Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-β-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-β-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-β-NalA–KOR–Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.

尽管 GPCR 结构的数量不断增加，并且肽设计方面取得了最新进展，但开发有效的技术来合理设计单个 GPCR 的高亲和力肽配体仍然是一个未解决的挑战。在这里，我们开发了一种计算方法来设计套索形大环化肽和小分子阿片类配体的缀合物。我们通过发现具有所需药理活性的κ阿片受体（KOR）的化学支架来证明其可行性。设计的从头环肽 (DNCP)-β-纳洛胺 (NalA) 在体外表现出有效的混合 KOR 激动/μ-阿片受体 (MOR) 拮抗作用、纳摩尔级结合亲和力、选择性和 KOR 功效偏差。体内功效概念验证研究表明，DNCP-β-NalA(1) 可在雄性小鼠中诱导有效的 KOR 介导的镇痛作用。利用 DNCP-β-NalA-KOR-Gi1 复合物的高分辨率冷冻电镜结构 (2.6 Å) 和分子动力学模拟来验证计算设计模型。这揭示了 ECL2/3 和 TM6/7 中控制 KOR 内在功效的残基网络。总的来说，我们的计算从头平台克服了超大型文库对接和虚拟小分子筛选活动中遇到的广泛先导优化，并为 GPCR 配体发现提供了创新。这可能会推动用于疼痛等医疗应用的下一代疗法的开发。