Progress on the development of Class A GPCR-biased ligands.

Class A G protein-coupled receptors (GPCRs) continue to garner interest for their essential roles in cell signalling and their importance as drug targets. Although numerous drugs in the clinic target these receptors, over 60% GPCRs remain unexploited. Moreover, the adverse effects triggered by the available unbiased GPCR modulators, limit their use and therapeutic value. In this context, the elucidation of biased signalling has opened up new pharmacological avenues holding promise for safer therapeutics. Functionally selective ligands favour receptor conformations facilitating the recruitment of specific effectors and the modulation of the associated pathways. This review surveys the current drug discovery landscape of GPCR-biased modulators with a focus on recent advances. Understanding the biological effects of this preferential coupling is at different stages depending on the Class A GPCR family. Therefore, with a focus on individual GPCR families, we present a compilation of the functionally selective modulators reported over the past few years. In doing so, we dissect their therapeutic relevance, molecular determinants and potential clinical applications.

N-Adamantyl-1-alkyl-4-oxo-1,4-dihydroquinoline-3-carboxamide Derivatives as Fluorescent Probes to Detect Microglia Activation through the Imaging of Cannabinoid Receptor Subtype 2 (CB2R).

Cannabinoid receptor subtype 2 (CB2R) is emerging as a pivotal biomarker to identify the first steps of inflammation-based diseases such as cancer and neurodegeneration. There is an urgent need to find specific probes that may result in green and safe alternatives to the commonly used radiative technologies, to deepen the knowledge of the CB2R pathways impacting the onset of the above-mentioned pathologies. Therefore, based on one of the CB2R pharmacophores, we developed a class of fluorescent N-adamantyl-1-alkyl-4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives spanning from the green to the near-infrared (NIR) regions of the light spectrum. Among the synthesized fluorescent ligands, the green-emitting compound 55 exhibited a favorable binding profile (strong CB2R affinity and high selectivity). Notably, this ligand demonstrated versatility as its use was validated in different experimental settings such as flow cytometry saturation, competitive fluorescence assays, and in vitro microglia cells mimicking inflammation states where CB2R are overexpressed.

Exploring Biginelli-based scaffolds as A2B adenosine receptor antagonists: Unveiling novel structure-activity relationship trends, lead compounds, and potent colorectal anticancer agents.

Antagonists of the A2B adenosine receptor have recently emerged as targeted anticancer agents and immune checkpoint inhibitors within the realm of cancer immunotherapy. This study presents a comprehensive evaluation of novel Biginelli-assembled pyrimidine chemotypes, including mono-, bi-, and tricyclic derivatives, as A2BAR antagonists. We conducted a comprehensive examination of the adenosinergic profile (both binding and functional) of a large compound library consisting of 168 compounds. This approach unveiled original lead compounds and enabled the identification of novel structure-activity relationship (SAR) trends, which were supported by extensive computational studies, including quantum mechanical calculations and free energy perturbation (FEP) analysis. In total, 25 molecules showed attractive affinity (Ki < 100 nM) and outstanding selectivity for A2BAR. From these, five molecules corresponding to the new benzothiazole scaffold were below the Ki < 10 nM threshold, in addition to a novel dual A2A/A2B antagonist. The most potent compounds, and the dual antagonist, showed enantiospecific recognition in the A2BAR. Two A2BAR selective antagonists and the dual A2AAR/A2BAR antagonist reported in this study were assessed for their impact on colorectal cancer cell lines. The results revealed a significant and dose-dependent reduction in cell proliferation. Notably, the A2BAR antagonists exhibited remarkable specificity, as they did not impede the proliferation of non-tumoral cell lines. These findings support the efficacy and potential that A2BAR antagonists as valuable candidates for cancer therapy, but also that they can effectively complement strategies involving A2AAR antagonism in the context of immune checkpoint inhibition.

Microscopy and spectroscopy approaches to study GPCR structure and function.

