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Drugs targeting adenosine receptors (AR) can provide treatment for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]pyridines L2–L10, A15, and A17as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]pyridine (A17) displaying the highest affinity at both receptors with a long residence time of binding, as determined using a NanoBRET-based assay. Two binding orientations of A17produce stable complexes inside the orthosteric binding area of A1R in molecular dynamics (MD) simulations, and we selected the most plausible orientation based on the agreement with alanine mutagenesis supported by affinity experiments. Interestingly, for drug design purposes, the mutation of L2506.51to alanine increased the binding affinity of A17at A1R. We explored the structure–activity relationships against A1R using alchemical binding free energy calculations with the thermodynamic integration coupled with the MD simulation (TI/MD) method, applied on the whole G-protein-coupled receptor–membrane system, which showed a good agreement (r= 0.73) between calculated and experimental relative binding free energies.

G-protein-coupled receptors (GPCRs) are membrane proteins that modulate physiology across human tissues in response to extracellular signals. GPCR-mediated signalling can differ because of changes in the sequence1,2or expression3of the receptors, leading to signalling bias when comparing diverse physiological systems4. An underexplored source of such bias is the generation of functionally diverse GPCR isoforms with different patterns of expression across different tissues. Here we integrate data from human tissue-level transcriptomes, GPCR sequences and structures, proteomics, single-cell transcriptomics, population-wide genetic association studies and pharmacological experiments. We show how a single GPCR gene can diversify into several isoforms with distinct signalling properties, and how unique isoform combinations expressed in different tissues can generate distinct signalling states. Depending on their structural changes and expression patterns, some of the detected isoforms may influence cellular responses to drugs and represent new targets for developing drugs with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling that considers how combinatorial expression of isoforms in a particular cell type, tissue or organism collectively influences receptor signalling and drug responses.

Adenosine A3receptor (A3R) is a promising drug target cancer and for a number of other conditions like inflammatory diseases, including asthma and rheumatoid arthritis, glaucoma, chronic obstructive pulmonary disease, and ischemic injury. Currently, there is no experimentally determined structure of A3R. We explored the binding profile of O4-{[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbonyl}-2-methyl-1,3-thiazole-4-carbohydroximamide (K18), which is a new specific and competitive antagonist at the orthosteric binding site of A3R. MD simulations and MM-GBSA calculations of the WT A3R in complex with K18 combined with in vitro mutagenic studies show that the most plausible binding conformation for the dichlorophenyl group of K18 is oriented toward trans-membrane helices (TM) 5, 6 and reveal important residues for binding. Further, MM-GBSA calculations distinguish mutations that reduce or maintain or increase antagonistic activity. Our studies show that selectivity of K18 toward A3R is defined not only by direct interactions with residues within the orthosteric binding area but also by remote residues playing a significant role. Although V1695.30is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid, K18 maintained antagonistic potency, in agreement with our previous results obtained for agonists binding profile investigation. Mutation of the direct interacting residue L903.32in the low region and the remote L2647.35in the middle/upper region to alanine increases antagonistic potency, suggesting an empty space in the orthosteric area available for increasing antagonist potency. These results approve the computational model for the description of K18 binding at A3R, which we previously performed for agonists binding to A3R, and the design of more effective antagonists based on K18.

Antagonism of the human adenosine A3receptor (hA3R) has potential therapeutic application. Alchemical relative binding free energy calculations of K18and K32suggested that the combination of a 3-(2,6-dichlorophenyl)-isoxazolyl group with 2-pyridinyl at the ends of a carbonyloxycarboximidamide group should improve hA3R affinity. Of the 25 new analogues synthesized, 37and 74showed improved hA3R affinity compared to K18(and K32). This was further improved through the addition of a bromine group to the 2-pyridinyl at the 5-position, generating compound 39. Alchemical relative binding free energy calculations, mutagenesis studies and MD simulations supported the compounds’ binding pattern while suggesting that the bromine of 39inserts deep into the hA3R orthosteric pocket, so highlighting the importance of rigidification of the carbonyloxycarboximidamide moiety. MD simulations highlighted the importance of rigidification of the carbonyloxycarboximidamide, while suggesting that the bromine of 39inserts deep into the hA3R orthosteric pocket, which was supported through mutagenesis studies 39also selectively antagonized endogenously expressed hA3R in nonsmall cell lung carcinoma cells, while pharmacokinetic studies indicated low toxicity enabling in vivo evaluation. We therefore suggest that 39has potential for further development as a high-affinity hA3R antagonist.

