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.

Design, synthesis, and in vitro and in vivo characterization of new memantine analogs for Alzheimer's disease.

Currently, of the few accessible symptomatic therapies for Alzheimer's disease (AD), memantine is the only N-methyl-d-aspartate receptor (NMDAR) blocker approved by the FDA. This work further explores a series of memantine analogs featuring a benzohomoadamantane scaffold. Most of the newly synthesized compounds block NMDARs in the micromolar range, but with lower potency than previously reported hit IIc, results that were supported by molecular dynamics simulations. Subsequently, electrophysiological studies with the more potent compounds allowed classification of IIc, a low micromolar, uncompetitive, voltage-dependent, NMDAR blocker, as a memantine-like compound. The excellent in vitro DMPK properties of IIc made it a promising candidate for in vivo studies in Caenorhabditis elegans (C. elegans) and in the 5XFAD mouse model of AD. Administration of IIc or memantine improved locomotion and rescues chemotaxis behavior in C. elegans. Furthermore, both compounds enhanced working memory in 5XFAD mice and modified NMDAR and CREB signaling, which may prevent synaptic dysfunction and modulate neurodegenerative progression.

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.

Synthesis, Pharmacological, and Biological Evaluation of 2-Furoyl-Based MIF-1 Peptidomimetics and the Development of a General-Purpose Model for Allosteric Modulators (ALLOPTML).

This work describes the synthesis and pharmacological evaluation of 2-furoyl-based Melanostatin (MIF-1) peptidomimetics as dopamine D2 modulating agents. Eight novel peptidomimetics were tested for their ability to enhance the maximal effect of tritiated N-propylapomorphine ([3H]-NPA) at D2 receptors (D2R). In this series, 2-furoyl-l-leucylglycinamide (6a) produced a statistically significant increase in the maximal [3H]-NPA response at 10 pM (11 ± 1%), comparable to the effect of MIF-1 (18 ± 9%) at the same concentration. This result supports previous evidence that the replacement of proline residue by heteroaromatic scaffolds are tolerated at the allosteric binding site of MIF-1. Biological assays performed for peptidomimetic 6a using cortex neurons from 19-day-old Wistar-Kyoto rat embryos suggest that 6a displays no neurotoxicity up to 100 µM. Overall, the pharmacological and toxicological profile and the structural simplicity of 6a makes this peptidomimetic a potential lead compound for further development and optimization, paving the way for the development of novel modulating agents of D2R suitable for the treatment of CNS-related diseases. Additionally, the pharmacological and biological data herein reported, along with >20 000 outcomes of preclinical assays, was used to seek a general model to predict the allosteric modulatory potential of molecular candidates for a myriad of target receptors, organisms, cell lines, and biological activity parameters based on perturbation theory (PT) ideas and machine learning (ML) techniques, abbreviated as ALLOPTML. By doing so, ALLOPTML shows high specificity Sp = 89.2/89.4%, sensitivity Sn = 71.3/72.2%, and accuracy Ac = 86.1%/86.4% in training/validation series, respectively. To the best of our knowledge, ALLOPTML is the first general-purpose chemoinformatic tool using a PTML-based model for the multioutput and multicondition prediction of allosteric compounds, which is expected to save both time and resources during the early drug discovery of allosteric modulators.

2-Oxaadamant-1-yl Ureas as Soluble Epoxide Hydrolase Inhibitors: In Vivo Evaluation in a Murine Model of Acute Pancreatitis.

In vivo pharmacological inhibition of soluble epoxide hydrolase (sEH) reduces inflammatory diseases, including acute pancreatitis (AP). Adamantyl ureas are very potent sEH inhibitors, but the lipophilicity and metabolism of the adamantane group compromise their overall usefulness. Herein, we report that the replacement of a methylene unit of the adamantane group by an oxygen atom increases the solubility, permeability, and stability of three series of urea-based sEH inhibitors. Most of these oxa-analogues are nanomolar inhibitors of both the human and murine sEH. Molecular dynamics simulations rationalize the molecular basis for their activity and suggest that the presence of the oxygen atom on the adamantane scaffold results in active site rearrangements to establish a weak hydrogen bond. The 2-oxaadamantane 22, which has a good solubility, microsomal stability, and selectivity for sEH, was selected for further in vitro and in vivo studies in models of cerulein-induced AP. Both in prophylactic and treatment studies, 22 diminished the overexpression of inflammatory and endoplasmic reticulum stress markers induced by cerulein and reduced the pancreatic damage.

A Novel NMDA Receptor Antagonist Protects against Cognitive Decline Presented by Senescent Mice.

