Binding and unbinding of potent melatonin receptor ligands: Mechanistic simulations and experimental evidence.

The labeled ligand commonly employed in competition binding studies for melatonin receptor ligands, 2-[125I]iodomelatonin, showed slow dissociation with different half-lives at the two receptor subtypes. This may affect the operational measures of affinity constants, which at short incubation times could not be obtained in equilibrium conditions, and structure-activity relationships, as the Ki values of tested ligands could depend on either interaction at the binding site or the dissociation path. To address these issues, the kinetic and saturation binding parameters of 2-[125I]iodomelatonin as well as the competition constants for a series of representative ligands were measured at a short (2 h) and a long (20 h) incubation time. Concurrently, we simulated by molecular modeling the dissociation path of 2-iodomelatonin from MT1 and MT2 receptors and investigated the role of interactions at the binding site on the stereoselectivity observed for the enantiomers of the subtype-selective ligand UCM1014. We found that equilibrium conditions for 2-[125I]iodomelatonin binding can be reached only with long incubation times, particularly for the MT2 receptor subtype, for which a time of 20 h approximates this condition. On the other hand, measured Ki values for a set of ligands including agonists, antagonists, nonselective, and subtype-selective compounds were not significantly affected by the length of incubation, suggesting that structure-activity relationships based on data collected at shorter time reflect different interactions at the binding site. Molecular modeling simulations evidenced that the slower dissociation of 2-iodomelatonin from the MT2 receptor can be related to the restricted mobility of a gatekeeper tyrosine along a lipophilic path from the binding site to the membrane bilayer. The enantiomers of the potent, MT2-selective agonist UCM1014 were separately synthesized and tested. Molecular dynamics simulations of the receptor-ligand complexes provided an explanation for their stereoselectivity as due to the preference shown by the eutomer at the binding site for the most abundant axial conformation adopted by the ligand in solution. These results suggest that, despite the slow-binding kinetics occurring for the labeled ligand, affinity measures at shorter incubation times give robust results consistent with known structure-activity relationships and with interactions taken at the receptor binding site.

Pharmacology of Adenosine Receptors: Recent Advancements.

Adenosine receptors (ARs) are widely acknowledged pharmacological targets yet are still underutilized in clinical practice. Their ubiquitous distribution in almost all cells and tissues of the body makes them, on the one hand, excellent candidates for numerous diseases, and on the other hand, intrinsically challenging to exploit selectively and in a site-specific manner. This review endeavors to comprehensively depict the substantial advancements witnessed in recent years concerning the development of drugs that modulate ARs. Through preclinical and clinical research, it has become evident that the modulation of ARs holds promise for the treatment of numerous diseases, including central nervous system disorders, cardiovascular and metabolic conditions, inflammatory and autoimmune diseases, and cancer. The latest studies discussed herein shed light on novel mechanisms through which ARs exert control over pathophysiological states. They also introduce new ligands and innovative strategies for receptor activation, presenting compelling evidence of efficacy along with the implicated signaling pathways. Collectively, these emerging insights underscore a promising trajectory toward harnessing the therapeutic potential of these multifaceted targets.

Pioneering first-in-class FAAH-HDAC inhibitors as potential multitarget neuroprotective agents.

Aiming to simultaneously modulate the endocannabinoid system (ECS) functions and the epigenetic machinery, we selected the fatty acid amide hydrolase (FAAH) and histone deacetylase (HDAC) enzymes as desired targets to develop potential neuroprotective multitarget-directed ligands (MTDLs), expecting to achieve an additive or synergistic therapeutic effect in oxidative stress-related conditions. We herein report the design, synthesis, and biological evaluation of the first-in-class FAAH-HDAC multitarget inhibitors. A pharmacophore merging strategy was applied, yielding 1-phenylpyrrole-based compounds 4a-j. The best-performing compounds (4c, 4f, and 4h) were tested for their neuroprotective properties in oxidative stress models, employing 1321N1 human astrocytoma cells and SHSY5 human neuronal cells. In our preliminary studies, compound 4h stood out, showing a balanced nanomolar inhibitory activity against the selected targets and outperforming the standard antioxidant N-acetylcysteine in vitro. Together with 4f, 4h was also able to protect 1321N1 cells from tert-butyl hydroperoxide or glutamate insult. Our study may provide the basis for the development of novel MTDLs targeting the ECS and epigenetic enzymes.

