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Background: Sigma receptors (Rs), initially classified as an additional class of opioid receptors, are now recognized as a unique entity with no homology to opioid receptors divided into two distinct subtypes, namely σ1R and σ2R. σ1R-targeting ligands have been conceived and explored for the treatment of various neurodegenerative disorders and neuropathic pain. Activation of the σ2R appears to be involved in the regulation of cellular proliferation and cell death. Objective: Up to now, the rational design of novel σ1R ligands was efficiently guided by computational methods, especially relying on homology modeling studies. Conversely, the limited number of in silico studies was applied in the search of σ2R-targeting compounds. Herein we explored several series of σ1R ligands, by computational methods, featuring variable selectivity profile towards σ1R and σ2R in order to gain useful information guiding the rational design of more selective ligands. Methods: Based on the recent X-ray crystallographic structure of the human σ1R, in-depth molecular docking studies on different series of σR ligands have been performed. These calculations have been followed by molecular dynamic simulations (MD) and two pharmacophore analyses, taking into account the activity levels towards σ1R and σ2R. Results: Structure-based studies revealed key contacts to be achieved in order to guide selectivity of σ1R-targeting compounds while the two pharmacophore models described the main features turning into effective σ1R or σ2R ligands. Conclusion: The applied computational approach allowed a more comprehensive exploration of the structure-activity relationship (SAR) within the herein analyzed σR ligands, deriving useful guidelines for the rational design of more selective compounds.

Sirtuins are NAD+-dependent deac(et)ylases with different subcellular localization. The sirtuins’ family is composed of seven members, named SIRT-1 to SIRT-7. Their substrates include histones and also an increasing number of different proteins. Sirtuins regulate a wide range of different processes, ranging from transcription to metabolism to genome stability. Thus, their dysregulation has been related to the pathogenesis of different diseases. In this review, we discussed the pharmacological approaches based on sirtuins’ modulators (both inhibitors and activators) that have been attempted in in vitro and/or in in vivo experimental settings, to highlight the therapeutic potential of targeting one/more specific sirtuin isoform(s) in cancer, neurodegenerative disorders and type 2 diabetes. Extensive research has already been performed to identify SIRT-1 and -2 modulators, while compounds targeting the other sirtuins have been less studied so far. Beside sections dedicated to each sirtuin, in the present review we also included sections dedicated to pan-sirtuins’ and to parasitic sirtuins’ modulators. A special focus is dedicated to the sirtuins’ modulators identified by the use of virtual screening.

Introduction: The greater interest in TAAR1-mediated potential for the treatment of different pathologies, especially those related to CNS disorders, has given a considerable boost to the search fo...

Magnolol and luteolin are two natural compounds recognized in several medicinal plants widely used in traditional medicine, including type 2 diabetes mellitus. This research aimed to determine the inhibitory activity of magnolol and luteolin on α-glucosidase activity. Their biological profile was studied by multispectroscopic methods along with inhibitory kinetic analysis and computational experiments. Magnolol and luteolin decreased the enzymatic activity in a concentration-dependent manner. With 0.075 µM α-glucosidase, the IC50 values were similar for both compounds (~ 32 µM) and significantly lower than for acarbose (815 μM). Magnolol showed a mixed-type antagonism, while luteolin showed a non-competitive inhibition mechanism. Thermodynamic parameters suggested that the binding of magnolol was predominantly sustained by hydrophobic interactions, while luteolin mainly exploited van der Waals contacts and hydrogen bonds. Synchronous fluorescence revealed that magnolol interacted with the target, influencing the microenvironment around tyrosine residues, and circular dichroism explained a rearrangement of the secondary structure of α-glucosidase from the initial α-helix to the final conformation enriched with β-sheet and random coil. Docking studies provided support for the experimental results. Altogether, the data propose magnolol, for the first time, as a potential α-glucosidase inhibitor and add further evidence to the inhibitory role of luteolin.

Cystic fibrosis (CF) is caused by different mutations related to the cystic fibrosis transmembrane regulator protein (CFTR), with F508del being the most common. Pioneering the development of CFTR modulators, thanks to the development of effective correctors or potentiators, more recent studies deeply encouraged the administration of triple combination therapeutics. However, combinations of molecules interacting with other proteins involved in functionality of the CFTR channel recently arose as a promising approach to address a large rescue of F508del-CFTR. In this context, the design of compounds properly targeting the molecular chaperone Hsp70, such as the allosteric inhibitor MKT-077, proved to be effective for the development of indirect CFTR modulators, endowed with ability to amplify the accumulation of the rescued protein. Herein we performed structure-based studies of a number of allosteric HSP70 inhibitors, considering the recent X-ray crystallographic structure of the human enzyme. This allowed us to point out the main interaction supporting the binding mode of MKT-077, as well as of the related analogues. In particular, cation-π and π–π stacking with the conserve residue Tyr175 deeply stabilized inhibitor binding at the HSP70 cavity. Molecular docking studies had been followed by QSAR analysis and then by virtual screening of aminoaryl thiazoles (I–IIIa) as putative HSP70 inhibitors. Their effectiveness as CFTR modulators has been verified by biological assays, in combination with VX-809, whose positive results confirmed the reliability of the whole applied computational method. Along with this, the “in-silico” prediction of absorption, distribution, metabolism, and excretion (ADME) properties highlighted, once more, that AATs may represent a chemical class to be further investigated for the rational design of novel combination of compounds for CF treatment.