A structure-based drug design pipeline that considers both thermodynamic and kinetic binding data of ligands against a receptor will enable the computational design of improved drug molecules. For unresolved GPCR-ligand complexes, a workflow that can apply both thermodynamic and kinetic binding data in combination with alpha-fold (AF)-derived or other homology models and experimentally resolved binding modes of relevant ligands in GPCR-homologs needs to be tested. Here, as test case, we studied a congeneric set of ligands that bind to a structurally unresolved G protein-coupled receptor (GPCR), the inactive human adenosine A3receptor (hA3R). We tested three available homology models from which two have been generated from experimental structures of hA1R or hA2AR and one model was a multistate alphafold 2 (AF2)-derived model. We applied alchemical calculations with thermodynamic integration coupled with molecular dynamics (TI/MD) simulations to calculate the experimental relative binding free energies and residence time (τ)-random accelerated MD (τ-RAMD) simulations to calculate the relative residence times (RTs) for antagonists. While the TI/MD calculations produced, for the three homology models, good Pearson correlation coefficients, correspondingly, r= 0.74, 0.62, and 0.67 and mean unsigned error (mue) values of 0.94, 1.31, and 0.81 kcal mol–1, the τ-RAMD method showed r= 0.92 and 0.52 for the first two models but failed to produce accurate results for the multistate AF2-derived model. With subsequent optimization of the AF2-derived model by reorientation of the side chain of R1735.34located in the extracellular loop 2 (EL2) that blocked ligand’s unbinding, the computational model showed r= 0.84 for kinetic data and improved performance for thermodynamic data (r= 0.81, mue = 0.56 kcal mol–1). Overall, after refining the multistate AF2 model with physics-based tools, we were able to show a strong correlation between predicted and experimental ligand relative residence times and affinities, achieving a level of accuracy comparable to an experimental structure. The computational workflow used can be applied to other receptors, helping to rank candidate drugs in a congeneric series and enabling the prioritization of leads with stronger binding affinities and longer residence times.

The adenosine A3receptor (A3R) binds adenosine and is a drug target against cancer cell proliferation. Currently, there is no experimental structure of A3R. Here, we have generated a molecular model of A3R in complex with two agonists, the nonselective 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-d-ribofuranuronamide (NECA) and the selective 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide (IB-MECA). Molecular dynamics simulations of the wild-type A3R in complex with both agonists, combined with in vitro mutagenic studies revealed important residues for binding. Further, molecular mechanics-generalized Born surface area calculations were able to distinguish mutations that reduce or negate agonistic activity from those that maintained or increased the activity. Our studies reveal that selectivity of IB-MECA toward A3R requires not only direct interactions with residues within the orthosteric binding area but also with remote residues. Although V1695.30is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid or alanine, the activity of IB-MECA increased by making new van der Waals contacts with TM5. This result may have implications in the design of new A3R agonists.

Direct reprogramming of human somatic cells toward induced pluripotent stem cells holds great promise for regenerative medicine and basic biology. We used a high‐throughput small interfering RNA screening assay in the initiation phase of reprogramming for 784 genes belonging to kinase and phosphatase families and identified 68 repressors and 22 effectors. Six new candidates belonging to the family of the G protein‐coupled receptors (GPCRs) were identified, suggesting an important role for this key signaling pathway during somatic cell‐induced reprogramming. Downregulation of one of the key GPCR effectors, endothelial differentiation GPCR5 (EDG5), impacted the maintenance of pluripotency, actin cytoskeleton organization, colony integrity, and focal adhesions in human embryonic stem cells, which were associated with the alteration in the RhoA‐ROCK‐Cofilin‐PAXILLIN‐actin signaling pathway. Similarly, downregulation of EDG5during the initiation stage of somatic cell‐induced reprogramming resulted in alteration of cytoskeleton, loss of human‐induced pluripotent stem cell colony integrity, and a significant reduction in partially and fully reprogrammed cells as well as the number of alkaline phosphatase positive colonies at the end of the reprogramming process. Together, these data point to an important role of EDG5 in the maintenance and acquisition of pluripotency. StemCells2019;37:318–331 Summary of the main findings generated from this study.

