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首页> 外文期刊>Journal of Medicinal Chemistry >Synthesis, Solution Structure, and Biological Evaluation of Urokinase Type Plasminogen Activator (uPA)-Derived Receptor Binding Domain Mimeties
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Synthesis, Solution Structure, and Biological Evaluation of Urokinase Type Plasminogen Activator (uPA)-Derived Receptor Binding Domain Mimeties

机译：尿激酶型纤溶酶原激活物（uPA）衍生的受体结合域模仿的合成，溶液结构和生物学评价。

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Tumor cell migration and metastasis in cancer are facilitated by interaction of the serine protease urokinase type plasminogen activator (uPA) with its receptor uPAR (CD 87). Overexpression of uPA and uPAR in cancer tissues is associated with a high incidence of disease recurrence and early death. In agreement with these findings, disruption of the protein-protein interaction between uPAR present on tumor cells and its ligand uPA evolved as an attractive intervention strategy to impair tumor growth and metastasis. For this, the uPAR antagonist cyclo[19,31][d-Cys~(19)]-uPA_(19-31) was optimized to efficiently interrupt binding of uPA to cellular uPAR. First, the disulfide bridge of this lead compound was shifted and then the modified peptide was shortened from the amino and carboxy terminus to generate cyclo [21,29][Cys~(21,29)]-uPA_(21-30). Next, Cyclo[21,29][d-Cys~(21)Cys~(29)]-uPA_(21-30) was yielded by changing the chirality of Cys~(21) to d-Cys~(21). For analysis of uPAR binding activity, we employed competitive flow cytofluorometric receptor binding assays, using FITC-uPA as the ligand and U937 promyeloid leukemia cells as the cellular source of uPAR. As demonstrated for cyclo[21,29][d-Cys~(21)Cys~(29)]-uPA_(21-30), the achieved peptide modifications maintained receptor binding activity (IC_(50) = 0.04 μM), which is close in order to that of the parent protein ligand, uPA (IC_(50) = 0.01 μM). A detailed NMR analysis with restrained and free molecular dynamics calculations in explicit H_2O exhibits a well-defined structure with characteristic features such as an ω-loop with two βI-turns about Lys~3, Tyr~4, Ser~6, and Asn~7. Hydrophobic clustering of the side chains of Tyr~4, Phe~5, Ile~8, and Trp~(10) is observed. Side chain mobility is analyzed with time-dependent distance restraints. The NMR structure of cyclo[21,29][d-Cys~(21)Cys~(29)]-uPA_(21-30) is very similar to the previously reported structure of the amino terminal fragment of uPA. Systematic point mutations led to cyclo[21,29][d-Cys~(21)Nle~(23)Cys~(29)]-uPA_(21-30), which still binds to uPAR but is resistant to proteolytic cleavage, e.g., by the tumor-associated serine proteases uPA and plasmin, and is stable in blood serum or plasma. In conclusion, small cyclic peptides were created, which mimic the structure and activity of the binding epitope of uPA to uPAR and which may serve as novel therapeutic agents in cancer metastasis.
机译：丝氨酸蛋白酶尿激酶型纤溶酶原激活物（uPA）与其受体uPAR（CD 87）的相互作用促进了肿瘤细胞的迁移和转移。 uPA和uPAR在癌症组织中的过表达与疾病复发和早期死亡的高发生率有关。与这些发现一致，肿瘤细胞上存在的uPAR与其配体uPA之间的蛋白质-蛋白质相互作用的破坏被发展为削弱肿瘤生长和转移的有吸引力的干预策略。为此，优化了uPAR拮抗剂环[19,31] [d-Cys〜（19）]-uPA_（19-31），以有效中断uPA与细胞uPAR的结合。首先，使该先导化合物的二硫键移位，然后将修饰的肽从氨基和羧基末端缩短，以生成环[21,29] [Cys〜（21,29）]-uPA_（21-30）。接下来，通过将Cys〜（21）的手性变为d-Cys〜（21），得到Cyclo [21，29] [d-Cys〜（21）Cys〜（29）]-uPA_（21-30）。为了分析uPAR结合活性，我们采用竞争性流式细胞荧光受体结合测定法，以FITC-uPA作为配体，U937早幼粒白血病细胞作为uPAR的细胞来源。如对环[21,29] [d-Cys〜（21）Cys〜（29）]-uPA_（21-30）所证明的，实现的肽修饰可维持受体结合活性（IC_（50）= 0.04μM），为了接近亲本蛋白配体的uPA（IC_（50）= 0.01μM），它是接近的。在明确的H_2O中进行的具有受限和自由分子动力学计算的详细NMR分析显示出结构明确的结构，具有特征特征，例如带有Lys〜3，Tyr〜4，Ser〜6和Asn〜的两个βI转向的ω环。 7。观察到Tyr〜4，Phe〜5，Ile〜8和Trp〜（10）的侧链疏水团聚。侧链流动性通过与时间相关的距离约束进行分析。环[21,29] [d-Cys〜（21）Cys〜（29）]-uPA_（21-30）的NMR结构与先前报道的uPA氨基末端片段的结构非常相似。系统性点突变导致环[21,29] [d-Cys〜（21）Nle〜（23）Cys〜（29）]-uPA_（21-30），其仍与uPAR结合，但对蛋白水解酶具有抗性，例如，通过肿瘤相关的丝氨酸蛋白酶uPA和纤溶酶，并且在血清或血浆中稳定。总之，创建了小的环状肽，其模仿了uPA与uPAR结合表位的结构和活性，并且可以作为癌症转移中的新型治疗剂。

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