Carfentanil is a ß-arrestin-biased agonist at the µ opioid receptor.

BACKGROUND AND PURPOSE: The illicit use of fentanyl-like drugs (fentanyls), which are µ opioid receptor agonists, and the many overdose deaths that result, has become a major problem. Fentanyls are very potent in vivo, leading to respiratory depression and death. However, the efficacy and possible signalling bias of different fentanyls is not clearly known. Here, we compared the relative efficacy and bias of a series of fentanyls. EXPERIMENTAL APPROACH: For agonist signalling bias and efficacy measurements, Bioluminescence Resonance Energy Transfer experiments were undertaken in HEK293T cells transiently transfected with µ opioid receptors, to assess Gi protein activation and ß-arrestin 2 recruitment. Agonist-induced cell surface receptor loss was assessed using an enzyme-linked immunosorbent assay, whilst agonist-induced G protein-coupled inwardly rectifying potassium channel current activation was measured electrophysiologically from rat locus coeruleus slices. Ligand poses in the µ opioid receptor were determined in silico using molecular dynamics simulations. KEY RESULTS: Relative to the reference ligand DAMGO, carfentanil was ß-arrestin-biased, whereas fentanyl, sufentanil and alfentanil did not display bias. Carfentanil induced potent and extensive cell surface receptor loss, whilst the marked desensitisation of G protein-coupled inwardly rectifying potassium channel currents in the continued presence of carfentanil in neurones was prevented by a GRK2/3 inhibitor. Molecular dynamics simulations suggested unique interactions of carfentanil with the orthosteric site of the receptor that could underlie the bias. CONCLUSIONS AND IMPLICATIONS: Carfentanil is a ß-arrestin-biased opioid drug at the µ receptor. It is uncertain how such bias influences in vivo effects of carfentanil relative to other fentanyls.

Interaction With the Lipid Membrane Influences Fentanyl Pharmacology.

Overdose deaths from fentanyl have reached epidemic proportions in the USA and are increasing worldwide. Fentanyl is a potent opioid agonist that is less well reversed by naloxone than morphine. Due to fentanyl's high lipophilicity and elongated structure we hypothesised that its unusual pharmacology may be explained by its interactions with the lipid membrane on route to binding to the µ-opioid receptor (MOPr). Through coarse-grained molecular dynamics simulations, electrophysiological recordings and cell signalling assays, we determined how fentanyl and morphine access the orthosteric pocket of MOPr. Morphine accesses MOPr via the aqueous pathway; first binding to an extracellular vestibule, then diffusing into the orthosteric pocket. In contrast, fentanyl may take a novel route; first partitioning into the membrane, before accessing the orthosteric site by diffusing through a ligand-induced gap between the transmembrane helices. In electrophysiological recordings fentanyl-induced currents returned after washout, suggesting fentanyl deposits in the lipid membrane. However, mutation of residues forming the potential MOPr transmembrane access site did not alter fentanyl's pharmacological profile in vitro. A high local concentration of fentanyl in the lipid membrane, possibly in combination with a novel lipophilic binding route, may explain the high potency and lower susceptibility of fentanyl to reversal by naloxone.

CFTR Modulators: From Mechanism to Targeted Therapeutics.

People with cystic fibrosis (CF) suffer from a multi-organ disorder caused by loss-of-function variants in the gene encoding the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Tremendous progress has been made in both basic and clinical sciences over the past three decades since the identification of the CFTR gene. Over 90% of people with CF now have access to therapies targeting dysfunctional CFTR. This success was made possible by numerous studies in the field that incrementally paved the way for the development of small molecules known as CFTR modulators. The advent of CFTR modulators transformed this life-threatening illness into a treatable disease by directly binding to the CFTR protein and correcting defects induced by pathogenic variants. In this chapter, we trace the trajectory of structural and functional studies that brought CF therapies from bench to bedside, with an emphasis on mechanistic understanding of CFTR modulators.

A small molecule CFTR potentiator restores ATP-dependent channel gating to the cystic fibrosis mutant G551D-CFTR.

BACKGROUND AND PURPOSE: Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators are small molecules developed to treat the genetic disease cystic fibrosis (CF). They interact directly with CFTR Cl- channels at the plasma membrane to enhance channel gating. Here, we investigate the action of a new CFTR potentiator, CP-628006 with a distinct chemical structure. EXPERIMENTAL APPROACH: Using electrophysiological assays with CFTR-expressing heterologous cells and CF patient-derived human bronchial epithelial (hBE) cells, we compared the effects of CP-628006 with the marketed CFTR potentiator ivacaftor. KEY RESULTS: CP-628006 efficaciously potentiated CFTR function in epithelia from cultured hBE cells. Its effects on the predominant CFTR variant F508del-CFTR were larger than those with the gating variant G551D-CFTR. In excised inside-out membrane patches, CP-628006 potentiated wild-type, F508del-CFTR, and G551D-CFTR by increasing the frequency and duration of channel openings. CP-628006 increased the affinity and efficacy of F508del-CFTR gating by ATP. In these respects, CP-628006 behaved like ivacaftor. CP-628006 also demonstrated notable differences with ivacaftor. Its potency and efficacy were lower than those of ivacaftor. CP-628006 conferred ATP-dependent gating on G551D-CFTR, whereas the action of ivacaftor was ATP-independent. For G551D-CFTR, but not F508del-CFTR, the action of CP-628006 plus ivacaftor was greater than ivacaftor alone. CP-628006 delayed, but did not prevent, the deactivation of F508del-CFTR at the plasma membrane, whereas ivacaftor accentuated F508del-CFTR deactivation. CONCLUSIONS AND IMPLICATIONS: CP-628006 has distinct effects compared to ivacaftor, suggesting a different mechanism of CFTR potentiation. The emergence of CFTR potentiators with diverse modes of action makes therapy with combinations of potentiators a possibility.

A tetrapeptide class of biased analgesics from an Australian fungus targets the µ-opioid receptor.

An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak (Ki low micromolar) µ-opioid agonists, which led to the design of bilorphin, a potent and selective µ-opioid receptor (MOPr) agonist (Ki 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit ß-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting ß-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics.

Drug Binding Poses Relate Structure with Efficacy in the µ Opioid Receptor.

The µ-opioid receptor (MOPr) is a clinically important G protein-coupled receptor that couples to Gi/o proteins and arrestins. At present, the receptor conformational changes that occur following agonist binding and activation are poorly understood. This study has employed molecular dynamics simulations to investigate the binding mode and receptor conformational changes induced by structurally similar opioid ligands of widely differing intrinsic agonist efficacy, norbuprenorphine, buprenorphine, and diprenorphine. Bioluminescence resonance energy transfer assays for Gi activation and arrestin-3 recruitment in human embryonic kidney 293 cells confirmed that norbuprenorphine is a high efficacy agonist, buprenorphine a low efficacy agonist, and diprenorphine an antagonist at the MOPr. Molecular dynamics simulations revealed that these ligands adopt distinct binding poses and engage different subsets of residues, despite sharing a common morphinan scaffold. Notably, norbuprenorphine interacted with sodium ion-coordinating residues W2936.48 and N1503.35, whilst buprenorphine and diprenorphine did not. Principal component analysis of the movements of the receptor transmembrane domains showed that the buprenorphine-bound receptor occupied a distinct set of conformations to the norbuprenorphine-bound receptor. Addition of an allosteric sodium ion caused the receptor and ligand to adopt an inactive conformation. The differences in ligand-residue interactions and receptor conformations observed here may underlie the differing efficacies for cellular signalling outputs for these ligands.