RasT开放构象新药物靶点的确证及其特异性靶向抑制剂的发现和分子机制研究

Targeted Ras appears to be hot topics in tumor therapy. However, effective drugs against Ras are not available currently. The open conformation of active Ras(RasT) intermediate for nucleotide exchange plays key roles during Ras proteins are activated, but their physiological functions are not yet clearly clarified. In our previous studies, the open conformation of RasT was first used as a new drug target to screen small molecules from NCI (National Cancer Institute) diversity compound libraries. As a result, three small-molecule inhibitors with high efficiency and low toxicity were successfully found by the two-dimension virtual drug screening strategy, which was established by ourselves, combined with the drug screening against pancreatic cancer cells in vitro. On the basis of our results as described above, the studies outlined in this application first aim at uncovering the targeting specificity and binding mechanism of the interactions between the selected three small-molecule inhibitors as drug probe molecules and the open conformation of RasT. Subsequently, this proposals are to aim at confirming the open conformation of RasT as a new drug target and acquiring specifically targeted inhibitors through using multidisciplinary approach. Aimed at the molecular mechanism of Ras activity modulated by the specifically targeted inhibitors selected, further, the proposed studies are to focus on testing the effects of the selected targeted inhibitors on the network regulation of Ras in pancreatic cnacer cells and on BALB/C nude mouse xenografts. The current proposal not only has an importantly theoretical significance on discovering a new drug target and its specifically targeted inhibitors and clarifying the pharmacologic effects of the open conformation of RasT, but also is with significant application value for an in-depth study of drug intervention mechanisms on Ras-related tumor therapy.

Ras的靶向治疗已成为肿瘤治疗领域的研究热点，目前并未获得有效的抗Ras药物。激活型Ras(RasT)在核苷酸交换过程中产生的中间体开放构象在Ras激活过程中起关键作用，但其生理功能并不清楚。前期工作中，申请人首次以RasT开放构象为药物靶点，通过自行建立的二维虚拟药物筛选技术，结合体外药物筛选，从NCI小分子多样性化合物库中鉴别出3个高效低毒的抗胰腺癌小分子抑制剂。本项目拟在此基础上，应用多学科交叉技术，以3个抑制剂为药物探针分子，研究其与RasT开放构象的靶向特异性和结合机理，确证新的药物靶点并获得特异性靶向抑制剂。对选定的靶向抑制剂进行胰腺癌细胞Ras网络调控和异种移植裸鼠模型研究，揭示其调控Ras活性的分子机制。本项目不但对于发现新药物靶点及其特异性靶向抑制剂、阐明RasT开放构象的药理学作用具有重要的理论意义，而且对于深入研究Ras相关肿瘤治疗的药物干预机制也具有重要应用价值。

Ras-GTP激活态有三个不同但处于均等集群构象的平衡态，其中之一为“非信号传递的开放构象”(简称“开放构象”)。因此，锁住这个“开放构象”的小分子理论上可以防止Ras激活下游效应因子并最终封闭Ras依赖的信号传递。我们使用蛋白结构分析和亲和性-特异性药物筛选策略在NCI小分子库中发现3个小分子化合物能够稳定这个“开放构象”。在本项目中，分子间相互作用实验证实NSC290956和NSC48693与“开放构象”有高特异性的亲和力；RNA干扰和细胞热位移实验证明KRas是NSC290956和NSC48693的细胞内靶点蛋白。NSC290956通过削弱胰腺癌细胞中KRas-GTP的表达水平对Ras下游信号通路进行有效抑制，进而抑制胰腺癌细胞生长和增殖。其对5种含KRas突变和野生型胰腺癌细胞48小时的IC50值范围为19.7-74.2μM，表现出对KRas突变的胰腺癌细胞具有较好的抗肿瘤选择性，而对野生型KRas胰腺癌细胞具有较低的抗肿瘤活性。然而，其对人正常肝细胞HL7702、胚肾细胞HEK293和人外周血细胞都没有明显的细胞生长抑制作用。NSC290956能阻滞癌细胞周期于G2期，对人正常细胞周期没有影响，进一步表明其对涉及KRas-GTP介导的靶向参与的癌细胞的选择性生物反应。BALB/c小鼠的肾被膜异种移植瘤模型研究表明，对模型鼠以68mg/kg的剂量持续腹腔给药两周，NSC290956没有降低小鼠的体重，表明具有较低的毒副作用，能完全抑制MIAPaCa-2肿瘤的生长。免疫组化分析表明，NSC290956下调肿瘤组织c-Raf和p-ERK蛋白的表达，增加TUNEL印迹水平。我们的研究揭示出NSC290956是突变KRas引发的胰腺癌细胞的特异性靶向抑制剂。在H358的非小细胞肺癌肿瘤移植裸鼠模型中，NSC290956有效地削弱了肿瘤生长，并具有与肿瘤细胞相同的体外作用机制，显示出NSC290956治疗KRas基因驱动的非小细胞肺癌的有效性。肿瘤细胞的能量代谢改变是癌症的标志现象。体外细胞实验表明，NSC48693诱导癌细胞凋亡通过扰动线粒体细胞代谢平衡而非改变糖酵解能量模式。动物实验证实，NSC48693减弱了肿瘤生长，其药理学作用模式与细胞实验一致。我们的研究结果为发展可靠的药理学Ras靶标和抑制剂产生了一个新的范例。

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