MEK抑制剂与NO供体偶联药物的设计、合成及其多靶点协同抗肿瘤作用评价

RAS/RAF/MEK/ERK is a key signaling pathway involved in the regulation of cell proliferation, survival, differentiation and apoptosis. Aberrant activation of this pathway contributes to various cancers and other diseases. MEK is one of the key kinases in this pathway and allosteric MEK inhibitors have demonstrated bona fide antitumor efficacy and low toxicity in clinics, particularly due to their high selectivities. Several MEK inhibitors have been approved for the treatment of metastatic melanoma as single agent, however, resistance often occurs within 6 to 7 months due to amplification of the upstream oncogenic driver of ERK signaling. Nitric oxide (NO) is a signaling molecule in many physiological and pathological processes. It has been reported that NO showed very good anti-proliferation activity in drug resistant tumor cells although severe side effects were also observed because of lacking good selectivity. Here in this project, we designed and synthesized hybrid molecules containing a MEK inhibitory motif and a NO donor motif as multitarget antitumor drugs to solve the resistance problem of current MEK inhibitors by selectively releasing NO in tumor cells. The designed hybrid drug can be metabolized to a pharmacologically active drug in the cell by interacting with the Cys122 in the MEK allosteric binding site, therefore, releasing the NO to kill drug resistant tumor cells. On the other hand, by utilizing the structure guided drug design strategy, we are hoping to obtain the first allosteric covalent MEK inhibitor, which is expected to improve the efficacy compared to current MEK inhibitors and more importantly, improve the activity against the drug resistant tumors together with the selective releasing of NO. In this proposal, we will optimize our hybrid drug to improve its efficacy and pharmacokinetics properties. We will perform biological evaluation and mechanistic studies in relevant cell models. We will confirm the covalent binding of our top three compounds. The high selectivity of the MEK inhibitor portion of the hybrid drug to aberrantly activated ERK pathway in the tumor cells can allow NO release at the tumor site selectively and reduce side effects. By combining a highly selective covalent MEK inhibitor and an NO donor, our designed hybrid drug can overcome resistance to MEK inhibitors. Our hybrid drug will have multitargeted and synergistic antitumor effects. This research is expected to lay the foundation for a highly efficient and safe drug with a new mechanism to treat MEK resistant tumors.

ERK通路对细胞增殖、分化、凋亡及肿瘤的发生、发展起着重要调控作用。MEK是该通路中的关键激酶，变构MEK抑制剂由于其特有的高选择性，在临床表现出良好的抗肿瘤疗效及低毒性，然而却因为机体产生获得性耐药而失去疗效。一氧化氮（NO）作为体内信号分子，具有抗耐药肿瘤作用，但缺乏靶向性。本项目设计NO供体与MEK抑制剂偶联杂合药物并进行合成、活性评价、机制阐明及结构优化，以解决MEK抑制剂耐药问题和NO供体靶向性问题。一方面利用MEK抑制剂对靶点激酶的高选择性将NO供体高选择性地带入肿瘤细胞，增强其靶向抗肿瘤作用，降低毒性；另一方面，根据结构导向设计有望得到首个作用在MEK变构位点的共价抑制剂，提高活性并克服因现有抑制剂诱导突变产生的耐药性，同时结合NO的抗耐药作用，克服MEK抑制剂获得性耐药问题。从而达到多靶点协同作用的效果，为创制具有高效、低毒、不易耐药等特点的新作用机制的抗肿瘤药物奠定基础。

ERK通路对细胞增殖、分化、凋亡及肿瘤的发生、发展起着重要调控作用。MEK是该通路中的关键激酶，变构MEK抑制剂由于其特有的高选择性，在临床表现出良好的抗肿瘤疗效及低毒性，一氧化氮（NO）作为体内信号分子，也具有抗肿瘤作用。本项目设计NO供体与MEK抑制剂偶联杂合药物并进行合成、活性评价、机制阐明及结构优化，达到多靶点协同作用的效果，为创制具有高效、低毒、不易耐药等特点的新作用机制的抗肿瘤药物奠定基础。本项目进行了MEK抑制剂与NO供体偶联物研究、MEK降解剂结构优化及其与NO供体偶联物研究。.MEK抑制剂与NO供体偶联物研究部分，设计、合成了一系列偶联物，在抗肿瘤作用研究中，对构效关系进行了总结分析，发现化合物78显示出较好的肿瘤细胞增殖抑制作用和较小的毒性。在MDA-MB-231细胞中的抗增殖活性最好，高于阳性对照化合物RO5126766 5倍，并且以剂量依赖和时间依赖的方式抑制了MEK和ERK的磷酸化。NO释放的评价实验也显示化合物78释放的NO量最多。说明NO释放能力和ERK/MAPK信号通路抑制作用产生协同的多靶点作用，是偶联化合物具有强抗肿瘤活性的原因。细胞凋亡实验和细胞周期实验显示化合物78与RO5126766的作用机制不同。在抗病毒作用研究中，化合物78 (18h)相比于索拉非尼可能具有更安全的治疗效果，可以明显地抑制EV71诱导的细胞病变及其在RD细胞中的复制。因此化合物78具有抗肿瘤和抗病毒的双重作用。.MEK降解剂与NO供体偶联物研究部分，经过多轮结构优化，发现了具有明显降解活性的MEK降解剂VC4，合成了MEK降解剂与NO供体偶联物并对活性及机制进行了研究。.本项目开发的50个MEK抑制剂与NO供体偶联物、MEK降解剂、MEK降解剂与NO供体偶联物及其作用机制研究均未见报道，这是针对MEK靶点开展的原创性研究，在抗肿瘤和抗病毒创新药物研发领域具有重要科学意义和巨大潜在应用价值。项目优化产生的2个结构新颖的具有进一步发展潜力的化合物78（18h）和VC4，为后续药物开发奠定了基础。.