Bialkowska Agnieszka, Crisp Melissa, Madoux Franck, Spicer Tim, Knapinska Ania, Mercer Becky, Bannister Thomas D., He Yuanjun, Chowdhury Sarwat, Cameron Michael, Yang Vincent W., Hodder Peter
Emory University (current affiliation Stony Brook University School of Medicine)
The Scripps Research Institute, Lead Identification Division, Jupiter, FL
Krüppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor that is highly expressed in rapidly dividing intestinal epithelial cells. KLF5 binds to GC-rich sequences in promoters of numerous genes, including cyclin D1, cyclin B1/Cdc2, and integrin-linked kinase. KLF5 mediates the transforming effects of oncogenic H-Ras, and as a result it is thought to be important in regulating colon cancer pathogenesis, thus KLF5 inhibitors are of interest in cancer chemotherapy. We used an ultra-high throughput screening (uHTS) strategy to identify and confirm KLF5-specific activity and potency of Molecular Libraries Small Molecule Repository (MLSMR) compounds, also using a cytotoxicity counterscreen with KLF5-deficient IEC-6 colon cell line to eliminate false positives. The SRIMSC performed the KLF5 and IEC-6 assays in dose response format, validating selected HTS hits for re-purchase and for follow-up studies. We initiated both structure activity relationship (SAR)-by-purchase and in-house synthesis campaigns. The assay provider evaluated hits and their analogs to determine potency, selectivity, and mechanism of action, establishing the necessary assays for proper evaluation (e.g., western blotting, cytotox evaluation, and cell cycle studies; all data were submitted to PubChem). The team ultimately chose to focus efforts on a lead series which provided a compound fully meeting the probe criteria for KLF5 inhibition: ML264, also designated CID 51003603, SID 117686865, and SR-03000002171. ML264 is highly active (IC = 29 nM is a cell-based assay for proliferation of DLD-1 cells, IC = 81 nM in a cell-based luciferase assay). It lacks cytotoxicity in the IEC-6 control cell line (IC >50 μM, <50% inhibition was observed at 100 μM). Robust activity was also seen in several other KLF5-expressing cell types as well (e.g., HCT116, IC = 560 nM; HT29, IC = 130 nM; SW620, IC = 430 nM). ML264 does not inhibit kinases associated with the KLF5 pathway, as determined using a panel of 47 selected kinases. Western blot analysis shows that ML264 significantly reduces KLF5 expression. These results demonstrate KLF5 target specificity. An NCI60 panel study using ML264 revealed that it induces death of most colon cancer cell lines, with cytotoxicity toward several other tumor cell lines as well. ML264 is chemically stable, unreactive with glutathione, has suitable aqueous solubility, is highly stable to mouse, rat, and human hepatic microsomes, has favorable properties associated with drug-likeness, and does not inhibit CYP enzymes. Due to these properties in concert with its high cellular potency and selectivity, ML264 is a good candidate for in vivo anticancer studies, with great potential for use in long time-course studies aimed to elucidate the role of KLF5 as a regulator of cellular proliferation and tumor formation in the intestinal epithelium.
Krüppel样因子5（KLF5）是一种含锌指的转录因子，在快速分裂的肠上皮细胞中高度表达。KLF5与众多基因启动子中富含GC的序列结合，这些基因包括细胞周期蛋白D1、细胞周期蛋白B1/Cdc2和整合素连接激酶。KLF5介导致癌性H-Ras的转化作用，因此被认为在调节结肠癌发病机制中起重要作用，所以KLF5抑制剂在癌症化疗中备受关注。我们采用超高通量筛选（uHTS）策略来鉴定和确认分子文库小分子储存库（MLSMR）化合物的KLF5特异性活性和效力，同时使用对KLF5缺陷的IEC-6结肠癌细胞系进行细胞毒性反筛选以消除假阳性。筛选资源整合与信息管理中心（SRIMSC）以剂量反应形式进行KLF5和IEC-6检测，验证所选的高通量筛选命中化合物以便重新购买和后续研究。我们启动了基于购买的构效关系（SAR）研究和内部合成项目。检测供应商评估命中化合物及其类似物以确定效力、选择性和作用机制，建立适当评估所需的检测方法（例如蛋白质印迹法、细胞毒性评估和细胞周期研究；所有数据都提交给了PubChem）。该团队最终选择专注于一个先导系列，该系列提供了一种完全符合KLF5抑制探针标准的化合物：ML264，也称为CID 51003603、SID 117686865和SR-03000002171。ML264具有高活性（在基于细胞的DLD-1细胞增殖检测中IC = 29 nM，在基于细胞的荧光素酶检测中IC = 81 nM）。它在IEC-6对照细胞系中无细胞毒性（IC >50 μM，在100 μM时观察到的抑制率<50%）。在其他几种表达KLF5的细胞类型中也观察到了强大的活性（例如，HCT116，IC = 560 nM；HT29，IC = 130 nM；SW620，IC = 430 nM）。使用一组47种选定的激酶进行检测发现，ML264不抑制与KLF5途径相关的激酶。蛋白质印迹分析表明，ML264显著降低KLF5表达。这些结果证明了KLF5的靶点特异性。使用ML264进行的NCI60细胞系研究表明，它能诱导大多数结肠癌细胞系死亡，对其他几种肿瘤细胞系也具有细胞毒性。ML264化学性质稳定，不与谷胱甘肽反应，具有合适的水溶性，对小鼠、大鼠和人肝微粒体高度稳定，具有与药物相似性相关的良好特性，并且不抑制CYP酶。由于这些特性以及其高细胞活性和选择性，ML264是体内抗癌研究的良好候选药物，在旨在阐明KLF5作为肠上皮细胞增殖和肿瘤形成调节因子作用的长期研究中具有巨大的应用潜力。
