基于JNK通路及cathepsin L酶双重机制的类肽类诱导自噬剂的设计、合成及抗肿瘤作用研究

More recently, inducing autophagy had emerged as a potential strategy for effective cancer therapies.Thus, exploitation of the targeted autophagy inducer has become a new hot-point in new drug research and development.In our research, asperphenamate and patriscabratine as natural peptidomimetic,isolated from Hordeum vulgare L. and Patrinia scabra Bunge respectively, were studied based on a natural product consensus pharmacophore strategy. By combination of biological test and molecular simulation we determined that autophagic cell death in MCF-7 cells was induced through cathepsin L inhibition and JNK pathway activation. Because the autophagy-related signal pathways and kinases had never been elucidated,it is meaningful to develop targeted autophagy inducer. Guided by previous molecular simulation results and structure-activity relationship, the derivatives were further designed and synthesized. The subsequent bioevaluation provides evidence for the protein-binding sites modulation and derivative design again. Based on it, accurate pharmacophore and structure-activity relationship were established. Meanwhile, the interaction between cathepsin L and JNK pathway in inducing autophagy will be elucidated. It lays a foundation for establishing a new drug target of inducing autophagy. After that, a nude mouse model of human breast cancer is established to test the antitumor activity in vivo. The innovative antitumor candidates with independent intellectual property rights are most likely to be obtained.

目前诱导细胞自噬已成为肿瘤治疗新的策略。因此，开发靶向性诱导自噬药物成为新药研究的又一热点。本课题前期运用天然产物共同药效团导向策略从传统抗肿瘤中药中垂钓出一对类肽类天然产物asperphenamate和patriscabratine。经生物测试和分子模拟方法研究确定其作用机制为JNK通路激活和cathepsin L激酶抑制双重作用诱导MCF-7细胞发生完全自噬。鉴于自噬信号通路及调控激酶尚未阐明，这一崭新机制对于诱导自噬药物开发意义重大。基于此，以前期分子模拟结果和构效关系为指导，进行衍生物设计和合成，进而以活性结果优化分子模拟活性位点的选取及修正化合物设计，建立准确的药效团和构效关系。阐明cathepsin L激酶与JNK通路在诱导自噬中的关系，为新的诱导自噬药物靶标的确立奠定基础。采用人肿瘤裸鼠模型，初步评价先导化合物的体内抗肿瘤作用，力争获得作用机制新颖、疗效确切的抗肿瘤候选药物。

日本分子细胞生物学家大隅良典因在细胞自噬机制研究领域的成就，获得2016年诺贝尔生理学或医学奖。显而易见，自噬已经越来越受到人们关注。研究表明，自噬在肿瘤发生和发展过程中的作用尤为明显，因此调控自噬已成为肿瘤治疗的新策略。迄今为止具有调控自噬作用的活性分子结构类型较为单一，同时与自噬相关的激酶也尚未阐明，因此开发新颖结构骨架结构的靶向性自噬调控分子成为抗肿瘤新药研究的又一热点。.本项研究以类二肽类天然产物asperphenamate 和patriscabratine为先导，以前期总结的构效关系为指导，进行衍生物设计合成，继而发现了B环磺酰化衍生物对于cathepsin L的抑制作用接近于阳性对照药E64。在调控肿瘤细胞自噬的研究中，发现此类化合物除了具有诱导乳腺癌细胞自噬作用之外，其对于宫颈癌和肝癌细胞也表现出了明显的自噬诱导作用。更令我们兴奋的研究是，此类化合物表现出了与阳性对照药相近的抑制肿瘤细胞迁移的作用，这也大大的增加了此类化合物的开发潜力。. 在自噬的生物学机制尚未阐明的情况下，本项研究提供了新颖结构类型的活性分子，并以此为基础，运用生物学和分子模拟相结合的方法确定了其作用的靶酶为cathepsin酶。为揭示其确切机制作了化学和生物学储备。此外，此类化合物的抗肿瘤细胞迁移作用的发现，也为开发多功能性的诱导自噬抗肿瘤候选药物提供可能。