Mapping the binding site of the 5-HT2A receptor using mutagenesis and ligand libraries: Insights into the molecular actions of psychedelics.

摘要

The serotonin 5-HT2A receptor, which is a G-protein coupled receptor (GPCR), resides primarily in the cerebral cortex and is important as a major target for certain neurological disease medications. Drugs that activate this receptor are considered psychedelics, and represent one of the oldest classes of psychoactive drugs known to man. The objective of these studies was to map how each structural class of psychedelics binds and activates the serotonin 5-HT2A, receptor using a combination of mutagenesis and a library of ligands. First, results indicate that serine 5.43 (S239) and asparagine 6.55 (N343) form a hydrogen bond with the 5-position of tryptamines and phenylalkylamines. Also, serine 5.46 (S242) and/or threonine 3.37 (T160) form a hydrogen bond with the N(1)-position of tryptamines and with the 2-position of phenylalkylamines. Results also indicate that the functional activation by the N(1)-position of LSD is mediated by T160, whereas the affinity of LSD is more mediated by S242. Furthermore, N343 and S239 were not found to be important for the binding of LSD, and in fact mutation of 6.55 in the D 1 receptor had no effect on LSD, but led to marked losses in affinity for all catechol ligands. Second, mutations of leucine 229 in extracellular loop 2 (EL2) of the 5-HT2A receptor affected the rigid diethylamide and other LSD analogs stereoselectively, whereas only mutations of alanine 230 had any effect on lisuride. Furthermore, mutation of serine 188 in EL2 of the D1 receptor showed a loss in affinity only with lisuride but not with LSD. Third, the efficacy of N-substituted tryptamines and phenylalkylamines in the 5-HT2A receptor is affected by mutations of serine 3.36 (S159) and tyrosine 7.43 (Y370), respectively, by possible coordination with conserved aspartate 3.32. Both S159 and Y370, however, seem to be involved with potency of N(6)-alkyl analogs of LSD. Finally, disruption of a postulated disulfide bridge by cysteine mutation in extracellular loop 3 (EL3) of the 5-HT2A receptor affects only the potency and efficacy of ligands but not their affinity. These results construct a map capable of predicting ligand-specific action at other GPCRs to guide design of new therapeutic agents.