Nα-acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification

Autor: Emma H. Kelley, Jerzy Osipiuk, Malgorzata Korbas, Michael Endres, Alayna Bland, Victoria Ehrman, Andrzej Joachimiak, Kenneth W. Olsen, Daniel P. Becker
Jazyk: angličtina
Rok vydání: 2024
Předmět:
Zdroj: Frontiers in Chemistry, Vol 12 (2024)
Druh dokumentu: article
ISSN: 2296-2646
DOI: 10.3389/fchem.2024.1415644
Popis: 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 Nɑ -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 N5, N5-di-methyl Nα-acetyl-L-ornithine (kcat/Km = 7.32 ± 0.94 × 104 M−1s−1). 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 (IC50 = 58.7 μM; Ki = 37.1 ± 0.85 μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC50 = 50.1 μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril Ki = 35.9 ± 5.1 μM). The active site structure of di-Zn EcArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli 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).
Databáze: Directory of Open Access Journals
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