Peptidic inhibitors of proteases are attracting increasing interest not only as drug candidates but also for studying the function and regulation mechanisms of these enzymes. Previously, we screened out a cyclic peptide inhibitor of human uPA [Formula: see text] and found that Ala substitution of P2 residue turns upain-1 to a substrate. To further investigate the effect of P2 residue on the peptide behavior transformation, we constructed upain-1-W3F, which has Phe replacement in the P2 position. We determined KD and Ki of upain-1-W3F and found that upain-1-W3F might still exist as an inhibitor. Furthermore, the high-resolution crystal structure of upain-1-W3F·uPA reveals that upain-1-W3F indeed stays as an intact inhibitor bind to uPA. We thus propose that the P2 residue plays a nonnegligible role in the conversion of upain-1 to a substrate. These results also proposed a strategy to optimize the pharmacological properties of peptide-based drug candidates by hydrophobicity and steric hindrance.Abbreviations : uPA: urokinase-type plasminogen activator; SPD: serine protease domain; S1 pocket: specific substrate-binding pocket.

中文翻译：

---

洞悉P2位置的残基可防止肽抑制剂被丝氨酸蛋白酶水解。

蛋白酶的肽抑制剂不仅作为候选药物而且用于研究这些酶的功能和调节机制都引起越来越多的关注。以前，我们筛选了人uPA的环肽抑制剂（配方：见正文），发现P2残基的Ala取代将upain-1变成底物。为了进一步研究P2残基对肽行为转化的影响，我们构建了upain-1-W3F，其在P2位置具有Phe替换。我们确定了upain-1-W3F的KD和Ki，发现upain-1-W3F可能仍作为抑制剂存在。此外，upain-1-W3F·uPA的高分辨率晶体结构揭示了upain-1-W3F实际上是作为完整抑制剂与uPA结合而保留的。因此，我们提出P2残基在upain-1向底物的转化中起不可忽略的作用。这些结果还提出了通过疏水性和空间位阻来优化基于肽的候选药物的药理学特性的策略。缩写：uPA：尿激酶型纤溶酶原激活剂；SPD：丝氨酸蛋白酶结构域；S1口袋：特定的底物结合口袋。