Larson Jacob E, Hardy P Brian, Schomburg Noah K, Wang Xiaodong, Kireev Dmitri, Rossman Kent L, Pearce Kenneth H
UNC Eshelman School of Pharmacy, Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
Department of Surgery, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599.
SLAS Discov. 2023 Jan;28(1):39-47. doi: 10.1016/j.slasd.2022.12.001. Epub 2022 Dec 21.
Mutations in the small GTPase protein KRAS are one of the leading drivers of cancers including lung, pancreatic, and colorectal, as well as a group of developmental disorders termed "Rasopathies". Recent breakthroughs in the development of mutant-specific KRAS inhibitors include the FDA approved drug Lumakras (Sotorasib, AMG510) for KRAS G12C-mutated non-small cell lung cancer (NSCLC), and MRTX1133, a promising clinical candidate for the treatment of KRAS G12D-mutated cancers. However, there are currently no FDA approved inhibitors that target KRAS mutations occurring at non-codon 12 positions. Herein, we focused on the KRAS mutant A146T, found in colorectal cancers, that exhibits a "fast-cycling" nucleotide mechanism as a driver for oncogenic activation. We developed a novel high throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay that leverages the reduced nucleotide affinity of KRAS A146T. As designed, the assay is capable of detecting small molecules that act to allosterically modulate GDP affinity or directly compete with the bound nucleotide. A pilot screen was completed to demonstrate robust statistics and reproducibility followed by a primary screen using a diversity library totaling over 83,000 compounds. Compounds yielding >50% inhibition of TR-FRET signal were selected as hits for testing in dose-response format. The most promising hit, UNC10104889, was further investigated through a structure activity relationship (SAR)-by-catalog approach in an attempt to improve potency and circumvent solubility liabilities. Overall, we present the TR-FRET platform as a robust assay to screen fast-cycling KRAS mutants enabling future discovery efforts for novel chemical probes and drug candidates.
小GTPase蛋白KRAS的突变是包括肺癌、胰腺癌和结直肠癌在内的多种癌症的主要驱动因素之一，也是一组称为“Rasopathies”的发育障碍的驱动因素。突变特异性KRAS抑制剂开发方面的最新突破包括美国食品药品监督管理局（FDA）批准的用于治疗KRAS G12C突变的非小细胞肺癌（NSCLC）的药物Lumakras（索托拉西布，AMG510），以及有望用于治疗KRAS G12D突变癌症的临床候选药物MRTX1133。然而，目前尚无FDA批准的针对非12密码子位置发生的KRAS突变的抑制剂。在此，我们聚焦于在结直肠癌中发现的KRAS突变体A146T，其表现出一种“快速循环”的核苷酸机制，作为致癌激活的驱动因素。我们开发了一种新型的高通量时间分辨荧光共振能量转移（TR-FRET）检测方法，该方法利用了KRAS A146T降低的核苷酸亲和力。按照设计，该检测方法能够检测通过变构调节GDP亲和力或直接与结合的核苷酸竞争而起作用的小分子。首先完成了初步筛选以证明可靠的统计学数据和可重复性，随后使用一个总计超过83,000种化合物的多样性文库进行了初次筛选。对TR-FRET信号抑制率>50%的化合物被选为用于剂量反应形式测试的命中化合物。最有前景的命中化合物UNC10104889通过基于目录的构效关系（SAR）方法进一步研究，以提高效力并规避溶解性问题。总体而言，我们展示了TR-FRET平台作为一种强大的检测方法，用于筛选快速循环的KRAS突变体，为未来发现新型化学探针和候选药物提供助力。