Targeting trace amine-associated receptor 1 (TAAR1) receptor continues to offer an intriguing opportunity to develop innovative therapies in different pharmacological settings. Pursuing our endeavors in the search for effective and safe human TAAR1 (hTAAR1) ligands, we synthesized a new series of 1-amidino-4-phenylpiperazine derivatives (1&ndash, 16) based on the application of a combined pharmacophore model/scaffold simplification strategy for an in-house series of biguanide-based TAAR1 agonists. Most of the novel compounds proved to be more effective than their prototypes, showing nanomolar EC50 values in functional activity at hTAAR1 and low general cytotoxicity (CC50 &gt, 80 &micro, M) when tested on the Vero-76 cell line. In this new series, the main determinant for TAAR1 agonism ability appears to result from the appropriate combination between the steric size and position of the substituents on the phenyl ring rather than from their different electronic nature, since both electron-withdrawing and electron donor groups are permitted. In particular, the ortho-substitution seems to impose a more appropriate spatial geometry to the molecule that entails an enhanced TAAR1 potency profile, as experienced, in the following order, by compounds 15 (2,3-diCl, EC50 = 20 nM), 2 (2-CH3, EC50 = 30 nM), 6 (2-OCH3, EC50 = 93 nM) and 3 (2-Cl, EC50 = 160 nM). Apart from the interest in them as valuable leads for the development of promising hTAAR1 agonists, these simple small molecules have further allowed us to identify the minimal structural requirements for producing an efficient hTAAR1 targeting ability.

Computational drug repositioning is of growing interest to academia and industry, for its ability to rapidly screen a huge number of candidates in silico (exploiting comprehensive drug datasets) together with reduced development cost and time. The potential of drug repositioning has not been fully evaluated yet for cystic fibrosis (CF), a disease mainly caused by deletion of Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. F508del-CFTR is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. CF is still a fatal disease. Nowadays, it is treatable by some CFTR-rescuing drugs, but new-generation drugs with stronger therapeutic benefits and fewer side effects are still awaited. In this manuscript we report about the results of a pilot computational drug repositioning screening in search of F508del-CFTR-targeted drugs performed on AIFA library by means of a dedicated computational pipeline and surface plasmon resonance binding assay to experimentally validate the computational findings.

Cystic fibrosis (CF) is the autosomal recessive disorder most recurrent in Caucasian populations. It is caused by different mutations in the cystic fibrosis transmembrane regulator protein (CFTR) gene, with F508del being the most common. During the last years, small-molecule therapy chosen to contrast CF relied on compounds that correct CFTR misfolding and ER retention (correctors such as VX-809), or defective channel gating (potentiators such as VX-770). Combination therapy with the two series of drugs has been applied, leading to the approval of several multi-drugs such as Orkambi. Despite this, this treatment proved to be only partially effective making the search for novel modulators an urgent need to contrast CF. Recently, we reported compound 2a as reference compound of a series of aminoarylthiazole-VX-809 hybrid derivatives exhibiting promising F508del-CFTR corrector ability. Herein, we report exploring the docking mode of the prototype VX-809 and of 2a in order to derive useful guidelines for the rational design of novel optimized analogues. To demonstrate experimentally their effective F508del-CFTR-binding and rescuing potential, the most promising derivatives had been synthesized and evaluated in biological assays including YFP functional assay on F508del-CFTR CFBE41o-cells, trans epithelial electrical resistance (TEER) and surface plasmon resonance (SPR). This multidisciplinary strategy led to the discovery of a second series of hybrids including 7j and 7m endowed with higher potency than the prototype.

Respiratory RNA viruses are responsible for recurrent acute respiratory illnesses that still represent a major medical need. Previously we developed a large variety of benzimidazole derivatives able to inhibit these viruses. Herein, two series of (thio)semicarbazone- and hydrazone-based benzimidazoles have been explored, by derivatizing 5-acetyl benzimidazoles previously reported by us, thereby evaluating the influence of the modification on the antiviral activity. Compounds 6, 8, 16 and 17, bearing the 5-(thio)semicarbazone and 5-hydrazone functionalities in combination with the 2-benzyl ring on the benzimidazole core structure, acted as dual inhibitors of influenza A virus and human coronavirus. For respiratory syncytial virus (RSV), activity is limited to the 5-thiosemicarbazone (25) and 5-hydrazone (22) compounds carrying the 2-[(benzotriazol-1/2-yl)methyl]benzimidazole scaffold. These molecules proved to be the most effective antiviral agents, able to reach the potency profile of the licensed drug ribavirin. The molecular docking analysis explained the SAR of these compounds around their binding mode to the target RSV F protein, revealing the key contacts for further assessment. The herein-investigated benzimidazole-based derivatives may represent valuable hit compounds, deserving subsequent structural improvements towards more efficient antiviral agents for the treatment of pathologies caused by these human respiratory viruses. ispartof: MOLECULES vol:25 issue:7 ispartof: location:Switzerland status: published

Cystic fibrosis transmembrane conductance regulator (CFTR)-rescuing drugs have already transformed cystic fibrosis (CF) from a fatal disease to a treatable chronic condition. However, new-generation drugs able to bind CFTR with higher specificity/affinity and to exert stronger therapeutic benefits and fewer side effects are still awaited. Computational methods and biosensors have become indispensable tools in the process of drug discovery for many important human pathologies. Instead, they have been used only piecemeal in CF so far, calling for their appropriate integration with well-tried CF biochemical and cell-based models to speed up the discovery of new CFTR-rescuing drugs. This review will give an overview of the available structures and computational models of CFTR and of the biosensors, biochemical and cell-based assays already used in CF-oriented studies. It will also give the reader some insights about how to integrate these tools as to improve the efficiency of the drug discovery process targeted to CFTR.

Aim: Targeting 5-HT1A receptor (5-HT1AR) as a strategy for CNS disorders and pain control. Methodology: A series of 1,3-dioxolane-based 2-heteroaryl-phenoxyethylamines was synthesized by a convergent approach and evaluated at α1-adrenoceptors and 5-HT1AR by binding and functional experiments. Absorption, distribution, metabolism, excretion and toxicity prediction studies were performed to explore the drug-likeness of the compounds. Results & conclusion: The most promising compound, the pyridin-4-yl derivative, emerged as a potent and selective 5-HT1AR agonist (pKi = 9.2; pD2 = 8.83; 5-HT1A/α1 = 135). In vitro it was able to permeate by passive diffusion MDCKII-MDR1 monolayer mimicking the blood–brain barrier and showed promising neuroprotective activity.

