Search

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.

96 p.-20 fig.-7 tab., Autophagy is a lysosome dependent cell survival mechanism and is central to the maintenance of organismal homeostasis in both physiological and pathological situations. Targeting autophagy in cancer therapy attracted considerable attention in the past as stress-induced autophagy has been demonstrated to contribute to both drug resistance and malignant progression and recently interest in this area has re-emerged. Unlocking the therapeutic potential of autophagy modulation could be a valuable strategy for designing innovative tools for cancer treatment. Microtubule-targeting agents (MTAs) are some of the most successful anti-cancer drugs used in the clinic to date. Scaling up our efforts to develop new anti-cancer agents, we rationally designed multifunctional agents 5a-l with improved potency and safety that combine tubulin depolymerising efficacy with autophagic flux inhibitory activity. Through a combination of computational, biological, biochemical, pharmacokinetic-safety, metabolic studies and SAR analyses we identified the hits 5i,k. These MTAs were characterised as potent pro-apoptotic agents and also demonstrated autophagy inhibition efficacy. To measure their efficacy at inhibiting autophagy, we investigated their effects on basal and starvation-mediated autophagic flux by quantifying the expression of LC3II/LC3I and p62 proteins in oral squamous cell carcinoma and human leukaemia through western blotting and by immunofluorescence study of LC3 and LAMP1 in a cervical carcinoma cell line. Analogues 5i and 5k, endowed with pro-apoptotic activity on a range of hematological cancer cells (including ex-vivo chronic lymphocytic leukaemia (CLL) cells) and several solid tumor cell lines, also behaved as late-stage autophagy inhibitors by impairing autophagosome-lysosome fusion., This research was funded by the European Union's Horizon 2020 (EU) Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No.721906-TRACT (TK, SM, MG, NMcC, GO’S, RT, VK, SG, DZ, GC, SB); Tuscany strategic project POR-FSE 2014-2020, ‘Medicina di Precisione e Malattie Rare’(MePreMaRe), (ACE-ESCC) (NR, GC, SB), and by “Epigenetic Hallmarks of Multiple Sclerosis” (acronym Epi-MS) (id:415, Merit Ranking Area ERC LS) in VALERE 2019 Program; V:ALERE 2020 – Progetto competitivo “CIRCE” in risposta al bando D.R. n. 138 del February 17, 2020 Program; Blueprint 282510; EPICHEMBIO CM1406; AIRC-17217; VALERE: Vanvitelli per la Ricerca; Campania Regional Government Technology Platform Lotta alle Patologie Oncologiche: iCURE; Campania Regional Government FASE2: IDEAL. MIUR, Proof of Concept POC01_00043. POR Campania FSE 2014-2020 ASSE III.

Cutaneous melanoma, the most aggressive form of skin cancer, is characterized by activating BRAF mutations. Despite the initial success of selective BRAF inhibitors, only few patients exhibited complete responses, whereas many showed disease progression. Melanoma is one of the few types of cancer in which p53 is not frequently mutated, but p53 inactivation can be indirectly achieved by a stable activation of MDM2 induced by a deletion in CDKN2A (Cyclin Dependent Kinase Inhibitor 2A) locus, encoding for p16INK4A and p14ARF, two tumor suppressor genes. In this study, we tested the efficacy of the previously synthesized tetra-substituted pyrrole derivatives, 8 g, 8 h and 8i, in melanoma cell lines, and we compared the effects of the most active of these, the 8i compound, with that exerted by Nutlin 3, a well-known inhibitor of p53-MDM2 interaction. The obtained results showed that 8i potentiates the inhibitory effect of Nutlin 3 and the combined use of 8i and Nutlin 3 triggers apoptosis and significantly impairs melanoma viability. Finally, the 8i compound reduces p53-MDM2 interaction and induces p53-HSP90 complex formation, suggesting that the observed raise in p53 transcriptional activity could be mediated by HSP90. Because the main feature of melanoma is the resistance to most chemotherapeutics, our studies suggest that the 8i tetra-substituted pyrrole derivative, restoring p53 functions and its transcriptional activities, may have potential application, at least as adjuvant, in the treatment of human melanoma.

