Improving the Selectivity of PACE4 Inhibitors through Modifications of the P1 Residue

Paired basic amino acid cleaving enzyme 4 (PACE4), a serine endoprotease of the proprotein convertases family, has been recognized as a promising target for prostate cancer. We previously reported a selective and potent peptide-based inhibitor for PACE4, named the multi-Leu peptide (Ac-LLLLRVKR-NH2 sequence), which was then modified into a more potent and stable compound named C23 with the following structure: Ac-dLeu-LLLRVK-Amba (Amba: 4-amidinobenzylamide). Despite improvements in both in vitro and in vivo profiles of C23, its selectivity for PACE4 over furin was significantly reduced. We examined other Arg-mimetics instead of Amba to regain the lost selectivity. Our results indicated that the replacement of Amba with 5-(aminomethyl)­picolinimidamide increased affinity for PACE4 and restored selectivity. Our results also provide a better insight on how structural differences between S1 pockets of PACE4 and furin could be employed in the rational design of selective inhibitors.

配对碱性氨基酸切割酶4（PACE4）是前蛋白转化酶家族的丝氨酸内切蛋白酶，已被认定为前列腺癌的极具潜力的治疗靶点。我们此前曾报道过一种针对PACE4的选择性强效肽类抑制剂，命名为多亮氨酸肽，其序列为Ac-LLLLRVKR-NH₂，随后将其修饰为一种更强效且稳定的化合物C23，其结构为Ac-dLeu-LLLRVK-Amba，其中Amba即4-脒基苯甲酰胺（4-amidinobenzylamide）。尽管C23的体外及体内特性均得到改善，但其相较于弗林蛋白酶（furin）对PACE4的选择性显著降低。我们转而研究其他精氨酸模拟物以恢复丧失的选择性。研究结果表明，将Amba替换为5-(氨甲基)吡啶甲脒可提升对PACE4的亲和力并恢复选择性。本研究结果还为如何利用PACE4与弗林蛋白酶S1口袋的结构差异，合理设计选择性抑制剂提供了更深入的见解。