An investigation of the activation of µ opioid receptors by fentanyls using in silico and in vitro approaches

Abstract

The µ opioid receptor (MOPr) is a clinically relevant drug target in anaesthesia and analgesia. However, drugs targeting MOPr are widely abused, often leading to overdose due to the respiratory depressive effects of opioids. The USA is currently experiencing an opioid epidemic, with a striking rise in fentanyl-related overdose deaths. Fentanyl is a highly potent synthetic opioid with many structural analogues, fentanyls are either taken intentionally, or are used to adulterate heroin.
The ability of different fentanyls to activate the MOPr, and how they interact with the receptor at a molecular level is not well understood. In this thesis, in silico methods are employed to explore how fentanyl and carfentanil interact with the MOPr. For both fentanyl and carfentanil, multiple stable potential binding poses, in two orientations were determined. In vitro signalling assays examining G protein activation, β-arrestin 2 recruitment and cell surface receptor loss were used to investigate the activation of MOPr by several fentanyls. From this it was shown that carfentanil recruited β-arrestin 2 and induced cell surface receptor loss with very high potency. Bias calculations revealed carfentanil’s β-arrestin bias and efficacy calculations revealed carfentanil’s low efficacy for G protein activation and high efficacy for β-arrestin 2 recruitment. The MDs generated for fentanyl and carfentanil were used to identify specific residues potentially involved in determining fentanyl’s binding orientation or interacted with carfentanil’s 4- carbomethoxy group. In silico mutagenesis was used to examine these residues of interest and shortlist several key residues that may affect the binding of fentanyl or carfentanil in vitro. The MOPr was then mutated in vitro, and assays were run to determine if any of the residues of interest affected fentanyl or carfentanil’s ability to activate the MOPr, with morphine used as a control.

Date of Award	24 Jan 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Eamonn P Kelly (Supervisor) & Richard B Sessions (Supervisor)