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 H 1 receptor (H 1 R) 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 H 1 R, current drugs can exhibit limited therapeutic efficacy, prompting researchers to look at H 1 R 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 H 1 R. 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 H 1 R inhibitor. In this study, we successfully measured the thermodynamic signatures of doxepin geometric isomers (E- and Z-isomers) to the H 1 R, 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 H 1 R 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 H 1 R binding than the E -isomer. They also identified a key threonine residue (Thr112 3.37 ) that contributed to this isomer-dependent selectivity. In the current study, the researchers performed a detailed thermodynamic analysis of the H 1 R-doxepin interaction to clarify this selectivity.
To this end, they synthesized two variants of H 1 R: a wild-type (H 1 R_WT) variant, which they used in the previous study, and a T112 3.37 V mutant, in which the Thr112 3.37 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 H 1 R_WT and the T112 3.37 V mutant; however, the enthalpic and entropic contributions differed. Binding to H 1 R_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 H 1 R_WT was associated with a larger enthalpic gain and a greater entropic penalty compared to the E -isomer. These differences were absent in the T112 3.37 V mutant. Furthermore, the binding energy of the Z -isomer was higher than that of the E -isomer for H 1 R_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 Thr112 3.37 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.
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Reference
DOI: 10.1021/acsmedchemlett.5c00696
About The Tokyo University of Science
Tokyo University of Science (TUS) is a well-known and respected university, and the largest science-specialized private research university in Japan, with four campuses in central Tokyo and its suburbs and in Hokkaido. Established in 1881, the university has continually contributed to Japan's development in science through inculcating the love for science in researchers, technicians, and educators.
With a mission of “Creating science and technology for the harmonious development of nature, human beings, and society," TUS has undertaken a wide range of research from basic to applied science. TUS has embraced a multidisciplinary approach to research and undertaken intensive study in some of today's most vital fields. TUS is a meritocracy where the best in science is recognized and nurtured. It is the only private university in Japan that has produced a Nobel Prize winner and the only private university in Asia to produce Nobel Prize winners within the natural sciences field.
Website: https://www.tus.ac.jp/en/mediarelations/
About Professor Mitsunori Shiroishi from Tokyo University of Science
Dr. Mitsunori Shiroishi graduated from the Department of Biomolecular Engineering at Tohoku University, Japan, in 1998 and completed his doctoral degree there in 2003. He joined Tokyo University of Science in April 2018, where he currently serves as a full-time Professor. He leads the Protein Engineering Laboratory as a principal investigator, where his team conducts cutting-edge research on protein science, focused mainly on immunology and cell receptors with pharmaceutical potential, including receptors, such as G-protein-coupled receptors. He has published over 70 research papers on these topics.
Laboratory website
Official TUS website
Funding information
This work was funded by JSPS KAKENHI (Grant Numbers: JP25709080 and JP19K06514), the Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED (Grant Number: JP24ama121033), and JST SPRING (Grant Number: JPMJSP2151).
ACS Medicinal Chemistry Letters
10.1021/acsmedchemlett.5c00696
Experimental study
Cells
Enthalpy–Entropy Trade-Off Underlies Geometric Isomer Selectivity in Histamine H1 Receptor–Doxepin Interaction
26-Jan-2026
The authors declare no competing financial interests.