The GPCR signalling cascade is a key pathway responsible for the signal transduction of a multitude of physical and chemical stimuli, including light, odorants, neurotransmitters and hormones. Understanding the structural and functional properties of the GPCR cascade requires direct observation of signalling processes in high spatial and temporal resolution, with minimal perturbation to endogenous systems. Optical microscopy and spectroscopy techniques are uniquely suited to this purpose because they excel at multiple spatial and temporal scales and can be used in living objects. Here, we review recent developments in microscopy and spectroscopy technologies which enable new insights into GPCR signalling. We focus on advanced techniques with high spatial and temporal resolution, single-molecule methods, labelling strategies and approaches suitable for endogenous systems and large living objects. This review aims to assist researchers in choosing appropriate microscopy and spectroscopy approaches for a variety of applications in the study of cellular signalling.

A Robust and Efficient FRET-Based Assay for Cannabinoid Receptor Ligands Discovery.

The identification of new modulators for Cannabinoid Receptors (CBRs) has garnered significant attention in drug discovery over recent years, owing to their manifold pathophysiological implications. In the context of hit identification, the availability of robust and sensitive high-throughput screening assays is essential to enhance the likelihood of success. In this study, we present the development and validation of a Tag-lite® binding assay designed for screening hCB1/hCB2 binding, employing a dual fluorescent ligand, CELT-335. Representative ligands for CBRs, exhibiting diverse affinity and functional profiles, were utilized as reference compounds to validate the robustness and efficiency of the newly developed Tag-lite® binding assay protocol. The homogeneous format, coupled with the sensitivity and optimal performance of the fluorescent ligand CELT-335, establishes this assay as a viable and reliable method for screening in hit and lead identification campaigns.

Pharmacological insights emerging from the characterization of a large collection of synthetic cannabinoid receptor agonists designer drugs.

Synthetic cannabinoid receptor agonists (SCRAs) constitute the largest and most defiant group of abuse designer drugs. These new psychoactive substances (NPS), developed as unregulated alternatives to cannabis, have potent cannabimimetic effects and their use is usually associated with episodes of psychosis, seizures, dependence, organ toxicity and death. Due to their ever-changing structure, very limited or nil structural, pharmacological, and toxicological information is available to the scientific community and the law enforcement offices. Here we report the synthesis and pharmacological evaluation (binding and functional) of the largest and most diverse collection of enantiopure SCRAs published to date. Our results revealed novel SCRAs that could be (or may currently be) used as illegal psychoactive substances. We also report, for the first time, the cannabimimetic data of 32 novel SCRAs containing an (R) configuration at the stereogenic center. The systematic pharmacological profiling of the library enabled the identification of emerging Structure-Activity Relationship (SAR) and Structure-Selectivity Relationship (SSR) trends, the detection of ligands exhibiting incipient cannabinoid receptor type 2 (CB2R) subtype selectivity and highlights the significant neurotoxicity of representative SCRAs on mouse primary neuronal cells. Several of the new emerging SCRAs are currently expected to have a rather limited potential for harm, as the evaluation of their pharmacological profiles revealed lower potencies and/or efficacies. Conceived as a resource to foster collaborative investigation of the physiological effects of SCRAs, the library obtained can contribute to addressing the challenge posed by recreational designer drugs.

Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells.

Dopamine receptors are G-protein-coupled receptors that are connected to severe neurological disorders. The development of new ligands targeting these receptors enables gaining a deeper insight into the receptor functioning, including binding mechanisms, kinetics and oligomerization. Novel fluorescent probes allow the development of more efficient, cheaper, reliable and scalable high-throughput screening systems, which speeds up the drug development process. In this study, we used a novel Cy3B labelled commercially available fluorescent ligand CELT-419 for developing dopamine D3 receptor-ligand binding assays with fluorescence polarization and quantitative live cell epifluorescence microscopy. The fluorescence anisotropy assay using 384-well plates achieved Z' value of 0.71, which is suitable for high-throughput screening of ligand binding. The assay can also be used to determine the kinetics of both the fluorescent ligand as well as some reference unlabeled ligands. Furthermore, CELT-419 was also used with live HEK293-D3R cells in epifluorescence microscopy imaging for deep-learning-based ligand binding quantification. This makes CELT-419 quite a universal fluorescence probe which has the potential to be also used in more advanced microscopy techniques resulting in more comparable studies.