Alzheimer's disease (AD) is the leading cause of dementia. Non-competitive N-Methyl-D-aspartate (NMDA) receptor antagonist memantine improved cognition and molecular alterations after preclinical treatment. Nevertheless, clinical results are discouraging. In vivo efficacy of the RL-208, a new NMDA receptor blocker described recently, with favourable pharmacokinetic properties was evaluated in Senescence accelerated mice prone 8 (SAMP8), a mice model of late-onset AD (LOAD). Oral administration of RL-208 improved cognitive performance assessed by using the three chamber test (TCT), novel object recognition test (NORT), and object location test (OLT). Consistent with behavioural results, RL-208 treated-mice groups significantly changed NMDAR2B phosphorylation state levels but not NMDAR2A. Calpain-1 and Caspase-3 activity was reduced, whereas B-cell lymphoma-2 (BCL-2) levels increased, indicating reduced apoptosis in RL-208 treated SAMP8. Superoxide Dismutase 1 (SOD1) and Glutathione Peroxidase 1 (GPX1), as well as a reduction of hydrogen peroxide (H2O2), was also determined in RL-208 mice. RL-208 treatment induced an increase in mature brain-derived neurotrophic factor (mBDNF), prevented Tropomyosin-related kinase B full-length (TrkB-FL) cleavage, increased protein levels of Synaptophysin (SYN) and Postsynaptic density protein 95 (PSD95). In whole, these results point out to an improvement in synaptic plasticity. Remarkably, RL-208 also decreased the protein levels of Cyclin-Dependent Kinase 5 (CDK5), as well as p25/p35 ratio, indicating a reduction in kinase activity of CDK5/p25 complex. Consequently, lower levels of hyperphosphorylated Tau (p-Tau) were found. In sum, these results demonstrate the neuroprotectant role of RL-208 through NMDAR blockade.

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.

Perturbation Theory/Machine Learning Model of ChEMBL Data for Dopamine Targets: Docking, Synthesis, and Assay of New l-Prolyl-l-leucyl-glycinamide Peptidomimetics.

Predicting drug-protein interactions (DPIs) for target proteins involved in dopamine pathways is a very important goal in medicinal chemistry. We can tackle this problem using Molecular Docking or Machine Learning (ML) models for one specific protein. Unfortunately, these models fail to account for large and complex big data sets of preclinical assays reported in public databases. This includes multiple conditions of assays, such as different experimental parameters, biological assays, target proteins, cell lines, organism of the target, or organism of assay. On the other hand, perturbation theory (PT) models allow us to predict the properties of a query compound or molecular system in experimental assays with multiple boundary conditions based on a previously known case of reference. In this work, we report the first PTML (PT + ML) study of a large ChEMBL data set of preclinical assays of compounds targeting dopamine pathway proteins. The best PTML model found predicts 50000 cases with accuracy of 70-91% in training and external validation series. We also compared the linear PTML model with alternative PTML models trained with multiple nonlinear methods (artificial neural network (ANN), Random Forest, Deep Learning, etc.). Some of the nonlinear methods outperform the linear model but at the cost of a notable increment of the complexity of the model. We illustrated the practical use of the new model with a proof-of-concept theoretical-experimental study. We reported for the first time the organic synthesis, chemical characterization, and pharmacological assay of a new series of l-prolyl-l-leucyl-glycinamide (PLG) peptidomimetic compounds. In addition, we performed a molecular docking study for some of these compounds with the software Vina AutoDock. The work ends with a PTML model predictive study of the outcomes of the new compounds in a large number of assays. Therefore, this study offers a new computational methodology for predicting the outcome for any compound in new assays. This PTML method focuses on the prediction with a simple linear model of multiple pharmacological parameters (IC50, EC50, Ki, etc.) for compounds in assays involving different cell lines used, organisms of the protein target, or organism of assay for proteins in the dopamine pathway.

Development of Fluorescent Probes that Target Serotonin 5-HT2B Receptors.

Some 5-HT2B fluorescent probes were obtained by tagging 1-(2,5-dimethoxy-4-iodophenyl)-propan-2-amine (DOI) with a subset of fluorescent amines. Some of the resulting fluorescent ligands showed excellent affinity and selectivity profiles at the 5-HT2B receptors (e.g. 12b), while retain the agonistic functional behaviour of the model ligand (DOI). The study highlighted the most salient features of the structure-activity relationship in this series and these were substantiated by a molecular modelling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human 5-HT2B receptor. One of the fluorescent ligands developed in this work, compound 12i, specifically labelled CHO-K1 cells expressing 5-HT2B receptors and not parental CHO-K1 cells in a concentration-dependent manner. 12i enables imaging and quantification of specific 5-HT2B receptor labelling in live cells by automated fluorescence microscopy as well as quantification by measurements of fluorescence intensity using a fluorescence plate reader.

Computer-aided structure-based design of multitarget leads for Alzheimer's disease.

Alzheimer's disease is a neurodegenerative pathology with unmet clinical needs. A highly desirable approach to this syndrome would be to find a single lead that could bind to some or all of the selected biomolecules that participate in the amyloid cascade, the most accepted route for Alzheimer disease genesis. In order to circumvent the challenge posed by the sizable differences in the binding sites of the molecular targets, we propose a computer-assisted protocol based on a pharmacophore and a set of required interactions with the targets that allows for the automated screening of candidates. We used a combination of docking and molecular dynamics protocols in order to discard nonbinders, optimize the best candidates, and provide a rationale for their potential as inhibitors. To provide a proof of concept, we proceeded to screen the literature and databases, a task that allowed us to identify a set of carbazole-containing compounds that initially showed affinity only for the cholinergic targets in our experimental assays. Two cycles of design based on our protocol led to a new set of analogues that were synthesized and assayed. The assay results revealed that the designed inhibitors had improved affinities for BACE-1 by more than 3 orders of magnitude and also displayed amyloid aggregation inhibition and affinity for AChE and BuChE, a result that led us to a group of multitarget amyloid cascade inhibitors that also could have a positive effect at the cholinergic level.