Background: Cystic fibrosis (CF) is the autosomal recessive disorder most common in Caucasian populations. It is caused by mutations in the cystic fibrosis transmembrane regulator protein (CFTR). CFTR is predominantly expressed at the apical plasma membranes of the epithelial cells lining several organs, and functions as a cAMP-regulated chloride/bicarbonate channel. To address the underlying causes of cystic fibrosis, two biomolecular activities are required, namely correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective opening of the CFTR channel. Objective: In our previous data, we demonstrated that some aminoarylthiazoles (AATs) have peculiar activity acting as correctors and as potentiator-like molecules. : Curiously, a compound called 1 has been shown to be markedly active as a potentiator. Now, we have further modified its scaffold at different portions, for the identification of molecules with improved potency and effectiveness on mutant CFTR. Methods: Starting from this active compound, we synthesized a small library trying to improve the activity as potentiators. To extrapolate the contribution of a particular structural portion to bioactivity, we selectively modified one portion at a time. Results: Our study has provided a structure-activity relationship (SAR) on AATs and led to the identification of some compounds, with a particular ability to act as CFTR potentiators. Conclusion: Two compounds 2 and 13 appear to be promising molecules and could be used for the future development of potentiators of the chloride transport defect in cystic fibrosis.

Recently we identified cycloguanil-like dihydrotriazine derivatives, which provided host-factor directed antiviral activity against influenza viruses and respiratory syncytial virus (RSV), by targeting the human dihydrofolate reductase (hDHFR) enzyme. In this context we deemed interesting to further investigate the structure activity relationship (SAR) of our first series of cycloguanil-like dihydrotriazines, designing two novel azaspiro dihydrotriazine scaffolds. The present study allowed the exploration of the potential chemical space, around these new scaffolds, that are well tolerated for maintaining the antiviral effect by means of interaction with the hDHFR enzyme. The new derivatives confirmed their inhibitory profile against influenza viruses, especially type B. In particular, the two best compounds shared potent antiviral activity (4: EC50 = 0.29 μM; 6: EC50 = 0.19 μM), which was comparable to that of zanamivir (EC50 = 0.14 μM), and better than that of ribavirin (EC50 = 3.2 μM). In addition, these two compounds proved to be also effective against RSV (4: EC50 = 0.40 μM, SI ≥ 250; 6: EC50 = 1.8 μM, SI ≥ 56), surpassing the potency and selectivity index (SI) of ribavirin (EC50 = 5.8 μM, SI > 43). By a perspective of these results, the above adequately substituted azaspiro dihydrotriazines may represent valuable hit compounds worthy of further structural optimization to develop improved host DHFR-directed antiviral agents., Graphical abstract Image 1, Highlights • Azaspiro dihydrotriazines proved to be active against influenza viruses. • The antiviral activity was related to the inhibition of the host factor DHFR. • Docking studies revealed two H-bonds with key residues I7 and S59 of hDHFR enzyme.

Nowadays, compounds targeting G-protein-coupled receptors (GPCRs) represent a large amount of clinically relevant drugs, depicting a leading role in the drug discovery panorama.Trace amine-associat...

Herein we continued our previous work on the development of CB2 ligands, reporting the design and synthesis of a series of benzimidazole-containing derivatives that were explored as selective CB2 ligands with binding affinity towards both CB1 and CB2 receptors. Seven out of eighteen compounds exhibited preferential binding ability to CB2 over CB1 receptors with potencies in the sub-micromolar or low micromolar range. In particular, we identified two promising hit compounds, the agonist 1-[2-(N,N-diethylamino)ethyl]-2-(4-ethoxybenzyl)-5-trifluoromethylbenzimidazole (3) (CB2: K(i) = 0.42 μM) and the inverse agonist/antagonist 1-butyl-2-(3,4-dichlorobenzyl)-5-trifluoromethylbenzimidazole (11) (CB2: K(i) = 0.37 μM). Docking studies also performed on other benzimidazoles reported in the literature supported the structure–activity relationship observed in this series of compounds and allowed the key contacts involved in the agonist and/or inverse agonist behaviour displayed by these derivatives to be determined. The in silico evaluation of ADMET properties suggested a favorable pharmacokinetic and safety profile, promoting the drug-likeness of these compounds towards a further optimization process.

Targeting the fusion (F) protein has been recognized as a fruitful strategy for the development of anti-RSV agents. Despite the considerable efforts so far put into the development of RSV F protein inhibitors, the discovery of adequate therapeutics for the treatment of RSV infections is still awaiting a positive breakthrough. Several benzimidazole-containing derivatives have been discovered and evaluated in clinical trials, with only some of them being endowed with a promising pharmacokinetic profile. In this context, we applied a computational study based on a careful analysis of a number of X-ray crystallographic data of the RSV F protein, in the presence of different clinical candidates. A deepen comparison of the related electrostatic features and H-bonding motifs allowed us to pave the way for the following molecular dynamic simulation of JNJ-53718678 and then to perform docking studies of the in-house library of potent benzimidazole-containing anti-RSV agents. The results revealed not only the deep flexibility of the biological target but also the most relevant and recurring key contacts supporting the benzimidazole F protein inhibitor ability. Among them, several hydrophobic interactions and π-π stacking involving F140 and F488 proved to be mandatory, as well as H-bonding to D486. Specific requirements turning in RSV F protein binding ability were also explored thanks to structure-based pharmacophore analysis. Along with this, in silico prediction of absorption, distribution, metabolism, excretion (ADME) properties, and also of possible off-target events was performed. The results highlighted once more that the benzimidazole ring represents a privileged scaffold whose properties deserve to be further investigated for the rational design of novel and orally bioavailable anti-RSV agents.

A new series of spirocyclic σ receptor (σR) ligands were prepared and studied. Most were found to have a high affinity and selectivity for σ1 R; three compounds were shown to be σ1 R agonists, while another proved to be the only σ1 R antagonist. Only one of the σ1 R agonists (BS148) also exhibited σ2 R selectivity and was able to inhibit the growth of metastatic malignant melanoma cell lines without affecting normal human melanocytes. The antiproliferative activity of this compound suggested an σ2 R agonist profile. Further, preliminary investigations indicated that the mechanism of metastatic malignant melanoma cell death induced by BS148 is due, at least in part, to apoptosis.

