ZAK激酶选择性小分子抑制剂的设计、合成及生物活性研究

Leucine-zipper and sterile-α motif kinase (ZAK) is a member of mixed-lineage kinase, which is overexpressed in cardiac cells. ZAK plays crucial roles in the regulation of cardiac hypertrophy caused by external stimuli and the particular cell signaling pathway. Thus, ZAK has been considered as emerging potential molecular target for treatment of myocardial hypertrophy and myocardial fibrosis and other diseases. Several small molecule Bcr-Abl inhibitors nilotinib, dasatinib, bofutinib and Raf inhibitor sorafenib, were reported to potently suppress the kinase activity of ZAK. However, the low selectivity of these molecules limits their potential as tools for the validation of ZAK as drug target. It is highly desirable to identify selective ZAK inhibitors as chemical probes to further validate ZAK as drug target..We have successfully designed and synthesized a series of N-(3-((1H-pyrazolo[3,4-b]pyridin-5-yl)ethynyl)-2,4-difluorophenyl)sulfonic amides as selective ZAK inhibitors. One of the most promising compounds L2 tightly binds with ZAK with Kd value of 0.39 nM and inhibits its enzymatic activity of ZAK with IC50 values of 9 nM, but is significantly less potent against other kinases in a profiling study on a panel of 468 different kinases at the concentration of 1000 nM. The compound showed good selectivity, especially for relatively high homology ZAK categories such as Bcr-Abl and B-Raf with both IC50 values of > 1 μM. On the basis of previous investigation, we will continue study SAR research of compound L2, and will design and synthesis of new generation of ZAK inhibitors using rational drug design strategy. Besides, we will also investigate the potential application of ZAK inhibitors for the treatment of myocardial hypertrophy and its related diseases. It hopes to obtain high activity and good selectivity ZAK inhibitors as research tools for further verification ZAK as drug target.

ZAK激酶在心肌细胞中过度表达，并参与调控特性肥大性心肌细胞的生长，是开发治疗心肌肥大等相关疾病的潜在新靶标。目前报道的ZAK抑制剂存在选择性较差或活性较弱等缺陷，迫切需要高选择性抑制剂作为工具来验证ZAK作为药物靶标的可能性。我们通过化合物库筛选，成功设计合成了首个ZAK激酶选择性小分子抑制剂L2。L2对ZAK的抑制IC50为9 nM，结合常数Kd为0.39 nM。L2在1000 nM浓度下对体外468种激酶测试显示较好的选择性，尤其是对ZAK同源性较高的蛋白如Bcr-Abl和B-Raf等抑制活性很弱(IC50 > 1 μM)。本项目在前期基础上，拟通过合理药物设计策略设计全新结构骨架的ZAK小分子抑制剂，并进行系统构效关系研究，针对优选化合物进而研究其对心肌肥大的潜在治疗作用，计划获得活性和特异性都较好的新型ZAK抑制剂，为进一步验证ZAK激酶作为药物靶标提供研究工具。

心肌肥大（HCM）是最常见的遗传性心血管疾病，终末期引发心率失常、心力衰竭、中风、心源性猝死等一系列反应。目前对心肌肥大发生的机制和最佳治疗方法仍不明确。激酶ZAK已被证明是治疗心肌肥大的新潜在靶标。ZAK的过度表达通过激活下游c-Jun N末端蛋白激酶（JNK）和p38丝裂原活化蛋白激酶（MAPK）途径与心脏肥大和心肌纤维化的发展密切相关。根据ZAK激酶在心肌肥大疾病中的作用，利用合理药物设计技术，总计合成杂环炔和三氮唑杂环两大类具有全新结构的，活性更强和选择性更高的ZAK小分子抑制剂，并进行构效关系研究，以及初步的成药性研究。总计合成了两大类约120个ZAK 小分子抑制剂，并进行的体外活性和作用机制研究，研究为抗心肌肥大药物研究提供基础。代表化合物10e与ZAK结合常数Kd值为8.0 nM，并对ZAK具有较强的激酶抑制活性，其IC50值为4.0 nM（对同源性蛋白BrafV600E抑制 IC50 >1uM），同时对468种激酶（包括403种非突变激酶）表现出很好的选择性。进一步的机制研究表明10e可以剂量依赖性地抑制ZAK诱导心肌肥大通路下游蛋白的磷酸化，通过阻断p38 / GATA-4和JNK / c-Jun信号传导，抑制剂心肌肥大相关多肽心房利钠因子ANP，及肥大标志物BNP。化合物10e还显示出良好的药代动力学特性，并且对自发性高血压大鼠（SHR）模型显示出有效的体内抗HCM活性。 执行期内，发表SCI论文6篇，申请发明专利2项。总计培养硕士研究生4名。