Tran John C, Hunsaker Thomas, Bell Christina, Ma Taylur P, Chan Emily, Larrocha Pablo Saenz-Lopez, Homyk Kelsey, Liu Liling, La Hank, Mao Jialin, de la Cruz Cecile C, Yu Kebing, Beresini Maureen, Forrest William F, Xiao Yang, Jang Anne, Samus Natalia, Stesco Nicholas Dupuis, Mentinova Marija, Parent Stephane, Pottiez Gwenael, Schirm Michael, Purkey Hans E, Liu Yichin, Merchant Mark
CellCarta, Montreal, Quebec H2X 3Y7, Canada.
Anal Chem. 2023 Mar 21;95(11):4834-4839. doi: 10.1021/acs.analchem.2c04417. Epub 2023 Mar 6.
The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.
共价药物抑制剂（如KRAS G12C抑制剂）所展现出的越来越多的机会，正推动着对质谱方法的需求，这些方法能够快速且可靠地测量用于药物发现研究与开发的治疗性药物活性。有效的前端样品制备对于从肿瘤中提取的蛋白质至关重要，但通常劳动强度大，且对于药效学（PD）研究中典型的大量样品而言不切实际。在此，我们描述了一种自动化且集成的样品制备方法，用于测量复杂肿瘤样品中KRAS G12C药物抑制剂烷基化的活性水平，该方法包括高通量去除去污剂和预浓缩，随后使用质谱进行定量。我们引入了一种稳健的检测方法，从七项研究中获得的平均批内变异系数（CV）为4%，批间CV为6%，这使我们能够了解KRAS G12C靶点占有率与小鼠肿瘤样品的治疗性PD效应之间的关系。此外，数据表明，候选药物GDC - 6036（一种KRAS G12C共价抑制剂）显示出剂量依赖性的靶点抑制（KRAS G12C烷基化）和MAPK途径抑制，这与MIA PaCa - 2胰腺异种移植模型中的高抗肿瘤效力相关。
