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G-protein coupled receptors (GPCRs) are the targets of over half of all prescribed drugs today. The UniProt database has records for about 800 proteins classified as GPCRs, but drugs have only been developed against 50 of these. Thus, there is huge potential in terms of the number of targets for new therapies to be designed. Several breakthroughs in GPCRs biased pharmacology, structural biology, modelling and scoring have resulted in a resurgence of interest in GPCRs as drug targets. Therefore, an international conference, sponsored by the Royal Society, with world-renowned researchers from industry and academia was recently held to discuss recent progress and highlight key areas of future research needed to accelerate GPCR drug discovery. Several key points emerged. Firstly, structures for all three major classes of GPCRs have now been solved and there is increasing coverage across the GPCR phylogenetic tree. This is likely to be substantially enhanced with data from x-ray free electron sources as they move beyond proof of concept. Secondly, the concept of biased signalling or functional selectivity is likely to be prevalent in many GPCRs, and this presents exciting new opportunities for selectivity and the control of side effects, especially when combined with increasing data regarding allosteric modulation. Thirdly, there will almost certainly be some GPCRs that will remain difficult targets because they exhibit complex ligand dependencies and have many metastable states rendering them difficult to resolve by crystallographic methods. Subtle effects within the packing of the transmembrane helices are likely to mask and contribute to this aspect, which may play a role in species dependent behaviour. This is particularly important because it has ramifications for how we interpret pre-clinical data. In summary, collaborative efforts between industry and academia have delivered significant progress in terms of structure and understanding of GPCRs and will be essential for resolving problems associated with the more difficult targets in the future. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) mediate several signaling pathways through a general mechanism that involves their activation, upholding a chain of events that lead to the release of molecules responsible for cytoplasmic action and further regulation. These physiological functions can be severely altered by mutations in GPCR genes. GPCRs subfamily A17 (dopamine, serotonin, adrenergic and trace amine receptors) are directly related with neurodegenerative diseases, and as such it is crucial to explore known mutations on these systems and their impact in structure and function. A comprehensive and detailed computational framework - MUG (Mutations Understanding GPCRs) - was constructed, illustrating key reported mutations and their effect on receptors of the subfamily A17 of GPCRs. We explored the type of mutations occurring overall and in the different families of subfamily A17, as well their localization within the receptor and potential effects on receptor functionality. The mutated residues were further analyzed considering their pathogenicity. The results reveal a high diversity of mutations in the GPCR subfamily A17 structures, drawing attention to the considerable number of mutations in conserved residues and domains. Mutated residues were typically hydrophobic residues enriched at the ligand binding pocket and known activating microdomains, which may lead to disruption of receptor function. MUG as an interactive web application is available for the management and visualization of this dataset. We expect that this interactive database helps the exploration of GPCR mutations, their influence, and their familywise and receptor-specific effects, constituting the first step in elucidating their structures and molecules at the atomic level., Competing Interests: The authors have no conflicts of interest to declare., (© 2022 The Author(s).)

Claudins (Cldns) define a family of transmembrane proteins that are the major determinants of the tight junction integrity and tissue selectivity. They promote the formation of either barriers or ion-selective channels at the interface between two facing cells, across the paracellular space. Multiple Cldn subunits form complexes that include cis- (intracellular) interactions along the membrane of a single cell and trans- (intercellular) interactions across adjacent cells. The first description of Cldn assemblies was provided by electron microscopy, while electrophysiology, mutagenesis and cell biology experiments addressed the functional role of different Cldn homologs. However, the investigation of the molecular details of Cldn subunits and complexes are hampered by the lack of experimental native structures, currently limited to Cldn15. The recent implementation of computer-based techniques greatly contributed to the elucidation of Cldn properties. Molecular dynamics simulations and docking calculations were extensively used to refine the first Cldn multimeric model postulated from the crystal structure of Cldn15, and contributed to the introduction of a novel, alternative, arrangement. While both these multimeric assemblies were found to account for the physiological properties of some family members, they gave conflicting results for others. In this review, we illustrate the major findings on Cldn-based systems that were achieved by using state-of-the-art computational methodologies. The information provided by these results could be useful to improve the characterization of the Cldn properties and help the design of new efficient strategies to control the paracellular transport of drugs or other molecules., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)

