Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site-Directed Methyl Labeling

G protein-coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein-stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope-labeled methyl probe, we show that the activity and the conformation state of stabilized neurotensin receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist-induced activity of the receptor. Furthermore, this assay can be used as a readout when re-introducing agonist-dependent signaling into a highly stabilized, and thus rigidified, receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively ¹⁹F-labeled receptors are not sufficient to resolve signal overlap for a more detailed analysis.     

Guidelines for the Synthesis of Small‐Molecule Irreversible Probes Targeting G Protein‐Coupled Receptors

Irreversible probes have been proven to be useful pharmacological tools in the study of structural and functional features in drug receptor pharmacology. They have been demonstrated to be particularly valuable for the isolation and purification of receptors. Furthermore, irreversible probes are helpful tools for the identification and characterization of binding sites, thereby supporting the advancement of rational drug design. In this Minireview, we provide insight into universal strategies and guidelines to successfully synthesize irreversible probes that target G protein‐coupled receptors (GPCRs). We provide an overview of commonly used chemoreactive and photoreactive groups, and make a comparison of their properties and potential applications. Furthermore, there is a particular focus on synthetic approaches to introduce these reactive groups based on commercially available reagents.     

Effect of Ions and Sequence Variants on the Antagonist Binding Properties of the Histamine H1 Receptor

The histamine H₁ receptor (H₁R) is a G protein-coupled receptor (GPCR) and represents a main target in the treatment of allergic reactions as well as inflammatory reactions and depressions. Although the overall effect of antagonists on H₁ function has been extensively investigated, rather little is known about the potential modulatory effect of ions or sequence variants on antagonist binding. We investigated the dynamics of a phosphate ion present in the crystal structure and of a sodium ion, for which we determined the position in the allosteric pocket by metadynamics simulations. Both types of ions exhibit significant dynamics within their binding site; however, some key contacts remain stable over the simulation time, which might be exploited to develop more potent drugs targeting these sites. The dynamics of the ions is almost unaffected by the presence or absence of doxepin, as also reflected in their small effect (less than 1 kcal·mol⁻¹) on doxepin binding affinity. We also examined the effect of four H₁R sequence variants observed in the human population on doxepin binding. These variants cause a reduction in doxepin affinity of up to 2.5 kcal·mol⁻¹, indicating that personalized medical treatments that take into account individual mutation patterns could increase precision in the dosage of GPCR-targeting drugs.     

短链脂肪酸作为信号分子在肠道炎症中的研究进展

肠道菌群代谢物短链脂肪酸在机体内可作为信号分子,在细胞外激活G蛋白偶联受体,在细胞内抑制组蛋白脱乙酰酶,起抗炎作用。本文就短链脂肪酸的来源、组成、合成、分布,短链脂肪酸作为信号分子在肠道炎症发生发展中的作用及其潜在分子机制进行综述。     

Selective Pharmacophore Models of Dopamine D1 and D2 Full Agonists Based on Extended Pharmacophore Features

This study is focused on the identification of structural features that determine the selectivity of dopamine receptor agonists toward D₁ and D₂ receptors. Selective pharmacophore models were developed for both receptors. The models were built by using projected pharmacophoric features that represent the main agonist interaction sites in the receptor (the Ser residues in TM5 and the Asp in TM3), a directional aromatic feature in the ligand, a feature with large positional tolerance representing the positively charged nitrogen in the ligand, and sets of excluded volumes reflecting the shapes of the receptors. The sets of D₁ and D₂ ligands used for modeling were carefully selected from published sources and consist of structurally diverse, conformationally rigid full agonists as active ligands together with structurally related inactives. The robustness of the models in discriminating actives from inactives was tested against four ensembles of conformations generated by using different established methods and different force fields. The reasons for the selectivity can be attributed to both geometrical differences in the arrangement of the features, e.g., different tilt angels of the π system, as well as shape differences covered by the different sets of excluded volumes. This work provides useful information for the design of new D₁ and D₂ agonists and also for comparative homology modeling of D₁ and D₂ receptors. The approach is general and could therefore be applied to other ligand–protein interactions for which no experimental protein structure is available.     