The identification of molecules whose biological activity can be properly modulated by light is a promising therapeutic approach aimed to improve drug selectivity and efficacy on the molecular target and to limit the side effects compared to traditional drugs. Recently, two photo-switchable diastereomeric benzodiazopyrrole derivatives 1RR and 1RS have been reported as microtubules targeting agents (MTAs) on human colorectal carcinoma p53 null cell line (HCT 116 p53-/-). Their IC50 was enhanced upon Light Emitting Diode (LED) irradiation at 435&thinsp, nm and was related to their cis form. Here we have investigated the photo-responsive behavior of the acid derivatives of 1RR and 1RS, namely, d1RR and d1RS, in phosphate buffer solutions at different pH. The comparison of the UV spectra, acquired before and after LED irradiation, indicated that the trans&rarr, cis conversion of d1RR and d1RS is affected by the degree of ionization. The apparent rate constants were calculated from the kinetic data by means of fast UV spectroscopy and the conformers of the putative ionic species present in solution (pH range: 5.7&ndash, 8.0) were modelled. Taken together, our experimental and theoretical results suggest that the photo-conversions of trans d1RR/d1RS into the corresponding cis forms and the thermal decay of cis d1RR/d1RS are dependent on the presence of diazonium form of d1RR/d1RS. Finally, a photo-reaction was detected only for d1RR after prolonged LED irradiation in acidic medium, and the resulting product was characterized by means of Liquid Chromatography coupled to High resolution Mass Spectrometry (LC-HRMS) and Nuclear Magnetic Resonance (NMR) spectroscopy.

In order to expand the class of diazocompounds able to act as photo-activable microtubule inhibitors the potential of azo-heteroarenes has been explored. In this paper we focus on the synthesis, physical properties and biological effects of methyl rac-2-(2-((E)-(4-((R)-2,3-dihydroxypropoxy)phenyl) diazenyl)-1H-pyrrol-1-yl)-3-hydroxypropanoate (1a) and methyl rac-2-(2-((E)-(4-((S)-2,3-dihydroxypropoxy)phenyl) diazenyl)-1H-pyrrol-1-yl)-3-hydroxypropanoate (1b). Preliminary biological studies on the HCT-116 p53-/- cancer cell line have shown that the weak antiproliferative action of the trans isomers of these molecules, especially of 1a, is enhanced upon LED light irradiation at 435 nm. On A375 cells the molecules have not shown any effect on cell viability either in the dark or under irradiation. Moreover, the two diastereomeric components of 1a as pure stereomers have been synthesized and characterized for their chemical-physical properties. Interestingly, upon irradiation, 1a has shown an antiproliferative activity on the HCT-116 p53-/- cells greater than that of the pure stereomers, 1RR and 1RS. Tubulin polymerization assay has also demonstrated that 1a, 1RR and 1RS inhibit tubulin aggregation mostly after exposure of the samples to LED light irradiation.

N-Palmitoyl-ethanolamine (PEA) is an anti-inflammatory component of egg yolk that is usually employed for the prevention of respiratory apparatus virus infection and then frequently used for its efficient anti-inflammatory and analgesic effects in experimental models of visceral, neuropathic, and inflammatory diseases. Nevertheless, data of its use in animal or human therapy are still scarce and further studies are needed. Herein, we report the biological evaluation of a small library of N-palmitoyl-ethanolamine analogues or derivatives, characterized by a protected acid function (either as palmitoyl amides or hexadecyl esters), useful to decrease their hydrolysis rate in vitro and prolong their biological activity. Two of these compounds-namely phenyl-carbamic acid hexadecyl ester (4) and 2-methyl-pentadecanoic acid (4-nitro-phenyl)-amide (5)-have shown good anti-inflammatory and antioxidant properties, without affecting the viability of J774A.1 macrophages. Finally, crystals suitable for X-ray analysis of compound 4 have been obtained, and its solved crystal structure is here reported. Our outcomes may be helpful for a rational drug design based on new PEA analogues/derivatives with improved biological properties.

