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Compounds combining dual inhibitory action against FAAH and cyclooxygenase (COX) may be potentially useful analgesics. Here, we describe a novel flurbiprofen analogue, N-(3-bromopyridin-2-yl)-2-(2-fluoro-(1,1'-biphenyl)-4-yl)propanamide (Flu-AM4). The compound is a competitive, reversible inhibitor of FAAH with a Ki value of 13 nM and which inhibits COX activity in a substrate-selective manner. Molecular modelling suggested that Flu-AM4 optimally fits a hydrophobic pocket in the ACB region of FAAH, and binds to COX-2 similarly to flurbiprofen. In vivo studies indicated that at a dose of 10 mg/kg, Flu-AM4 was active in models of prolonged (formalin) and neuropathic (chronic constriction injury) pain and reduced the spinal expression of iNOS, COX-2, and NFκB in the neuropathic model. Thus, the present study identifies Flu-AM4 as a dual-action FAAH/substrate-selective COX inhibitor with anti-inflammatory and analgesic activity in animal pain models. These findings underscore the potential usefulness of such dual-action compounds.

G-protein-coupled receptors (GPCRs) are the molecular target of 40% of marketed drugs and the most investigated structures to develop novel therapeutics. Different members of the GPCRs superfamily can modulate the same cellular process acting on diverse pathways, thus representing an attractive opportunity to achieve multitarget drugs with synergic pharmacological effects. Here, we present a series of compounds with dual activity toward cysteinyl leukotriene receptor 1 (CysLT1R) and G-protein-coupled bile acid receptor 1 (GPBAR1). They are derivatives of REV5901─the first reported dual compound─with therapeutic potential in the treatment of colitis and other inflammatory processes. We report the binding mode of the most active compounds in the two GPCRs, revealing unprecedented structural basis for future drug design studies, including the presence of a polar group opportunely spaced from an aromatic ring in the ligand to interact with Arg792.60 of CysLT1R and achieve dual activity.

Inhibition of fatty acid amide hydrolase (FAAH) reduces the gastrointestinal damage produced by non-steroidal anti-inflammatory agents such as sulindac and indomethacin in experimental animals, suggesting that a dual-action FAAH-cyclooxygenase (COX) inhibitor could have useful therapeutic properties. Here, we have investigated 12 novel amide analogues of ibuprofen as potential dual-action FAAH/COX inhibitors. N-(3-Bromopyridin-2-yl)−2-(4-isobutylphenyl)propanamide (Ibu-AM68) was found to inhibit the hydrolysis of [3H]anandamide by rat brain homogenates by a reversible, mixed-type mechanism of inhibition with a Ki value of 0.26 µM and an α value of 4.9. At a concentration of 10 µM, the compound did not inhibit the cyclooxygenation of arachidonic acid by either ovine COX-1 or human recombinant COX-2. However, this concentration of Ibu-AM68 greatly reduced the ability of the COX-2 to catalyse the cyclooxygenation of the endocannabinoid 2-arachidonoylglycerol. It is concluded that Ibu-AM68 is a dual-acting FAAH/substrate-selective COX inhibitor.

