Dayal Neetu, Chaudhuri Riddhi, Yeboah Kofi Simpa, Brauer Nickolas R, Sintim Herman O
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA.
Purdue Institute for Drug Discovery, Purdue University, 720 Clinic Drive, West Lafayette, IN 47907, USA.
Pharmaceuticals (Basel). 2024 Dec 10;17(12):1660. doi: 10.3390/ph17121660.
The protein kinases CLK and ROCK play key roles in cell growth and migration, respectively, and are potential anticancer targets. ROCK inhibitors have been approved by the FDA for various diseases and CLK inhibitors are currently being trialed in the clinic as anticancer agents. Compounds with polypharmacology are desired, especially in oncology, due to the potential for high efficacy as well as addressing resistance issues. In this report, we have identified and characterized novel, boron-containing dual CLK/ROCK inhibitors with promising anticancer properties. A library of boronic acid-based CLK/ROCKi was synthesized via Povarov/Doebner-type multicomponent reactions. Kinase inhibition screening and cancer cell viability assays were performed to identify the hit compounds. To gain insights into the probable binding modes of the compounds to the kinases, docking studies were performed. Cell cycle analysis, qPCR and immunoblotting were carried out to further characterize the mode(s) of action of the lead candidates. At 25 nM, the top compounds and inhibited CLK1 and 2 and ROCK2 at greater than 70%. While also inhibited CLK4, the C1 methylated analog did not inhibit CLK4. Antitumor effects of the top compounds were evaluated and dose-response analysis indicated potent inhibition of renal cancer and leukemia cell growth. Immunoblotting results indicated that the top compounds induce DNA damage via upregulation of p-H2AX. Moreover, flow cytometry results demonstrated that the top compounds promote cell cycle arrest in the renal cancer cell line, Caki-1. qPCR and immunoblotting analysis upon dosing indicated suppression of cyclin D/Rb oncogenic pathway upon compound treatment. Novel boronic acid-containing pyrazolo[4,3-]quinoline-based dual CLK/ROCK inhibitors were identified. The so-called "magic methylation" design approach was used to tune CLK selectivity. Additionally, the findings demonstrate potent in vitro anticancer activity of the lead candidates against renal cancer and leukemia. This adds to the growing list of boron-containing compounds that display biological activities.
蛋白激酶CLK和ROCK分别在细胞生长和迁移中起关键作用，是潜在的抗癌靶点。ROCK抑制剂已获美国食品药品监督管理局（FDA）批准用于治疗多种疾病，CLK抑制剂目前正在临床中作为抗癌药物进行试验。由于具有高效的潜力以及能够解决耐药性问题，尤其在肿瘤学领域，人们期望获得具有多药理学特性的化合物。在本报告中，我们鉴定并表征了具有有望的抗癌特性的新型含硼双CLK/ROCK抑制剂。通过Povarov/Doebner型多组分反应合成了基于硼酸的CLK/ROCKi文库。进行了激酶抑制筛选和癌细胞活力测定以鉴定命中化合物。为了深入了解化合物与激酶可能的结合模式，进行了对接研究。进行了细胞周期分析、定量聚合酶链反应（qPCR）和免疫印迹以进一步表征先导候选物的作用模式。在25 nM时，顶级化合物 和 对CLK1和2以及ROCK2的抑制率大于70%。虽然 也抑制CLK4，但C1甲基化类似物 不抑制CLK4。评估了顶级化合物的抗肿瘤作用，剂量反应分析表明其对肾癌和白血病细胞生长有强效抑制作用。免疫印迹结果表明，顶级化合物通过上调p-H2AX诱导DNA损伤。此外，流式细胞术结果表明，顶级化合物促进肾癌细胞系Caki-1中的细胞周期停滞。给药后的qPCR和免疫印迹分析表明，化合物处理后细胞周期蛋白D/Rb致癌途径受到抑制。鉴定出了新型含硼的基于吡唑并[4,3-]喹啉的双CLK/ROCK抑制剂。采用所谓的“神奇甲基化”设计方法来调节CLK选择性。此外，研究结果证明了先导候选物对肾癌和白血病具有强大的体外抗癌活性。这增加了显示生物活性的含硼化合物的数量。