Development of Fluorescent 4-[4-(3H-Spiro[isobenzofuran-1,4'-piperidin]-1'-yl)butyl]indolyl Derivatives as High-Affinity Probes to Enable the Study of σ Receptors via Fluorescence-Based Techniques.

Sigma (σ) receptor subtypes, σ1 and σ2, are targets of wide pharmaceutical interest. The σ2 receptor holds promise for the development of diagnostics and therapeutics against cancer and Alzheimer's disease. Nevertheless, little is known about the mechanisms activated by the σ2 receptor. To contribute to the exploitation of its therapeutic potential, we developed novel specific fluorescent ligands. Indole derivatives bearing the N-butyl-3H-spiro[isobenzofuran-1,4'-piperidine] portion were functionalized with fluorescent tags. Nanomolar-affinity fluorescent σ ligands, spanning from green to red to near-infrared emission, were obtained. Compounds 19 (σ pan affinity) and 29 (σ2 selective), which displayed the best compromise between pharmacodynamic and photophysical properties, were investigated in flow cytometry, confocal, and live cell microscopy, demonstrating their specificity for the σ2 receptor. To the best of our knowledge, these are the first red-emitting fluorescent σ2 ligands, validated as powerful tools for the study of σ2 receptors via fluorescence-based techniques.

N-adamantyl-anthranil amide derivatives: New selective ligands for the cannabinoid receptor subtype 2 (CB2R).

Cannabinoid type 2 receptor (CB2R) is a G-protein-coupled receptor that, together with Cannabinoid type 1 receptor (CB1R), endogenous cannabinoids and enzymes responsible for their synthesis and degradation, forms the EndoCannabinoid System (ECS). In the last decade, several studies have shown that CB2R is overexpressed in activated central nervous system (CNS) microglia cells, in disorders based on an inflammatory state, such as neurodegenerative diseases, neuropathic pain, and cancer. For this reason, the anti-inflammatory and immune-modulatory potentials of CB2R ligands are emerging as a novel therapeutic approach. The design of selective ligands is however hampered by the high sequence homology of transmembrane domains of CB1R and CB2R. Based on a recent three-arm pharmacophore hypothesis and latest CB2R crystal structures, we designed, synthesized, and evaluated a series of new N-adamantyl-anthranil amide derivatives as CB2R selective ligands. Interestingly, this new class of compounds displayed a high affinity for human CB2R along with an excellent selectivity respect to CB1R. In this respect, compounds exhibiting the best pharmacodynamic profile in terms of CB2R affinity were also evaluated for the functional behavior and molecular docking simulations provided a sound rationale by highlighting the relevance of the arm 1 substitution to prompt CB2R action. Moreover, the modulation of the pro- and anti-inflammatory cytokines production was also investigated to exert the ability of the best compounds to modulate the inflammatory cascade.

Exploring the Effect of Halogenation in a Series of Potent and Selective A2B Adenosine Receptor Antagonists.

The modulation of the A2B adenosine receptor is a promising strategy in cancer (immuno) therapy, with A2BAR antagonists emerging as immune checkpoint inhibitors. Herein, we report a systematic assessment of the impact of (di- and mono-)halogenation at positions 7 and/or 8 on both A2BAR affinity and pharmacokinetic properties of a collection of A2BAR antagonists and its study with structure-based free energy perturbation simulations. Monohalogenation at position 8 produced potent A2BAR ligands irrespective of the nature of the halogen. In contrast, halogenation at position 7 and dihalogenation produced a halogen-size-dependent decay in affinity. Eight novel A2BAR ligands exhibited remarkable affinity (Ki < 10 nM), exquisite subtype selectivity, and enantioselective recognition, with some eutomers eliciting sub-nanomolar affinity. The pharmacokinetic profile of representative derivatives showed enhanced solubility and microsomal stability. Finally, two compounds showed the capacity of reversing the antiproliferative effect of adenosine in activated primary human peripheral blood mononuclear cells.