Graphical abstract Pelargonidin down-regulates Ace2 expression and inhibits binding of the SARS-Cov-2 virus on the host cell ACE2 receptor. Inflammatory stimuli lead to the release of TNF-α which binds to its receptor present on epithelial cells of the colon. The activation of TNF-α receptor induces an activation of NF-kB that migrates to the nucleus where activates the transcription of several genes including Il-6 and Ace2. Pelargonidin exerts a protective effect through AHR which blocks NF-kB translocation into the nucleus (indicated with a red line because this mechanism has not been demonstrated in this study but in previous studies [57,58]) and thereby inhibiting the expression of Ace2. Pelargonidin also directly inhibits the binding of the SARS-Cov-2 virus to the ACE2 receptor., The severe acute respiratory syndrome (SARS)-CoV-2 is the pathogenetic agent of Corona Virus Induced Disease (COVID)19. The virus enters the human cells after binding to the angiotensin converting enzyme (ACE)2 receptor in target tissues. ACE2 expression is induced in response to inflammation. The colon expression of ACE2 is upregulated in patients with inflammatory bowel disease (IBD), highlighting a potential risk of intestinal inflammation in promoting viral entry in the human body. Because mechanisms that regulate ACE2 expression in the intestine are poorly understood and there is a need of anti-SARS-CoV-2 therapies, we have settled to investigate whether natural flavonoids might regulate the expression of Ace2 in intestinal models of inflammation. The results of these studies demonstrated that pelargonidin activates the Aryl hydrocarbon Receptor (AHR) in vitro and reverses intestinal inflammation caused by chronic exposure to high fat diet or to the intestinal braking-barrier agent TNBS in a AhR-dependent manner. In these two models, development of colon inflammation associated with upregulation of Ace2 mRNA expression. Colon levels of Ace2 mRNA were directly correlated with Tnf-α mRNA levels. Molecular docking studies suggested that pelargonidin binds a fatty acid binding pocket on the receptor binding domain of SARS-CoV-2 Spike protein. In vitro studies demonstrated that pelargonidin significantly reduces the binding of SARS-CoV-2 Spike protein to ACE2 and reduces the SARS-CoV-2 replication in a concentration-dependent manner. In summary, we have provided evidence that a natural flavonoid might hold potential in reducing intestinal inflammation and ACE2 induction in the inflamed colon in a AhR-dependent manner.

Fatty acid amide hydrolase (FAAH) has a key role in the control of the cannabinoid signaling, through the hydrolysis of the endocannabinoids anandamide and in some tissues 2-arachidonoylglycerol. FAAH inhibition represents a promising strategy to activate the cannabinoid system, since it does not result in the psychotropic and peripheral side effects characterizing the agonists of the cannabinoid receptors. Here we present the discovery of a novel class of profen derivatives, the N-(heteroaryl)-2-(4-((2-(trifluoromethyl)pyridin-4-yl)amino)phenyl)propanamides, as FAAH inhibitors. Enzymatic assays showed potencies toward FAAH ranging from nanomolar to micromolar range, and the most compounds lack activity toward the two isoforms of cyclooxygenase. Extensive structure-activity studies and the definition of the binding mode for the lead compound of the series are also presented. Kinetic assays in rat and mouse FAAH on selected compounds of the series demonstrated that slight modifications of the chemical structure could influence the binding mode and give rise to competitive (TPA1) or non-competitive (TPA14) inhibition modes.

It is well recognized that the non-canonical DNA structures known as G-quadruplexes (G4s) have a potential anticancer significance and several compounds have been discovered and evaluated as promising G4 binders with anticancer activity. Here, starting from a promising hit with an indolo-naphthyridine scaffold, a small series of five indolo-naphthyridine based derivatives have been designed and evaluated as G4-targeting compounds. FRET biophysical studies were performed on multiple DNA G4 structures, leading to the identification of a multi-target G4 stabilizer with a slight preference for the c-KIT1 and a good G4 over duplex selectivity. The good affinity of this compound against c-KIT1 G4 was also confirmed by SPR and MST experiments, while biological assays revealed its cytotoxic activity on tumour cells. Finally, Molecular Dynamics simulations helped to elucidate the stabilization effect of the selected compound against the c-KIT1 G4.

Herein, we reported on the synthesis of cpIPP, which is a new structurally-reduced analogue of cyclic ADP-ribose (cADPR), a potent Ca2+-releasing secondary messenger that was firstly isolated from sea urchin eggs extracts. To obtain cpIPP the “northern” ribose of cADPR was replaced by a pentyl chain and the pyrophosphate moiety by a phophono-phosphate anhydride. The effect of the presence of the new phosphono-phosphate bridge on the intracellular Ca2+ release induced by cpIPP was assessed in PC12 neuronal cells in comparison with the effect of the pyrophosphate bridge of the structurally related cyclic N1-butylinosine diphosphate analogue (cbIDP), which was previously synthesized in our laboratories, and with that of the linear precursor of cpIPP, which, unexpectedly, revealed to be the only one provided with Ca2+ release properties.

