Kelley Emma H, Osipiuk Jerzy, Korbas Malgorzata, Endres Michael, Bland Alayna, Ehrman Victoria, Joachimiak Andrzej, Olsen Kenneth W, Becker Daniel P
Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, IL, United States.
Structural Biology Center, Argonne National Laboratory, X-ray Science Division, Lemont, IL, United States.
Front Chem. 2024 Jul 11;12:1415644. doi: 10.3389/fchem.2024.1415644. eCollection 2024.
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).
细菌对抗生素的耐药性日益增强，因此迫切需要新型抗生素来对抗抗生素耐药性。哺乳动物不表达对细菌生存和生长至关重要的酶——N - 乙酰 - L - 鸟氨酸脱乙酰酶（ArgE），因此ArgE是一个有前景的新型抗生素药物靶点，因为抑制剂不会产生基于机制的毒性。设计并验证了一种基于茚三酮的新检测方法，该方法包括底物类似物N，N - 二甲基 - N - 乙酰 - L - 鸟氨酸的合成（k/K = 7.32 ± 0.94 × 10⁻⁵ M⁻¹s⁻¹）。这种新检测方法能够筛选在紫外区域有吸收的潜在抑制剂，因此优于已有的214 nm检测方法。使用这种基于茚三酮的新检测方法，证实卡托普利是一种ArgE抑制剂（IC₅₀ = 58.7 μM；Kd = 37.1 ± 0.85 μM），并且鉴定出一些苯基硼酸衍生物为抑制剂，包括4 - （二乙氨基）苯基硼酸（IC₅₀ = 50.1 μM）。还使用SYPRO Orange染料对选定的抑制剂与ArgE进行热迁移分析，以观察在抑制剂（卡托普利Kd = 35.9 ± 5.1 μM）存在下酶的稳定性。使用X射线吸收光谱法确认了二锌ArgE的活性位点结构，并且我们报道了ArgE的两个X射线晶体结构。总之，我们描述了一种用于ArgE的基于茚三酮的新检测方法的开发、卡托普利和苯基硼酸作为ArgE抑制剂的鉴定、ArgE + 卡托普利的热迁移研究以及首次发表的ArgE的两个晶体结构（单锌和二锌）。
