Binding characteristics of the doxepin E/Z-isomers to the histamine H₁ receptor revealed by receptor-bound ligand analysis and molecular dynamics study

Doxepin is an antihistamine and tricyclic antidepressant that binds to the histamine H₁ receptor (H₁R) with high affinity. Doxepin is an 85:15 mixture of the E- and Z-isomers. The Z-isomer is well known to be more effective than the E-isomer, whereas based on the crystal structure of the H₁R/doxepin complex, the hydroxyl group of Thr112^3.37 is close enough to form a hydrogen bond with the oxygen atom of the E-isomer. The detailed binding characteristics and reasons for the differences remain unclear. In this study, we analyzed doxepin isomers bound to the receptor following extraction from a purified H₁R protein complexed with doxepin. The ratio of the E- and Z-isomers bound to wild-type (WT) H₁R was 55:45, indicating that the Z-isomer was bound to WT H₁R with an approximately 5.2-fold higher affinity than the E-isomer. For the T112^3.37V mutant, the E/Z ratio was 89:11, indicating that both isomers have similar affinities. Free energy calculations using molecular dynamics (MD) simulations also reproduced the experimental results of the relative binding free energy differences between the isomers for WT and T112^3.37V. Furthermore, MD simulations revealed that the hydroxyl group of T112^3.37 did not form hydrogen bonds with the E-isomer, but with the adjacent residues in the binding pocket. Analysis of the receptor-bound doxepin and MD simulations suggested that the hydroxyl group of T112^3.37 contributes to the formation of a chemical environment in the binding pocket, which is slightly more favorable for the Z-isomer without hydrogen bonding with doxepin.