Lin Hanfeng, Yang Bin, Ding Lang, Yang Yen-Yu, Holt Matthew V, Jung Sung Yun, Zhang Bing, Wang Meng C, Wang Jin
bioRxiv. 2025 Jun 22:2025.06.19.660637. doi: 10.1101/2025.06.19.660637.
Covalent inhibitors are an emerging class of therapeutics, but methods to comprehensively profile their binding kinetics and selectivity across the proteome have been limited. Here we introduce COOKIE-Pro (COvalent Occupancy KInetic Enrichment via Proteomics), an unbiased method for quantifying irreversible covalent inhibitor binding kinetics on a proteome-wide scale. COOKIE-Pro uses a two-step incubation process with mass spectrometry-based proteomics to determine and values for covalent inhibitors against both on-target and off-target proteins. We validated COOKIE-Pro using the BTK inhibitors spebrutinib and ibrutinib, accurately reproducing known kinetic parameters and identifying both expected and novel off-targets. The method revealed that spebrutinib has over 10-fold higher potency for TEC kinase compared to its intended target BTK. We further demonstrate that COOKIE-Pro is compatible with streamlined cysteine activity-based protein profiling (SLC-ABPP) datasets, enabling efficient conversion of competition ratios to meaningful kinetic parameters. By providing a comprehensive view of covalent inhibitor binding across the proteome, COOKIE-Pro represents a powerful new tool for optimizing the potency and selectivity of covalent drugs during preclinical development.
共价抑制剂是一类新兴的治疗药物，但全面分析其在蛋白质组中的结合动力学和选择性的方法一直有限。在此，我们介绍了COOKIE-Pro（通过蛋白质组学进行共价占据动力学富集），这是一种在蛋白质组范围内量化不可逆共价抑制剂结合动力学的无偏方法。COOKIE-Pro采用两步孵育过程结合基于质谱的蛋白质组学，以确定共价抑制剂针对靶标和脱靶蛋白的 $k_{on}$ 和 $k_{off}$ 值。我们使用布鲁顿酪氨酸激酶（BTK）抑制剂司布替尼和伊布替尼对COOKIE-Pro进行了验证，准确再现了已知的动力学参数，并识别出预期和新的脱靶蛋白。该方法表明，与预期靶标BTK相比，司布替尼对酪氨酸激酶（TEC）的效力高出10倍以上。我们进一步证明，COOKIE-Pro与简化的基于半胱氨酸活性的蛋白质谱分析（SLC-ABPP）数据集兼容，能够将竞争比有效地转化为有意义的动力学参数。通过全面了解共价抑制剂在蛋白质组中的结合情况，COOKIE-Pro代表了一种在临床前开发过程中优化共价药物效力和选择性的强大新工具。
