针对黑色素瘤靶点BRAF的II型不可逆抑制剂的开发及其防止BRAF信号通路负反馈机制的功能探究

Melanoma is a highly malignant cutaneous cancer with a high death rate which accounts for about 75% death for skin cancers. It is most common in the population living in the sunny climates. Accumulating basic research and clinical evidence have demonstrated that BRAF kinase bearing V600E is the predominate tumourignenesis driver for the melanoma and has been serving as the validated drug discovery target. Currently the most popular BARF based targeted therapy for melanoma is PLX4032 and GSK2118436 which are both reversible type I kinase inhibitors. However, the drug induced signaling pathway negative feedback response and signaling bypass would render the drug resistance in a short period during the treatment thus has greatly limited their clinical application. In addition, treatment of these drugs can induce the squamous cell carcinoma as a severe mechanism based side effect. Theoretically, these mechanism based drug resistance and side effects are difficult to avoid by developing a new pharmacophore with the same reaction mechanism. Therefore, a new mechanism based BRAF inhibitor is highly desired to meet the unmet Melanoma medical need. Hereby we propose to take full advantage of irreversible inhibitors' ability to degrade targeted protein resulting the more completed shutting down of signaling pathway and hence possibly prevent the negative feedback response of the signaling pathway to develop an irreversible type II BRAF kinase inhibitor by targeting the BARF Cys531 located in the hinge area from a type II kinase inhibitor chemical scaffold aided by structure based irreversible kinase inhibitor design approach. Thereby explore its ability and mechanism to prevent the signaling feedback response and pave the way for the new functional mechanism based melanoma therapies

激酶BRAF是一个经过临床验证的治疗黑色素瘤的有效药物作用靶点，目前临床上主要采用第一代BRAF激酶可逆I型抑制剂PLX4032以及GSK2118436进行治疗。但是它们在临床上的使用会迅速产生由信号通路负反馈或者信号通路规避造成的适应性抗药性从而导致失去药效，同时还会产生诱导扁平细胞癌生成等副作用。这些机理性的副作用很难通过开发一个新的相同作用机理的抑制剂来解决，因此临床上急需一种新型机理的针对BRAF的药物。我们拟利用不可逆抑制剂能够降解靶点蛋白从而更加彻底的阻断信号通路进而可能阻断上下游的信号负反馈作用的特点，以II型激酶抑制剂的设计开发理念为基础，采用结构药学设计不可逆抑制剂的方法，利用BRAF激酶hinge区域的半胱氨酸531，开发新型的不可逆II型抑制剂，并探究其抑制BRAF-MEK-ERK信号通路的负反馈作用的效果和机制，为开发新型抗耐药性的黑色素瘤治疗药物奠定基础。

黑色素瘤是皮肤癌中最致命的一种恶性肿瘤，BRAF激酶属于RAF激酶家族，是一个经过临床验证的针对黑色素瘤的有效药物作用靶点。针对目前临床上治疗黑色素瘤的靶向药物的适应性抗药性等问题，我们在本项目中综合使用基于结构的药物设计、分子生物学等多种研究手段，发现了一系列新型的BRAF激酶抑制剂如LXX-7-48/122/128等化合物。在更全面的药物学表征研究中，我们进一步发现了高活性的II型BRAF抑制剂LXX-7-128能够显著抑制BRAF介导的MAPK信号通路，诱导细胞发生凋亡并将细胞分裂阻滞在G0-G1期。在A375（BRAF V600E）黑色素瘤细胞小鼠药效模型上，LXX-7-128展现出剂量依赖性的肿瘤抑制效果（100 mpk剂量组的抑瘤率达到80%）。作用机制研究表明LXX-7-128不仅对于BRAF-V600E突变，而且对野生型的BRAF、CRAF激酶也有较强的抑制作用，说明其不仅可以作用于由BRAF-V600E突变所导致的MAPK信号通路过度活化的黑色素瘤，对于由RAS突变所导致的MAPK信号通路过度活化的其他癌症也有抑制作用，而PLX-4032等已知BRAF抑制剂在携带RAS突变的肿瘤中没有作用。进一步在具有RAS突变的癌症细胞上的研究结果表明LXX-7-128在蛋白、细胞、动物模型三种不同的层次上与已上市药物PLX-4032相比能够显著的抑制肿瘤的生长。药物代谢动力学研究和初步安全性评价显示LXX-7-128具有良好的成药性潜力，目前正在向临床前研究推进。该成果为研发能够治疗包括黑色素瘤在内的多种实体瘤的新型BRAF抑制剂奠定了理论和实验基础。目前标注本资助项目编号的相关科研成果已发表SCI论文6篇，发明专利正在申请中。项目执行期间，项目负责人获得中科院青年科学家奖和中国药学会-以岭生物医药青年奖，培养毕业博士和硕士研究生各1名，出站博士后1名。

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