ABSTRACTIntroduction: Glioblastoma multiforme (GBM) is the most common and aggressive malignant glioma, with patients having a median survival of just over one year. Current chemotherapies, with surgery and radiotherapy, provide only minor patient benefit. There is a great need to discover and develop novel therapies for this devastating disease.Areas covered: The patent literature reveals novel therapies, providing insights into emerging GBM therapeutics. We have used the Google and USPTO patent databases to generate a detailed landscape of patents and patent applications from companies active in the areas of glioma and/or GBM. Specific patents have been grouped into six areas: novel compounds; treatments and therapeutic targets; combination therapies; immunotherapies; delivery methods; and biomarkers for diagnosis and prognosis.Expert opinion: There has been a steady increase in the number of patents on GBM over the last five years. Despite many new compounds being developed and patented for a broad range of cancers, only a small percentage of these are specifically targeted to GBM. Notable trends in the patent literature include both the development of combination therapies to combat the heterogeneous nature of GBM, and the use of immunotherapies building on the promise of cancer vaccines and CAR T-cell therapy.

We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER) to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTAΔ), follow ER-associated degradation (ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the Hrd1p complex. In the case of native RTA, we established that dislocation also depends on other components of the classical ERAD-L pathway as well as an ongoing ER–Golgi transport. However, the dislocation pathways deviate strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTAΔ, although the involvement of individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the proteasome RP, can discriminate between structural features of the same substrate.

This study reveals that components of the yeast ERAD-L pathway can discriminate between two subtly different forms of the same toxin substrate. Although precytosolic requirements are similar for both toxin structures, there is a divergence in fate on the cytosolic face of the ER membrane.

A series of benzyloxy and phenoxy derivatives of the adenosine receptor agonists N6-cyclopentyl adenosine (CPA) and N6-cyclopentyl 5′-N-ethylcarboxamidoadenosine (CP-NECA) were synthesized, and their potency and selectivity were assessed. We observed that the most potent were the compounds with a halogen in the metaposition on the aromatic ring of the benzyloxy- or phenoxycyclopentyl substituent. In general, the NECA-based compounds displayed greater A1R selectivity than the adenosine-based compounds, with N6-2-(3-bromobenzyloxy)cyclopentyl-NECA and N6-2-(3-methoxyphenoxy)cyclopentyl-NECA showing ∼1500-fold improved A1R selectivity compared to NECA. In addition, we quantified the compounds’ affinity and kinetics of binding at both human and rat A1R using a NanoBRET binding assay and found that the halogen substituent in the benzyloxy- or phenoxycyclopentyl moiety seems to confer high affinity for the A1R. Molecular modeling studies suggested a hydrophobic subpocket as contributing to the A1R selectivity displayed. We believe that the identified selective potent A1R agonists are valuable tool compounds for adenosine receptor research.

Yeast provides an attractive system in which to study proprotein processing. Many of their processing events are remarkably similar to those in higher eukaryotes and their amenability to experimental manipulation permits approaches that are not always feasible in multicellular organisms. Analysing the biosynthesis of the yeast mating pheromones has been particularly rewarding and has provided insights into both the proteolytic aspects of processing and other post-translational events such as prenylation and carboxymethylation.

Many cellular responses are initiated by the binding of an extracellular ligand to receptors at the cell surface. The removal of these ligands, or agonists, would therefore be expected to contribute to the process by which cells recover from stimulation. While dilution in the extracellular medium reduces the concentration of most agonists, many cells have developed specific mechanisms for removing particular ligands. One of the more effective mechanisms is to degrade the extracellular agonist and here we review the production and action of the enzymes responsible for the degradation of a number of these agonists.

Despite being a target for about one-third of approved drugs, G protein-coupled receptors (GPCRs) still represent a tremendous reservoir for therapeutic strategies against diseases. For example, several cardiovascular and central nervous system conditions could benefit from clinical agents that activate the adenosine 1 receptor (A1R); however, the pursuit of A1R agonists for clinical use is usually impeded by both on- and off-target side effects. One of the possible strategies to overcome this issue is the development of positive allosteric modulators (PAMs) capable of selectively enhancing the effect of a specific receptor subtype and triggering functional selectivity (a phenomenon also referred to as bias). Intriguingly, besides enforcing the effect of agonists upon binding to an allosteric site, most of the A1R PAMs display intrinsic partial agonism and orthosteric competition with antagonists. To rationalize this behavior, we simulated the binding of the prototypical PAMs PD81723 and VCP171, the full-agonist NECA, the antagonist 13B, and the bitopic agonist VCP746. We propose that a single PAM can bind several A1R sites rather than a unique allosteric pocket, reconciling the structure–activity relationship and the mutagenesis results.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.