The manuscript deals with the design, synthesis and biological evaluation of novel benzoxazinone-based and indole-based compounds as multifunctional neuroprotective agents. These compounds inhibit human adenosine kinase (hAK) and human glycogen synthase kinase 3 beta (hGSK-3β) enzymes. Computational analysis based on a molecular docking approach underlined the potential structural requirements for simultaneously targeting both proteins' allosteric sites. In silico hints drove the synthesis of appropriately decorated benzoxazinones and indoles (5a-s, and 6a-c) and biochemical analysis revealed their behavior as allosteric inhibitors of hGSK-3β. For both our hit 4 and the best compounds of the series (5c,l and 6b) the potential antioxidant profile was assessed in human neuroblastoma cell lines (IMR 32, undifferentiated and neuronal differentiated), by evaluating the protective effect of selected compounds against H2O2 cytotoxicity and reactive oxygen species (ROS) production. Results showed a strong efficacy of the tested compounds, even at the lower doses, in counteracting the induced oxidative stress (50 μM of H2O2) and in preventing ROS formation. In addition, the tested compounds did not show any cytotoxic effect determined by the LDH release, at the concentration range analyzed (from 0.1 to 50 μM). This study allowed the identification of compound 5l, as the first dual hAK/hGSK-3β inhibitor reported to date. Compound 5l, which behaves as an effective antioxidant, holds promise for the development of new series of potential therapeutic agents for the treatment of neurodegenerative diseases characterized by an innovative pharmacological profile.

The novel tetrasubstituted pyrrole derivatives 8g, 8h, and 8i showed selective cytotoxicity against M14 melanoma cells at low micromolar concentration. Structure???activity relationships (SARs) indicated the presence of three aromatic substituents on the pyrrole core as necessary for biological activity. Computational studies strongly suggest that the peculiar 3D orientation of these substituents is able to reproduce the hydrophobic side chains in LxxLL-like protein recognition motifs. Biological results showed altered p53 expression and nuclear translocation in cells sensitive to the compounds, suggesting p53 involvement in their anticancer mechanism of action. Unfortunately, because of poor solubility of the active analogues, it was not possible to perform further investigation by NMR techniques. Pharmacophore models were generated and used to perform 3D searches in molecular databases. Results indicated that two compounds share the same pharmacological profile and the same pharmacophoric features with our new derivatives, and one of them inhibited MDM2???MDM4 heterodimer formation.

Aims/Objective: Phosphonium salts are compounds whose structural characteristics enable them to cross the plasma and mitochondrial membrane with ease. Cancer cells have higher plasma membrane potentials than normal cells; phosphonium salts selectively accumulate in the mitochondria of neoplastic cells and inhibit mitochondrial function. METHOD In the present work, we investigated the cytotoxic activity of lipophilic phosphonium salt (11- methoxy11-oxo-undecyl) triphenylphosphonium bromide (MUTP) as well as of the two new phosphine oxide salts, 3,3'-(methylphosphoryl) dibenzenaminium chloride (SBAMPO) and 3,3' (phenylphosphoryl) dibenzenaminium chloride (SBAPPO) on the proliferation of breast cancer cell line (MCF-7) and human uterin cervix adenocarcinoma cells (HeLa). RESULT We showed that only MUTP exhibits antiproliferative effects on both cell lines, without affecting the normal breast epithelial cell proliferation. More specifically, we demonstrated that MUTP treatment of breast cancer cells is associated with impaired cell-cycle progression and metabolically induces mitochondrial damage and triggers apoptotic cell death in MCF-7 and HeLa cells. Taken together, these findings suggest that MUTP may be capable of selectively targeting neoplastic cell growth and therefore has potential applications as anticancer agent.

