Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
J Am Chem Soc. 2012 Jun 27;134(25):10345-8. doi: 10.1021/ja303400u. Epub 2012 Jun 12.
The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.
小分子抑制剂的开发用于干扰酶的功能，需要进行测定以确认抑制剂在体内与酶的靶标相互作用。对于缺乏已知生物标志物（例如内源性底物和产物）来报告其抑制作用的特征描述不佳的酶，确定体内靶标占有率可能特别具有挑战性。在这里，我们描述了一种用于在动物模型中测量可逆抑制剂与酶结合的竞争性基于活性的蛋白质分析（ABPP）方法。该方法成功的关键是使用反应性适中的基于活性的探针，以便可以在体内测量与可逆抑制剂的靶标占有率竞争。我们将竞争性 ABPP 策略应用于评估一类新描述的哌嗪酰胺类可逆抑制剂对丝氨酸水解酶 LYPLA1 和 LYPLA2 的抑制作用，这两种酶缺乏选择性、体内有效的抑制剂。竞争性 ABPP 鉴定出单独的哌嗪酰胺类抑制剂，可在小鼠中选择性抑制 LYPLA1 或 LYPLA2。总之，适应性强、反应性适中的探针可用于评估动物模型中可逆抑制剂的靶标占有率，以促进用于体内表征酶功能的小分子探针的发现。
