The c-Src inhibitor eCF506 diminishes opioid tolerance and reduces β-arrestin2 recruitment.

BACKGROUND AND PURPOSE: Opioids, such as morphine, are effective analgesics, but their persistent use is compromised by tolerance. In mice, tolerance is attenuated by β-arrestin2 depletion or Src-family kinase inhibitors, which may act through off-target effects. We tested the effect of eCF506 a conformationally selective and potent c-Src inhibitor on μ-opioid receptor signalling and trafficking. EXPERIMENTAL APPROACH: Morphine antinociception and tolerance were assessed in C57BL/6J mice following oral eCF506. μ receptor signalling was examined in cells expressing recombinant μ receptors by measuring agonist-evoked inhibition of cAMP accumulation, β-arrestin2 recruitment, receptor phosphorylation, surface expression and internalisation following exposure to eCF506 (0.01-1 μM). The role of c-Src was further tested using targeted degradation and expression of mutant constructs. KEY RESULTS: Oral eCF506 (20 or 80 mg kg-1) had no effect on acute morphine antinociception but inhibited the development of tolerance in mice. In vitro, c-Src inhibition increased receptor surface expression and reduced agonist-induced internalisation, without altering agonist-evoked C-terminal phosphorylation. eCF506 did not affect DAMGO- or morphine-evoked inhibition of cAMP accumulation but reduced their efficacies for recruiting β-arrestin2 in a time- and concentration-dependent manner. This effect was reproduced by targeted degradation of c-Src and required the catalytic domain, as it was attenuated in cells over-expressing kinase-inactive Src250-536 (K298M). CONCLUSIONS AND IMPLICATIONS: These findings suggest that selective inhibition of c-Src by eCF506 prolongs opioid antinociception by limiting β-arrestin2-dependent regulation and trafficking of μ receptors, while preserving G-protein signalling, identifying c-Src as a mechanistically distinct target for modulating opioid tolerance.