Aggregation of β-amyloid peptides into senile plaques has been identified as one of the hallmarks of Alzheimer’s disease. An attractive therapeutic strategy for Alzheimer’s disease is the inhibition of the soluble β-amyloid aggregation using synthetic β-sheet breaker peptides that are capable of binding Aβ but are unable to become part of a β-sheet structure. As the early stages of the Aβ aggregation process are supposed to occur close to the neuronal membrane, it is strategic to define the β-sheet breaker peptide positioning with respect to lipid bilayers. In this work, we have focused on the interaction between the β-sheet breaker peptide acetyl-LPFFD-amide, iAβ5p, and lipid membranes, studied by ESR spectroscopy, using either peptides alternatively labeled at the C- and at the N-terminus or phospholipids spin-labeled in different positions of the acyl chain. Our results show that iAβ5p interacts directly with membranes formed by the zwitterionic phospholipid dioleoyl phosphatidylcholine and this interaction is modulated by inclusion of cholesterol in the lipid bilayer formulation, in terms of both peptide partition coefficient and the solubilization site. In particular, cholesterol decreases the peptide partition coefficient between the membrane and the aqueous medium. Moreover, in the absence of cholesterol, iAβ5p is located between the outer part of the hydrophobic core and the external hydrophilic layer of the membrane, while in the presence of cholesterol it penetrates more deeply into the lipid bilayer. Copyright c � 2010 European Peptide Society and John Wiley & Sons, Ltd.

We report the synthesis, antioxidant and antiproliferative activity and a QSAR analysis of synthetic diphenylpropionamide derivatives. Synthesis of these compounds was achieved by direct condensation of 2,2- and 3,3-diphenylpropionic acid and appropriate amines using 1-propylphoshonic acid cyclic anhydride (PPAA) as catalyst. Compound structures were elucidated by NMR analysis and their melting points were measured. The in vitro antioxidant activity of these compounds was tested by evaluating the amount of scavenged ABTS radical and estimating ROS and NO production in LPS stimulated J774.A1 macrophages. All compounds were tested for their effect on viability of cells and results demonstrated that they are not toxic towards the cell lines used. The cytotoxic activity of all compounds was evaluated by a Brine Shrimp Test.

The antitumor activity shown by many platinum complexes has produced a strong interest in research of new organometallic compounds having anticancer action. Among the many metal compounds synthesized and tested, those based on titanium have received considerable attention because of their cytotoxic activity against solid tumors. Particularly, new titanocene compounds containing aromatic groups linked to the Cp (cyclopentadienyl ring, C5H5) have been synthetized, such as the titanocene Y (bis-[(p-methoxybenzyl)cyclopentadienyl]titanium dichloride) that displayed promising medium-high cytotoxic activity on breast cancer cell lines. Other titanocene complexes recently synthesized, obtained by replacing the substituent methoxy-aryl of cyclopentadienes of titanocene Y with ethenyl-methoxide or ethenyl-phenoxide, showed increased cytotoxic activity on breast cancer cell lines being more stable compounds. In this paper, we report that new titanocene complexes holding lipophilic groups, for instance a methyl group on benzyl carbon, exhibit improved antiproliferative effect on breast cancer cell line MCF-7. Similar results have been obtained introducing a 5-methoxy naphthyl group to further stabilize the titanocene complexes. These inhibitory effects on breast cancer cells have been ascribed to human topoisomerase I and II inhibition as demonstrated by specific enzymatic assays.

