Evan S. O’Brien, Junzheng Wang, Parthasaradhireddy Tanguturi, Mengchu Li, Elizabeth White, Yuki Shiimura, Barnali Paul, Kevin Appourchaux, Kaavya Krishna Kumar, Weijiao Huang, Susruta Majumdar, John R. Traynor, John M. Streicher, Chunlai Chen, Brian K. Kobilka

bioRxiv - Biophysics

DOI: 10.64898/2026.02.20.707058

Abstract

Opioid agonists such as morphine and fentanyl exert analgesic effects by binding and activating the µ-opioid receptor (µOR), yet agonism of the µOR causes a slate of serious side effects. µOR-mediated addiction and respiratory depression are the major causes of the current opioid overdose crisis, largely driven by the explosion in illicit use of fentanyl, a potent opioid receptor full agonist. Given these serious side effects (and high resulting societal cost), molecules that act as analgesics with distinct mechanisms of action are of great interest. Positive allosteric modulators (PAMs) of the µOR have the potential to avoid many off-target side effects of conventional opioid orthosteric agonists by enhancing the signaling properties of natural opioid peptide systems. We used a DNA-encoded chemical library screening approach to selectively discover active-state-specific µOR PAMs. Two out of 3 selected prospective PAMs displayed the anticipated enhancement in agonist activity. The most effective of these compounds enhanced the activity of all orthosteric opioid agonists tested, including the native opioid peptide met-enkephalin. Little is known about the underlying dynamic basis of allosteric modulation of Family A GPCRs like the µOR. To that end, we used single-molecule fluorescence resonance energy transfer experiments to detail the impact that our novel µOR PAM has on the dynamic activation behavior of a key region on the intracellular face of the receptor. Our results here provide both a new chemical scaffold that acts as a µOR PAM and detailed pharmacological and dynamic insights into its mechanism of action.