Serine protease zymogen activation by small molecules

本项目的目标是确定选择性激活类糜蛋白酶丝氨酸蛋白酶酶原的小分子探针。虽然酶原被广泛地通过蛋白水解法去除前肽来处理，但这种激活机制在生物系统中很难控制。因此，许多蛋白水解酶的生物学功能仍然不清楚。在这里，我们建议筛选NIH小分子文库集合，以识别激活类糜蛋白酶样丝氨酸蛋白酶(CSP)酶原的小分子探针，而不需要对酶原进行蛋白分解处理。利用共振能量转移技术建立并优化了CSP活性化合物的高通量筛选方法。在二次筛选试验中，与相关家族成员相比表现出高效力选择性和结构相似的蛋白酶的化合物将被下调选择。非蛋白水解酶原激活将使用正交法进行验证。从二次筛选中挑选出的最佳热门将经过两到三轮药物化学，以进一步提高其效力和选择性。这项工作产生的化学探针将用于研究丝氨酸蛋白酶是如何被激活的，并研究CSP在肿瘤转移和突触可塑性中的作用。

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.