Serine protease zymogen activation by small molecules

本项目的目的是确定小分子探针，选择性激活糜蛋白酶样丝氨酸蛋白酶酶原。虽然酶原广泛地通过蛋白水解去除前肽来加工，但是这种激活机制在生物系统中难以控制。因此，许多蛋白酶的生物学功能仍然不清楚。在这里，我们建议筛选NIH的小分子文库收集，以确定小分子探针，激活糜蛋白酶样丝氨酸蛋白酶（CSP）酶原，而不需要蛋白水解加工的酶原。CSP活化化合物的高通量筛选测定已经开发并使用共振能量转移进行优化。相对于相关家族成员和结构相似的蛋白酶表现出高效选择性的化合物将在二级筛选试验中被下调选择。将使用正交试验验证非蛋白水解酶原激活。从二次筛选中选出的最佳命中物将进行两到三轮药物化学，以进一步提高其效力和选择性。本工作所产生的化学探针将用于研究丝氨酸蛋白酶如何被激活，以及CSP在肿瘤转移和突触可塑性中的功能。

The objective of this project is to identify small molecule probes that selectively activate a chymotrypsin-like serine protease zymogen. Although zymogens are widely processed by proteolytic removal of a propeptide, this mechanism of activation is difficult to control in biological systems. Consequently, the biological functions of many proteases remain obscure. Here we propose to screen the NIH small molecule library collection to identify small molecule probes that activate a chymotrypsin-like serine protease (CSP) zymogen, without the requirement for proteolytic processing of the proenzyme. A high-throughput screening assay for CSP activating compounds has been developed and optimized using resonance energy transfer. Compounds that exhibit high potency selectivity over related family members, and structurally similar proteases will be downselected in secondary screening assays. Non-proteolytic zymogen activation will be verified using orthogonal assays. The best hits downselected from secondary screens will be subjected to two to three rounds of medicinal chemistry to further improve their potency and selectivity. The chemical probes arising from this work will be applied to study how serine proteases can be activated, and to investigate the functions of CSP in tumor metastasis and synaptic plasticity.