Interaction Networks within Disease-Associated GαS Variants Characterized by an Integrative Biophysical Approach.

Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide exchange is regulated by the formation of G protein-G protein-coupled receptor (GPCR) complexes. Single point mutations in the Gα subunit of G proteins bypass this interaction, leading to loss-of-function or constitutive gain-of-function, which is closely linked with the onset of multiple diseases. Despite the recognized significance of Gα mutations in disease pathology, structural information for most variants is lacking, potentially due to inherent protein dynamics that pose challenges for crystallography. To address this, we leveraged an integrative spectroscopic and computational approach to structurally characterize seven of the most frequently observed clinically-relevant mutations in the stimulatory Gα subunit, GαS. A previously proposed allosteric model of Gα activation linked structural changes in the nucleotide binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in GαS by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics (MD) simulations, which observed changes in interaction networks between GαS switch regions. Further, saturation-transfer difference NMR (STD-NMR) spectroscopy was applied to observe changes in nucleotide interactions with residues within the nucleotide binding site. These data have enabled testing of predictions regarding how mutations in GαS result in loss or gain of function and evaluation of proposed structural mechanisms. The integration of experimental and computational data allowed us to propose a more nuanced classification of mechanisms underlying GαS gain-of-function and loss-of-function mutations.

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.

Visualizing the impact of disease-associated mutations on G protein-nucleotide interactions.

Activation of G proteins stimulates ubiquitous intracellular signaling cascades essential for life processes. Under normal physiological conditions, nucleotide exchange is initiated upon the formation of complexes between a G protein and G protein-coupled receptor (GPCR), which facilitates exchange of bound GDP for GTP, subsequently dissociating the trimeric G protein into its Gα and Gßγ subunits. However, single point mutations in Gα circumvent nucleotide exchange regulated by GPCR-G protein interactions, leading to either loss-of-function or constitutive gain-of-function. Mutations in several Gα subtypes are closely linked to the development of multiple diseases, including several intractable cancers. We leveraged an integrative spectroscopic and computational approach to investigate the mechanisms by which seven of the most frequently observed clinically-relevant mutations in the α subunit of the stimulatory G protein result in functional changes. Variable temperature circular dichroism (CD) spectroscopy showed a bimodal distribution of thermal melting temperatures across all GαS variants. Modeling from molecular dynamics (MD) simulations established a correlation between observed thermal melting temperatures and structural changes caused by the mutations. Concurrently, saturation-transfer difference NMR (STD-NMR) highlighted variations in the interactions of GαS variants with bound nucleotides. MD simulations indicated that changes in local interactions within the nucleotide-binding pocket did not consistently align with global structural changes. This collective evidence suggests a multifaceted energy landscape, wherein each mutation may introduce distinct perturbations to the nucleotide-binding site and protein-protein interaction sites. Consequently, it underscores the importance of tailoring therapeutic strategies to address the unique challenges posed by individual mutations.

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.

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.

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.

Slow conformational dynamics of the human A2A adenosine receptor are temporally ordered.

A more complete depiction of protein energy landscapes includes the identification of different function-related conformational states and the determination of the pathways connecting them. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), at the single-molecule level. Slow, reversible conformational exchange was observed among three different fluorescence emission states populated for agonist-bound A2AAR. Transitions among these states predominantly occurred in a specific order, and exchange between inactive and active-like conformations proceeded through an intermediate state. Models derived from molecular dynamics simulations with available A2AAR structures rationalized the relative fluorescence emission intensities for the highest and lowest emission states but not the transition state. This suggests that the functionally critical intermediate state required to achieve activation is not currently visualized among available A2AAR structures.

Deciphering conformational selectivity in the A2A adenosine G protein-coupled receptor by free energy simulations.

Transmembranal G Protein-Coupled Receptors (GPCRs) transduce extracellular chemical signals to the cell, via conformational change from a resting (inactive) to an active (canonically bound to a G-protein) conformation. Receptor activation is normally modulated by extracellular ligand binding, but mutations in the receptor can also shift this equilibrium by stabilizing different conformational states. In this work, we built structure-energetic relationships of receptor activation based on original thermodynamic cycles that represent the conformational equilibrium of the prototypical A2A adenosine receptor (AR). These cycles were solved with efficient free energy perturbation (FEP) protocols, allowing to distinguish the pharmacological profile of different series of A2AAR agonists with different efficacies. The modulatory effects of point mutations on the basal activity of the receptor or on ligand efficacies could also be detected. This methodology can guide GPCR ligand design with tailored pharmacological properties, or allow the identification of mutations that modulate receptor activation with potential clinical implications.

