G-protein-coupled receptors (GPCRs) represent one of the most expansive and diverse families of cell surface proteins, playing essential roles in recognizing a broad spectrum of signaling molecules including hormones, neurotransmitters, and pharmacological agents. These receptors orchestrate critical physiological processes, from sensory perception to immune defense, making them a central focus in drug discovery efforts. Strikingly, over 30% of drugs on the market today exert their effects through interactions with GPCRs, underscoring their pharmaceutical significance. Among the myriad GPCR subtypes, the histamine H1 receptor (H1R) is pivotal in the pathophysiology of allergic responses, inflammation, and neurological functions like wakefulness and cognition.
Despite the widespread targeting of H1R by antihistamines, clinical efficacy remains sometimes suboptimal, prompting researchers to rethink the paradigms underpinning drug design for these receptors. Traditional approaches have emphasized optimizing the binding affinity or energy between ligand and receptor. However, a more nuanced understanding now emphasizes the imperative role of thermodynamic components—enthalpy and entropy—in modulating ligand-receptor interaction specificity and efficacy. This thermodynamic profiling is particularly enriched by the concept of enthalpy-entropy compensation, a delicate balance that governs molecular recognition but has been experimentally elusive in membrane proteins like GPCRs.
Addressing this critical knowledge gap, a multidisciplinary team led by Professor Mitsunori Shiroishi at Tokyo University of Science embarked on a methodically rigorous investigation into the thermodynamics underlying H1R ligand binding. Their study, which integrates experimental thermodynamics and computational molecular dynamics, centers on doxepin, a well-characterized H1R antagonist known for its dual existence as E- and Z-geometric isomers. These isomers differ subtly in configuration but exhibit markedly divergent pharmacological profiles, making them ideal candidates for examining the enthalpic and entropic forces at play during receptor interaction.
Doxepin, originally developed as a tricyclic antidepressant, also serves as a potent antihistamine by inhibiting H1R. Previous research by this group revealed that the Z-isomer of doxepin binds H1R with roughly fivefold greater affinity than the E-isomer, a distinction attributed to interaction with the threonine residue Thr112^3.37 located within the receptor’s transmembrane domain. This residue’s contribution suggested a positional role in dictating isomer specificity, setting the stage for an in-depth thermodynamic characterization.
To dissect these binding nuances, the team engineered two receptor variants: the wild-type H1R (H1R_WT) and a mutant form wherein Thr112 was substituted with valine (T112^3.37V). Employing isothermal titration calorimetry (ITC), an experimental technique capable of precisely measuring binding enthalpy and entropy, alongside sophisticated molecular dynamics simulations, the researchers quantified and modeled ligand interactions with both receptor forms. This dual approach provided an unprecedented thermodynamic perspective on GPCR-ligand affinity and specificity that extends beyond conventional binding energy metrics.
Remarkably, while the binding free energy of doxepin exhibited no significant difference between the H1R_WT and the T112^3.37V mutant, the underlying thermodynamic signatures diverged substantially. The wild-type receptor’s ligand binding was dominated by favorable enthalpic contributions, indicative of strong, specific molecular interactions such as hydrogen bonding or van der Waals contacts. Contrarily, the mutant receptor displayed attenuated enthalpy gains balanced by increased entropic contributions, suggesting a shift towards less rigid, more dynamically disordered complexes possibly due to altered side-chain interactions at the binding site.
In particular, the Z-isomer’s interaction with H1R_WT showed pronounced enthalpic advantages coupled with significant entropic penalties relative to the E-isomer, revealing that binding is a thermodynamically costly conformationally restrictive event but ultimately favored by stabilizing interactions. This intricate enthalpy-entropy interplay was conspicuously absent in the mutant receptor, highlighting Thr112^3.37’s critical role in shaping the receptor’s ligand binding landscape and its capacity to discriminate between isomers via thermodynamic tuning.
Molecular dynamics simulations further corroborated these findings, illustrating that the Z-isomer’s higher affinity arises from conformational constraints imposed on the receptor-ligand complex, directly linking the experimental thermodynamic observations with structural dynamics. The simulations depicted a binding pocket contoured by Thr112^3.37 fostering a more ordered and energetically favorable interaction with the Z-isomer, while the mutant receptor’s altered binding pocket accommodated the isomers more indiscriminately, consistent with the thermodynamic data.
These insights into the enthalpy-entropy trade-off in GPCR-ligand interactions profoundly impact rational drug design strategies. By recognizing the importance of conformational restrictions and flexibility within ligand molecules and their targeted receptors, medicinal chemists can optimize compounds not just for binding strength but for kinetic stability and selectivity, potentially reducing side effects and improving therapeutic windows. The ability to finely tune thermodynamic parameters heralds a new frontier in the development of next-generation pharmaceuticals that adeptly exploit subtle molecular interactions.
Moreover, the integrated methodological framework combining ITC and molecular dynamics simulations demonstrated in this study offers a robust platform for probing protein-ligand interactions beyond H1R. Given the ubiquity of GPCRs in human physiology and their involvement in various pathological states, extending this approach could accelerate the discovery of more effective drugs tailored through thermodynamic optimization. This pioneering work underscores the vital necessity of blending experimental and computational techniques to unravel complex biomolecular phenomena with translational potential.
Beyond the immediate implications for antihistamine drug development, the study promotes a broader perspective on the interplay of entropy and enthalpy in biological recognition processes. The findings suggest that even minute conformational variations in ligands can dramatically shift the thermodynamic balance, highlighting the intricacies of molecular recognition in membrane proteins. Such an understanding is not only relevant for GPCR pharmacology but also for diverse protein-drug and protein-protein interactions critical to health and disease.
In conclusion, this groundbreaking research elucidates the molecular and thermodynamic principles governing the selective binding of doxepin isomers to the histamine H1 receptor. It reveals how a single amino acid residue mediates the delicate enthalpy-entropy equilibrium that underpins ligand specificity, providing a compelling mechanistic narrative that could inform the rational design of improved therapeutics. The work by Professor Shiroishi and colleagues marks a significant advancement in GPCR biophysics, promising to impact the broader fields of pharmacology and drug development profoundly.
Subject of Research: Cells
Article Title: Enthalpy–Entropy Trade-Off Underlies Geometric Isomer Selectivity in Histamine H1 Receptor–Doxepin Interaction
News Publication Date: 26-Jan-2026
References: DOI: 10.1021/acsmedchemlett.5c00696
Image Credits: Professor Mitsunori Shiroishi from Tokyo University of Science, Japan
Keywords
Drug design, Pharmacology, Medicinal chemistry, G protein coupled receptors, Receptor proteins, Antihistamines, Thermodynamics, Molecular dynamics