The antitumor activity shown by many platinum complexes has produced a strong interest in research of new organometallic compounds having anticancer action. Among the many metal compounds synthesized and tested, those based on titanium have received considerable attention because of their cytotoxic activity against solid tumors. Particularly, new titanocene compounds containing aromatic groups linked to the Cp (cyclopentadienyl ring, C5H5) have been synthetized, such as the titanocene Y (bis-[(p-methoxybenzyl)cyclopentadienyl]titanium dichloride) that displayed promising medium-high cytotoxic activity on breast cancer cell lines. Other titanocene complexes recently synthesized, obtained by replacing the substituent methoxy-aryl of cyclopentadienes of titanocene Y with ethenyl-methoxide or ethenyl-phenoxide, showed increased cytotoxic activity on breast cancer cell lines being more stable compounds. In this paper, we report that new titanocene complexes holding lipophilic groups, for instance a methyl group on benzyl carbon, exhibit improved antiproliferative effect on breast cancer cell line MCF-7. Similar results have been obtained introducing a 5-methoxy naphthyl group to further stabilize the titanocene complexes. These inhibitory effects on breast cancer cells have been ascribed to human topoisomerase I and II inhibition as demonstrated by specific enzymatic assays.

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.

The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells.

The human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitor pyrrolopyridooxazepinone (PPO) derivative, (+/-)-PPO294, was shown to be active toward wild type and mutated HIV-1 RT and to act synergistically in combination with 3'-azido-3'-deoxythymidine (Campiani, G., Morelli, E., Fabbrini, M., Nacci, V., Greco, G., Novellino, E., Ramunno, A., Maga, G., Spadari, S., Caliendo, G., Bergamini, A., Faggioli, E., Uccella, I., Bolacchi, F., Marini, S., (1999) J. Med. Chem. 42, 4462-4470). The (+/-)-PPO294 racemate was resolved into its pure enantiomers, and the absolute configuration was determined by x-ray analysis. Only one enantiomer, (R)-(-)-PPO464, displayed antiviral activity against both the wild type and the K103N mutant HIV-1 RT and was found to interact exclusively with the reaction intermediate formed by RT complexed with both the DNA and the nucleotide substrates. Being the first compound of its class to display this behavior, (R)-(-)-PPO464 is the representative of a novel generation of nonnucleoside inhibitors. (R)-(-)-PPO464 showed significant synergism when tested in combination with other RT inhibitors and efficiently inhibited viral replication when tested against the laboratory strain HIV-1 IIIB or against either wild type or multidrug-resistant clinical isolates. Pharmacokinetic studies in mice and rats showed a more favorable profile for (R)-(-)-PPO464 than for the corresponding racemate. (R)-(-)-PPO464 was also found to easily cross the blood-brain barrier. The coadministration of the HIV-1 protease inhibitor ritonavir increased the bioavailability of (R)-(-)-PPO464, having little effect on its plasma and brain elimination rates.

Quinoxalinylethylpyridylthioureas (QXPTs) represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 6-FQXPT (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis of novel heteroarylethylpyridylthioureas which were tested as anti-HIV agents. Several compounds proved to be potent broad-spectrum enzyme inhibitors and significantly inhibited HIV-1 replication in vitro. Their potency depends on the substituents and the nature of the heterocyclic skeleton linked to the ethyl spacer, and structure-activity relationships are discussed in terms of the possible interaction with the RT binding site. Although the new QXPTs analogues show potent antiviral activity, none of the compounds tested overcome the pharmacokinetic disadvantages inherent to ethylpyridylthioureidic antiviral agents, which in general have very low oral bioavailability. Through an integrated effort involving synthesis, docking studies, and biological and pharmacokinetic evaluation, we investigated the structural dependence of the poor bioavailability and rapid clearance within the thioureidic series of antivirals. Replacing the ethylthioureidic moiety with a hydrazine linker led to a new antiviral lead, offering promising pharmacological and pharmacokinetic properties in terms of antiviral activity and oral bioavailability.

