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

Abstract

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.