Cystic Fibrosis (CF) is one of the most common life shortening conditions in Caucasians. CF is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene which result in reduced or altered CFTR functionality. Several microRNAs (miRNAs) downregulate the expression of CFTR, thus causing or exacerbating the symptoms of CF. In this context, the design of anti-miRNA agents represents a valid functional tool, but its translation to the clinic might lead to unpredictable side effects because of the interference with the expression of other genes regulated by the same miRNAs. Herein, for the first time, is proposed the use of peptide nucleic acids (PNAs) to protect specific sequences in the 3’UTR (untranslated region) of the CFTR messenger RNA (mRNA) by action of miRNAs. Two PNAs (7 and 13 bases long) carrying the tetrapeptide Gly-SerP-SerP-Gly at their C-end, fully complementary to the 3’UTR sequence recognized by miR-509-3p, have been synthesized and the structural features of target PNA/RNA heteroduplexes have been investigated by spectroscopic and molecular dynamics studies. The co-transfection of the pLuc-CFTR-3´UTR vector with different combinations of PNAs, miR-509-3p, and controls in A549 cells demonstrated the ability of the longer PNA to rescue the luciferase activity by up to 70% of the control, thus supporting the use of suitable PNAs to counteract the reduction in the CFTR expression.

Identification of new molecular scaffolds structurally unrelated to known antimalarials may represent a valid strategy to overcome resistance of P. falciparum (Pf) to currently available drugs. We describe herein the investigation of a new polycyclic pharmacophore, related to clotrimazole, to develop innovative antimalarial agents. This study allowed us to discover compounds characterized by a high in vitro potency, particularly against Pf CQ-resistant strains selectively targeting free heme, which are easy to synthesize by low-cost synthetic strategies.

Pyrrolobenzoxazepinones (PBOs) represent a new class of human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 5. Molecular modeling studies based on the X-ray structures of HIV-1 RT prompted the synthesis of novel analogues which were tested as anti-HIV agents. The PBO derivatives specifically designed to target the highly conserved amino acid residues within the beta12-beta13 hairpin, namely primer grip, proved to be very potent against the most common mutant enzymes, including the highly resistant K103N mutant strain. Structure-activity relationships (SARs) are discussed in terms of a possible interaction with the RT binding site, depending on the nature of the substituents at C-6. Among the pyrrolobenzoxazepines investigated, 15c appeared to be the most promising NNRTI of the series characterized by potent antiviral activity, broad spectrum, and low cytotoxicity. 15c showed synergistic antiviral activity with AZT.

We have recently developed five novel pyrrolo-1,5-benzoxazepines as proapoptotic agents. Their JNK-dependent induction of apoptosis in tumor cells suggested their potential as novel anticancer agents. The core structure of the apoptotic agent 6 was investigated, and the SARs were expanded with the design and synthesis of several analogues. To define the apoptotic mechanism of the new compounds and the localization of their drug target, two analogues of 6 were designed and synthesized to delineate events leading to JNK activation. The cell-penetrating compound 16 induced apoptosis in tumor cells, while its nonpenetrating analogue, 17, was incapable of inducing apoptosis or activating JNK. Plasma membrane permeabilization of tumor cells resulted in 17-induced JNK activation, suggesting that the pyrrolo-1,5-benzoxazepine molecular target is intracellular. Interestingly, compound 6 displayed cytotoxic activity against a panel of human tumor cell lines but demonstrated negligible toxicity in vivo with no effect on the animals' hematology parameters.

2'-Deoxy-8-(propyn-1-yl)adenosine has been incorporated in synthetic oligodeoxyribonucleotides and its influence on thermal stability of duplex and quadruplex structures investigated by UV, CD and 1H NMR. The obtained results seem to indicate that the presence of the modified base negatively affects the stability of double stranded DNA whereas remarkably increases the stability of parallel quadruplex structures. (C) 2000 Elsevier Science Ltd.

