抗神经病理性痛候选新药钩吻素子作为TSPO别构调节剂的研究

The discovery of allosteric modulator drugs of proteins is an important breakthrough in the field of new medicine development. Koumine, a new drug candidate, possesses the significant antagonistic effect on neuropathic pain, but its deep understanding of mechanisms is to be undertaken urgently. The 18 kDa translocator protein (TSPO) may become a new important therapeutic target on neuropathic pain. Our preliminary study has demonstrated that koumine may be a positive allosteric modulator of protein TSPO, which needs further confirmation. Based on this finding, the present project is going to confirm that koumine is a positive allosteric modulator of TSPO by using approaches about pharmacology and chemical biology. Firstly, we are going to explore how koumine enhances the affinity of TSPO interacting with its orthosteric ligands including TSPO endogenous ligands. Subsequently, we will investigate whether koumine could promotes the efficacy or potency of function of TSPO orthosteric ligands in cell models. In expect of confirming that koumine is a positive allosteric modulator of protein TSPO, finally, we are plan to study the cooperative effects of koumine and TSPO agonists on neuropathic pain in models of rats. This project is undertaken to establish foundation for proposing the new efficiency of koumine against neuropathic pain, and provide a scientific basis for developing new allosteric modulator drugs of TSPO. It will also provide the new mode of drug action for the development of new therapeutic drugs against neuropathic pain.

发现蛋白别构调节剂药物为近年来新药创制的重大突破。钩吻素子具有显著抗神经病理性痛(NP)作用，成药特征良好，其作用机制亟待深入研究。TSPO蛋白可成为治疗NP的重要靶点。我们前期工作提示，钩吻素子可能是TSPO正性别构调节剂，但有待确证。鉴此，本项目拟采用药理学、化学生物学技术手段，首先在分子水平从增强TSPO正位配体亲和力角度，探讨钩吻素子对TSPO的别构调节；进而在细胞模型上，论证钩吻素子可否增强TSPO正位配体功能的效价或效能；最后在大鼠NP模型上，分析钩吻素子可否增强TSPO激动剂的镇痛效应，从多个角度较为全面阐释钩吻素子对TSPO的别构调节作用，确证其为TSPO正性别构调节剂。本研究将为创制新药钩吻素子奠定理论基础，为开发新型TSPO别构调节剂药物提供科学依据，并为防治NP新药研发提供新的药物作用方式。

发现蛋白别构调节剂药物为近年来新药创制的重大突破。钩吻素子具有显著抗神经病理性痛(NP)作用，作用机制亟待深入研究。TSPO蛋白可成为治疗NP的重要靶点。我们前期工作提示，钩吻素子可能是TSPO正性别构调节剂，但有待确证。本项目首先采用放射配体结合分析法，通过平衡增强实验发现10-6 M、10-10 M钩吻素子可显著增强3H-PK11195与TSPO的结合；在上述研究基础上，分析10-6 M、10-10 M钩吻素子对3H-PK11195与TSPO解离动力学的影响，结果观察到10-6 M、10-10 M钩吻素子均可显著降低3H-PK11195与TSPO的解离速率，提示钩吻素子为3H-PK11195与TSPO结合亲和力的正性别构调节剂。其次采用表面等离子共振技术研究钩吻素子与TSPO脂蛋白体的亲和力，观察到钩吻素子与TSPO脂蛋白体结合的平衡解离常数KD值为0.70±0.28 nM，表明钩吻素子为TSPO高亲和力的配体；进而分析钩吻素子对PK11195与TSPO脂蛋白体结合亲和力的影响，发现10-5 M钩吻素子可显著提高PK11195与TSPO的亲和力，再次从亲和力角度证明钩吻素子对TSPO的正性别构调节作用。然后选用T98G人胶质瘤细胞模型，观察到PK11195可剂量依赖性地抑制T98G人胶质瘤细胞的增殖，1 μM-600 μM 钩吻素子单用对T98G细胞的增殖未有影响，但与75 μM（～EC20）PK11195合用时可剂量依赖性地增强PK11195抑制细胞增殖的作用，提示钩吻素子为PK11195抑制T98G人胶质瘤细胞增殖的正性别构调节剂。最后在大鼠坐骨神经慢性束缚损伤（CCI）模型中，钩吻素子及TSPO配体Ro5-4864各自单用均可产生显著的镇痛效应，作用呈剂量依赖性；在此基础上，选用无镇痛效应的0.28 mg/kg钩吻素子与Ro5-4864合用，结果观察到0.28 mg/kg钩吻素子可显著增强Ro5-4864 0.0625，0.125，0.25，0.5 mg/kg剂量组的镇痛效应，提示钩吻素子是Ro5-4864在CCI模型中镇痛效应的正性别构调节剂。综上，本项目表明钩吻素子对TSPO存在别构调节作用，别构调节TSPO可能是钩吻素子发挥镇痛作用的重要方式。这些研究成果为发展原创性候选新药钩吻素子奠定了理论基础，并为开发新型TSPO别构调节剂药物提供了科学依据。