Identification of A2BAR as a potential target in colorectal cancer using novel fluorescent GPCR ligands.

G-protein coupled receptors (GPCRs) have been largely targeted in a wide range of diseases, but few therapies have been directed against GPCRs in the field of cancer, partly because of the lack of effective target identification strategies. Here, using colorectal cancer (CRC) as a model, we explored the gene expression of a panel of GPCRs in tumor and stromal cells, identifying specific gene sets defining each cellular compartment. We selected the adenosine receptor 2B (A2BAR), specifically expressed in cancer cell lines compared with stromal cells, to explore the use of fluorescent ligands that can be used for target visualization. Fluorescent probes allowed semi-quantitative receptor mapping in living cells and validated the specific expression of A2BAR in CRC cell lines. As well, fluorescent ligands were effective at monitoring real-time A2BAR receptor labeling using live-imaging modalities, and displayed high efficiency when used to label complex 3D cellular systems such as tumor spheroids. Finally, we validated A2BAR as a potential pharmacological tool in CRC, using selective antagonists, finding a reduction in tumor cell proliferation. This proof-of-concept study suggests the use of fluorescent ligands for GPCR characterization through imaging, and as possible new tools used for target validation in drug screening methodologies.

A2B adenosine receptor antagonists rescue lymphocyte activity in adenosine-producing patient-derived cancer models.

BACKGROUND: Adenosine is a metabolite that suppresses antitumor immune response of T and NK cells via extracellular binding to the two subtypes of adenosine-2 receptors, A2ARs. While blockade of the A2AARs subtype effectively rescues lymphocyte activity, with four A2AAR antagonists currently in anticancer clinical trials, less is known for the therapeutic potential of the other A2BAR blockade within cancer immunotherapy. Recent studies suggest the formation of A2AAR/A2BAR dimers in tissues that coexpress the two receptor subtypes, where the A2BAR plays a dominant role, suggesting it as a promising target for cancer immunotherapy. METHODS: We report the synthesis and functional evaluation of five potent A2BAR antagonists and a dual A2AAR/A2BAR antagonist. The compounds were designed using previous pharmacological data assisted by modeling studies. Synthesis was developed using multicomponent approaches. Flow cytometry was used to evaluate the phenotype of T and NK cells on A2BAR antagonist treatment. Functional activity of T and NK cells was tested in patient-derived tumor spheroid models. RESULTS: We provide data for six novel small molecules: five A2BAR selective antagonists and a dual A2AAR/A2BAR antagonist. The growth of patient-derived breast cancer spheroids is prevented when treated with A2BAR antagonists. To elucidate if this depends on increased lymphocyte activity, immune cells proliferation, and cytokine production, lymphocyte infiltration was evaluated and compared with the potent A2AAR antagonist AZD-4635. We find that A2BAR antagonists rescue T and NK cell proliferation, IFNγ and perforin production, and increase tumor infiltrating lymphocytes infiltration into tumor spheroids without altering the expression of adhesion molecules. CONCLUSIONS: Our results demonstrate that A2BAR is a promising target in immunotherapy, identifying ISAM-R56A as the most potent candidate for A2BAR blockade. Inhibition of A2BAR signaling restores T cell function and proliferation. Furthermore, A2BAR and dual A2AAR/A2BAR antagonists showed similar or better results than A2AAR antagonist AZD-4635 reinforcing the idea of dominant role of the A2BAR in the regulation of the immune system.

Exploring Non-orthosteric Interactions with a Series of Potent and Selective A3 Antagonists.