Protein conformational fluctuations are critical for biological functions, although the relationship between protein motion and function has yet to be fully explored. By a thorough bioinformatics analysis of cholinesterases (ChEs), we identified specific hot spots, responsible for protein fluctuations and functions, and those active-site residues that play a role in modulating the cooperative network among the key substructures. This drew the optimization of our design strategy to discover potent and reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase (hAChE and hBuChE) that selectively interact with specific protein substructures. Accordingly, two tricyclic moieties differently spaced by functionalized linkers were investigated as molecular yardsticks to probe the finest interactions with specific hot spots in the hChE gorge. A number of SAR trends were identified, and the multisite inhibitors 3a and 3d were found to be the most potent inhibitors of hBuChE and hAChE known to date.

The design, synthesis, and antiplasmodial activity of antimalarial heterodimers based on the 1,4-bis(3-aminopropyl)piperazine linker is reported. In this series key structural elements derived from quinoline antimalarials were coupled to fragments capable of coordinating metal ions. Biological evaluation included determination of activity against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains. Some of the novel compounds presented high activity in vitro against chloroquine-resistant strains, more potent than chloroquine and clotrimazole. Computational studies revealed that the activity is likely due to the ability of the compounds to assume a multisite iron coordinating geometry.

We describe herein the development of novel huperzine A−tacrine hybrids characterized by 3-methylbicyclo[3.3.1]non-3-ene scaffolds. These compounds were specifically designed to establish tight interactions, through different binding modes, with the midgorge recognition sites of human acetylcholinesterase (hAChE: Y72, D74) and human butyrylcholinesterase (hBuChE: N68, D70) and their catalytic or peripheral sites. Compounds 5a−c show a markedly improved biological profile relative to tacrine and huperzine A.

Plakortin (1) is a remarkably simple 1,2-dioxane derivative, extracted from the marine sponge Plakortis simplex, showing a submicromolar activity against chloroquine-resistant strains of Plasmodium falciparum. Using plakortin as a novel antimalarial hit, we have prepared a series of semisynthetic derivatives in order to gain insights into the structural requirements of simple 1,2-dioxanes for exhibiting antimalarial activity. Their synthesis, spectroscopic and computational analysis, and in vitro antimalarial activity are herein reported. Results obtained, besides confirming the crucial role of the cycloperoxide functionality, revealed other structural features critical for antimalarial activity, namely the “Western” alkyl side chain, the dioxane ring conformation, and the absolute configuration of the stereogenic carbons on the 1,2-dioxane ring, when affecting the bioactive ring conformation.

Using rational drug design to develop atypical antipsychotic drug candidates, we generated novel and metabolically stable pyrrolobenzazepines with an optimized pK(i) 5-HT(2A)/D(2) ratio. 5a, obtained by a new palladium-catalyzed three-step synthesis, was selected for further pharmacological and biochemical investigations and showed atypical antipsychotic properties in vivo. 5a was active on conditioned avoidance response at 0.56 mg/kg, it had low cataleptic potential and proved to be better than ST1899, clozapine, and olanzapine, representing a new clinical candidate.

