TRANSITION-STATE ANALOG INHIBITORS OF THIOL PROTEASES

本提案涉及硼对半胱氨酸蛋白酶的抑制作用。 酸过渡态类似物。硼酸是众所周知的阻断剂 丝氨酸和金属水解酶。它们还没有被研究过与 硫醇酶。早些时候观察这种禁忌的尝试都失败了。 在这个实验室最近完成的初步工作中，它已经显示出 组织蛋白酶C(二肽基肽酶I，E.C.3.4.14.1) 被芳基硼酸抑制。 我们打算研究硼酸对硫醇蛋白水解酶的抑制。 精氨酸和苯丙氨酸的类似物。酶抑制作用的pH依赖性 这些酶与丝氨酸已有的类似数据相结合 蛋白水解酶有望帮助显示硫醇和丝氨酸蛋白酶在 它们的酰化和脱酰化机理。使用的酶将是 木瓜蛋白酶是一种被广泛研究的具有精氨酸特异性的硫醇蛋白酶 以及组织蛋白酶C，这是一种研究较少的硫醇蛋白水解酶，具有 S1亚基上芳香族氨基酸的专一性。 这项研究将涉及多肽硼酸的化学合成： 组织蛋白酶C为Gly-BoroPhe，木瓜蛋白酶为Ala-Ala-Phe-BoroArg。这将是 之后是对这些影响的稳态动力学研究 在不同的pH值下，化合物对相应的酶有抑制作用。(BoroPhe是 苯丙氨酸的硼酸类似物，用硼取代 羧酸碳原子；硼精氨酸是类似的精氨酸衍生物。) 小分子抑制剂对硫醇蛋白水解酶的抑制作用 生物学上有趣的话题，因为它已经被证明是多肽 醛既能抑制丝氨酸酶，又能抑制硫醇蛋白水解酶，可延缓发病。 肌肉营养不良动物模型中肌肉退化的研究。这 疾病与肌肉蛋白水解酶水平升高有关，包括 因此，组织蛋白酶C的特异性抑制物可能是药物 对这种疾病的研究很有用。这项研究包括第一个 含硼精氨酸多肽的制备。成功地合成了这样的化合物 从长远来看，多肽将使研究它们的 与生物学上有趣的类胰蛋白酶丝氨酸蛋白酶的相互作用。 例如凝血级联反应的酶，以及纤溶酶 和纤溶酶原激活剂。

This proposal deals with the inhibition of cysteine proteases by boronic acid transition state analogs. Boronic acids are well-known inhibitors of serine and metallo-hydrolases. They have not been studied in relation to thiol enzymes. Earlier attempts to observe such inhibitions had failed. In preliminary work done recently in this laboratory, it has been shown that the thiol protease cathepsin C (dipeptidyl peptidase I, E.C. 3.4.14.1) is inhibited by arylboronic acids. We intend to study the inhibition of thiol proteases by boronic acid analogs of arginine and phenylalanine. The pH-dependence of inhibition for these enzymes combined with similar data already available for the serine proteases is expected to help show how thiol and serine proteases differ in their mechanisms of acylation and deacylation. The enzymes used will be papain an extensively studied thiol protease with an argining specificity at the S1 subsite, and cathepsin C, a less-well studied thiol protease with a specificity for aromatic amino acids in the S1 subsite. This study will involve the chemical synthesis of peptide boronic acids: Gly-BoroPhe for cathepsin C, and Ala-Ala-Phe-BoroArg for papain. This will be followed by a steady-state kinetic investigation of the effects of these compounds on the appropriate enzyme at different pH values. (BoroPhe is the boronic acid analog of phenylalanine, with boron substituting for the carboxylic acid carbon atom; BoroArg is the analogous arginine derivative.) The inhibition of thiol proteases by small-molecule inhibitors is a biologically interesting topic since it has been shown that peptide aldehydes, which inhibit both serine and thiol proteases, delay the onset of muscle deterioration in animal models of muscular dystrophy. This disease is associated with elevated levels of muscle proteinases, including cathepsin C. Therefore, specific inhibitors of cathepsin C may be agents useful in the study of this disease. This study includes the first preparation of peptides containing BoroArg. Successful synthesis of such peptides will make it possible, in the longer term, to study their interaction with biologically interesting trypsin-like serine proteases. Examples are the enzymes of the blood-clotting cascade, as well as plasmin and plasminogen activator.