G-protein-coupled receptors represent main targets of several clinically relevant drugs, playing nowadays a leading part for further drug discovery process. Trace amine-associated receptor's family (TAARs) assumed an intriguing role as druggable target in medicinal chemistry, being TAAR1 the most investigated. Indeed, related ligands proved to be intertwined in several circuits involved in pathological pathways or therapeutic routes. Herein, we highlight relevant efforts in the search of novel agonists, focusing on responsiveness featured by different chemotypes toward rodent and human TAAR1, in order to explore species-specificity preferences. We also discuss the main strategies guiding so far the design of new TAAR1 agonists, giving a perspective of the structure-based methodologies aimed at deriving new insights for more potent and selective derivatives.

The 5-hydroxytryptamine (5-HT1A) receptors represent an attractive target in drug discovery. In particular, 5-HT1A agonists and partial agonists are deeply investigated for their potential role in the treatment of anxiety, depression, ischaemic brain disorder and more recently, of pain. On the other hand, 5-HT1A antagonists have been revealed promising compounds in cognition disorders and, lately, in cancer. Thus, the discovery of 5HT1A ligands is nowadays an appealing research activity in medicinal chemistry. In this work, Comparative Molecular Fields Analysis (CoMFA) and Comparative Molecular Similarity Index Analysis (CoMSIA) were applied on an in-house library of 5-HT1A ligands bearing different chemical scaffolds in order to elucidate their affinity and selectivity for the target. Following this procedure, a number of structural modifications have been drawn for the development of much more effective 5-HT1AR ligands.

Positive inotropic agents are fundamental in the treatment of heart failure; however, their arrhythmogenic liability and the increased myocardial oxygen demand strongly limit their therapeutic utility. Pursuing our study on cardiovascular activities of lupin alkaloid derivatives, several 2-(4-substituted-phenyl)-2-dehydrosparteines and 2-(4-substituted-phenyl)sparteines were prepared and tested for inotropic and chronotropic activities on isolated guinea pig atria. Four compounds (6b, 6e, 7b, and 7f) exhibited significant inotropism that, at the higher concentrations, was followed by negative inotropism or toxicity. Compound 7e (2-(4-tolyl)sparteine) exhibited a steep dose-depending inotropic activity up to the highest concentration tested (300 µM) with an Emax of 116.5 ± 3.4% of basal force, proving less potent but much more active in comparison to the highest concentrations tested of digoxin and milrinone having Emax of 87.5 ± 3.1% and 52.2 ± 1.1%, respectively. Finally, docking studies suggested that the relevant sparteine derivatives could target the sigma-1 receptor, whose involvement in cardiac activity is well documented.

Cystic fibrosis (CF) is the autosomal recessive disorder most recurrent in Caucasian populations. To combat this disease, many life-prolonging therapies are required and deeply investigated, including the development of the so-called cystic fibrosis transmembrane conductance regulator (CFTR) modulators, such as correctors and potentiators. Combination therapy with the two series of drugs led to the approval of several multi-drug effective treatments, such as Orkambi, and to the recent promising evaluation of the triple-combination Elexacaftor-Tezacaftor-Ivacaftor. This scenario enlightened the effectiveness of the multi-drug approach to pave the way for the discovery of novel therapeutic agents to contrast CF. The recent X-crystallographic data about the human CFTR in complex with the well-known potentiator Ivacaftor (VX-770) opened the possibility to apply a computational study aimed to explore the key features involved in the potentiator binding. Herein, we discussed molecular docking studies performed onto the chemotypes so far discussed in the literature as CFTR potentiator, reporting the most relevant interactions responsible for their mechanism of action, involving Van der Waals interactions and &pi, &ndash, &pi, stacking with F236, Y304, F305 and F312, as well as H-bonding F931, Y304, S308 and R933. This kind of positioning will stabilize the effective potentiator at the CFTR channel. These data have been accompanied by pharmacophore analyses, which promoted the design of novel derivatives endowed with a main (hetero)aromatic core connected to proper substituents, featuring H-bonding moieties. A highly predictive quantitative-structure activity relationship (QSAR) model has been developed, giving a cross-validated r2 (r2cv) = 0.74, a non-cross validated r2 (r2ncv) = 0.90, root mean square error (RMSE) = 0.347, and a test set r2 (r2pred) = 0.86. On the whole, the results are expected to gain useful information to guide the further development and optimization of new CFTR potentiators.

Previous studies have investigated the relevance and structure-activity relationships (SARs) of pyrazole derivatives in relation with cannabinoid receptors, and the series of tricyclic 1,4-dihydroindeno[1,2-c]pyrazoles emerged as potent CB2 receptor ligands. In the present study, novel 1,4-dihydroindeno[1,2-c]pyrazole and 1H-benzo[g]indazole carboxamides containing a cyclopropyl or a cyclohexyl substituent were designed and synthesized to evaluate the influence of these structural modifications towards CB1 and CB2 receptor affinities. Among these derivatives, compound 15 (6-cyclopropyl-1-(2,4-dichlorophenyl)-N-(adamantan-1-yl)-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide) showed the highest CB2 receptor affinity (Ki = 4 nM) and remarkable selectivity (KiCB1/KiCB2 = 2232), whereas a similar affinity, within the nM range, was seen for the fenchyl derivative (compound 10: Ki = 6 nM), for the bornyl analogue (compound 14: Ki = 38 nM) and, to a lesser extent, for the aminopiperidine derivative (compound 6: Ki = 69 nM). Compounds 10 and 14 were also highly selective for the CB2 receptor (KiCB1/KiCB2 > 1000), whereas compound 6 was relatively selective (KiCB1/KiCB2 = 27). The four compounds were also subjected to GTPγS binding analysis showing antagonist/inverse agonist properties (IC50 for compound 14 = 27 nM, for 15 = 51 nM, for 10 = 80 nM and for 6 = 294 nM), and this activity was confirmed for the three more active compounds in a CB2 receptor-specific in vitro bioassay consisting in the quantification of prostaglandin E2 release by LPS-stimulated BV2 cells, in the presence and absence of WIN55,212-2 and/or the investigated compounds. Modelling studies were also conducted with the four compounds, which conformed with the structural requirements stated for the binding of antagonist compounds to the human CB2 receptor.

