-acetyl-L-ornithine deacetylase from and a ninhydrin-based assay to enable inhibitor identification.

Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog , -di-methyl -acetyl-L-ornithine (k/K = 7.32 ± 0.94 × 10 Ms). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214 nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC = 58.7 μM; K = 37.1 ± 0.85 μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC = 50.1 μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril K = 35.9 ± 5.1 μM). The active site structure of di-Zn ArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn).