Identification of Small Molecules that Disrupt Signaling between ABL and Its Positive Regulator RIN1

Constitutively active BCR-ABL kinase fusions are causative mutations in the pathogenesis of hematopoietic neoplasias including chronic myelogenous leukemia (CML). Although these fusions have been successfully targeted with kinase inhibitors, drug-resistance and relapse continue to limit long-term survival, highlighting the need for continued innovative drug discovery. We developed a time-resolved Förster resonance energy transfer (TR-FRET) -based assay to identify compounds that disrupt stimulation of the ABL kinase by blocking its ability to bind the positive regulator RIN1. This assay was used in a high throughput screen (HTS) of two small molecule libraries totaling 444,743 compounds. 708 confirmed hits were counter-screened to eliminate off-target inhibitors and reanalyzed to prioritize compounds with IC50 values below 10 μM. The CML cell line K562 was then used to identify five compounds that decrease MAPK1/3 phosphorylation, which we determined to be an indicator of RIN1-dependent ABL signaling. One of these compounds is a thiadiazole, and the other four are structurally related acyl piperidine amides. Notably, these five compounds lower cellular BCR-ABL1 kinase activity by blocking a positive regulatory interaction rather than directly inhibiting ABL catalytic function.组成型活化的BCR-ABL激酶融合变异是包括慢性髓系白血病（chronic myelogenous leukemia, CML）在内的造血系统恶性肿瘤发病机制中的致病性突变。尽管这类融合变异已可通过激酶抑制剂实现靶向治疗，但耐药性与复发仍持续制约患者的长期生存，这凸显了持续开展创新性药物研发的必要性。我们开发了一种基于时间分辨福斯特共振能量转移（time-resolved Förster resonance energy transfer, TR-FRET）的检测方法，用于筛选可通过阻断ABL激酶与正调控因子RIN1结合、从而破坏ABL激酶激活通路的小分子化合物。我们利用该检测方法对总计包含444743个化合物的两个小分子化合物库开展了高通量筛选（high throughput screen, HTS）。对708个经确认的阳性命中化合物进行了复筛以剔除脱靶抑制剂，并重新分析以优先选取半数抑制浓度（half maximal inhibitory concentration, IC50）低于10 μM的化合物。随后我们使用慢性髓系白血病细胞系K562，筛选出5种可降低MAPK1/3磷酸化水平的化合物，我们证实该磷酸化水平可作为RIN1依赖型ABL信号通路的检测指标。其中1种化合物为噻二唑类，其余4种均为结构相似的酰基哌啶酰胺类化合物。值得注意的是，这5种化合物并非直接抑制ABL激酶的催化活性，而是通过阻断正调控相互作用来降低细胞内BCR-ABL1激酶的活性。