G protein-coupled receptors (GPCRs) are promising drug targets because they play a large role in physiological processes by modulating diverse signaling pathways in the human body. The GPCR-mediated signaling pathways are regulated by four types of ligands-agonists, neutral antagonists, partial agonists, and inverse agonists. Once each type of ligand is bound to the binding site, it activates, deactivates, or does not perturb signaling by shifting the conformational ensemble of GPCRs. Predicting the ligand's effect on the conformation at the binding moment could be a powerful screening tool for rational GPCR drug design. Here, we detected conformational differences by capturing the spatiotemporal residue pair pattern of the ligand-bound β2-adrenergic receptor (β2AR) using a 3-dimensional residual network, 3D-ResNets. The network was trained with the time series of protein distance maps extracted from hundreds of molecular dynamics (MD) simulation trajectories of ten β2AR-ligand complexes. The MD system was constructed with a lipid bilayer embedded in an inactive β2AR X-ray crystal structure and solvated with explicit water molecules. To train the network, three hyperparameters were tested, and it was found that the number of MD trajectories in the training set significantly affected the model's accuracy. The classification of agonists and neutral antagonists was successful, but inverse agonists were not. Between the agonists and antagonists, different residue pair patterns were spotted on the extracellular loop segment. This result demonstrates the potential application of a 3-D neural network in GPCR drug screening, as well as an analysis tool for protein functional dynamics., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Abstract: In view of the natural resistance to infection by HIV and occasional delayed clinical manifestation of the disease, as also the fact that the virus is able to enter only cells that express CD4 and a co-receptor, we initiated a search for a soluble co-receptor that might compete with its membrane counterpart. Using a sandwich ELISA system, a soluble human CCR5 receptor (sCCR5) was indeed detected in the circulation. Immunoprecipitation of sCCR5-positive plasma samples from Israelis of Ethiopian and non-Ethiopian origin with mAb 2D7, a conformation-dependent anti-CCR5 antibody, revealed the presence of a ∼22kDa protein. A panel of antibodies directed against the membrane receptor was used to characterize the structure of the soluble CCR5: mAb CTC8, recognizing the N-terminal sequence of the protein,10YDIN13; “multidomain” mAbs FAB181B and FAB183B that are dependent upon the presence of Q93 and D95 in ECL1 and K171 and E172 in ECL2A, and mAb FAB182B, recognizing the stretch 184YSQYQF189, which spans the C-terminal part of the second extracellular loop. The presence of short soluble CCR5 in human plasma has not been previously described. Among HIV-negative non-Ethiopian Israelis, 20.4% were sCCR5-positive, as against only 10.5% in HIV-positives. However, 7.1% of HIV-negative Ethiopian Israelis were sCCR5 positive, as were 5.6% HIV-positives. Plasma concentrations of MIP-1β, the CCR5 agonist, were twice as high in sCCR5-positives (140.8 ± 25.8pg/ml) as in the sCCR5-negatives (77.6 ± 11.0pg/ml, P = 0.0157). A significant positive correlation between plasma levels of sCCR5 and MIP-1β was found (Fig. 4, r = 0.8, P < 0.0001). [Copyright &y& Elsevier]

The chemokine receptor CCR5 is the receptor for several chemokines and major coreceptor for R5 human immunodeficiency virus type-1 strains entry into cell. Three-dimensional models of CCR5 were built by using homology modeling approach and 1 ns molecular dynamics (MD) simulation, because studies of site-directed mutagenesis and chimeric receptors have indicated that the N-terminus (Nt) and extracellular loops (ECLs) of CCR5 are important for ligands binding and viral fusion and entry, special attention was focused on disulfide bond function, conformational flexibility, hydrogen bonding, electrostatic interactions, and solvent-accessible surface area of Nt and ECLs of this protein part. We found that the extracellular segments of CCR5 formed a well-packet globular domain with complex interactions occurred between them in a majority of time of MD simulation, but Nt region could protrude from this domain sometimes. The disulfide bond Cys20–Cys269 is essential in controlling specific orientation of Nt region and maintaining conformational integrity of extracellular domain. RMS comparison analysis between conformers revealed the ECL1 of CCR5 stays relative rigid, whereas the ECL2 and Nt are rather flexible. Solvent-accessible surface area calculations indicated that the charged residues within Nt and ECL2 are often exposed to solvent. Integrating these results with available experimental data, a two-step gp120-CCR5 binding mechanism was proposed. The dynamic interaction of CCR5 extracellular domain with gp120 was emphasized. [Copyright &y& Elsevier]

Lysine-288 in the glucagon-like peptide-1 receptor was predicted to be ideally positioned to play a role in hormone binding. Subsequent mutation of Lys-288 to Ala or Leu greatly reduced hormone affinity, while substitution with Arg had minimal effect. Compared to wild type, the Lys288-Ala receptor had a reduced affinity for three peptide ligands with complete N-terminal sequences but not for their N-truncated analogues. Hence, the role of this positively charged residue, which is conserved at the equivalent position in all other Family B receptors, was determined to be important for receptor interaction with the N-terminal eight residues of peptide agonists. [Copyright &y& Elsevier]

