Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California at San Francisco, 1700 4th Street, San Francisco, CA 94143, USA.
Chem Biol Drug Des. 2012 Feb;79(2):209-15. doi: 10.1111/j.1747-0285.2011.01261.x. Epub 2011 Nov 28.
The development of highly selective small molecule inhibitors for individual caspases, a class of cysteine-dependent aspartate-specific proteases, has been challenging due to conservation of the active site. Previously, we discovered an allosteric site at the dimer interface of caspases-3, -7, and -1 using disulfide trapping. Here, we show this approach can generate selective tethered ligands and inhibitors for caspase-5, which is remarkable considering its high sequence similarity to caspase-1. Among the 62 hits of a screen of ∼15 000 thiol-containing fragments, a naphthyl-thiazole-containing molecule was identified that selectively inhibited and labeled the allosteric cysteine in the p10 subunit of caspase-5, but caused very little inhibition or labeling of caspase-1. Interestingly, some of allosteric tethered compounds to caspase-5 did not inhibit its enzymatic activity, suggesting that thiol-labeling itself is not sufficient to drive inhibition. These studies validate an allosteric site on caspase-5 and provide a useful starting point to develop selective compounds to probe the role of caspase-5 separate from caspase-1 in the innate immune response.
由于活性位点的保守性，针对半胱氨酸依赖性天冬氨酸特异性蛋白酶家族中个别胱天蛋白酶的高选择性小分子抑制剂的开发一直具有挑战性。先前，我们使用二硫键捕获在胱天蛋白酶-3、-7 和 -1 的二聚体界面上发现了一个别构位点。在这里，我们展示了这种方法可以生成针对 caspase-5 的选择性连接配体和抑制剂，这是值得注意的，因为它与 caspase-1 的序列高度相似。在对约 15000 个含硫片段进行的筛选中，发现了一种含有萘基噻唑的分子，它选择性地抑制和标记了 caspase-5 的 p10 亚基中的别构半胱氨酸，但对 caspase-1 的抑制或标记作用很小。有趣的是，一些别构连接化合物对 caspase-5 没有抑制其酶活性，这表明硫标记本身不足以驱动抑制。这些研究验证了 caspase-5 上的一个别构位点，并为开发选择性化合物提供了有用的起点，以研究 caspase-5 在先天免疫反应中与 caspase-1 分离的作用。
