Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor
Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor
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DOI：
10.1073/pnas.96.22.12257
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发表时间：
1999-10-26
影响因子：
11.1
通讯作者：
White, KB
中科院分区：
文献类型：
--
作者：
Andrade-Gordon, P;Mayanoff, BE;White, KB
蛋白水解酶激活受体(PARs)代表一个独特的七个跨膜G蛋白偶联受体家族，它们被酶切后暴露出一个截短的细胞外N末端，作为一个被拴住的激活配体。PAR-1被丝氨酸蛋白酶α-凝血酶裂解并激活，在各种组织(如血小板和血管细胞)中表达，并参与与止血、增殖和组织损伤相关的细胞反应。我们已经发现了一系列有效的PAR-I模拟多肽拮抗剂，以RWJ-56110为例。利用PAR-I激动肽表位SFLLRN的重要功能基团之间的空间关系，设计并合成了固定在刚性分子支架上的合适基团的候选配体。通过化学库的固相平行合成，对原型RWJ-53052进行了鉴定和优化。RWJ-56110是一种有效的、选择性的PAR-1拮抗剂，缺乏PAR-1激动剂和凝血酶抑制活性。它与PAR-1结合，干扰PAR-1的钙动员和细胞功能(血小板聚集；细胞增殖)，对PAR-2、PAR-3或PAR-4没有影响。经流式细胞仪检测，RWJ-56110可直接抑制PAR-1的激活和内化，而不影响其N端的切割作用。在高浓度的α-凝血酶时，RWJ-56110完全阻断了人血管细胞的激活反应，尽管在人的血小板中没有；而在高浓度的SFLLRN-NH2时，RWJ-56110阻断了这两种细胞的激活反应。因此，凝血酶独立于PAR-I激活人血小板，即通过PAR-4激活，这一点我们通过聚合酶链式反应分析证实。选择性的PAR-1拮抗剂，如RWJ-56110，可以作为研究PAR的有用工具，并可能在治疗血栓形成和再狭窄方面具有治疗潜力。
Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-I, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-I agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH2, RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-I, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.