Mechanism of µ Receptor Activation by SR-17018 Conferring G-Protein Signaling Bias

1. Distinct mechanism of µ receptor activation by SR-17018 confers G-protein signalling bias Dr. Samuel Singleton Postdoc TGH lab, University of Dundee SSingleton001@dundee.ac.uk

2. Background Improved analgesia with reduced RD, tolerance and OIC

3. Additional limitations associated with in vitro models Spare receptors Spare receptors Partial agonists are able to achieve maximum responses in amplified pathways (with fractional occupancy) to give the appearance of full efficacy. This often leads to the false impression of signalling bias.

4. Reconciling differences in stoichiometry reveals lower efficacy FDA approved 2020 MORPHINE Does partial agonism also explain the diverse signalling outcomes of whole panels of agonists? DAMGO (ref) Morphine Fentanyl* Tianeptine Oxycodone Buprenorphine* TRV130** PZM21** Herkinorin** U4791e bromadoline** U-47700 (U4) SR-17018** * Previous designation of bias

5. Approaches to measure opioid signalling pathways Linear β-arrestin2 recruitment assay Highly amplified cAMP accumulation assay Human Efficacies and potencies assessed in Chinese Hamster Ovarian cells stably overexpressing human µ receptors and β-arrestin2 Number of available receptors is reduced by 1-hr exposure to an irreversible antagonist (β-funaltrexamine; β-FNA)

6. Apparent bias observed in cells with full µ receptor availability Linear β-arrestin2 recruitment assay Highly amplified cAMP accumulation assay 100 100 120 120 DAMGO Morphine Less potent Efficacy 100 100 Oxycodone 80 80 TRV130 PZM21 Buprenorphine Fentanyl % Maximum RLU % Maximum RLU cAMP (% control) cAMP (% control) 80 80 60 60 Potency 60 60 40 40 Herkinorin 40 40 SR17018 20 20 U47700 U47931e Tianeptine 20 20 Less potent 0 0 0 0 10 1000 0.001 0.1 1 100 0.01 0.0001 10 1000 0.001 0.1 1 100 0.01 0.0001 10 0.1 0.00001 0.001 1 100 0.0001 0.01 [Agonist] µM 10 0.00001 0.001 [Agonist] µM 0.1 1 0.0001 0.01 0.000001 [Agonist] µM [Agonist] µM DAMGO >>> Tianeptine = fentanyl = morphine = SR-17018 = Herkinorin = Oxycodone = U47700 >> U47931e = TRV130 = PZM21 > Buprenorphine DAMGO = Tianeptine= fentanyl = morphine = SR-17018 = Herkinorin = Oxycodone = U47700 = U47931e = PZM21 >> Rank order: TRV130 > Buprenorphine

7. Limiting receptor availability reduces µ agonist efficacy < 100 nM 100 nM < < 100 nM µ 100 nM < + β-FNA µ < 100 nM < 100 nM < 100 nM < 100 nM + β-FNA µ + β-FNA < 100 nM < 100 nM < 100 nM 100 nM < µ *** First significant reduction in efficacy from control (0 nM)

8. Partial agonism revealed by reducing receptor availability 120 120 120 120 < 100 nM < 100 nM 100 nM < 100 nM < 100 100 100 100 cAMP (% control) cAMP (% control) cAMP (% control) cAMP (% control) 80 80 80 80 60 60 60 60 40 40 40 40 [-FNA] nM: 0 100 [-FNA] nM: 0 100 [-FNA] nM: [-FNA] nM: 20 20 20 20 0 100 0 100 0 0 0 0 10 0.00001 0.001 0.1 1 100 0.0001 0.01 10 0.001 0.1 1 100 0.0001 0.01 10 10 0.00001 0.001 0.1 0.00001 0.001 0.1 1 100 1 100 0.0001 0.01 0.0001 0.01 0.000001 0.000001 µ [Oxycodone] M [DAMGO] M [Morphine] M [Fentanyl] M 120 120 120 120 100 100 100 100 cAMP (% control) cAMP (% control) cAMP (% control) cAMP (% control) + β-FNA (100 nM) 80 80 80 80 60 60 60 60 < 100 nM < 100 nM < 100 nM < 100 nM 40 40 40 40 µ [-FNA] nM: 0 100 [-FNA] nM: [-FNA] nM: [-FNA] nM: 20 20 20 20 0 100 0 100 0 100 0 0 0 0 10 10 0.00001 0.001 0.1 0.00001 0.001 0.1 1 100 1 100 10 0.0001 0.01 0.0001 0.01 0.001 0.1 1 100 0.0001 0.01 10 0.00001 0.001 0.1 1 0.000001 0.0001 0.01 [Herkinorin] M [TRV130] M [PZM21] M [Buprenorphine] M 120 120 120 120 100 100 100 100 cAMP (% control) cAMP (% control) cAMP (% control) cAMP (% control) 80 80 80 80 60 60 60 60 < 100 nM < 100 nM 100 nM < < 100 nM 40 40 40 40 [-FNA] nM: [-FNA] nM: 0 100 [-FNA] nM: 0 100 [-FNA] nM: 0 100 20 20 20 20 0 100 0 0 0 0 10 0.1 0.001 1 100 0.0001 0.01 10 0.001 0.1 1 100 0.0001 0.01 10 1000 0.001 0.1 1 100 0.01 10 1000 0.001 0.1 1 100 0.01 [U47700] M [U47931e] M [SR17018] M [Tianeptine] M