A study was performed on structure-activity relationships of a series of phenol-derived, CVFM analogs, inhibitors of Ras Farnesyltransferase (FTase). The effect of various substituents on the phenol ring was examined, while the VFM moiety of the potent inhibitor CVFM was kept constant. The FTase inhibitory activity, reported as IC 50 in table I , was influenced by both the chemical properties and the relative position of the substituents on the phenolic ring. The most active compounds in this series contained a chloro or bromine substituent on the phenolic ring. Subsequently we have tested the effects of these FTase inhibitors on the anchorage-dependent growth of two rat epithelial cell lines, FRTL-5 and the same line v-Ha-ras transformed. While most of the compounds were inactive, two showed a growth inhibitory effect: compound 4 was active against normal as well against transformed cells while derivative 13 was active only against transformed cells.

Aldose reductase (ALR2) is a crucial enzyme in the development of the major complications of diabetes mellitus. Very recently it has been demonstrated that the ARL2 inhibitor, fidarestat, significantly prevents inflammatory signals (TNF-α, LPS) that cause cancer (colon, breast, prostate and lung), metastasis, asthma, and other inflammatory diseases. Currently, fidarestat is in phase III clinical trial for diabetic neuropathy and was found to be safe. Thus the finding of novel, potent ARL2 inhibitors is today more than in the past in great demand as they can pave the way for a novel therapeutic approach for a number of diseases besides the diabetes. Herein, starting from the virtual screening-derived ALR2 inhibitor S12728 (1), a rational receptor-based lead optimization has been undertaken. The design and synthetic efforts here reported led to the discovery of several new compounds endowed with low micromolar/submicromolar activities. © 2012 Elsevier Masson SAS. All rights reserved.

Abeta (16-35) is the hydrophobic central core of beta-amyloid peptide, the main component of plaques found in the brain tissue of Alzheimer's disease patients. Depending on the conditions present, beta-amyloid peptides undergo a conformational transition from random coil or alpha-helical monomers, to highly toxic beta-sheet oligomers and aggregate fibrils. The behavior of beta-amyloid peptide at plasma membrane level has been extensively investigated, and membrane charge has been proved to be a key factor modulating its conformational properties. In the present work we probed the conformational behavior of Abeta (16-35) in response to negative charge modifications of the micelle surface. CD and NMR conformational analyses were performed in negatively charged pure SDS micelles and in zwitterionic DPC micelles "doped" with small amounts of SDS. To analyze the tendency of Abeta (16-35) to interact with these micellar systems, we performed EPR experiments on three spin-labeled analogues of Abeta (16-35), bearing the methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl) methanethiolsulfonate spin label at the N-terminus, in the middle of the sequence and at the C-terminus, respectively. Our conformational data show that, by varying the negative charge of the membrane, Abeta (16-35) undergoes a conformational transition from a soluble helical-kink-helical structure, to a U-turn shaped conformation that resembles protofibril models.

Starting from the prototypic compound 4, we describe new, potent, and broad-spectrum pyrrolobenzo(py-rido)oxazepinones antivirals. A biochemical and enzymological investigation was performed for defining their mechanism of inhibition at either recombinant HIV-1 RT wild type and non-nucleoside reverse transcriptase inhibitors (NNRTIs)-resistant mutants. For the novel compounds (S)-(+)-5 and (S)-(-)-7,a clear-cut stereoselective mechanism of enzyme inhibition was found. Molecular modeling studies were performed for revealing the underpinnings of this behavior. © 2009 American Chemical Society.

In the present study, 11 novel N -(3,3-diphenyl)propyl-2,2-diphenylacetamide derivatives ( 4a – d and 9a – g ) and six triphenylacetamides ( 10a – c and 11a – c ) were synthesized and tested as ligands of cannabinoid CB 1 and CB 2 receptors. All compounds exhibited affinity for CB 1 and CB 2 receptors. Four compounds ( 4b , 9a , 9b , and 11a ) showed selectivity for CB 1 versus CB 2 receptors, although only the N -(3,3-diphenyl)propyl-2,2-diphenylacetamide ( 4b ) can be considered a potent CB 1 ligand ( K i = 58 nM). It was 140-fold selective over CB 2 receptors ( K i = 7800 nM) and behaved as an inverse agonist by stimulating forskolin-induced cAMP formation in mouse N18TG2 neuroblastoma cells. This compound is the first of a novel class of tetraphenyl CB 1 ligands that, in view of its easy synthesis and high affinity for CB 1 receptors and despite its sterical hindrance, will be useful for the design of new blockers of this therapeutically exploitable receptor type.