Several recent studies have focused on a detailed analysis of the trace amine-associated receptor type 5 (TAAR5) pharmacology, up to now revealing only a limited number of species-specific ligands, which are also active towards other TAAR receptors. In this context, we developed our work on TAAR5 applying a structure-based computational protocol, revolving around homology modeling and virtual screening calculations. In detail, mTAAR5 and hTAAR5 homology models were built, in order to explore any pattern of structural requirements which could be involved in species-specific differences. Successively, the mTAAR5 model was employed to perform a virtual screening of an in-house library of compounds, including different five-membered ring derivatives, linked to a phenyl ring through a flexible or a rigid basic moiety. The computational protocol applied allowed to select a number of chemical scaffolds that were tested in a biological assay leading to the discovery of the first two mTAAR5 antagonists.

A series of sulfenamide and sulfonamide derivatives was synthesized and evaluated for the affinity at CB1 and CB2 receptors. The N-bornyl-S-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide, compound 11, displayed good affinity and high selectivity for CB1 receptors (Ki values of 44.6 nM for CB1 receptors and >40 μM for CB2 receptors, respectively). The N-isopinocampheyl-sulfenamide 12 and its sulfonamide analogue 22 showed similar selectivity for CB1 receptors with Ki values of 75.5 and 73.2 nM, respectively. These novel compounds behave as antagonists/inverse agonists at CB1 receptor in the [35S]-GTPγS binding assays, and none showed adequate predictive blood–brain barrier permeation, exhibiting low estimated LD50. However, testing compound 12 in a supraspinal analgesic test (hot-plate) revealed that it was as effective as the classic CB1 receptor antagonist rimonabant, in reversing the analgesic effect of a cannabinoid agonist.

Cystic fibrosis (CF) is mainly caused by the deletion of Phe 508 (DeltaF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. New drugs able to rescue DeltaF508-CFTR trafficking are eagerly awaited. An integrated bioinformatics and surface plasmon resonance (SPR) approach was here applied to investigate the rescue mechanism(s) of a series of CFTR-ligands including VX809, VX770 and some aminoarylthiazole derivatives (AAT). Computational studies tentatively identified a large binding pocket in the DeltaF508-CFTR nucleotide binding domain-1 (NBD1) and predicted all the tested compounds to bind to three sub-regions of this main pocket. Noticeably, the known CFTR chaperone keratin-8 (K8) seems to interact with some residues located in one of these sub-pockets, potentially interfering with the binding of some ligands. SPR results corroborated all these computational findings. Moreover, for all the considered ligands, a statistically significant correlation was determined between their binding capability to DeltaF508-NBD1 measured by SPR and the pockets availability measured by computational studies. Taken together, these results demonstrate a strong agreement between the in silico prediction and the SPR-generated binding data, suggesting a path to speed up the identification of new drugs for the treatment of cystic fibrosis.

Phosphodiesterases (PDEs) regulate the intracellular levels of cAMP and cGMP. The great clinical success of the PDE5 inhibitors, Sildenafil (Viagra), Vardenafil (Levitra) and Tadalafil (Cialis) has led to an increasing interest for this class of enzymes. Recent studies have shown a correlation between tumor growth and PDE5 overexpression, making PDE5-selective inhibitors promising candidates for cancer treatment. The search for such inhibitors rests today on radioactive assays. In this work, we exploit the conserved catalytic domain of the enzyme and propose a faster and safer method for detecting the binding of ligands and evaluate their affinities. The new approach takes advantage of Forster Resonance Energy Transfer (FRET) between, as the donor, a fluorescein-like diarsenical probe able to covalently bind a tetracysteine motif fused to the recombinant PDE5 catalytic domain and, as the acceptor, a rhodamine probe covalently bound to the pseudosubstrate cGMPS. The FRET efficiency decreases when a competitive ligand binds the PDE5 catalytic site and displaces the cGMPS-rhodamine conjugate. We have structurally investigated the PDE5/cGMPS-rhodamine complex by molecular modelling and have used the FRET signal to quantitatively characterize its binding equilibrium. Competitive displacement experiments were carried out with tadalafil and cGMPS. An adaptation of the competitive-displacement equilibrium model yielded the affinities for PDE5 of the incoming ligands, nano- and micromolar, respectively.

The design of novel chemical classes acting towards several G-protein-coupled receptors (GPCRs) represents a leading strategy in drug discovery, aimed at deriving effective and safe candidates for further assessment. During the last years, TAAR1 arose as a promising druggable target in medicinal chemistry, being of interest in the treatment of several pathologies, such as neuropsychiatric disorders, type 2 diabetes and obesity. Nevertheless, the limited number of known potent and selective ligands and the species-specificity responsiveness exhibited by those derivatives nowadays available make the discovery of novel compounds a challenging task. Herein, we discuss the development of two quantitative-structure activity relationship (QSAR) models around the agonism ability experienced by different chemo-types toward murine and human TAAR1 (m/hTAAR1) with the aim at deciphering some clues involved in their species-specificity responsiveness. Qualitatively, these information were evaluated guiding for the synthesis of novel ligands, which proved to feature selective agonism ability with respect to the mTAAR1 and hTAAR1 orthologues.

The most common CF mutation, F508del, impairs the processing and gating of CFTR protein. This deletion results in the improper folding of the protein and its degradation before it reaches the plasma membrane of epithelial cells. Present correctors, like VX809 only induce a partial rescue of the mutant protein. Our previous studies reported a class of compounds, called aminoarylthiazoles (AATs), featuring an interesting activity as correctors. Some of them show additive effect with VX809 indicating a different mechanism of action. In an attempt to construct more interesting molecules, it was thought to generate chemically hybrid compounds, blending a portion of VX809 merged to the thiazole scaffold. This approach was guided by the development of QSAR analyses, which were performed based on the F508del correctors so far disclosed in the literature. This strategy was aimed at exploring the key requirements turning in the corrector ability of the collected derivatives and allowed us to derive a predictive model guiding for the synthesis of novel hybrids as promising correctors. The new molecules were tested in functional and biochemical assays on bronchial CFBE41o-cells expressing F508del-CFTR showing a promising corrector activity.

