Evaluation of a Novel Series of Mechanism-Based Inhibitors of Glutamate Racemase Isozymes

Evaluation of a Novel Series of Mechanism-Based Inhibitors of Glutamate Racemase Isozymes

Jenna Stoltzfus, Nicholas Vance, M. Ashley Spies

Abstract: There is a desperate need for the discovery of novel antibiotics against pathogenic bacteria due to the rise of antibiotic resistance. Many bacteria rely on glutamate racemase (GR) isozymes which isomerize L-glutamate to produce D-glutamate which is necessary for cell wall maintenance and replication. Inhibition of GR reduces bacterial growth and causes cell wall lysis, making it an attractive drug target. Due to GR isozymes inherent flexibility, few compounds have been developed that effectively attenuate GR activity.

Irreversible inhibition through covalent modification has historically been successful mechanism for inhibiting enzymes (e.g. Β-lactam antibiotics and salicylic acid) and could be a potential avenue for drugs targeting GR. We have discovered a series of potential mechanism based inhibitors of various GR isozymes through docking experiments. We hypothesize that the catalytic cysteines are forming a covalent adduct with the compounds, which contain an electrophilic Michael acceptor. These compounds effects on GR isozymes were assessed by enzyme kinetics, cell kill assays, and surface plasmon resonance experiments. It was determined from the kinetics data that the compounds are effectively inhibiting GR in a time-dependent fashion. Some scaffolds display more potent time-based inhibition, indicating that some may be forming more productive Michaelis complexes. These results indicate that inhibiting GR through covalent modification could be an effective strategy for the development of novel antibiotics.