Xue Liang, van Kalken Daniel, James Erika M, Giammo Giulia, Labenski Matthew T, Cantin Susan, Fahnoe Kelly, Worm Karin, Wang Zhigang, Corin Alan F
Leads Discovery & Optimization, Bristol Myers Squibb, 250 Water Street, Cambridge, Massachusetts 02141, United States.
ACS Pharmacol Transl Sci. 2024 Aug 1;7(8):2507-2515. doi: 10.1021/acsptsci.4c00326. eCollection 2024 Aug 9.
Establishing target engagement is fundamental to effective target-based drug development. It paves the way for efficient medicinal chemistry design and definitive answers about target validation in the clinic. For irreversible targeted covalent inhibitor (TCI) drugs, there is a unique opportunity to establish and quantify the target engagement or occupancy. This is typically accomplished by using a covalent molecular probe, often a TCI analogue, derivatized to allow unoccupied target sites to be tracked; the difference of total sites minus unoccupied sites yields the occupied sites. When such probes are not available or the target is not readily accessible to covalent probes, another approach is needed. Receptor tyrosine-protein kinase erbB-2 (HER2) occupancy by afatinib presents such a case. Available HER2 covalent probes were unable to consistently modify HER2 after sample preparation, resulting in inadequate data. We demonstrate an alternative quantitative probe-free occupancy (PFO) method. It employs the immunoprecipitation of HER2 and direct mass spectrometer analysis of the cysteine-containing peptide that is targeted and covalently occupied by afatinib. Nontarget HER2 peptides provide normalization to the total protein. We show that HER2 occupancy by afatinib correlates directly to the inhibition of the receptor tyrosine kinase activity in NCI-N87 cells in culture and using those cells in a mouse tumor xenograft mode.
建立靶点结合对于基于靶点的有效药物开发至关重要。它为高效的药物化学设计以及临床中关于靶点验证的确切答案铺平了道路。对于不可逆靶向共价抑制剂（TCI）药物，存在建立和量化靶点结合或占有率的独特机会。这通常通过使用共价分子探针来实现，该探针通常是TCI类似物，经过衍生化处理以追踪未被占据的靶点位点；总位点减去未被占据的位点之差即为被占据的位点。当此类探针不可用或共价探针难以接近靶点时，就需要另一种方法。阿法替尼对受体酪氨酸蛋白激酶erbB-2（HER2）的占有率就属于这种情况。现有的HER2共价探针在样品制备后无法持续修饰HER2，导致数据不足。我们展示了一种替代性的无探针定量占有率（PFO）方法。它采用HER2的免疫沉淀以及对被阿法替尼靶向并共价占据的含半胱氨酸肽段进行直接质谱分析。非靶向HER2肽段用于对总蛋白进行标准化。我们表明，阿法替尼对HER2的占有率与培养的NCI-N87细胞中受体酪氨酸激酶活性的抑制直接相关，并且在小鼠肿瘤异种移植模型中使用这些细胞时也是如此。