GPCRs——一类值得关注的潜在杀虫剂新靶标

G蛋白偶联受体(GPCRs)是人类药物的重要作用靶标,参与人体内多种重要的生理功能。同样地,GPCRs在昆虫体内也同样发挥着重要的作用。综述了昆虫GPCRs的研究进展,并探讨了昆虫GPCRs可能成为新的杀虫剂靶标的前景。     

Predicted Ligands for the Human Urotensin‐II G Protein‐Coupled Receptor with Some Experimental Validation

Human Urotensin‐II (U‐II) is the most potent mammalian vasoconstrictor known. 1 Thus, a U‐II antagonist would be of therapeutic value in a number of cardiovascular disorders. 2 Here, we describe our work on the prediction of the structure of the human U‐II receptor (hUT₂R) using GEnSeMBLE (GPCR Ensemble of Structures in Membrane BiLayer Environment) complete sampling Monte Carlo method. With the validation of our predicted structures, we designed a series of new potential antagonists predicted to bind more strongly than known ligands. Next, we carried out R‐group screening to suggest a new ligand predicted to bind with 7 kcal mol^?1 better energy than 1‐{2‐[4‐(2‐bromobenzyl)‐4‐hydroxypiperidin‐1‐yl]ethyl}‐3‐(thieno[3,2‐b]pyridin‐7‐yl)urea, the designed antagonist predicted to have the highest affinity for the receptor. Some of these predictions were tested experimentally, validating the computational results. Using the pharmacophore generated from the predicted structure for hUT₂R bound to ACT‐058362, we carried out virtual screening based on this binding site. The most potent hit compounds identified contained 2‐(phenoxymethyl)‐1,3,4‐thiadiazole core, with the best derivative exhibiting an IC₅₀ value of 0.581 μM against hUT₂R when tested in vitro. Our efforts identified a new scaffold as a potential new lead structure for the development of novel hUT₂R antagonists, and the computational methods used could find more general applicability to other GPCRs.     

Novel BQCA- and TBPB-Derived M1 Receptor Hybrid Ligands: Orthosteric Carbachol Differentially Regulates Partial Agonism

Recently, investigations of the complex mechanisms of allostery have led to a deeper understanding of G protein-coupled receptor (GPCR) activation and signaling processes. In this context, muscarinic acetylcholine receptors (mAChRs) are highly relevant due to their exemplary role in the study of allosteric modulation. In this work, we compare and discuss two sets of putatively dualsteric ligands, which were designed to connect carbachol to different types of allosteric ligands. We chose derivatives of TBPB [1-(1′-(2-tolyl)-1,4′-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one] as M₁-selective putative bitopic ligands, and derivatives of benzyl quinolone carboxylic acid (BQCA) as an M₁ positive allosteric modulator, varying the distance between the allosteric and orthosteric building blocks. Luciferase protein complementation assays demonstrated that linker length must be carefully chosen to yield either agonist or antagonist behavior. These findings may help to design biased signaling and/or different extents of efficacy.     

Antibody Therapies Targeting Complex Membrane Proteins

In analyses of protein families that may serve as drug targets, membrane-associated G-protein-coupled receptors (GPCRs) dominate, followed by ion channels, transporters, and—to a lesser extent—membrane-bound enzymes. However, various challenges put such membrane proteins among key groups of underutilized opportunities for the application of therapeutic antibodies. Antibodies hold the promise of exquisite specificity, as they are able to target even specific conformations of a particular membrane protein, as well as adaptability through engineering into various antibody formats. However, the ease of raising and isolating specific, effective antibodies targeting membrane proteins depends on many factors. In particular, the generation of specific antibodies is easier when targeting larger, simpler, extracellular domains with greater uniqueness of amino acid sequence. The rareness of such ideal conditions is illustrated by the limited number of approved biologics for targeting GPCRs and other complex membrane proteins. Challenges in developing antibodies to complex membrane proteins such as GPCRs, ion channels, transporters, and membrane-bound enzymes can be addressed by the design of the antigen, antibody-generation strategies, lead optimization technologies, and antibody modalities. A better understanding of the membrane proteins being targeted would facilitate mechanism-based drug discovery. This review describes the advantages and challenges of targeting complex membrane proteins with antibodies and discusses the preparation of membrane protein antigens and antibody generation, illustrated by select examples of success.