Achieving ligand subtype selectivity within highly homologous subtypes of G-protein-coupled receptor (GPCR) is critical yet challenging for GPCR drug discovery, primarily due to the unclear mechanism underlying ligand subtype selectivity, which hampers the rational design of subtype-selective ligands. Herein, we disclose an unusual molecular mechanism of entropy-driven ligand recognition in cannabinoid (CB) receptor subtypes, revealed through atomic-level molecular dynamics simulations, cryoelectron microscopy structure, and mutagenesis experiments. This mechanism is attributed to the distinct conformational dynamics of the receptor\'s orthosteric pocket, leading to variations in ligand binding entropy and consequently, differential binding affinities, which culminate in specific ligand recognition. We experimentally validated this mechanism and leveraged it to design ligands with enhanced or ablated subtype selectivity. One such ligand demonstrated favorable pharmacokinetic properties and significant efficacy in rodent inflammatory analgesic models. More importantly, it is precisely due to the high subtype selectivity obtained based on this mechanism that this ligand does not show addictive properties in animal models. Our findings elucidate the unconventional role of entropy in CB receptor subtype selectivity and suggest a strategy for rational design of ligands to achieve entropy-driven subtype selectivity for many pharmaceutically important GPCRs.

Prostaglandin F2α (PGF2α) and thromboxane A2 (TXA2) are endogenous arachidonic acid metabolites, modulating diverse physiological processes including inflammation and cardiovascular homeostasis through activating PGF2α receptor (FP) and TXA2 receptor (TP). Ligands targeting FP and TP have demonstrated efficacy in treating conditions like glaucoma and cardiovascular diseases in humans, as well as reproductive-related diseases in animals. Here, we present five cryoelectron microscopy structures illustrating FP and TP in complex with Gq and bound to PGF2α (endogenous ligand), latanoprost acid (a clinical drug), and two other synthetic agonists. Combined with mutational and functional studies, these structures reveal not only structural features for the specific recognition of endogenous ligands and attainment of receptor selectivity of FP and TP but also the common mechanisms of receptor activation and Gq protein coupling. The findings may enrich our knowledge of ligand recognition and signal transduction of the prostanoid receptor family and facilitate rational ligand design toward these two receptors.

The G-protein-coupled human cannabinoid receptor 1 (CB1) is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. The structures of CB1-Gi complexes in synthetic agonist-bound forms have been resolved to date. However, the commercial drug recognition and Gq coupling mechanisms of CB1 remain elusive. Herein, the cryo-electron microscopy (cryo-EM) structure of CB1-Gq complex, in fenofibrate-bound form, at near-atomic resolution, is reported. The structure elucidates the delicate mechanisms of the precise fenofibrate recognition and Gq protein coupling by CB1 and will facilitate future drug discovery and design.

OBJECTIVE: The P2X3 receptor, a trimeric ionotropic purinergic receptor, has emerged as a potential therapeutic target for refractory chronic cough (RCC). Nevertheless, gefapixant/AF-219, the only marketed P2X3 receptor antagonist, might lead taste disorders by modulating the human P2X2/3 (hP2X2/3) heterotrimer. Hence, in RCC drug development, compounds exhibiting strong affinity for the hP2X3 homotrimer and a weak affinity for the hP2X2/3 heterotrimer hold promise. An example of such a molecule is sivopixant/S-600918, a clinical Phase II RCC candidate with a reduced incidence of taste disturbance compared to gefapixant. Sivopixant and its analogue, (3-(4-([3-chloro-4-isopropoxyphenyl]amino)-3-(4-methylbenzyl)-2,6-dioxo-3,6-dihydro-1,3,5-triazin-1(2H)-yl)propanoic acid (DDTPA), exhibit both high affinity and high selectivity for hP2X3 homotrimers, compared with hP2X2/3 heterotrimers. The mechanism underlying the druggable site and its high selectivity remains unclear.
METHODS: To analyse mechanisms that distinguish this drug candidate from other inhibitors of the P2X3 receptors we used a combination of chimera construction, site covalent occupation, metadynamics, mutagenesis and whole-cell recording.
RESULTS: The high affinity and selectivity of sivopixant/DDTPA for hP2X3 receptors was determined by the tri-symmetric site located close to the upper vestibule. Substitution of only four amino acids inside the upper body domain of hP2X2 with those of hP2X3, enabled the hP2X2/3 heterotrimer to exhibit a similar level of apparent affinity for sivopixant/DDTPA as the hP2X3 homotrimer.
CONCLUSIONS: From the receptor-ligand recognition perspective, we have elucidated the molecular basis of novel RCC clinical candidates\' cough-suppressing properties and reduced side effects, offering a promising approach to the discovery of novel drugs that specifically target P2X3 receptors.

Lectin receptor-like kinases (LecRKs) locate on the cell membrane and play diverse roles in perceiving environmental factors in higher plants. Studies have demonstrated that LecRKs are involved in plant development and response to abiotic and biotic stresses. In this review, we summarize the identified ligands of LecRKs in Arabidopsis, including extracellular purine (eATP), extracellular pyridine (eNAD+), extracellular NAD+ phosphate (eNADP+) and extracellular fatty acids (such as 3-hydroxydecanoic acid). We also discussed the posttranslational modification of these receptors in plant innate immunity and the perspectives of future research on plant LecRKs.

