Design and structural validation of peptide–drug conjugate ligands of the kappa-opioid receptor

Abstract

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.

Peptide–Drug Conjugates: An Emerging Direction for the Next Generation of Peptide Therapeutics

“…Recently, PDCs have been designed to target the KOR to generate compounds with high receptor specificity, while also reducing analgesic side effects. 136 In this study, the crystal structure of KOR complexed with a ligand, MP1104, 137 was used to computationally design a series of PDCs that link KOR ligands based on MP1104 to thioether cyclized peptides designed to interact with the extracellular loops of KOR. Before small-molecule conjugation, the computationally designed peptides were assessed in cell-based assays which demonstrated that the compounds were unable to displace an orthosteric ligand at concentrations as high as 10 μM.…”

“…Recently, PDCs have been designed to target the KOR to generate compounds with high receptor specificity, while also reducing analgesic side effects . In this study, the crystal structure of KOR complexed with a ligand, MP1104, was used to computationally design a series of PDCs that link KOR ligands based on MP1104 to thioether cyclized peptides designed to interact with the extracellular loops of KOR.…”

“…Molecules with unknown binding partners are noted with a question mark. (A) The cyclic peptide DNCP was linked to β-NalA through an L-Thr-D-Phe linker 136. (B) Example of a betulinic acid peptide conjugate where betulinic acid is linked to an azido peptide directly through a C−C bond 142.…”

Decoding the κ Opioid Receptor (KOR): Advancements in Structural Understanding and Implications for Opioid Analgesic Development

“…Among the agonist-bound structures, six are bound with small molecules MP1104 (PDB ID: 6B73) [ 54 ], nalfurafine (PDB ID: 7YIT) [ 38 ], GR89696 (PDB ID: 8DZR, 8DZS) [ 55 ], and momSalB (PDB ID: 8DZP, 8DZQ) [ 55 ]. The remaining three are bound with short peptides dynorphin (PDB ID: 7Y1F, 8F7W) and de novo cyclic peptide(DNCP)-β-naloxamine (NalA) (PDB ID: 8FEG) [ 56 , 57 , 58 ]. X-ray diffraction methods were employed in determining two antagonist-bound [ 52 , 53 ] and two agonist-bound structures [ 38 , 54 ].…”

Mechanism of antagonist ligand binding to REV-ERBα

“…Dysregulation of circadian rhythm has been associated with various health conditions, including metabolic disorders, cardiovascular diseases, and certain cancers 19 – 21 . Recent studies highlighted their role in neurological disorders such as cognitive diseases 22 . Although REV-ERBs share similar molecular domain organization to most nuclear hormone receptors, they are distinctive because they lack the carboxy-terminal helix-12 activation function 2 (AF-2) region.…”