基于机制导向的新型MKK3抗溃疡性结肠炎小分子抑制剂的设计与药物研究

Ulcerative colitis (UC) is a chronic and significant autoimmune disease of the intestine, which causes a malignant rate of colon cancer of up to 40%. A large number of clinical studies have confirmed that human over-activated MKK3 is a target for effective and hot research of anti-UC drugs. It has been reported that the targeted MKK3 inhibitor is limited to a small molecule of a small structural type, and has high toxicity and poor drug-like properties, and the MKK3 inhibitor drug has been blank at home and abroad. MKK3 acts as a "switch" that can be activated by the upstream kinase MEKKs through its "V" type active domain by ATP action, which in turn conducts the downstream p38 protein, resulting in the release of inflammatory factors. Previous pre-research found that the natural priviledged skeleton ketone chiral small molecule (R)-TML104 has better MKK3 enzyme inhibition (IC50 = 58 nM) and shows that it can compete with ATP for the "V" structure (KDMKK3 = 73 nM), but its anti-UC effect in vivo is too weak (60 mg/kg). This project is to find a small molecule inhibitor with good pharmacodynamics and better activity than ATP combined with MKK3 "V" domain, and (R)-TML104 as the lead, based on the mechanism guidance, focus on the "binding domain" and "solvent region" of the "V" domain in MKK3 to enhance the activity and improve the drug-like properties by using the design principles of electron isosterism and skeleton transition, synthesizing novel MKK3 small molecule inhibitors. It reveals its interaction mode with target and its influence on the differentiation and function of macrophage phenotype, and lays a foundation for the discovery of a safe and effective anti-UC small molecule drug.

溃疡性结肠炎（UC）是一种慢性重大自身免疫性疾病，其致结肠癌恶变率高达40%。大量临床研究证实过度活化的MKK3是抗UC药物有效和热点研究靶点。目前已报道靶向MKK3抑制剂仅限于结构类型少量的小分子，且毒性大、成药性差，至今MKK3抑制剂药物国内外是空白。MKK3作为“开关”被上游激酶MEKKs通过ATP作用其“V”型结构域激活，信号传递给下游p38蛋白导致炎症。预研发现，天然优势骨架茚酮手性小分子(R)-TML104，具有一定MKK3酶竞争结合和抑制作用（KD=73nM,IC50=58nM），但体内抗UC药效较弱（60mg/kg）。本项目聚焦靶点MKK3的“V”型结构域的“结合区”增强活性和“溶剂区”改善成药性进行优化，合成新型活性优于ATP与MKK3结合且体内药效好的小分子抑制剂，揭示其与靶点相互作用模式及对巨噬细胞表型分化和功能影响，为发现机制明确安全有效的抗UC小分子药物奠定基础。

本项目药用植物金雀根中发现的具有一定潜力抗结肠炎活性的茚酮类化合物ZC-K-6（TNFα, IC50 = 46 μM）为先导，基于其抗炎机制为导向，围绕增加其活性，改善其成药性差以及增强其口服生物利用度等方面开展了药物化学工作。一方面，针对其作用信号通路的新靶标，开展原创药物发现工作，获得了FIC小分子候选化合物(R)-STU104。机制研究发现它通过调控TAK1，并调节抑制其下游蛋白MKK3的磷酸化（IC50 MKK3 = 4 μM，KDMKK3 = 71 nM），进而选择性抑制结肠炎症细胞内MKK3下游信号通路上p38→MnK1→MK2→elF4E所有蛋白的磷酸化，并通过调节降低它们磷酸化水平而最终达到抑制了炎症因子TNFα的释放，从而达到体内抗UC的效果。因此，避免了抗体类药物直接作用于TNFα，完全抑制TNFα正常功能的缺陷，大大改善抗体类药物免疫抑制和全身感染的毒副作用。另一方面，针对该优选小分子化合物开展安全有效临床前候选药物发现工作。主要开展的内容包括：其具有优秀的TNFα抑制活性（IC50 = 0.58μM）；化学稳定性和水溶性好，大鼠口服F为43.8%，T1/2为1.18h，小鼠口服F为33%，T1/2为8.67小时；安全性好，小鼠最大耐受剂量LD50大于500 mg/kg，治疗指数TI大于50;体内动物药效优异，在小鼠DSS诱导急性，及白介素10基因敲除鼠慢性两种模型中，在优于或相同药效时，(R)-STU104给药剂量仅为临床一线对照药美沙拉嗪的1/5，且未见明显毒副作用，治疗窗口良好。以上药物发现工作，具有自主知识产权，有望填补国内临床缺乏TNFα小分子抑制剂抗结肠炎I类小分子药物的空白。