Potent Inhibition of Nicotinamide N-Methyltransferase by Alkene-Linked Bisubstrate Mimics Bearing Electron Deficient Aromatics.

Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide (vitamin B3) to generate 1-methylnicotinamide (MNA). NNMT overexpression has been linked to a variety of diseases, most prominently human cancers, indicating its potential as a therapeutic target. The development of small-molecule NNMT inhibitors has gained interest in recent years, with the most potent inhibitors sharing structural features based on elements of the nicotinamide substrate and the S-adenosyl-l-methionine (SAM) cofactor. We here report the development of new bisubstrate inhibitors that include electron-deficient aromatic groups to mimic the nicotinamide moiety. In addition, a trans-alkene linker was found to be optimal for connecting the substrate and cofactor mimics in these inhibitors. The most potent NNMT inhibitor identified exhibits an IC50 value of 3.7 nM, placing it among the most active NNMT inhibitors reported to date. Complementary analytical techniques, modeling studies, and cell-based assays provide insights into the binding mode, affinity, and selectivity of these inhibitors.

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.

Design, synthesis, HER2 inhibition and anticancer evaluation of new substituted 1,5-dihydro-4,1-benzoxazepines.

A series of 11 new substituted 1,5-dihydro-4,1-benzoxazepine derivatives was synthesised to study the influence of the methyl group in the 1-(benzenesulphonyl) moiety, the replacement of the purine by the benzotriazole bioisosteric analogue, and the introduction of a bulky substituent at position 6 of the purine, on the biological effects. Their inhibition against isolated HER2 was studied and the structure-activity relationships have been confirmed by molecular modelling studies. The most potent compound against isolated HER2 is 9a with an IC50 of 7.31 µM. We have investigated the effects of the target compounds on cell proliferation. The most active compound (7c) against all the tumour cell lines studied (IC50 0.42-0.86 µM) does not produce any modification in the expression of pro-caspase 3, but increases the caspase 1 expression, and promotes pyroptosis.

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.

Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Benzopyran-Based Inhibitors.

Inhibition of the insulin-regulated aminopeptidase (IRAP) improves memory and cognition in animal models. The enzyme has recently been crystallized and several series of inhibitors reported. We herein focused on one series of benzopyran-based inhibitors of IRAP known as the HFI series, with unresolved binding mode to IRAP, and developed a robust computational model to explain the structure-activity relationship (SAR) and potentially guide their further optimization. The binding model here proposed places the benzopyran ring in the catalytic binding site, coordinating the Zn2+ ion through the oxygen in position 3, in contrast to previous hypothesis. The whole series of HFI compounds was then systematically simulated, starting from this binding mode, using molecular dynamics and binding affinity estimated with the linear interaction energy (LIE) method. The agreement with experimental affinities supports the binding mode proposed, which was further challenged by rigorous free energy perturbation (FEP) calculations. Here, we found excellent correlation between experimental and calculated binding affinity differences, both between selected compound pairs and also for recently reported experimental data concerning the site directed mutagenesis of residue Phe544. The computationally derived structure-activity relationship of the HFI series and the understanding of the involvement of Phe544 in the binding of this scaffold provide valuable information for further lead optimization of novel IRAP inhibitors.

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.

Nitrogen-Walk Approach to Explore Bioisosteric Replacements in a Series of Potent A2B Adenosine Receptor Antagonists.

A systematic exploration of bioisosteric replacements for furan and thiophene cores in a series of potent A2BAR antagonists has been carried out using the nitrogen-walk approach. A collection of 42 novel alkyl 4-substituted-2-methyl-1,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxylates, which contain 18 different pentagonal heterocyclic frameworks at position 4, was synthesized and evaluated. This study enabled the identification of new ligands that combine remarkable affinity (Ki < 30 nM) and exquisite selectivity. The structure-activity relationship (SAR) trends identified were substantiated by a molecular modeling study, based on a receptor-driven docking model and including a systematic free energy perturbation (FEP) study. Preliminary evaluation of the CYP3A4 and CYP2D6 inhibitory activity in optimized ligands evidenced weak and negligible activity, respectively. The stereospecific interaction between hA2BAR and the eutomer of the most attractive novel antagonist (S)-18g (Ki = 3.66 nM) was validated.

X-Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A2A Adenosine Receptor Antagonists.

We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X-ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2A AR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2A AR were experimentally determined and investigated through a cycle of ligand-FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X-ray crystallography of the A2A AR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2A AR, an emerging target in immuno-oncology.

Characterization of cancer-related somatic mutations in the adenosine A2B receptor.

