Departamento de Biofísica, Universidade Federal de São Paulo, 04044-020, São Paulo, SP, Brazil.
Centro Interdisciplinar de Investigação Bioquímica (CIIB), Universidade de Mogi das Cruzes, 08780-911, Mogi das Cruzes, SP, Brazil.
Biochem Biophys Res Commun. 2020 Feb 5;522(2):368-373. doi: 10.1016/j.bbrc.2019.11.097. Epub 2019 Nov 21.
Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (NEL, EC 3.4.24.16) are closely related zinc-dependent metalo-oligopeptidases, which take part in the metabolism of oligopeptides (from 5 to 17 amino acid residues) inside and outside cells. Both peptidases are ubiquitously distributed in tissues. TOP is one of the main intracellular peptide-processing enzymes being important for the antigen selection in the MHC Class I presentation route, while NEL function has been more associated with the extracellular degradation of neurotensin. Despite efforts being made to develop specific inhibitors for these peptidases, the most used are: CPP-Ala-Ala-Tyr-PABA, described by Orlowski et al. in 1988, and CPP-Ala-Aib-Tyr-PABA (JA-2) that is an analog more resistant to proteolysis, which development was made by Shrimpton et al. in 2000. In the present work, we describe other analogs of these compounds but, with better discriminatory capacity to inhibit specifically NEL or TOP. The modifications introduced in these new analogs were based on a key difference existent in the extended binding sites of NEL and TOP: the negatively charged Glu residue of TOP corresponds to the positively charged Arg residue of NEL. These residues are in position to interact with the residue at the P' and/or P' of their substrates (mimicked by the Ala-Ala/P1'-P2' residues of the CPP-Ala-Ala-Tyr-PABA). Therefore, exploring this single difference, the following compounds were synthesized: CPP-Asp-Ala-Tyr-PABA, CPP-Arg-Ala-Tyr-PABA, CPP-Ala-Asp-Tyr-PABA, CPP-Ala-Arg-Tyr-PABA. Confirming the predictions, the replacement of each non-charged residue of the internal portion Ala-Ala by a charged residue Asp or Arg resulted in compounds with higher selectivity for NEL or TOP, especially due to the electrostatic attraction or repulsion by the NEL Arg or TOP Glu residue. The CPP-Asp-Ala-Tyr-PABA and CPP-Ala-Asp-Tyr-PABA presented higher affinities for NEL, and, the CFP-Ala-Arg-Tyr-PABA showed higher affinity for TOP.
组织蛋白酶 M（Cathepsin M，CAT M，EC 3.4.23.50）和组织蛋白酶 L（Cathepsin L，CAT L，EC 3.4.23.51）是两种密切相关的锌依赖性金属肽酶，它们参与细胞内外寡肽（5 到 17 个氨基酸残基）的代谢。这两种肽酶在组织中广泛分布。CAT M 是主要的细胞内肽酶加工酶之一，对于 MHC 类 I 呈递途径中的抗原选择很重要，而 CAT L 的功能更多地与神经降压素的细胞外降解有关。尽管人们努力开发针对这些肽酶的特异性抑制剂，但最常用的是：1988 年由 Orlowski 等人描述的 CPP-Ala-Ala-Tyr-PABA，以及 2000 年由 Shrimpton 等人开发的对蛋白水解更具抗性的 CPP-Ala-Aib-Tyr-PABA（JA-2）。在本工作中，我们描述了这些化合物的其他类似物，但它们具有更好的鉴别能力，能够特异性抑制 NEL 或 TOP。这些新类似物中的修饰是基于 NEL 和 TOP 的扩展结合位点存在的关键差异：TOP 的带负电荷的 Glu 残基对应于 NEL 的带正电荷的 Arg 残基。这些残基位于与它们底物的 P'和/或 P'相互作用的位置（由 CPP-Ala-Ala-Tyr-PABA 的 Ala-Ala/P1'-P2'残基模拟）。因此，探索这一单一差异，我们合成了以下化合物：CPP-Asp-Ala-Tyr-PABA、CPP-Arg-Ala-Tyr-PABA、CPP-Ala-Asp-Tyr-PABA、CPP-Ala-Arg-Tyr-PABA。证实了预测，内部 Ala-Ala 部分的每个非带电残基被带电荷的残基 Asp 或 Arg 取代，导致化合物对 NEL 或 TOP 的选择性更高，尤其是由于 NEL Arg 或 TOP Glu 残基的静电吸引或排斥。CPP-Asp-Ala-Tyr-PABA 和 CPP-Ala-Asp-Tyr-PABA 对 NEL 具有更高的亲和力，而 CFP-Ala-Arg-Tyr-PABA 对 TOP 具有更高的亲和力。
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