靶向MyD88抑制剂的设计、合成及其对脓毒症的药理作用研究

Sepsis, a clinical syndrome occurring in patients following infection or injury, is the most frequent cause of death in hospitalized patients. Treating sepsis remains problematic, as no effective anti-sepsis drug is currently available. Sepsis is associated with the activation of multiple inflammatory pathways within the host. Myeloid differentiating factor 88 (MYD88) as an anchor to recruit signaling proteins to the (TLR/IL-1R) receptors is associated with induction of innate immune response. It also becomes an important target in the research and development of therapeutic drugs for sepsis..As homodimerization of MyD88 and its interaction with the receptors require the TIR domain, this region is targeted for drug design. There were several peptides and peptidomimetic compounds have been developed as MyD88 inhibitors. There is a lack of known low molecular weight inhibitors of MyD88 that bind on the dimeric interface of TIR domain, block dimerization of MyD88, and inhibit downstream signaling..To date, the crystal structure of a dimeric TIR-TIR domain of MyD88 have not been identified and only the monomeric structure was known, but the dimeric structure of other adaptor proteins containing a TIR domain, such as MAL, has been demonstrated. In our previous studies, we modeled a structural model of the MyD88 dimer by homology modeling and molecular dynamic simulation. We analyzed the structural information of the dimeric model to find a pocket on the TIR domain surface of MyD88 amenable to binding small molecules. We performed computational screening of commercially-available small molecule library. A small set of top-ranked compounds were tested in the biological assay. The pyrazolopyrimidine derivatives were most potent. About 10 pyrazolopyrimidine derivatives are thereby designed and synthesized, the most promising compound of which, MI-161, shows inhibition effect on MyD88 in the biological tests..In this project, we aim to develop potent inhibitor of MyD88 based on pyrazolopyrimidine skeleton. A variety of pyrazolopyrimidine derivatives will be designed and synthesized. To verify their inhibition to MyD88, a series of biochemical analysis and a cell-based reporter assay will be performed. On the basis of their in vitro profile, 3-4 compounds are selected to investigate the anti-inflammatory effect in septic rats induced by cecal ligation and puncture (CLP). Molecular mechanisms of the inhibitors disrupting MyD88 homodimeric formation will be claritied by SPR, ITC, secreted alkaline phosphatase (SEAP) assays and co-immunoprecipitation assay. Protein crystallization of the protein-ligand complex and X-ray diffraction experiments are performed to provide vivid sight and understanding. These mechanisms might hold the key to future therapeutic strategies.

脓毒症是由感染引发的全身性炎症反应综合征，目前尚无有效治疗药物。MyD88是TLR4信号通路中关键的TIR接头蛋白，在介导急性炎症通路中起重要作用，是治疗脓毒症的潜在靶点。若阻断MyD88 TIR域二聚化会影响其下游信号传导，抑制促炎因子的释放。目前尚无其二聚化的晶体报道，作用位点不明确，难以对其i靶向设计抑制剂。本课题组前期利用同源建模和分子动力学模拟，以同源蛋白MAL为模板，构建MyD88 TIR域二聚化模型，并假设其二聚化模型的接触界面是抑制剂的作用位点。经过虚拟筛选、结构优化，得到抗炎活性较好的化合物MI-161。本课题将进一步靶向设计、合成不同系列的MI-161衍生物，筛选它们抗炎活性。检测抑制剂对MyD88下游信号通路的影响；用SPR、ITC和免疫共沉淀检验抑制剂对蛋白二聚的抑制作用机制；最后检查抑制剂对脓毒症模型大鼠的治疗作用。本研究将为脓毒症药物设计提供新的靶点和先导结构。

TLRs信号通路异常激活与炎症、癌症以及自身免疫性疾病密切相关。作为TLRs信号通路中枢蛋白的MyD88，已被证实可作为信号通路异常激活所致疾病的治疗靶点。本项目通过探究MyD88 TIR蛋白质的动力学特征来构建TIR-TIR同源二聚模型。在此同源二聚模型上，对Enamine拟肽化合物库进行虚拟筛选，筛出41个尚未见国内外文献报道的新化合物，并购买获得化合物实体。采用LPS刺激小鼠原代腹腔巨噬细胞释放炎症因子TNF-α和IL-6的实验模型，对41个化合物进行抗炎活性的筛选。其中5个化合物可以剂量依赖性的抑制炎症因子IL-6和TNF-α的释放，被视为苗头化合物。. 进一步分析苗头化合物和MyD88蛋白对接模型，我们保留苗头化合物中共性部分，引入两侧疏水结构，由此得到先导化合物。将先导化合物分为三个优化改造片段，设计合成了系列一、系列二、系列三等三类小分子抑制剂，分别为21、26、34个。通过对合成的化合物进行初步毒性实验评估，对这80个活性化合物在10μM条件下对LPS刺激的抗炎因子TNF-α以及IL-6的抑制效率评价进行抗炎活性初筛。筛选得到4个活性化合物对IL-6的IC50值分别为13.64、2.44、17.22、18.58 μM，TNF-a的IC50值分别为23.37、6.53、19.71、26.47μM。. 最终以两个新型化合物M-20和M-28进行活性测试和机理研究。两化合物均展现出体内外抗炎作用，并且针对脓毒症诱导下的急性肺损伤小鼠保护效果良好，或有助于治疗其他MyD88介导的炎症性疾病。本研究所发现的小分子抑制剂，不同于以往的肽类及其改造修饰化合物，其以全新的骨架结构揭示了吡唑啉[3,4-b]吡啶骨架的可能性，丰富了MyD88抑制剂的骨架多样性，也为未来MyD88相关的研发提供了良好的基础。