Novel 1,4-dihydropyrazolo[3,4-a]pyrrolizine-, 4,5-dihydro-1H-pyrazolo[4,3-g]indolizine- and 1,4,5,6-tetrahydropyrazolo[3,4-c]pyrrolo[1,2-a]azepine-3-carboxamide based compounds were designed and synthesized for cannabinoid CB1 and CB2 receptor interaction. Any of the new synthesized compounds showed high affinity for CB2 receptor with Ki values superior to 314 nM, whereas some of them showed moderate affinity for CB1 receptor with Ki values inferior to 400 nM. 7-Chloro-1-(2,4-dichlorophenyl)-N-(homopiperidin-1-yl)-4,5-dihydro-1H-pyrazolo[4,3-g]indolizine-3-carboxamide (2j) exhibited good affinity for CB1 receptor (KiCB1 = 81 nM) and the highest CB2/CB1 selectively ratio (>12). Docking studies carried out on such compounds were performed by using the hCB1 X-ray in complex with the close pyrazole analogue AM6538, and disclosed specific pattern of interactions related to the tricyclic pyrrolopyrazole scaffolds as CB1 ligands. This article is protected by copyright. All rights reserved.

PDE4 inhibitors have been largely studied because of their promising therapeutic effects concerning inflammation and neurodegenerative dysfunctions, such as depression, schizophrenia and Alzheimer's diseases. In this context, the PDE4B isoform proved to be particularly involved in the activation of inflammatory responses, while the PDE4D subfamily is more associated with neuropathologies. The clinical use of PDE4 inhibitors was restricted by the presence of prominent side effects probably due to their non-specific action across the different isoforms. Therefore, this work deals with the development of 3D-QSAR models, supported by molecular docking studies, to identify the key requirements underlying selective PDE4B or PDE4D inhibition. The results highlighted the ligand-based approach as a promising tool to guide the rational design of novel PDE4 inhibitors endowed with high affinity and selectivity profiles. The alignment of compound 1-85 and the model A statistical results are depicted.

CB1 and CB2 receptors belong to the large family of G-protein coupled receptors (GPCRs), being involved in a wide variety of signal transduction processes. In this context, CB2 selective compounds were described in the literature to be active in different neuropathic and inflammatory pain models, also showing beneficial effects as neuroprotective agents. Indeed, CB2 proved to be up-regulated in reactive microglial cells in Alzheimer's disease (AD) and Huntington's, suggesting a promising therapeutic panorama for CB2 inverse agonists/antagonists. At present, the development of new selective CB2 antagonists is prevented by an unclear depiction of the reference antagonist SR144528 binding mode. Indeed, a few number of models concerning the CB2 receptor antagonist binding site have been proposed, leading sometimes to contradictory results. In this context, a specific hCB2 ligand-based homology model in the presence of the antagonist SR144528 was built. Notably, the refined model also allowed us to explore the pyrazole scaffold as prototype for CB2 ligand recognition and also to elucidate the CB1/CB2 structure–activity relationship of an in-house series of analogues we previously published. The derived information is expected to be a useful tool for guiding a much more focused and reliable CB2 antagonist design.

Recently, 1-(1,4-dioxaspiro[4,5]dec-2-ylmethyl)-4-(2-methoxyphenyl)piperazine (1) was reported as a potent 5-HT1AR agonist with a moderate 5-HT1AR selectivity. In an extension of this work a series of derivatives of 1, obtained by combining different heterocyclic rings with a more flexible amine chain, was synthesized and tested for binding affinity and activity at 5-HT1AR and α1 adrenoceptors. The results led to the identification of 14 and 15 as novel 5-HT1AR partial agonists, the first being outstanding for selectivity (5-HT1A/α1d = 80), the latter for potency (pD2 = 9.58) and efficacy (Emax = 74%). Theoretical studies of ADME properties shows a good profile for the entire series and MDCKII-MDR1 cells permeability data predict a good BBB permeability of compound 15, which possess a promising neuroprotective activity. Furthermore, in mouse formalin test, compound 15 shows a potent antinociceptive activity suggesting a new strategy for pain control.

We have identified a series of 1-aryl-4,6-diamino-1,2-dihydrotriazines, structurally related to the antimalarial drug cycloguanil, as new inhibitors of influenza A and B virus and respiratory syncytial virus (RSV) via targeting of the host dihydrofolate reductase (DHFR) enzyme. Most analogues proved active against influenza B virus in the low micromolar range, and the best compounds (11, 13, 14 and 16) even reached the sub-micromolar potency of zanamivir (EC50 = 0.060 μM), and markedly exceeded (up to 327 times) the antiviral efficacy of ribavirin. Activity was also observed for two influenza A strains, including a virus with the S31N mutant form of M2 proton channel, which is the most prevalent resistance mutation for amantadine. Importantly, the compounds displayed nanomolar activity against RSV and a superior selectivity index, since the ratio of cytotoxic to antiviral concentration was >10,000 for the three most active compounds 11, 14 and 16 (EC50 ∼0.008 μM), far surpassing the potency and safety profile of the licensed drug ribavirin (EC50 = 5.8 μM, SI > 43)., Graphical abstract Image 1, Highlights • 4,6-Diamino-1,2-dihydrotriazines were found active against influenza A and B viruses. • 4,6-Diamino-1,2-dihydrotriazines displayed high antiviral activity against RSV virus. • Their mechanism of action was related to the inhibition of the host (human) DHFR.

