Mu opioid receptor (µOR) agonists like fentanyl have long been used for pain management, but are considered a major public health concern due to their adverse side effects, including lethal overdose.¹ To design safer therapeutics, we report a conceptually novel approach targeting conserved sodium (Na⁺) binding site², observed in µOR³ and many other class A GPCRs, by bitopic fentanyl derivatives functionalized via a linker with a positively charged guanidino group. Cryo-EM structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the ligand’s guanidine and the key Asp^2.50 residue in the Na⁺ site. While the lead bitopics maintain nanomolar potency and high efficacy at Gi subtypes, they show strongly reduced arrestin recruitment, one also shows the lowest Gz-efficacy among the panel of µOR agonists, including partial and biased, morphinan and fentanyl analogs. In mice, the best bitopic ligand displayed µOR dependent antinociception with attenuated adverse effects supporting the µOR Na⁺ site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands engaging the Na⁺ pocket in class A GPCRs can be designed to control their efficacy and functional selectivity profiles for G_i/o/z subtypes and arrestins, thus modulating their in vivo pharmacology.

芬太尼等 Mu 阿片受体 (μOR) 激动剂长期以来一直用于疼痛治疗，但由于其不良副作用（包括致命过量）而被认为是主要的公共卫生问题。 ¹为了设计更安全的治疗方法，我们报告了一种针对保守钠 (Na ⁺ ) 结合位点²的概念性新颖方法，这种方法在 µOR ³和许多其他 A 类 GPCR 中观察到，通过通过带有带正电荷的胍基的连接体进行功能化的双位芬太尼衍生物。最有效的双位配体与 µOR 复合物的冷冻电镜结构突出了配体的胍和 Na ⁺位点中关键的 Asp ^2.50残基之间的关键相互作用。虽然先导双位药物在 Gi 亚型上保持纳摩尔效力和高效，但它们显示出抑制蛋白募集大大减少，其中一种还显示 µOR 激动剂组中最低的 Gz 功效，包括部分和偏向、吗啡喃和芬太尼类似物。在小鼠中，最好的双位配体表现出 µOR 依赖性镇痛作用，并减弱了副作用，支持 µOR Na ⁺位点作为设计更安全镇痛药的潜在目标。总的来说，我们的研究表明，可以设计与 A 类 GPCR 中的 Na ⁺口袋结合的双位配体来控制其对 G _i/o/z亚型和视紫红质抑制蛋白的功效和功能选择性特征，从而调节其体内药理学。