Riboswitches are bacterial mRNA structure elements regulating either transcription or translation of downstream genes in response to high-affinity binding of a low molecular weight ligand. Among this diverse group of RNA structures, the class-I preQ1 sensing riboswitches (QSW) stand out since they are the smallest known natural riboswitches. The preQ1 sensing riboswitches combine ligand sensing and functional control within a single structural domain that adopts a pseudoknot conformation encapsulating both the cognate ligand and the ribosome binding site. preQ1 sensing riboswitches also occur in thermophilic bacteria. In these cases, their tertiary structures have to be stable even at temperatures above 60 °C to be functional at the organism\'s optimal growth temperatures. Despite the available high-resolution structures of these riboswitches, it is not yet understood which tertiary interactions are primarily responsible for their exceptional temperature stability. Here, we show that an intricate three-dimensional network of non-canonical interactions involving various non-neighboring nucleobases is the origin of the riboswitch\'s thermostability. An essential part of this network is a so far undetected stably protonated cytidine. It is characterized by an exceptional high pKA value of >9.7 and could be unambiguously identified through the application of modern heteronuclear detected NMR experiments. Thus, the presence or absence of a single proton can modulate the formation of an RNA tertiary structure and ligand binding capacity under extreme environmental conditions.

Mas-related G protein-coupled receptor (MRGPR) family members play important roles in the sensation of noxious stimuli and represent novel targets for the treatment of itch and pain. MRGPRs recognize a diversity of agonists and display complicated downstream signaling profiles, high sequence diversity across species, and many polymorphisms in humans. The recent structural advances on MRGPRs reveal unique structural features and diverse agonist recognition modes of this receptor family, which should facilitate the structure-based drug discovery at MRGPRs. In addition, the newly discovered ligands also provide valuable tools to explore the function and the therapeutic potential of MRGPRs. In this review, we discuss these progresses in our understanding of MRGPRs and highlight the challenges and potential opportunities for the future drug discovery at these receptors.

Nucleotide-binding and leucine-rich repeat (NLR) proteins are critical intracellular immune receptors in both animals and plants. Perception of pathogen-derived or stress-associated signals induces NLR oligomerization to form multiprotein complexes called inflammasomes in animals or resistosomes in plants to mediate host immune response. Significant progress has been made during the past few years in our understanding of NLR biology, particularly the structural perspective of these two types of NLR-containing complexes. In this article, we review the latest advances in our structural knowledge of how NLR inflammasomes and resistosomes are activated and assembled and how the structural information provides insight into their distinct mechanisms of action. Commonalities and differences between NLR inflammasomes and resistosomes are also discussed.

TLR5, as a member of Toll-like receptors (TLRs) family in mammals, is responsible for recognizing bacterial flagellin and initiating innate immunity, but its function is still unclear in fish species. In this study, two family members of TLR5 were cloned and identified from Sinocyclocheilus grahami (S. grahami), named sgTLR5a and sgTLR5b. The length of coding sequence of sgTLR5a and sgTLR5b is 2,622 bp and 2,658 bp, encoding 873 and 885 amino acids, respectively. Molecular phylogenetic analysis indicates that sgTLR5a and sgTLR5b have the closest genetic relationship with TLR5M (membrane-type) of Cyprinus carpio and Schizothorax prenanti, respectively. sgTLR5a and sgTLR5b were widely expressed in various tested tissues, of which the expression levels were the highest in skin tissue. After stimulations of Aeromonas hydrophila (A. hydrophila) and flagellin, the expression levels of sgTLR5a and sgTLR5b in liver, spleen and head kidney tissues were strongly up-regulated, but LPS stimulation only increased the expression of sgTLR5b in these tissues. The luciferase reporter assay displayed that sgTLR5a and sgTLR5b could specifically recognize bacterial flagellin and A. hydrophila and activate the downstream NF-κB signaling pathway in HEK293T cells. Moreover, the overexpression of sgTLR5a and sgTLR5b in EPC cells up-regulated the expression levels of IL-8 and TNF. sgTLR5a and sgTLR5b were observed to locate in the intracellular region by confocal microscope. Interestingly, we found that the NF-κB signaling pathway was positively regulated by co-transfecting sgTLR5a or sgTLR5b with TLR trafficking chaperone sgUNC93B1. In conclusion, our results reveal sgTLR5a and sgTLR5b may play an important role in antibacterial response by activating the NF-κB signaling pathway.

Riboswitches are RNA-structured elements that modulate gene expression by changing their conformation in response to specific metabolite ligand binding. Therefore, the biological functions of riboswitches mainly depend on the switching of secondary and three-dimensional structures in the presence and absence of the metabolite ligands. However, the binding mechanisms of cognate ligands to riboswitches are still not well understood. Here, we have introduced how to use explicit solvent molecular dynamics (MD) simulation to observe the binding process of cognate ligand to add adenine riboswitch aptamer at the atomic level. In addition, we have analyzed the driving factors of the binding process and calculated the binding free energy based on the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) method.