Respiratory syncytial virus (RSV) has been identified as a main cause of hospitalisation in infants and children. To date, the current therapeutic arsenal is limited to ribavirin and palivizumab with variable efficacy. In this work, starting from a number of in-house series of previously described anti-RSV agents based on the benzimidazole scaffold, with the aim at gaining a better understanding of the related chemical features involved in potency and safety profiles, we applied a computational study including two focussed comparative molecular fields analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The results allowed us to derive useful suggestions for the design of derivatives and also to set up statistical models predicting the potency and selectivity index (SI = CC50/EC50) of any new analogue prior to synthesis. Accordingly, here, we discuss preliminary results obtained through the applied exhaustive QSAR analyses, leading to design and synthesise more effective anti-RSV agents.

In the last few years, cannabinoid type-2 receptor (CBR) selective ligands have shown a great potential as novel therapeutic drugs in several diseases. With the aim of discovering new selective cannabinoid ligands, a series of pyridazinone-4-carboxamides was designed and synthesized, and the new derivatives tested for their affinity toward the hCBR and hCBR. The 6-(4-chloro-3-methylphenyl)-2-(4-fluorobenzyl)-N-(cis-4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide (9) displayed high CB-affinity (KCB = 2.0 ± 0.81 nM) and a notable selectivity (KCB/KCB > 2000). In addition, 9 and other active new synthesized entities have demonstrated to behave as CBR inverse agonists in [S]-GTPγS binding assay. ADME predictions of the newly synthesized CBR ligands suggest a favourable pharmacokinetic profile. Docking studies disclosed the specific pattern of interactions of these derivatives. Our results support that pyridazinone-4-carboxamides represent a new promising scaffold for the development of potent and selective CBR ligands., JFR and NJ thanks the MINECO (Plan Nacional de Biomedicina) for the joint grant SAF2015-68580-C2 (1-R to JFR and 2-R to NJ) and the “Programa de Biomedicina, Comunidad de Madrid” for the grant S2011/BMD-2308

Phosphodiesterase 11 (PDE11) is the latest isoform of the PDEs family to be identified, acting on both cyclic adenosine monophosphate and cyclic guanosine monophosphate. The initial reports of PDE11 found evidence for PDE11 expression in skeletal muscle, prostate, testis, and salivary glands; however, the tissue distribution of PDE11 still remains a topic of active study and some controversy. Given the sequence similarity between PDE11 and PDE5, several PDE5 inhibitors have been shown to cross-react with PDE11. Accordingly, many non-selective inhibitors, such as IBMX, zaprinast, sildenafil, and dipyridamole, have been documented to inhibit PDE11. Only recently, a series of dihydrothieno[3,2-d]pyrimidin-4(3H)-one derivatives proved to be selective toward the PDE11 isoform. In the absence of experimental data about PDE11 X-ray structures, we found interesting to gain a better understanding of the enzyme-inhibitor interactions using in silico simulations. In this work, we describe a computational approach based on homology modeling, docking, and molecular dynamics simulation to derive a predictive 3D model of PDE11. Using a Graphical Processing Unit architecture, it is possible to perform long simulations, find stable interactions involved in the complex, and finally to suggest guideline for the identification and synthesis of potent and selective inhibitors.

The multistep preparation of the new 10-substituted 2-(1-piperazinyl)pyrimido[1,2- a ]benzimidazol-4(10 H )-ones 6a – o , and of the two isomers 10-ethyl-2-(diethylamino)pyrimido[1,2- a ]benzimidazol-4(10 H )-one 6p and 10-ethyl-4-(diethylamino)pyrimido[1,2- a ]benzimidazol-2(10 H )-one 13 , as well as the in vitro evaluation of their inhibitory activity on human platelet aggregation induced in platelet-rich plasma by ADP, collagen or the Ca 2+ ionophore A23187 were here described. Nine out of fifteen 2-(1-piperazinyl)derivatives ( 6g – o ) showed good inhibitory properties towards all the platelet aggregation agonists used. Moreover, a molecular modelling study has been performed on two of the best compounds of this series ( 6i and 6o ) to confirm in silico their interactions with the catalytic site of human platelet PDE3, using the X-ray data of the PDE3B isoform in complex with an inhibitor.

Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor that belongs to the family of TAAR receptors and responds to a class of compounds called trace amines, such as β-phenylethylamine (β-PEA) and 3-iodothyronamine (T(1)AM). The receptor is known to have a very rich pharmacology and could be also activated by other classes of compounds, including adrenergic and serotonergic ligands. It is expected that targeting TAAR1 could provide a novel pharmacological approach to correct monoaminergic dysfunctions found in several brain disorders, such as schizophrenia, depression, attention deficit hyperactivity disorder and Parkinson's disease. Only recently, the first selective TAAR1 agonist RO5166017 has been identified. To explore the molecular mechanisms of protein-agonist interaction and speed up the identification of new chemical entities acting on this biomolecular target, we derived a homology model for the hTAAR1. The putative protein-binding site has been explored by comparing the hTAAR1 model with the β(2)-adrenoreceptor binding site, available by X-ray crystallization studies, and with the homology modelled 5HT(1A) receptor. The obtained results, in tandem with docking studies performed with RO5166017, β-PEA and T(1)AM, provided an opportunity to reasonably identify the hTAAR1 key residues involved in ligand recognition and thus define important starting points to design new agonists.

Trace amines (TAs) are endogenous neuromodulators that play a functional role in the synaptic transmission within central nervous system (CNS), targeting trace amine-associated receptors (TAARs). Starting from our previous computational studies on TAAR1 and TAAR5 interactions with the unselective ligand 3-iodothyronamine (T1AM), we investigated the functional activity at murine and human TAAR1 and murine TAAR5 receptors of twenty-seven biguanide-based derivatives, including six newly synthesized compounds. Phenyl (BIG2, BIG4, BIG8 and BIG22) or benzyl (BIG10-BIG16) biguanides were found to be selective murine and human TAAR1 agonists with potencies in nanomolar or low micromolar range, respectively. In particular, compounds BIG2 and BIG12-BIG14 were the most promising and they could be considered valuable lead compounds worthy of further investigations. In addition to the interest for developing more effective human TAAR1 ligands, the disclosed here potent murine TAAR1 agonists could offer suitable tools for studying the pharmacology of TAAR1 receptor.