Recently we reported the pharmacological characterization of the 9,10-dihydropyrrolo[1,3]benzothiazepine derivative (S)-(+)-8 as a novel atypical antipsychotic agent. This compound had an optimum pK(i) 5-HT(2A)/D(2) ratio of 1.21 (pK(i) 5-HT(2A) = 8.83; pK(i) D(2) = 7.79). The lower D(2) receptor affinity of (S)-(+)-8 compared to its enantiomer was explained by the difficulty in reaching the conformation required to optimally fulfill the D(2) pharmacophore. With the aim of finding novel atypical antipsychotics we further investigated the core structure of (S)-(+)-8, synthesizing analogues with specific substituents; the structure-activity relationship (SAR) study was also expanded with the design and synthesis of other analogues characterized by a pyrrolo[2,1-b][1,3]benzothiazepine skeleton, substituted on the benzo-fused ring or on the pyrrole system. On the 9,10-dihydro analogues the substituents introduced on the pyrrole ring were detrimental to affinity for dopamine and for 5-HT(2A) receptors, but the introduction of a double bond at C-9/10 on the structure of (S)-(+)-8 led to a potent D(2)/5-HT(2A) receptor ligand with a typical binding profile (9f, pK(i) 5-HT(2A)/D(2) ratio of 1.01, log Y = 8.43). Then, to reduce D(2) receptor affinity and restore atypicality on unsaturated analogues, we exploited the effect of specific substitutions on the tricyclic system of 9f. Through a molecular modeling approach we generated a novel series of potential atypical antipsychotic agents, with optimized 5HT(2A)/D(2) receptor affinity ratios and that were easier to synthesize and purify than the reference compound (S)-(+)-8. A number of SAR trends were identified, and among the analogues synthesized and tested in binding assays, 9d and 9m were identified as the most interesting, giving atypical log Y scores respectively 4.98 and 3.18 (pK(i) 5-HT(2A)/D(2) ratios of 1.20 and 1.30, respectively). They had a multireceptor affinity profile and could be promising atypical agents. Compound 9d, whose synthesis is easier and whose binding profile is atypical (log Y score similar to that of olanzapine, 3.89), was selected for further biological investigation. Pharmacological and biochemical studies confirmed an atypical antipsychotic profile in vivo. The compound was active on conditioned avoidance response at 1.1 mg/kg, a dose 100-times lower than that required to cause catalepsy (ED(50) >90 mg/kg), it induced a negligible increase of prolactin serum levels after single and multiple doses, and antagonized the cognitive impairment induced by phencyclidine. In conclusion, the pharmacological profile of 9d proved better than clozapine and olanzapine, making this compound a potential clinical candidate.

We present and examine the efficacy of a novel benzoxepin-based scaffold for modulation of the human estrogen receptor. Receptor tolerance of this new molecular scaffold is examined through presentation of experimentally determined antiproliferative effects on human MCF-7 breast tumor cells and measured binding affinities. The effect of functional group substitution on the benzoxepin scaffold is explored through a brief computational structure-activity relationship investigation with molecular simulation.

The synthesis, pharmacological evaluation, and structure-activity relationships (SARs) of a series of novel arylalkylpiperazines structurally related to BP897 (3) are described. In binding studies, the new derivatives were tested against a panel of dopamine, serotonin, and noradrenaline receptor subtypes. Focusing mainly on dopamine D(3) receptors, SAR studies brought to light a number of structural features required for high receptor affinity and selectivity. Several heteroaromatic systems were explored for their dopamine receptor affinities, and combinations of synthesis, biology, and molecular modeling, were used to identify novel structural leads for the development of potent and selective D(3) receptor ligands. Introduction of an indole ring linked to a dichlorophenylpiperazine system provided two of the most potent and selective ligands known to date (D(3) receptor affinity in the picomolar range). The intrinsic pharmacological properties of a subset of potent D(3) receptor ligands were also assessed in [(35)S]-GTPgammaS binding assays. Evidence from animal studies, in particular, has highlighted the dopaminergic system's role in how environmental stimuli induce drug-seeking behavior. We therefore tested two novel D(3) receptor partial agonists and a potent D(3)-selective antagonist in vivo for their effect in the cocaine-seeking behavior induced by reintroduction of cocaine-associated stimuli after a long period of abstinence, and without any further cocaine. Compound 5 g, a nonselective partial D(3) receptor agonist with a pharmacological profile similar to 3, and 5p, a potent and selective D(3) antagonist, reduced the number of active lever presses induced by reintroduction of cocaine-associated stimuli. However, 5q, a highly potent and selective D(3) partial agonist, did not have any effect on cocaine-seeking behavior. Although brain uptake studies are needed to establish whether the compounds achieve brain concentrations comparable to those active in vitro on the D(3) receptor, our experiments suggest that antagonism at D(2) receptors might significantly contribute to the reduction of cocaine craving by partial D(3) agonists.

