Drug discovery to counteract antinociceptive tolerance with mu-opioid receptor endocytosis

Morphine antinociceptive tolerance is highly correlated with its poor ability to promote mu-opioid–receptor (MOR) endocytosis. Our objective was to discover a novel positive allosteric modulator of MOR to enhance morphine-induced MOR endocytosis. We used high-throughput screening to identify several cardiotonic steroids as positive allosteric modulators of morphine-induced MOR endocytosis having high potency and efficacy, independently of Na + /K + -ATPase inhibition. Convallatoxin was found to enhance morphine-induced MOR endocytosis through an adaptor protein 2/clathrin-dependent mechanism without regulating G protein- or β -arrestin-mediated pathways. Both F243 and I292 residues of MOR were essential to the effect of convallatoxin on MOR endocytosis. Co-treatment with chronic morphine and convallatoxin reduced morphine tolerance in animal models of acute thermal pain and chronic inflammatory pain. Acute convallatoxin administration reversed morphine tolerance in morphine-tolerant mice. These findings suggest that cardiotonic steroids are potentially therapeutic for morphine side effects and open a new avenue for the study of MOR trafficking.