Abstract：
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 Ki 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 (Ki = 0.20 µM) and murine orthologue (Ki = 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.
摘要：
人尿激酶型纤溶酶原激活物（huPA）的抑制作用，一种在细胞周蛋白水解中起重要作用的丝氨酸蛋白酶，是降低肿瘤细胞侵袭和转移活性的有希望的策略。然而,由于huPA与其他旁系丝氨酸蛋白酶的结构相似性高,因此选择性小分子huPA抑制剂的产生已被证明具有挑战性.产生更多特异性疗法的努力已经导致对huPA具有高得多的选择性的基于环肽的抑制剂的开发。虽然后者的属性是需要的，保留直系同源小鼠给在临床前小鼠模型中测试抑制剂带来困难。在这项工作中,我们应用了基于达尔文进化论的方法来鉴定对小鼠uPA（muPA）具有更好交叉反应性的huPA的噬菌体编码双环肽抑制剂。最佳选择的双环肽（UK132）抑制huPA和muPA，Ki值为0.33和12.58µM，分别。这种抑制作用似乎对uPA是特异性的，如UK132仅微弱地抑制一组结构相似的丝氨酸蛋白酶。第二个环的去除或被体外未进化的环取代导致单环和双环肽类似物的效力低于UK132。此外，用噬菌体选择中未使用的不同小分子交换1,3,5-三-（溴甲基）-苯，导致效力降低80倍，揭示了支化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导致双环肽UK140对huPA（Ki=0.20µM）和鼠直向同源物（Ki=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，在这项工作中开发的双环肽抑制剂可能为开发有效和选择性的抗转移疗法提供了一种新型的人和鼠交叉反应性线索。