9. Limiting receptor reserve reveals correlation in efficacy between assays Partial receptor availability Full receptor availability EMAX cAMP (100 nM β-FNA) 80 100 EMAX cAMP (no β-FNA) 80 60 µ 60 µ 40 40 20 r = 0.76 p < 0.05 20 r = 0.56 p > 0.05 0 0 0 20 40 60 80 0 20 40 60 80 EMAX-arrestin2 (no β-FNA) EMAX-arrestin2 (no β-FNA) All agonists able to achieve similar maximum response True efficacy

10. Competitive antagonism reveals distinct activation by SR-17018 Agonist Antagonist DAMGO SR-17018 120 120 Same EMAX 100 cAMP accumulation 100 cAMP (%control) cAMP (%control) 80 80 60 60 [Cyprodime] [Cyprodime] 40 40 0 10 100 1000 0 10 100 1000 20 20 Lower potency 0 0 10 0.001 0.1 1 100 0.01 0.0001 10 0.1 0.001 1 0.0001 0.01 If agonist/antagonist are competing for the same site then increasing [agonist] can overcome antagonism. [SR-17018] M [DAMGO] M DAMGO is competing for the same site as cyprodime as is SR-17018 SR-17018 is competing for the same site as DAMGO

11. Competitive antagonism reveals distinct activation by SR-17018 Agonist Antagonist DAMGO SR-17018 [Cyprodime] 0 1 10 100 [Cyprodime] 0 10 100 1000 Lower EMAX 100 100 β-arrestin2 recruitment % Maximum response % Maximum response 80 80 60 60 40 40 20 20 Same potency 0 0 10 1000 0.1 0.001 1 100 0.0001 0.01 10 1000 0.1 0.001 1 100 0.0001 0.01 If agonist/antagonist are competing for different sites then increasing [agonist] has no effect. [SR-17018] M [DAMGO] M DAMGO is competing for the same site as cyprodime whereas SR-17018 is not SR-17018 is competing for a different site to DAMGO

12. Competitive agonism reveals distinct activation by SR-17018 Fentanyl Morphine TRV130 SR-17018 120 120 120 120 100 100 100 100 80 80 80 80 % Max RLU % Max RLU % Max RLU % Max RLU 60 60 60 60 40 40 40 40 20 20 20 20 0 0 0 0 10 10 10 0.001 0.1 0.001 0.1 0.001 0.1 1 100 1 100 1 100 0.0001 0.01 0.0001 0.01 0.0001 0.01 10 0.001 0.1 1 100 0.01 0.0001 [Fentanyl] M [Morphine] M [TRV130] M [SR-17018] µM Unlike other orthosteric agonists, SR-17018 fails to compete with DAMGO suggesting a non-competitive activation method

13. Summary 1. Agonists appearing as biased (against β-arrestin2 recruitment) have lower efficacy when spare receptors are removed. 2. Low efficacy can explain the improved side effect of recently developed biased agonists (Gillis et al, 2020; 32234959). 3. SR-17018 is the most promising novel µ agonist BUT: A. It has characteristic signs of being a partial agonist in vitro B. Causes non-competitive activation (is this bias ?) C. Still causes detrimental effects in mice (Hill et al., 2023; 37489013) 4. If opioids analogous to SR-17018 are to be exploited clinically it will be important for future studies to test whether its effects are reversed by traditional antagonists e.g, naloxone

14. Acknowledgements TGH Lab contributions: Professor Tim G. Hales (t.g.hales@dundee.ac.uk) Dr. Daniel Baptista-Hon (Macau UST, China) Contributions by: Clara Dieterle Dr. David Walker Andrew S. Oswald Greta Rosenqvist Tyko Runeberg Laura Robertson Dr. Shuvam Sarkar Taylor McCarthy