In Vitro Pharmacology of Mitragynine at α-Adrenoceptors

Mitragynine is a psychoactive alkaloid in Mitragyna speciosa with unique polypharmacology at G protein-coupled receptors. In addition to its well-known partial agonist activity at opioid receptors, mitragynine is an antagonist at human α2A-adrenoceptors (α2ARs), as measured in an in vitro GTPγS G protein assay. Mitragynine's in vitro α2AR antagonist pharmacology contrasts with rat behavioral pharmacology studies that suggest mitragynine behaves as an in vivo agonist at rat α₂Rs. This study investigates this apparent discrepancy using recombinant α-adrenoceptors and a range of orthogonal signal transducers. We evaluated whether mitragynine activates any of seven Gαi/o proteins coupled to α2A, α2B, and α2CRs, as well as Gαq and Gα₁₁ coupled to α1AR. Additionally, we examined rat and human α2AR-mediated cAMP inhibition, α2AR-mediated β-arrestin2 recruitment, and tested α₂R or α₁R-mediated ERK phosphorylation in wild-type, β-arrestin 1/2 knockout, and Gαq/11 knockout cells. Finally, we report binding and enzyme-inhibition profiling results for mitragynine and its major metabolites, 7-Hydroxymitragynine and 9-Hydroxycorynantheidine, at 99 targets. The results did not support the hypothesis that mitragynine (or its primary metabolites) activates α₂Rs, but, aligned with our previous GTPγS results, demonstrate that mitragynine is a low-potency, competitive α2AR antagonist at Gαi1, cAMP, and β-arrestin2 transducers. However, we show that mitragynine is a low-potency (EC₅₀ ∼3 μM), partial agonist at α1AR-Gα₁₁ and stimulates ERK phosphorylation via Gαq/11-coupled α₁Rs, supporting in vivo studies that suggest mitragynine is an α₁R agonist. Nevertheless, the agonist effects of mitragynine at α1AR-Gα₁₁ were modest compared to clonidine, a partial agonist control that also activated all α₂R transducers. Mitragynine's dual α2AR antagonist/α₁R partial agonist pharmacology might contribute to mitragynine's psychostimulant-like properties.