics. We have succeeded in difficult and time-consuming biology and biomolecular NMR spectroscopy studies to define how, at a molecular level, E. faecium PBP5 achieves resistance to β-lactam antibiotics. Here, we are now utilizing these data in multiple manners. In Aim 1 we will leverage these data to understand how stem peptides (i.e., PBP substrates) bind to PBPs as well as identify which stem peptide residues are necessary for this interaction. We will also show how peptidoglycan, directly extracted from E. faecium, binds to PBP5. Lastly, we will perform physiological E. faecium experiments to translate our NMR based results into physiological studies. In Aim 2 we will build on a tour-de-force effort that required the determination of nearly 1000 PBP crystal structures. This effort resulted in the identification of 15 novel small fragments that bind the PBP4, including a pocket in the periphery of the β-lactam binding site. We have established a fragment optimization pipeline, combining fragment derivatization, SPR, crystallography, NMR and biological assays to identify stronger binders for iterative compound development into a potent inhibitor. Results from these efforts will then be leveraged for the development of novel β-lactam-derived inhibitors via fragment linking. Together, our rigorous studies will reveal the structural and functional details of enterococcal low-affinity PBP function, providing the molecular basis for their interaction with stem peptides and peptidoglycan. Critically, we have also initiated a translational effort to identify and already molecularly characterize fragments that bind to novel binding pockets in the PBP active site cleft, setting the possibility for the generation of novel antibiotics.