Activity-based protein profiling (ABPP) has been used extensively to discover and optimize selective inhibitors of enzymes. Here, we show that ABPP can also be implemented to identify the converse—small-molecule enzyme activators. Using a kinetically controlled, fluorescence polarization-ABPP assay, we identify compounds that stimulate the activity of LYPLAL1—a poorly characterized serine hydrolase with complex genetic links to human metabolic traits. We apply ABPP-guided medicinal chemistry to advance a lead into a selective LYPLAL1 activator suitable for use in vivo. Structural simulations coupled to mutational, biochemical and biophysical analyses indicate that this compound increases LYPLAL1’s catalytic activity likely by enhancing the efficiency of the catalytic triad charge-relay system. Treatment with this LYPLAL1 activator confers beneficial effects in a mouse model of diet-induced obesity. These findings reveal a new mode of pharmacological regulation for this large enzyme family and suggest that ABPP may aid discovery of activators for additional enzyme classes.

基于活性的蛋白质分析 (ABPP) 已被广泛用于发现和优化酶的选择性抑制剂。在这里，我们展示了 ABPP 也可以用于识别相反的小分子酶激活剂。使用动力学控制的荧光偏振-ABPP 分析，我们确定了刺激 LYPLAL1 活性的化合物，LYPLAL1 是一种特征不佳的丝氨酸水解酶，与人类代谢特征具有复杂的遗传联系。我们应用 ABPP 指导的药物化学将先导推进到适合体内使用的选择性 LYPLAL1 激活剂中。结合突变、生化和生物物理分析的结构模拟表明，该化合物可能通过提高催化三联体电荷中继系统的效率来增加 LYPLAL1 的催化活性。用这种 LYPLAL1 激活剂治疗对饮食诱导的肥胖小鼠模型产生了有益的影响。这些发现揭示了这一大酶家族的一种新的药理调节模式，并表明 ABPP 可能有助于发现其他酶类的激活剂。