Caffeine for Prevention of Alzheimer's Disease: Is the A2A Adenosine Receptor Its Target?

Alzheimer's disease (AD) is the most prevalent kind of dementia with roughly 135 million cases expected in the world by 2050. Unfortunately, current medications for the treatment of AD can only relieve symptoms but they do not act as disease-modifying agents that can stop the course of AD. Caffeine is one of the most widely used drugs in the world today, and a number of clinical studies suggest that drinking coffee may be good for health, especially in the fight against neurodegenerative conditions such as AD. Experimental works conducted "in vivo" and "in vitro" provide intriguing evidence that caffeine exerts its neuroprotective effects by antagonistically binding to A2A receptors (A2ARs), a subset of GPCRs that are triggered by the endogenous nucleoside adenosine. This review provides a summary of the scientific data supporting the critical role that A2ARs play in memory loss and cognitive decline, as well as the evidence supporting the protective benefits against neurodegeneration that may be attained by caffeine's antagonistic action on these receptors. They are a novel and fascinating target for regulating and enhancing synaptic activity, achieving symptomatic and potentially disease-modifying effects, and protecting against neurodegeneration.

Development of potent and selective FAAH inhibitors with improved drug-like properties as potential tools to treat neuroinflammatory conditions.

The neuroprotective performance against neuroinflammation of the endocannabinoid system (ECS) can be remarkably improved by indirect stimulation mediated by the pharmacological inhibition of the key ECS catabolic enzyme fatty acid amide hydrolase (FAAH). Based on our previous works and aiming to discover new selective FAAH inhibitors , we herein reported a new series of carbamate-based FAAH inhibitors (4a-t) which showed improved drug disposition properties compared to the previously reported analogues 2a-b. The introduction of ionizable functions allowed us to obtain new FAAH inhibitors of nanomolar potency characterized by good water solubility and chemical stability at physiological pH. Interesting structure-activity relationships (SARs), deeply analyzed by molecular docking and molecular dynamic (MD) simulations, were obtained. All the newly developed inhibitors showed an excellent selectivity profile evaluated against monoacylglycerol lipase and cannabinoid receptors. The reversible mechanism of action was determined by a rapid dilution assay. Absence of toxicity was confirmed in mouse fibroblasts NIH3T3 (for compounds 4e, 4g, 4n-o, and 4s) and in human astrocytes cell line 1321N1 (for compounds 4e, 4n, and 4s). The absence of undesired cardiac effects was also confirmed for compound 4n. Selected analogues (compounds 4e, 4g, 4n, and 4s) were able to reduce oxidative stress in 1321N1 astrocytes and exhibited notable neuroprotective effects when tested in an ex vivo model of neuroinflammation.

Update on the recent development of allosteric modulators for adenosine receptors and their therapeutic applications.

Adenosine receptors (ARs) have been identified as promising therapeutic targets for countless pathological conditions, spanning from inflammatory diseases to central nervous system disorders, from cancer to metabolic diseases, from cardiovascular pathologies to respiratory diseases, and beyond. This extraordinary therapeutic potential is mainly due to the plurality of pathophysiological actions of adenosine and the ubiquitous expression of its receptors. This is, however, a double-edged sword that makes the clinical development of effective ligands with tolerable side effects difficult. Evidence of this is the low number of AR agonists or antagonists that have reached the market. An alternative approach is to target allosteric sites via allosteric modulators, compounds endowed with several advantages over orthosteric ligands. In addition to the typical advantages of allosteric modulators, those acting on ARs could benefit from the fact that adenosine levels are elevated in pathological tissues, thus potentially having negligible effects on normal tissues where adenosine levels are maintained low. Several A1 and various A3AR allosteric modulators have been identified so far, and some of them have been validated in different preclinical settings, achieving promising results. Less fruitful, instead, has been the discovery of A2A and A2BAR allosteric modulators, although the results obtained up to now are encouraging. Collectively, data in the literature suggests that allosteric modulators of ARs could represent valuable pharmacological tools, potentially able to overcome the limitations of orthosteric ligands.

Synthesis of Unsymmetrical Squaramides as Allosteric GSK-3ß Inhibitors Promoting ß-Catenin-Mediated Transcription of TCF/LEF in Retinal Pigment Epithelial Cells.

