Highly Multiplexed Kinase Profiling in Spleen with Targeted Mass Spectrometry Reveals Kinome Plasticity across Species

Early attrition of drug candidates, including kinase inhibitors, often occurs due to issues that arise during preclinical safety and efficacy evaluation. This problem may be exacerbated by the fact that these studies might fail to consider the basic physiological differences that could exist between human patients and animal models. We report the development of a targeted mass spectrometry-based assay capable of monitoring >50 different kinases using peptides conserved in humans and the key preclinical species used in drug development (mouse, rat, dog, and cynomolgus monkey). These methods were then used to profile interspecies kinome variability in spleen with three of the current techniques used in targeted proteomics (MRM, PRM, and IS-PRM). IS-PRM provides the highest number of kinase identifications, and the results indicate that while this initial set of kinases exhibits high correlation between species for this tissue type, distinct species-specific differences do exist, especially within the cyclin-dependent kinase family. An initial screen in two species with the kinase inhibitor dasatinib in competition with the chemoproteomic kinase-binding probe XO44 demonstrated how the targeted methods can be further applied to study species-specific inhibitor occupancy profiles. Understanding such differences could help rationalize the findings of preclinical studies and have major implications for the selection of these animals as models in kinase drug development.包括激酶抑制剂（kinase inhibitors）在内的药物候选分子早期研发失败，常因临床前安全性与有效性评价过程中出现的问题所致。此类问题可能因以下事实进一步恶化：这些研究往往未考虑人类患者与动物模型之间可能存在的基础生理差异。本研究报道了一种基于靶向质谱（mass spectrometry）的检测方法的开发过程，该方法可利用人类及药物研发中常用的关键临床前实验动物（小鼠、大鼠、犬、食蟹猴）体内保守的肽段，对超过50种不同的激酶进行监测。随后，我们利用靶向蛋白质组学（targeted proteomics）的三种主流技术——多反应监测（MRM, Multiple Reaction Monitoring）、平行反应监测（PRM, Parallel Reaction Monitoring）及内标平行反应监测（IS-PRM, Internal Standard-Parallel Reaction Monitoring），对脾脏组织中的种间激酶组变异度进行了分析。结果显示，内标平行反应监测可鉴定到最多数量的激酶；尽管该初始激酶集合在该组织类型中呈现出较高的种间相关性，但确实存在显著的物种特异性差异，尤其是在细胞周期蛋白依赖性激酶家族（cyclin-dependent kinase family）中。我们针对两种实验动物开展了初步筛选实验：使用激酶抑制剂达沙替尼（dasatinib）与化学蛋白质组学激酶结合探针XO44进行竞争性结合实验，验证了该靶向检测方法可进一步用于研究物种特异性的抑制剂结合占据谱。明晰此类差异有助于阐释临床前研究的实验结果，同时对选择此类动物作为激酶类药物研发的模型动物具有重要意义。