Despite the considerable successes of highly active antiretroviral therapy, new classes of therapeutic agents are still urgently needed. Unfortunately, the emergence of antiviral resistance and drug toxicity remain challenging obstacles to successful treatment in many HIV-1-infected individuals. HIV-1 entry is a multi-step process that is an attractive target for the development of new classes of therapeutic agents. Considerable progress has been made in the understanding of HIV-1 cell entry, enabling the design of specific agents that can inhibit each step of cellular entry. A number of promising agents have commenced clinical trials, including the attachment inhibitor PRO 542, co-receptor inhibitor AMD3100 and fusion inhibitor T-20. A greater number of HIV-1 entry inhibitors are in preclinical development. This review outlines the mechanisms involved in HIV-1 entry and the sites of action of specific HIV-1 inhibitors. [Copyright &y& Elsevier]

Olfactory receptor (OR) 1A2 is the member of largest superfamily of G protein-coupled receptors (GPCRs). OR1A2 is an ectopically expressed receptor with only 13 known ligands, implicated in reducing hepatocellular carcinoma progression, with enormous therapeutic potential. We have developed a two-stage screening approach to identify novel putative ligands of OR1A2. We first used a pharmacophore model based on atomic property field (APF) to virtually screen a library of 5942 human metabolites. We then carried out structure-based virtual screening (SBVS) for predicting the potential agonists, based on a 3D homology model of OR1A2. This model was developed using a biophysical approach for template selection, based on multiple parameters including hydrophobicity correspondence, applied to the complete set of available GPCR structures to pick the most appropriate template. Finally, the membrane-embedded 3D model was refined by molecular dynamics (MD) simulations in both the apo and holo forms. The refined model in the apo form was selected for SBVS. Four novel small molecules were identified as strong binders to this olfactory receptor on the basis of computed binding energies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

The epithelial tight junction determines the paracellular water and ion movement in the intestine and also prevents uptake of larger molecules, including antigens, in an uncontrolled manner. Claudin-2, one of the 27 mammalian claudins regulating that barrier function, forms a paracellular channel for small cations and water. It is typically expressed in leaky epithelia like proximal nephron and small intestine and provides a major pathway for the paracellular transport of sodium, potassium, and fluid. In intestinal inflammation (Crohn's disease, ulcerative colitis), immune-mediated diseases (celiac disease), and infections (HIV enteropathy), claudin-2 is upregulated in small and large intestine and contributes to diarrhea via a leak flux mechanism. In parallel to that upregulation, other epithelial and tight junctional features are altered and the luminal uptake of antigenic macromolecules is enhanced, for which claudin-2 may be partially responsible through induction of tight junction strand discontinuities.

Background: Development of functional monoclonal antibodies against intractable GPCR targets., Results: Identification of structured peptides mimicking the ligand binding site, their use in panning to enrich for a population of binders, and the subsequent challenge of this population with receptor overexpressing cells leads to functional monoclonal antibodies., Conclusion: The combination of techniques provides a successful strategic approach for the development of functional monoclonal antibodies against CXCR2 in a relatively small campaign., Significance: The presented combination of techniques might be applicable for other, notoriously difficult, GPCR targets., Summary: The CXC chemokine receptor-2 (CXCR2) is a member of the large 'family A' of G-protein-coupled-receptors and is overexpressed in various types of cancer cells. CXCR2 is activated by binding of a number of ligands, including interleukin 8 (IL-8) and growth-related protein α (Gro-α). Monoclonal antibodies capable of blocking the ligand-receptor interaction are therefore of therapeutic interest; however, the development of biological active antibodies against highly structured GPCR proteins is challenging. Here we present a combination of techniques that improve the discovery of functional monoclonal antibodies against the native CXCR2 receptor. The IL-8 binding site of CXCR2 was identified by screening peptide libraries with the IL-8 ligand, and then reconstructed as soluble synthetic peptides. These peptides were used as antigens to probe an antibody fragment phage display library to obtain subpopulations binding to the IL-8 binding site of CXCR2. Further enrichment of the phage population was achieved by an additional selection round with CXCR2 overexpressing cells as a different antigen source. The scFvs from the CXCR2 specific phage clones were sequenced and converted into monoclonal antibodies. The obtained antibodies bound specifically to CXCR2 expressing cells and inhibited the IL-8 and Gro-α induced ß-arrestin recruitment with IC50 values of 0.3 and 0.2 nM, respectively, and were significantly more potent than the murine monoclonal antibodies (18 and 19 nM, respectively) obtained by the classical hybridoma technique, elicited with the same peptide antigen. According to epitope mapping studies, the antibody efficacy is largely defined by N-terminal epitopes comprising the IL-8 and Gro-α binding sites. The presented strategic combination of in vitro techniques, including the use of different antigen sources, is a powerful alternative for the development of functional monoclonal antibodies by the classical hybridoma technique, and might be applicable to other GPCR targets.