以PI4KIIα为靶点抗肿瘤抑制剂的筛选及优化

We have previously reported that PI4KIIαis a new tumor regulator （Oncogene,2010）, and suppressed PI4KIIα expression can enhanced the anti-tumor effect of EGFR targeted therapy (Cell death and disease, under review). All above suggested that PI4KIIα could be an potential breast cancer target. However, there is no PI4KIIα specific inhibitors available still now. Recently, we solved the crystal structure of PI4KIIα ( Nature Communications， 2014）, and then initially screened the ATP competitive inhibitors of PI4KIIα by using virtual screening and high-throug enzyme activity screening methods which were optimized by our own group. We have got 2 potential PI4KIIα specific inhibitors now. In this project, we will first confirm and optimize the existing PI4KIIα inhibitors. In order to improve the efficiency of the inhibitors, the crystal structure of PI4KIIα complexed with its inhibitors will be solved to identify the interaction site, and then optimizing virtual screening could be taken. The kinase panel analysis, comparing the difference of transcriptomics between PI4KIIα inhibitors treatment and PI4KIIα knock down cells, shRNA method combined with cellular PI4P detection assay will be used to confirm the specific of the inhibitors. Furthermore, we will use tumor xenograft model (including patient-derivedxenograft （PDX） model) to confirm the anti-tumor effect of selected inhibitors. Finally, we will enhance the bioavailability and reduce the toxicity by drug chemistry modifying and enclosed the inhibitors into nanocarriers. The final goal is getting a low toxicity and high effect PI4KIIα targeted anti-tumor inhibitors.

本课题组前期工作表明PI4KIIα是一个潜在的乳腺癌治疗靶点（Oncogen,2010;Cell death and disease, under review）。但是截至目前仍未有PI4KIIα特异抑制剂报道。为解决这一问题我们解析了PI4KIIα的晶体结构（Nature Communications，2014）,并结合虚拟药物设计和本课题组自主完善的高通量PI4KIIα酶活筛选技术，得到了2个抑制效率较好的PI4KIIα抑制剂。基于此本项目将通过蛋白小分子复合体结构指导虚拟药物设计的方法提高小分子抑制效率；酶谱分析，转录组学调控比对及小分子抑制效果对PI4KIIα依赖性来确定其特异性；通过免疫缺陷鼠肿瘤异源移植模型（多种细胞系及PDX模型）来确定其抗肿瘤效果；通过水溶性改进，纳米载体包装提高其的生物利用度，降低其生物毒性;最终筛选到PI4KIIα特异高效的抗肿瘤抑制剂。

在我们的前期工作中揭示PI4KIIα是一个重要的肿瘤调控靶点，但是截至目前仍未有PI4KIIα亚型特异的抑制剂可用。本项目的主要目的是综合生物化学，细胞生物学，生物物理学，计算机虚拟筛选等手段筛选到PI4KIIα的亚型特异性抑制剂；利用裸鼠异源移植肿瘤确定所筛抑制剂的抗肿瘤效果；并进一步通过药物化学性质改良提高抑制剂生物利用度，降低毒性，提高化合物的生物利用度和成药性。目前，我们已经解析了PI4KIIα的晶体结构, 并利用高通量虚拟筛选结合实验验证对PI4KIIα的抑制剂进行了筛选优化，得到了一个PI4KIIα的亚型特异抑制剂，PI-273。通过生化酶学动力学检测确定筛选出的最终抑制剂PI-273是一个PI竞争性抑制剂而不是ATP竞争性抑制剂。通过生化酶谱分析， PI4KIIa基因完全敲除细胞系，CRISPR全基因组文库富集筛选技术确定抑制剂的特异性；进而在动物水平上通过肿瘤异源移植模型来确定其抗肿瘤效果；该研究促进了PI4KII a在肿瘤治疗里的应用。