L-Glutamate is the major excitatory neurotransmitter in mammalian central nervous system, and excitatory amino acid transporters (EAATs) are essential for terminating synaptic excitation and for maintaining extracellular glutamate concentration below toxic levels. Although the structure of these channel-like proteins has not been yet reported, their membrane topology has been hypothesised based on biochemical and protein sequence analyses. In the case of an inadequate clearance from synaptic cleft and from the extrasynaptic space, glutamate behaves as a potent neurotoxin, and it may be related to several neurodegenerative pathologies including epilepsy, ischemia, amyotrophic lateral sclerosis, and Alzheimer disease. The recent boom of glutamate is demonstrated by the enormous amount of publications dealing with the function of glutamate, with its role on modulation of synaptic transmission throughout the brain, mainly focusing: i). on the structure of its receptors, ii). on molecular biology and pharmacology of Glu transporters, and iii). on the role of glutamate uptake and reversal uptake in several neuropathologies. This review will deal with the recent and most interesting published results on Glu transporters membrane topology, Glu transporters physiopathological role and Glu transporters medicinal chemistry, highlighting the guidelines for the development of potential neuroprotective agents targeting neuronal high-affinity sodium-dependent glutamate transporters.

Tacrine-based AChE and BuChE inhibitors were designed by investigating the topology of the active site gorge of the two enzymes. The homobivalent ligands characterized by a nitrogen-bridged atom at the tether level could be considered among the most potent and selective cholinesterase inhibitors described to date. The nitrogen-containing homobivalent ligands 3e,g and the sulfur-containing 3h validated the hypothesis of extra sites of interaction in the AChE and BuChE active site gorges.

The prototypical dopamine and serotonin antagonist (+/-)-7-chloro-9-(4-methylpiperazin-1-yl)-9,10-dihydropyrrolo[2,1-b][1,3]benzothiazepine (5) was resolved into its R and S enantiomers via crystallization of the diastereomeric tartaric acid salts. Binding studies confirmed that the (R)-(-)-enantiomer is a more potent D(2) receptor antagonist than the (S)-(+)-enantiomer, with almost identical affinity at the 5-HT(2) receptor ((S)-(+)-5, log Y = 4.7; (R)-(-)-5, log Y = 7.4). These data demonstrated a significant stereoselective interaction of 5 at D(2) receptors. Furthermore, enantiomer (S)-(+)-5 (ST1460) was tested on a panel of receptors; this compound showed an intriguing binding profile characterized by high affinity for H(1) and the alpha(1) receptor, a moderate affinity for alpha(2) and D(3) receptors, and low affinity for muscarinic receptors. Pharmacological and biochemical investigation confirmed an atypical pharmacological profile for (S)-(+)-5. This atypical antipsychotic lead has low propensity to induce catalepsy in rat. It has minimal effect on serum prolactin levels, and it has been selected for further pharmacological studies. (S)-(+)-5 increases the extracellular levels of dopamine in the rat striatum after subcutaneous administration. By use of 5 as the lead compound, a novel series of potential atypical antipsychotics has been developed, some of them being characterized by a stereoselective interaction at D(2) receptors. A number of structure-activity relationships trends have been identified, and a possible explanation is advanced in order to account for the observed stereoselectivity of the enantiomer of (+/-)-5 for D(2) receptors. The molecular structure determination of the enantiomers of 5 by X-ray diffraction and molecular modeling is reported.

Along with nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) have gained a definitive and important place in the treatment of HIV-1 infections, and are in rapid development. These compounds can be grouped into two classes: the first generation NNRTIs, mainly discovered by random screening, and the second generation NNRTIs, developed as a result of comprehensive strategies involving molecular modelling, rationale-based drug synthesis, biological and pharmacokinetic evaluations. The recent boom of NNRTIs is mainly due to their antiviral potency, high specificity and low toxicity. The rapid emergence of drug-resistant HIV-1 strains induced by the first generation drugs is a disadvantage bypassed, in part, by the broad spectrum second generation NNRTIs. Starting from the first generation, this review will focus on the second generation NNRTIs dealing with the recent and most interesting published results, highlighting the guidelines for the development of a third generation of NNRTIs.

Two three-dimensional receptor interaction models for EAAT substrates and nontransportable inhibitors have been developed, and new glutamate (Glu) and aspartate (Asp) analogues have been synthesized. The analogues 1a and 3 represent novel lead compounds for the development of EAAT substrates and nontransportable inhibitors, selective for EAATs over iGluRs, as possible neuroprotective agents useful to minimize the progression of chronic or acute neurodegenerative diseases. The role played by the protonatable amine function in the interaction with EAATs has been discussed.