Recent studies have further investigated the trace amine-associated receptor type 2 (TAAR2) pharmacology, revealing its role not only at the olfactory sensory neurons but also at the immune system, being expressed in human leucocytes. In particular, the ability of this receptor to bind the unselective TAAR ligand 3-iodo-thyronamine (T1 AM) was elucidated, making in the meanwhile the discovery of selective compounds a urgent need to derive much more suitable tools for studying TAARs. In this context, we developed our work on TAAR2 applying a structure-based computational protocol, including TAAR2 homology modelling and T1 AM docking studies. The results were compared with those we previously obtained about TAAR1, in order to point out new insights guiding for selectivity between TAAR1 and TAAR2. The in silico strategy applied allowed us to provide for the first time thorough TAAR2 homology models, which are expected to be useful tools for a further design process of more selective TAAR ligands.

Alzheimer′s disease has recently emerged as a possible field of application for PDE4D inhibitors (PDE4DIs). The great structure similarity among the various PDE4 isoforms and, furthermore, the lack of the full length crystal structure of the enzyme, impaired the rational design of new selective PDE4DIs. In this paper, with the aim of exploring new insights into the PDE4D binding, we tackled the problem by performing a computational study based on docking simulations combined with molecular dynamics (D‐MD). Our work uniquely identified the binding mode and the key residues involved in the interaction with a number of in‐house catechol iminoether derivatives, acting as PDE4DIs. Moreover, the new binding mode was tested using a series of analogues previously reported by us and it was used to confirm their key structural features to allow PDE4D inhibition. The binding model disclosed within the current computational study may prove to be useful to further advance the design and synthesis of novel, more potent and selective, PDE4D inhibitors.

The trace amine-associated receptor 1 (TAAR1) is a G-protein-coupled receptors (GPCR) potently activated by a variety of molecules besides trace amines (TAs), including thyroid hormone-derivatives like 3-iodothyronamine (T1AM), catechol-O-methyltransferase products like 3-methoxytyramine, and amphetamine-related compounds. Accordingly, TAAR1 is considered a promising target for medicinal development. To gain more insights into TAAR1 physiological functions and validation of its therapeutic potential, we recently developed a new class of thyronamine-like derivatives. Among them compound SG2 showed high affinity and potent agonist activity at mouse TAAR1. In the present work, we describe design, synthesis, and SAR study of a new series of compounds (1-16) obtained by introducing specific structural changes at key points of our lead compound SG2 skeleton. Five of the newly synthesized compounds displayed mTAAR1 agonist activity higher than both SG2 and T1AM. Selected diphenylmethane analogues, namely 1 and 2, showed potent functional activity in in vitro and in vivo models.

Herein we report the synthesis and biological activity of new sigma receptor (σR) ligands obtained by combining different substituted five-membered heterocyclic rings with appropriate σR pharmacophoric amines. Radioligand binding assay, performed on guinea pig brain membranes, identified 25b (1-(1,4-dioxaspiro[4.5]decan-2-ylmethyl)-4-benzylpiperazine) as the most interesting compound of the series, displaying high affinity and selectivity for σ1R (pKiσ1 = 9.13; σ1/σ2 = 47). The ability of 25b to modulate the analgesic effect of the κ agonist (-)-U-50,488H and μ agonist morphine was evaluated in vivo by radiant heat tail-flick test. It exhibited anti-opioid effects on both κ and μ receptor-mediated analgesia, suggesting an agonistic behavior at σ1R. Docking studies were performed on the theoretical σ1R homology model. The present work represents a new starting point for the design of more potent and selective σ1R ligands.

Pyrazolo[5,1-f][1,6]naphthyridine-carboxamide derivatives were synthesized and evaluated for the affinity at CB1 and CB2 receptors. Based on the AgOTf and proline-cocatalyzed multicomponent methodology, the ethyl 5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (12) and ethyl 5-(2,4-dichlorophenyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (13) intermediates were synthesized from the appropriate o-alkynylaldehydes, p-toluenesulfonyl hydrazide and ethyl pyruvate. Most of the novel compounds feature a p-tolyl (8a–i) or a 2,4-dichlorophenyl (8j) motif at the C5-position of the tricyclic pyrazolo[5,1-f][1,6]naphthyridine scaffold. Structural variation on the carboxamide moiety at the C2-position includes basic monocyclic, terpenoid and adamantine-based amines. Among these derivatives, compound 8h (N-adamant-1-yl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxamide) exhibited the highest CB2 receptor affinity (Ki = 33 nM) and a high degree of selectivity (KiCB1/KiCB2 = 173:1), whereas a similar trend in the near nM range was seen for the bornyl analogue (compound 8f, Ki = 53 nM) and the myrtanyl derivative 8j (Ki = 67 nM). Effects of 8h, 8f and 8j on forskolin-stimulated cAMP levels were determined, showing antagonist/inverse agonist properties for such compounds. Docking studies conducted for these derivatives and the reference antagonist/inverse agonist compound 4 (SR144528) disclosed the specific pattern of interactions probably related to the pyrazolo[5,1-f][1,6]naphthyridine scaffold as CB2 inverse agonists.

Trace amine associated receptor 1 (TAAR1) is a G protein coupled receptor (GPCR) expressed in brain and periphery activated by a wide spectrum of agonists that include, but are not limited to, trace amines (TAs), amphetamine-like psychostimulants, and endogenous thyronamines such as thyronamine (T0AM) and 3-iodothyronamine (T1AM). Such polypharmacology has made it challenging to understand the role and the biology of TAAR1. In an effort to understand the molecular basis of TAAR1 activation, we rationally designed and synthesized a small family of thyronamine derivatives. Among them, compounds 2 and 3 appeared to be a good mimic of the parent endogenous thyronamine, T0AM and T1AM, respectively, both in vitro and in vivo. Thus, these compounds offer suitable tools for studying the physiological roles of mouse TAAR1 and could represent the starting point for the development of more potent and selective TAAR1 ligands.