The glycogen synthase kinase 3ß (GSK-3ß) is a ubiquitous enzyme that is a validated target for the development of potential therapeutics useful in several diseases including retinal degeneration. Aiming at developing an innovative class of allosteric inhibitors of GSK-3ß potentially useful for retinal degeneration, we explored the class of squaramides. The developed compounds (6 a-l) were obtained through a nontoxic one-pot synthetic protocol, which employs low-cost goods and avoids any purification step. Ethanol was used as the reaction solvent, simultaneously allowing the pure reaction products' recovery (by precipitation). Out of this set of squaramides, 6 j stood out, from computational and enzymatic converging data, as an ATP non-competitive inhibitor of GSK-3ß of micromolar potency. When engaged in cellular studies using retinal pigment epithelial cells (ARPE-19) transfected with a luciferase reporter gene under the control of T-cell factor/lymphoid enhancer factor (TCF/LEF) binding sites, 6 j was able to dose-dependently induce ß-catenin nuclear accumulation, as shown by the increased luciferase activity at a concentration of 2.5 µM.

Polypharmacological Approaches for CNS Diseases: Focus on Endocannabinoid Degradation Inhibition.

Polypharmacology breaks up the classical paradigm of "one-drug, one target, one disease" electing multitarget compounds as potential therapeutic tools suitable for the treatment of complex diseases, such as metabolic syndrome, psychiatric or degenerative central nervous system (CNS) disorders, and cancer. These diseases often require a combination therapy which may result in positive but also negative synergistic effects. The endocannabinoid system (ECS) is emerging as a particularly attractive therapeutic target in CNS disorders and neurodegenerative diseases including Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), stroke, traumatic brain injury (TBI), pain, and epilepsy. ECS is an organized neuromodulatory network, composed by endogenous cannabinoids, cannabinoid receptors type 1 and type 2 (CB1 and CB2), and the main catabolic enzymes involved in the endocannabinoid inactivation such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). The multiple connections of the ECS with other signaling pathways in the CNS allows the consideration of the ECS as an optimal source of inspiration in the development of innovative polypharmacological compounds. In this review, we focused our attention on the reported polypharmacological examples in which FAAH and MAGL inhibitors are involved.

Adenosine Receptors in Neuropsychiatric Disorders: Fine Regulators of Neurotransmission and Potential Therapeutic Targets.

Adenosine exerts an important role in the modulation of central nervous system (CNS) activity. Through the interaction with four G-protein coupled receptor (GPCR) subtypes, adenosine subtly regulates neurotransmission, interfering with the dopaminergic, glutamatergic, noradrenergic, serotoninergic, and endocannabinoid systems. The inhibitory and facilitating actions of adenosine on neurotransmission are mainly mediated by A1 and A2A adenosine receptors (ARs), respectively. Given their role in the CNS, ARs are promising therapeutic targets for neuropsychiatric disorders where altered neurotransmission represents the most likely etiological hypothesis. Activating or blocking ARs with specific pharmacological agents could therefore restore the balance of altered neurotransmitter systems, providing the rationale for the potential treatment of these highly debilitating conditions. In this review, we summarize and discuss the most relevant studies concerning AR modulation in psychotic and mood disorders such as schizophrenia, bipolar disorders, depression, and anxiety, as well as neurodevelopment disorders such as autism spectrum disorder (ASD), fragile X syndrome (FXS), attention-deficit hyperactivity disorder (ADHD), and neuropsychiatric aspects of neurodegenerative disorders.

Adenosine and Inflammation: Here, There and Everywhere.

Adenosine is a ubiquitous endogenous modulator with the main function of maintaining cellular and tissue homeostasis in pathological and stress conditions. It exerts its effect through the interaction with four G protein-coupled receptor (GPCR) subtypes referred as A1, A2A, A2B, and A3 adenosine receptors (ARs), each of which has a unique pharmacological profile and tissue distribution. Adenosine is a potent modulator of inflammation, and for this reason the adenosinergic system represents an excellent pharmacological target for the myriad of diseases in which inflammation represents a cause, a pathogenetic mechanism, a consequence, a manifestation, or a protective factor. The omnipresence of ARs in every cell of the immune system as well as in almost all cells in the body represents both an opportunity and an obstacle to the clinical use of AR ligands. This review offers an overview of the cardinal role of adenosine in the modulation of inflammation, showing how the stimulation or blocking of its receptors or agents capable of regulating its extracellular concentration can represent promising therapeutic strategies for the treatment of chronic inflammatory pathologies, neurodegenerative diseases, and cancer.