A library of potent and highly A3AR selective pyrimidine-based compounds was designed to explore non-orthosteric interactions within this receptor. Starting from a prototypical orthosteric A3AR antagonist (ISVY130), the structure-based design explored functionalized residues at the exocyclic amide L1 region and aimed to provide additional interactions outside the A3AR orthosteric site. The novel ligands were assembled through an efficient and succinct synthetic approach, resulting in compounds that retain the A3AR potent and selective profile while improving the solubility of the original scaffold. The experimentally demonstrated tolerability of the L1 region to structural functionalization was further assessed by molecular dynamics simulations, giving hints of the non-orthosteric interactions explored by these series. The results pave the way to explore newly functionalized A3AR ligands, including covalent drugs and molecular probes for diagnostic and delivery purposes.

Optimization of 2-Amino-4,6-diarylpyrimidine-5-carbonitriles as Potent and Selective A1 Antagonists.

We herein document a large collection of 108 2-amino-4,6-disubstituted-pyrimidine derivatives as potent, structurally simple, and highly selective A1AR ligands. The most attractive ligands were confirmed as antagonists of the canonical cyclic adenosine monophosphate pathway, and some pharmacokinetic parameters were preliminarilly evaluated. The library, built through a reliable and efficient three-component reaction, comprehensively explored the chemical space allowing the identification of the most prominent features of the structure-activity and structure-selectivity relationships around this scaffold. These included the influence on the selectivity profile of the aromatic residues at positions R4 and R6 of the pyrimidine core but most importantly the prominent role to the unprecedented A1AR selectivity profile exerted by the methyl group introduced at the exocyclic amino group. The structure-activity relationship trends on both A1 and A2AARs were conveniently interpreted with rigorous free energy perturbation simulations, which started from the receptor-driven docking model that guided the design of these series.

Similarities and differences upon binding of naturally occurring Δ9-tetrahydrocannabinol-derivatives to cannabinoid CB1 and CB2 receptors.

We have here assessed, using Δ9-tetrahydrocannabinol (Δ9-THC) for comparison, the effect of Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and of Δ9-tetrahydrocannabivarin (Δ9-THCV) that is mediated by human versions of CB1, CB2, and CB1-CB2 receptor functional units, expressed in a heterologous system. Binding to the CB1 and CB2 receptors was addressed in living cells by means of a homogeneous assay. A biphasic competition curve for the binding to the CB2 receptor, was obtained for Δ9-THCV in cells expressing the two receptors. Signaling studies included cAMP level determination, activation of the mitogen-activated protein kinase pathway and ß-arrestin recruitment were performed. The signaling triggered by Δ9-THCA and Δ9-THCV via individual receptors or receptor heteromers disclosed differential bias, i.e. the bias observed using a given phytocannabinoid depended on the receptor (CB1, CB2 or CB1-CB2) and on the compound used as reference to calculate the bias factor (Δ9-THC, a selective agonist or a non-selective agonist). These results are consistent with different binding modes leading to differential functional selectivity depending on the agonist structure, and the state (monomeric or heteromeric) of the cannabinoid receptor. In addition, on studying Gi-coupling we showed that Δ9-THCV and Δ9-THCA and Δ9-THCV were able to revert the effect of a selective CB2 receptor agonist, but only Δ9-THCV, and not Δ9-THCA, reverted the effect of arachidonyl-2'-chloroethylamide (ACEA 100 nM) a selective agonist of the CB1 receptor. Overall, these results indicate that cannabinoids may have a variety of binding modes that results in qualitatively different effects depending on the signaling pathway that is engaged upon cannabinoid receptor activation.

Identification of V6.51L as a selectivity hotspot in stereoselective A2B adenosine receptor antagonist recognition.

