G-protein-coupled receptors (GPCRs) are one of the largest families of cell surface proteins in the human body that recognize hormones, neurotransmitters, and drugs. These receptors regulate a wide range of physiological processes and are the targets of more than 30% of currently marketed drugs. The histamine H1 receptor (H1R) is one such GPCR subtype that plays a key role in mediating allergic reactions, inflammation, vascular permeability, airway constriction, wakefulness, and cognitive functions in the human body. While antihistamines primarily target H1R, current drugs can exhibit limited therapeutic efficacy, prompting researchers to look at H1R ligands from new perspectives.
Recently, the importance of drug design based not only on the affinity or binding energy between a compound and its target protein, but also on its components, enthalpy, and entropy, has been recognized as crucial for rational drug design. In particular, enthalpy–entropy compensation has emerged as a key concept for understanding ligand selectivity and isomer specificity. However, direct experimental measurement of these thermodynamic parameters has been limited to cell surface proteins, such as GPCRs.
Addressing this gap, a research team led by Professor Mitsunori Shiroishi from the Department of Life System Engineering, Tokyo University of Science (TUS), Japan, systematically investigated the binding thermodynamics of the H1R. The team included Mr. Hiroto Kaneko (first-year doctoral student) and Associate Professor Tadashi Ando from TUS, among others. Their study was published online in ACS Medicinal Chemistry Letters on January 26, 2026.
"Doxepin is a compound that has been widely used as an H1R inhibitor. In this study, we successfully measured the thermodynamic signatures of doxepin geometric isomers (E- and Z-isomers) to the H1R, prepared via a budding yeast expression system, using isothermal titration calorimetry and molecular dynamics simulations," explains Prof. Shiroishi.
Doxepin, a tricyclic antidepressant, is also a potent antihistamine targeting H1R and exists as a mixture of E- and Z-isomers. In a previous study, the team reported that the Z-isomer exhibits approximately five times higher affinity for H1R binding than the E-isomer. They also identified a key threonine residue (Thr1123.37) that contributed to this isomer-dependent selectivity. In the current study, the researchers performed a detailed thermodynamic analysis of the H1R-doxepin interaction to clarify this selectivity.
To this end, they synthesized two variants of H1R: a wild-type (H1R_WT) variant, which they used in the previous study, and a T1123.37V mutant, in which the Thr1123.37 residue is swapped with a different amino acid. Their interaction was tested first with doxepin (E- and Z-isomer mixture) and then with individual E- and Z-isomers.
The results showed no differences in binding energy for interactions of doxepin between H1R_WT and the T1123.37V mutant; however, the enthalpic and entropic contributions differed. Binding to H1R_WT was predominantly enthalpy-driven, whereas binding to the mutant receptor showed a reduced enthalpic contribution accompanied by a relatively larger entropic contribution.
Notably, binding of the Z-isomer to H1R_WT was associated with a larger enthalpic gain and a greater entropic penalty compared to the E-isomer. These differences were absent in the T1123.37V mutant. Furthermore, the binding energy of the Z-isomer was higher than that of the E-isomer for H1R_WT, while in the case of the mutant receptor, the binding energies of both isomers were comparable, consistent with the findings of the previous study. These observations underscore the role of Thr1123.37 in balancing the enthalpic gains and entropic losses during ligand binding, as well as the more pronounced effect in the interaction with the Z-isomer.
To further understand the molecular basis of this selectivity, the researchers conducted molecular dynamics simulations, which showed that the high-affinity binding of the Z-isomer arises from conformational restrictions, consistent with the observed high enthalpy and reduced entropy associated with binding.
"These mechanistic insights into the enthalpy-entropy trade-off in GPCR-ligand interactions highlight the importance of considering conformational constraints and flexibility in designing ligands with optimized thermodynamic properties," remarks Prof. Shiroishi. "This could lead to the development of drugs with improved selectivity, reduced side effects, and longer-lasting therapeutic effects. Moreover, our approach, combining thermodynamic analysis with molecular dynamics simulations, can be applied to other GPCRs and proteins, aiding rational drug design."
The study implies that tiny molecular conformations can shift the entropy–enthalpy balance, and understanding these concepts could aid in designing effective therapeutics, while reducing off-target effects and maintaining higher efficacy. Additionally, the entropy–enthalpy balance could be observed not only in GPCR-based interactions, but also in other proteins and drugs.
Overall, this study provides new insights into the thermodynamic principles governing GPCR–ligand interactions and offers a valuable framework for the development of more effective therapeutics.
