Exploiting new approaches for selective inhibition of trypsins

项目摘要/摘要 人胰酶亚型，胰酶1，胰酶2，和中胰蛋白酶，都是与 癌症和胰腺炎的疾病过程，并可能提供可行的治疗靶点。胰酶属于一种 具有相似活性部位拓扑结构的胰酶样酶大家族，因此现有的抑制剂缺乏 选择性。需要选择性胰酶抑制剂和异构体选择性胰酶抑制剂 药理学工具，以更好地定义这些单独的酶在疾病中的功能，并评估 胰酶抑制作为临床前疾病模型的一种治疗策略。在这个项目中，我们将采取一个 多管齐下，开发新的策略，有效和选择性地抑制每个人类 胰酶亚型。(1)我们的初步数据揭示了一种先前未被怀疑的自动抑制构象 具有配体靶向的变构位点的中胰蛋白酶，可用于抑制作用。我们将使用高- 产能虚拟筛选和基于结构的点击到领先优化，以开发潜力和选择性 中胰蛋白酶的变构抑制剂。我们还将使用结构和分子动力学分析来评估 是否类似的策略可能对胰蛋白酶1和2具有潜力。(2)我们发表的研究表明， 库尼茨结构域可以被改造成产生更具选择性的基于蛋白的类胰蛋白酶抑制剂 通过使用酵母表面展示(YSD)平台进行定向进化。为了进一步优化这种 抑制剂，我们试图生成结合特异性景观的全面地图，可以，对于任何 抑制物内突变的可能组合，预测对抑制物亲和力和 对一组目标蛋白酶的专一性。我们将通过集成YSD组合来完成这项任务 使用下一代测序(NGS)、机器学习(ML)方法筛选文库，以及 能够定量预测多个潜在相互作用的影响的实验校准 一种抑制物的突变。这些数据将使我们能够确定最有效和最有选择性的库尼茨结构域 这可以通过靶向每一种人类胰酶来实现。(3)我们的初步数据显示 能够同时与两个分子结合的双价抑制剂增强胰酶亲和力 中胰蛋白酶。在这里，我们将剖析负责这些亲和力增强和设计的机制 利用这些信息开发更有效和更有选择性的多价胰酶的策略 抑制剂。除了制定三个相辅相成的战略外，每一个战略都具有很高的潜力 生产所需的人类胰酶选择性抑制剂，我们的项目将提供更广泛的见解，有助于 其他许多重要的类胰蛋白酶的抑制剂的未来发展。最后，新的方法论 这里开发的用于绘制蛋白质-蛋白质相互作用(PPI)亲和力和特异性的图景将是广泛的 用于表征支配官能团亲和力和选择性的序列和结构约束的效用 许多其他不同系统中的蛋白质相互作用。

PROJECT SUMMARY/ABSTRACT The human trypsin isoforms, trypsin 1, trypsin 2, and mesotrypsin, are proteases that have been implicated in disease processes in cancer and pancreatitis, and may offer viable therapeutic targets. Trypsins belong to a large family of trypsin-like enzymes with similar active site topology, and hence existing inhibitors lack selectivity. There is a need for selective trypsin inhibitors and isoform-selective trypsin inhibitors as pharmacological tools to better define the functions of these individual enzymes in disease, and to evaluate trypsin inhibition as a therapeutic strategy in preclinical models of disease. In this project, we will take a multipronged approach to develop new strategies for potent and selective inhibition of each of the human trypsin isoforms. (1) Our preliminary data reveal a previously unsuspected auto-inhibited conformation of mesotrypsin with a ligand-targetable allosteric site that may be exploited for inhibitory effect. We will use high- throughput virtual screening and structure-based hit-to-lead optimization to develop potent and selective allosteric inhibitors of mesotrypsin. We will also use structural and molecular dynamics analyses to evaluate whether similar strategies may hold potential for trypsins 1 and 2. (2) Our published studies have shown that Kunitz domains can be engineered to create more selective protein-based inhibitors of trypsin-like proteases by using a yeast surface display (YSD) platform for directed evolution. To enable further optimization of such inhibitors, we seek to generate comprehensive maps of the binding specificity landscapes that can, for any possible combination of mutations within an inhibitor, predict the consequences on inhibitor affinity and specificity toward a set of target proteases. We will accomplish this task by integrating YSD combinatorial library screening with next-generation sequencing (NGS), machine-learning (ML) approaches, and experimental calibration to enable quantitative prediction of the impact of multiple potentially interacting mutations of an inhibitor. These data will enable us to identify the most potent and selective Kunitz domains that can be achieved for targeting each of the human trypsins. (3) Our preliminary data demonstrate an enhancement of trypsin affinity by bivalent inhibitors capable of binding simultaneously to two molecules of mesotrypsin. Here, we will dissect the mechanisms responsible for these affinity enhancements and design strategies to exploit this information toward development of more potent and selective polyvalent trypsin inhibitors. In addition to developing three complementary strategies, each of which has high potential to produce the desired selective inhibitors of human trypsins, our project will provide broader insights that can aid future development of inhibitors for many other important trypsin-like proteases. Finally, the novel methodology developed here for mapping protein-protein interaction (PPI) affinity and specificity landscapes will be of broad utility for characterizing the sequence and structural constraints governing affinity and selectivity of functional protein interactions in many other diverse systems.