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

## TL;DR

Researchers developed a new assay to identify inhibitors of a bacterial enzyme, ArgE, and found captopril and phenylboronic acids as potential antibiotic candidates.

## Contribution

A novel ninhydrin-based assay for ArgE was developed, enabling inhibitor screening and leading to the discovery of new inhibitors.

## Key findings

- Captopril inhibits ArgE with an IC50 of 58.7 μM and Ki of 37.1 ± 0.85 μM.
- Phenylboronic acid derivatives, including 4-(diethylamino)phenylboronic acid, were identified as ArgE inhibitors.
- X-ray crystal structures of E. coli ArgE (mono-Zn and di-Zn) were reported for the first time.

## Abstract

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 N
ɑ -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 N
5, N
5-di-methyl N
α-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).