In cancer, G protein-coupled receptors (GPCRs) are involved in tumor progression and metastasis. In this study we particularly examined one GPCR, the adenosine A2B receptor. This receptor is activated by high concentrations of its endogenous ligand adenosine, which suppresses the immune response to fight tumor progression. A series of adenosine A2B receptor mutations were retrieved from the Cancer Genome Atlas harboring data from patient samples with different cancer types. The main goal of this work was to investigate the pharmacology of these mutant receptors using a 'single-GPCR-one-G protein' yeast assay technology. Concentration-growth curves were obtained with the full agonist NECA for the wild-type receptor and 15 mutants. Compared to wild-type receptor, the constitutive activity levels in mutant receptors F141L4.61, Y202C5.58 and L310P8.63 were high, while the potency and efficacy of NECA and BAY 60-6583 on Y202C5.58 was lower. A 33- and 26-fold higher constitutive activity on F141L4.61 and L310P8.63 was reduced to wild-type levels in response to the inverse agonist ZM241385. These constitutively active mutants may thus be tumor promoting. Mutant receptors F259S6.60 and Y113F34.53 showed a more than one log-unit decrease in potency. A complete loss of activation was observed in mutant receptors C29R1.54, W130C4.50 and P249L6.50. All mutations were characterized at the structural level, generating hypotheses of their roles on modulating the receptor conformational equilibrium. Taken together, this study is the first to investigate the nature of adenosine A2B receptor cancer mutations and may thus provide insights in mutant receptor function in cancer.

Evolution of Angiotensin Peptides and Peptidomimetics as Angiotensin II Receptor Type 2 (AT2) Receptor Agonists.

Angiotensin II receptor type 1 and 2 (AT1R and AT2R) are two G-protein coupled receptors that mediate most biological functions of the octapeptide Angiotensin II (Ang II). AT2R is upregulated upon tissue damage and its activation by selective AT2R agonists has become a promising approach in the search for new classes of pharmaceutical agents. We herein analyzed the chemical evolution of AT2R agonists starting from octapeptides, through shorter peptides and peptidomimetics to the first drug-like AT2R-selective agonist, C21, which is in Phase II clinical trials and aimed for idiopathic pulmonary fibrosis. Based on the recent crystal structures of AT1R and AT2R in complex with sarile, we identified a common binding model for a series of 11 selected AT2R agonists, consisting of peptides and peptidomimetics of different length, affinity towards AT2R and selectivity versus AT1R. Subsequent molecular dynamics simulations and free energy perturbation (FEP) calculations of binding affinities allowed the identification of the bioactive conformation and common pharmacophoric points, responsible for the key interactions with the receptor, which are maintained by the drug-like agonists. The results of this study should be helpful and facilitate the search for improved and even more potent AT2R-selective drug-like agonists.

Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro-Oxindole Dihydroquinazolinones as IRAP Inhibitors.

Insulin-regulated aminopeptidase (IRAP) is a new potential macromolecular target for drugs aimed for treatment of cognitive disorders. Inhibition of IRAP by angiotensin IV (Ang IV) improves the memory and learning in rats. The majority of the known IRAP inhibitors are peptidic in character and suffer from poor pharmacokinetic properties. Herein, we present a series of small non-peptide IRAP inhibitors derived from a spiro-oxindole dihydroquinazolinone screening hit (pIC50 5.8). The compounds were synthesized either by a simple microwave (MW)-promoted three-component reaction, or by a two-step one-pot procedure. For decoration of the oxindole ring system, rapid MW-assisted Suzuki-Miyaura cross-couplings (1 min) were performed. A small improvement of potency (pIC50 6.6 for the most potent compound) and an increased solubility could be achieved. As deduced from computational modelling and MD simulations it is proposed that the S-configuration of the spiro-oxindole dihydroquinazolinones accounts for the inhibition of IRAP.

Trifluorinated Pyrimidine-Based A2B Antagonists: Optimization and Evidence of Stereospecific Recognition.

We report the identification of two subsets of fluorinated nonxanthine A2B adenosine receptor antagonists. The novel derivatives explore the effect of fluorination at different positions of two pyrimidine-based scaffolds. The most interesting ligands combine excellent hA2B affinity (Ki < 15 nM) and remarkable subtype selectivity. The results of functional cAMP experiments confirmed the antagonistic behavior of representative ligands. The compounds were designed on the basis of previous molecular models of the stereoselective binding of the parent scaffolds to the hA2B receptor, and we herein provide refinement of such models with the fluorinated compounds, which allows the explanation of the spurious effects of the fluorination at the different positions explored. These models are importantly confirmed by a synergistic study combining chiral HPLC, circular dichroism, diastereoselective synthesis, molecular modeling, and X-ray crystallography, providing experimental evidence toward the stereospecific interaction between optimized trifluorinated stereoisomers and the hA2B receptor.