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Abstract：
Cystatins are classical competitive inhibitors of C1 family cysteine proteases (papain family). Phytocystatin superfamily shares high sequence homology and typical tertiary structure with conserved glutamine-valine-glycine (Q-X-V-X-G) loop blocking the active site of C1 proteases. Here, we develop a cysteine-bounded cyclic peptide (CYS-cIHL) and linear peptide (CYS-IHL), using the conserved inhibitory hairpin loop amino acid sequence. Using an in silico approach based on modeling, protein-peptide docking, molecular dynamics simulations and calculation of free energy of binding, we designed and validated inhibitory peptides against falcipain-2 (FP-2) and -3 (FP-3), cysteine proteases from the malarial parasite Plasmodium falciparum. Falcipains are critical hemoglobinases of P. falciparum that are validated targets for the development of antimalarial therapies. CYS-cIHL was able to bind with micromolar affinity to FP-2 and modulate its binding with its substrate, hemoglobin in in vitro and in vivo assays. CYS-cIHL could effectively block parasite growth and displayed antimalarial activity in culture assays with no cytotoxicity towards human cells. These results indicated that cyclization can substantially increase the peptide affinity to the target. Furthermore, this can be applied as an effective strategy for engineering peptide inhibitory potency against proteases.
摘要：
胱抑素是C1家族半胱氨酸蛋白酶(木瓜蛋白酶家族)的经典竞争性抑制剂。植物胱抑素超家族具有高度的序列同源性和典型的三级结构，保守的谷氨酰胺-缬氨酸-甘氨酸（Q-X-V-X-G）环阻断C1蛋白酶的活性位点。这里,我们开发了半胱氨酸结合的环肽（CYS-cIHL）和线性肽（CYS-IHL），使用保守的抑制性发夹环氨基酸序列。使用基于建模的计算机模拟方法，蛋白质-肽对接，分子动力学模拟和结合自由能的计算，我们设计并验证了针对falcipain-2（FP-2）和-3（FP-3）的抑制肽，来自疟疾寄生虫恶性疟原虫的半胱氨酸蛋白酶。Falcipains是恶性疟原虫的关键血红蛋白酶，是开发抗疟疾疗法的有效靶标。CYS-cIHL能够以微摩尔亲和力与FP-2结合并调节其与底物的结合，血红蛋白在体外和体内测定。CYS-cIHL可以有效地阻止寄生虫的生长，并在培养试验中显示出抗疟疾活性，对人细胞没有细胞毒性。这些结果表明环化可以显著增加肽对靶标的亲和力。此外，这可以作为工程肽抑制蛋白酶效力的有效策略。