The four adenosine receptors (ARs) A1AR, A2AAR, A2BAR, and A3AR are G protein-coupled receptors (GPCRs) for which an exceptional amount of experimental and structural data is available. Still, limited success has been achieved in getting new chemical modulators on the market. As such, there is a clear interest in the design of novel selective chemical entities for this family of receptors. In this work, we investigate the selective recognition of ISAM-140, a recently reported A2BAR reference antagonist. A combination of semipreparative chiral HPLC, circular dichroism and X-ray crystallography was used to separate and unequivocally assign the configuration of each enantiomer. Subsequently affinity evaluation for both A2A and A2B receptors demonstrate the stereospecific and selective recognition of (S)-ISAM140 to the A2BAR. The molecular modeling suggested that the structural determinants of this selectivity profile would be residue V2506.51 in A2BAR, which is a leucine in all other ARs including the closely related A2AAR. This was herein confirmed by radioligand binding assays and rigorous free energy perturbation (FEP) calculations performed on the L249V6.51 mutant A2AAR receptor. Taken together, this study provides further insights in the binding mode of these A2BAR antagonists, paving the way for future ligand optimization.

Potent and Subtype-Selective Dopamine D2 Receptor Biased Partial Agonists Discovered via an Ugi-Based Approach.

Using a previously unexplored, efficient, and versatile multicomponent method, we herein report the rapid generation of novel potent and subtype-selective DRD2 biased partial agonists. This strategy exemplifies the search for diverse and previously unexplored moieties for the secondary/allosteric pharmacophore of the common phenyl-piperazine scaffold. The pharmacological characterization of the new compound series led to the identification of several ligands with excellent DRD2 affinity and subtype selectivity and remarkable functional selectivity for either the cAMP (22a and 24d) or the ß-arrestin (27a and 29c) signaling pathways. These results were further interpreted on the basis of molecular models of these ligands in complex with the recent DRD2 crystal structures, highlighting the critical role of the secondary/allosteric pharmacophore in modulating the functional selectivity profile.

3,4-Dihydropyrimidin-2(1H)-ones as Antagonists of the Human A2B Adenosine Receptor: Optimization, Structure-Activity Relationship Studies, and Enantiospecific Recognition.

We present and thoroughly characterize a large collection of 3,4-dihydropyrimidin-2(1H)-ones as A2BAR antagonists, an emerging strategy in cancer (immuno) therapy. Most compounds selectively bind A2BAR, with a number of potent and selective antagonists further confirmed by functional cyclic adenosine monophosphate experiments. The series was analyzed with one of the most exhaustive free energy perturbation studies on a GPCR, obtaining an accurate model of the structure-activity relationship of this chemotype. The stereospecific binding modeled for this scaffold was confirmed by resolving the two most potent ligands [(±)-47, and (±)-38 Ki = 10.20 and 23.6 nM, respectively] into their two enantiomers, isolating the affinity on the corresponding (S)-eutomers (Ki = 6.30 and 11.10 nM, respectively). The assessment of the effect in representative cytochromes (CYP3A4 and CYP2D6) demonstrated insignificant inhibitory activity, while in vitro experiments in three prostate cancer cells demonstrated that this pair of compounds exhibits a pronounced antimetastatic effect.

Novel Antiproliferative Biphenyl Nicotinamide: NMR Metabolomic Study of its Effect on the MCF-7 Cell in Comparison with Cisplatin and Vinblastine.

A 1H-NMR-based metabolomic study was performed on MCF-7 cell lines treated with a novel nicotinamide derivative (DT-8) in comparison with two drugs characterized by a well-established mechanism of action, namely the DNA-metalating drug cisplatin (cis-diamminedichloridoplatinum(II), CDDP) and the antimitotic drug vinblastine (vinblastine, VIN). The effects of the three compounds, each one at the concentration corresponding to the IC50 value, were investigated, with respect to the controls (K), by the 1H-NMR of cells lysates and multivariate analysis (MVA) of the spectroscopic data. Relevant differences were found in the metabolic profiles of the different treatments with respect to the controls. A large overlap of the metabolic profiles in DT-8 vs. K and VIN vs. K suggests a similar biological response and mechanism of action, significantly diverse with respect to CDDP. On the other hand, DT8 seems to act by disorganizing the mitotic spindle and ultimately blocking the cell division, through a mechanism implying methionine depletion and/or S-adenosylmethionine (SAM) limitation.