F. Hoffmann-La Roche Ltd, Pharma Research & Early Development, Discovery Technologies, Basel, Switzerland.
MAbs. 2012 Jul-Aug;4(4):497-508. doi: 10.4161/mabs.20452. Epub 2012 Jul 1.
The serine protease chymase (EC = 3.4.21.39) is expressed in the secretory granules of mast cells, which are important in allergic reactions. Fynomers, which are binding proteins derived from the Fyn SH3 domain, were generated against human chymase to produce binding partners to facilitate crystallization, structure determination and structure-based drug discovery, and to provide inhibitors of chymase for therapeutic applications. The best Fynomer was found to bind chymase with a KD of 0.9 nM and koff of 6.6x10 (-4) s (-1) , and to selectively inhibit chymase activity with an IC 50 value of 2 nM. Three different Fynomers were co-crystallized with chymase in 6 different crystal forms overall, with diffraction quality in the range of 2.25 to 1.4 Å resolution, which is suitable for drug design efforts. The X-ray structures show that all Fynomers bind to the active site of chymase. The conserved residues Arg15-Trp16-Thr17 in the RT-loop of the chymase binding Fynomers provide a tight interaction, with Trp16 pointing deep into the S1 pocket of chymase. These results confirm the suitability of Fynomers as research tools to facilitate protein crystallization, as well as for the development of assays to investigate the biological mechanism of targets. Finally, their highly specific inhibitory activity and favorable molecular properties support the use of Fynomers as potential therapeutic agents.
糜蛋白酶（EC=3.4.21.39）是一种丝氨酸蛋白酶，存在于肥大细胞的分泌颗粒中，在过敏反应中起着重要作用。Fynomer 是一种来源于 Fyn SH3 结构域的结合蛋白，它被用来针对人源糜蛋白酶生成结合伴侣，以促进结晶、结构确定和基于结构的药物发现，并提供糜蛋白酶抑制剂用于治疗应用。研究发现，最佳的 Fynomer 与人源糜蛋白酶的 KD 值为 0.9 nM，koff 值为 6.6x10(-4)s(-1)，并对糜蛋白酶活性具有选择性抑制作用，IC50 值为 2 nM。三种不同的 Fynomer 与糜蛋白酶共结晶，共获得 6 种不同晶体形式，衍射质量在 2.25 到 1.4 Å 分辨率范围内，适合药物设计工作。X 射线结构表明，所有的 Fynomer 都结合在糜蛋白酶的活性部位。糜蛋白酶结合 Fynomer 的 RT 环中的保守残基 Arg15-Trp16-Thr17 提供了紧密的相互作用，其中 Trp16 指向糜蛋白酶的 S1 口袋深处。这些结果证实了 Fynomer 作为研究工具的适用性，可促进蛋白质结晶，并可用于开发研究靶标生物学机制的测定方法。最后，它们具有高度特异性的抑制活性和有利的分子特性，支持将 Fynomer 用作潜在的治疗剂。
