Frommlet Alexandra, Nguyen Lan K, Saabye Matt, Endres Nicholas F, Mulvihill Melinda M, Quinn John G
Department of Biochemical and Cellular Pharmacology, Genentech, Inc., South San Francisco, California 94080, United States.
Confluence, Saint Louis, Missouri 63110, United States.
ACS Omega. 2024 Dec 16;9(52):51508-51514. doi: 10.1021/acsomega.4c08774. eCollection 2024 Dec 31.
We measure the fundamental rate constants of internally discovered KRAS G12C inhibitors to demonstrate how kinetic analyses can be integrated with standard biochemical and cell-based assays for more optimal biophysical compound prioritization. In this proof-of-principle study, we characterize three irreversible covalent inhibitors targeting the mutant cysteine at the switch II binding pocket. We estimate the three fundamental kinetic rate constants ( , , ) that define the contributions of affinity and inactivation to the overall alkylation rate for a more complete biophysical characterization. These parameters are typically unavailable and are generally approximated by a single overall alkylation rate constant ( ), where the relative contributions of affinity and inactivation remain unknown. We demonstrate that the alkylation rate constant sacrifices valuable mechanistic information leading to higher risk of suboptimal compound prioritization. Estimation of the three fundamental kinetic rate constants was made possible by developing label-free surface plasmon resonance (SPR) methodologies adapted to measure transient binding using standard SPR equipment. Binding enthalpy was measured by Eyring transition state analysis, which can also benefit compound prioritization. We illustrate how these methodologies can enable more reliable prioritization of lead-like compounds when combined with standard orthogonal assays in a typical lead optimization setting.
我们测量了内部发现的KRAS G12C抑制剂的基本速率常数，以展示如何将动力学分析与标准生化和基于细胞的检测相结合，从而更优化地进行生物物理化合物的优先级排序。在这项原理验证研究中，我们表征了三种靶向开关II结合口袋处突变半胱氨酸的不可逆共价抑制剂。我们估计了定义亲和力和失活对总体烷基化速率贡献的三个基本动力学速率常数（ 、 、 ），以进行更完整的生物物理表征。这些参数通常无法获得，通常由单个总体烷基化速率常数（ ）近似，其中亲和力和失活的相对贡献仍然未知。我们证明，烷基化速率常数牺牲了有价值的机制信息，导致次优化合物优先级排序的风险更高。通过开发适用于使用标准SPR设备测量瞬态结合的无标记表面等离子体共振（SPR）方法，可以估计三个基本动力学速率常数。通过艾林过渡态分析测量结合焓，这也有助于化合物的优先级排序。我们说明了在典型的先导优化环境中，当这些方法与标准正交检测相结合时，如何能够更可靠地对类先导化合物进行优先级排序。
