Enzymatic Characterization of Bacterial Enzymes and Inhibitors as Potential Antibiotics with New Mechanisms of Action

Bacteria have become increasingly resistant to antibiotics, therefore there is an urgent need for new drug classes of antibiotics to help fight antibiotic infections. To this end, our research is focused on inhibitors of dizinc metalloenzymes N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE), an enzyme in the lysine biosynthesis pathway, N-acetyl-L-ornithine deacetylase enzyme (ArgE), an enzyme in the arginine biosynthesis pathway, and sodium-dependent NADH: ubiquinone oxidoreductase (Na+-NQR) enzyme, a respiratory complex enzyme as promising drug targets. DapE, ArgE, and Na+-NQR and are only present in bacteria, including ESKAPE pathogens that can cause potentially deadly infections, thus inhibitors of DapE, ArgE, and Na+-NQR offer promise as potential antibiotics with a new mechanism of action without mechanism-based side effects in humans. A high-throughput screen against Haemophilus influenzae DapE (HiDapE) previously provided several hits in different chemical classes. Structure activity relationships (SAR) were developed for several series synthesized and tested against DapE in our ninhydrin-based DapE assay. Inhibitors were also tested in a thermal shift assay using SYPRO Orange dye against DapE from several bacterial species to observe the stability of the enzyme impacted by inhibitors binding. Additionally, our group’s ninhydrin-based assay has been updated and improved utilizing a new substrate for DapE. We are also interested in ArgE as an antibiotic target. We have elucidated the active site structure of a di-Zn Escherichia coli ArgE (EcArgE) using X-ray absorption spectroscopy. Several classes of compounds were screened against ArgE, and SAR is being followed to design new inhibitors against ArgE. A new N,N-dimethyl-N-acetylornithine substrate was designed and synthesized in our lab enabling development of a ninhydrin-based ArgE assay which enabled testing inhibitors that absorb at shorter wavelengths. In-house DapE inhibitors were evaluated for ArgE inhibition, and SAR was determined with a series of boronic acid inhibitors. Selected inhibitors were also tested in a thermal shift assay using SYPRO Orange dye against EcArgE to observe the stability of the enzyme in the presence of inhibitors. We have also targeted Na+-NQR, where we have synthesized clofazimine-like phenazine derivatives and tested them with in-cellular assays along with calculating EC50s.