Mishra Manasi, Singh Vigyasa, Tellis Meenakshi B, Joshi Rakesh S, Singh Shailja
Department of Life Sciences, School of Natural Sciences, Shiv Nadar University, Gautam Buddha Nagar 201314, India.
Special Centre for Molecular Medicine, Jawahar Lal Nehru University, New Delhi 110067, India.
Pharmaceuticals (Basel). 2020 Dec 23;14(1):7. doi: 10.3390/ph14010007.
Clan C1A or 'papain superfamily' cysteine proteases are key players in many important physiological processes and diseases in most living systems. Novel approaches towards the development of their inhibitors can open new avenues in translational medicine. Here, we report a novel design of a re-engineered chimera inhibitor Mco-cysteine protease inhibitor (CPI) to inhibit the activity of C1A cysteine proteases. This was accomplished by grafting the cystatin first hairpin loop conserved motif (QVVAG) onto loop 1 of the ultrastable cyclic peptide scaffold McoTI-II. The recombinantly expressed Mco-CPI protein was able to bind with micromolar affinity to papain and showed remarkable thermostability owing to the formation of multi-disulphide bonds. Using an in silico approach based on homology modelling, protein-protein docking, the calculation of the free-energy of binding, the mechanism of inhibition of Mco-CPI against representative C1A cysteine proteases (papain and cathepsin L) was validated. Furthermore, molecular dynamics simulation of the Mco-CPI-papain complex validated the interaction as stable. To conclude, in this McoTI-II analogue, the specificity had been successfully redirected towards C1A cysteine proteases while retaining the moderate affinity. The outcomes of this study pave the way for further modifications of the Mco-CPI design for realizing its full potential in therapeutics. This study also demonstrates the relevance of ultrastable peptide-based scaffolds for the development of novel inhibitors via grafting.
C1A家族或“木瓜蛋白酶超家族”半胱氨酸蛋白酶是大多数生命系统中许多重要生理过程和疾病的关键参与者。开发其抑制剂的新方法可为转化医学开辟新途径。在此，我们报告了一种重新设计的嵌合抑制剂Mco-半胱氨酸蛋白酶抑制剂（CPI）的新设计，以抑制C1A半胱氨酸蛋白酶的活性。这是通过将胱抑素的第一个发夹环保守基序（QVVAG）嫁接到超稳定环肽支架McoTI-II的环1上来实现的。重组表达的Mco-CPI蛋白能够以微摩尔亲和力与木瓜蛋白酶结合，并且由于形成了多个二硫键而表现出显著的热稳定性。使用基于同源建模、蛋白质-蛋白质对接、结合自由能计算的计算机模拟方法，验证了Mco-CPI对代表性C1A半胱氨酸蛋白酶（木瓜蛋白酶和组织蛋白酶L）的抑制机制。此外，Mco-CPI-木瓜蛋白酶复合物的分子动力学模拟验证了这种相互作用是稳定的。总之，在这种McoTI-II类似物中，特异性已成功重新定向至C1A半胱氨酸蛋白酶，同时保留了适度的亲和力。本研究结果为进一步修饰Mco-CPI设计以实现其在治疗中的全部潜力铺平了道路。本研究还证明了超稳定肽基支架通过嫁接开发新型抑制剂的相关性。
