Mazzocato Ylenia, Perin Stefano, Morales-Sanfrutos Julia, Romanyuk Zhanna, Pluda Stefano, Acquasaliente Laura, Borsato Giuseppe, De Filippis Vincenzo, Scarso Alessandro, Angelini Alessandro
Department of Molecular Sciences and Nanosystems, Ca' Foscari University of Venice, Via Torino 155, 30172 Venice, Italy.
Proteomics Unit, Spanish National Cancer Research Centre (CNIO), C. de Melchor Fernández Almagro 3, 28029 Madrid, Spain.
Bioorg Med Chem. 2023 Nov 15;95:117499. doi: 10.1016/j.bmc.2023.117499. Epub 2023 Oct 12.
The inhibition of human urokinase-type plasminogen activator (huPA), a serine protease that plays an important role in pericellular proteolysis, is a promising strategy to decrease the invasive and metastatic activity of tumour cells. However, the generation of selective small molecule huPA inhibitors has proven to be challenging due to the high structural similarity of huPA to other paralogue serine proteases. Efforts to generate more specific therapies have led to the development of cyclic peptide-based inhibitors with much higher selectivity against huPA. While this latter property is desired, the sparing of the orthologue murine poses difficulties for the testing of the inhibitor in preclinical mouse model. In this work, we have applied a Darwinian evolution-based approach to identify phage-encoded bicyclic peptide inhibitors of huPA with better cross-reactivity towards murine uPA (muPA). The best selected bicyclic peptide (UK132) inhibited huPA and muPA with K values of 0.33 and 12.58 µM, respectively. The inhibition appears to be specific for uPA, as UK132 only weakly inhibits a panel of structurally similar serine proteases. Removal or substitution of the second loop with one not evolved in vitro led to monocyclic and bicyclic peptide analogues with lower potency than UK132. Moreover, swapping of 1,3,5-tris-(bromomethyl)-benzene with different small molecules not used in the phage selection, resulted in an 80-fold reduction of potency, revealing the important structural role of the branched cyclization linker. Further substitution of an arginine in UK132 to a lysine resulted in a bicyclic peptide UK140 with enhanced inhibitory potency against both huPA (K = 0.20 µM) and murine orthologue (K = 2.79 µM). By combining good specificity, nanomolar affinity and a low molecular mass, the bicyclic peptide inhibitor developed in this work may provide a novel human and murine cross-reactive lead for the development of a potent and selective anti-metastatic therapy.
人尿激酶型纤溶酶原激活剂（huPA）是一种在细胞周围蛋白水解中起重要作用的丝氨酸蛋白酶，抑制它是降低肿瘤细胞侵袭和转移活性的一种有前景的策略。然而，由于huPA与其他同源丝氨酸蛋白酶的高度结构相似性，生成选择性小分子huPA抑制剂已被证明具有挑战性。致力于开发更特异性疗法已导致基于环肽的抑制剂的发展，其对huPA具有更高的选择性。虽然需要后一种特性，但该抑制剂对小鼠同源物的保留给在临床前小鼠模型中测试该抑制剂带来了困难。在这项工作中，我们应用了基于达尔文进化的方法来鉴定对鼠uPA（muPA）具有更好交叉反应性的噬菌体编码的huPA环肽抑制剂。最佳选择的环肽（UK132）分别以0.33和12.58μM的K值抑制huPA和muPA。这种抑制似乎对uPA具有特异性，因为UK132仅微弱抑制一组结构相似的丝氨酸蛋白酶。用一个未在体外进化的环替换第二个环导致单环和环肽类似物，其效力低于UK132。此外，用噬菌体筛选中未使用的不同小分子替换1,3,5-三（溴甲基）苯导致效力降低80倍，揭示了支链环化接头的重要结构作用。将UK132中的精氨酸进一步替换为赖氨酸产生了环肽UK140，其对huPA（K = 0.20μM）和小鼠同源物（K = 2.79μM）的抑制效力增强。通过结合良好的特异性、纳摩尔亲和力和低分子量，这项工作中开发的环肽抑制剂可能为开发有效和选择性抗转移疗法提供一种新型的人和小鼠交叉反应性先导物。