There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the accumulation of beta-amyloid peptides into senile plaques, one of the hallmarks of Alzheimer's disease (AD). With the aim to clarify the molecular basis of the interaction between amyloid peptides and cellular membranes, we investigated the interaction between a cytotoxic fragment of Abeta(1-42), i.e., Abeta(25-35), and phospholipid bilayer membranes. These systems were studied by Electron Paramagnetic Resonance (EPR) spectroscopy, using phospholipids spin-labeled on the acyl chain. The effect of inclusion of charged phospholipids or/and cholesterol in the bilayer composition was considered in relation to the peptide/membrane interaction. The results show that Abeta(25-35) inserts in bilayers formed by the zwitterionic phospholipid dilauroyl phosphatidylcholine (DLPC), positioning between the outer part of the hydrophobic core and the external hydrophilic layer. This process is not significantly influenced by the inclusion of the anionic phospholipid phosphatidylglycerol (DLPG) in the bilayer, indicating the peptide insertion to be driven by hydrophobic rather than electrostatic interactions. Cholesterol plays a fundamental role in regulating the peptide/membrane association, inducing a membrane transition from a fluid-disordered to a fluid-ordered phase. At low cholesterol content, in the fluid-disordered phase, the insertion of the peptide in the membrane causes a displacement of cholesterol towards the more external part of the membrane. The crowding of cholesterol enhances its rigidifying effect on this region of the bilayer. Finally, the cholesterol-rich fluid-ordered membrane looses the ability to include Abeta(25-35).

6-(2-Phenylethyl)nicotine (1b; K(i)=15 nM) was unexpectedly found to bind at alpha4beta2 nicotinic cholinergic (nACh) receptors. Although this compound failed to produce nicotine-like agonist action in several functional assays, 1b antagonized the antinociceptive effects of nicotine (mouse tail-flick assay) in a dose-dependent fashion when administered via an intrathecal route.

An examination of several 3-(2-aminoethyl)pyridine analogs suggests that they likely orient at α4β2 nicotinic cholinergic receptors in a different fashion than their correspondingly substituted nicotine analogs.

We have previously described a novel class of nonnucleoside reverse transcriptase (RT) inhibitors, the pyrrolobenzoxazepinone (PBO) and the pyridopyrrolooxazepinone (PPO) derivatives, which were effective inhibitors of human immunodeficiency virus type 1 (HIV-1) RT, either wild type or carrying known drug resistance mutations (G. Campiani et al., J. Med. Chem. 42:4462-4470, 1999). The lead compound of the PPO class, ( R )-(−)-PPO464, was shown to selectively target the ternary complex formed by the viral RT with its substrates nucleic acid and nucleotide (G. Maga et al., J. Biol. Chem. 276:44653-44662, 2001). In order to better understand the structural basis for this selectivity, we exploited some PBO analogs characterized by various substituents at C-3 and by different inhibition potencies and drug resistance profiles, and we studied their interaction with HIV-1 RT wild type or carrying the drug resistance mutations L100I and V106A. Our kinetic and thermodynamic analyses showed that the formation of the complex between the enzyme and the nucleotide increased the inhibition potency of the compound PBO354 and shifted the free energy (energy of activation, ΔG # ) for inhibitor binding toward more negative values. The V106A mutation conferred resistance to PBO 354 by increasing its dissociation rate from the enzyme, whereas the L100I mutation mainly decreased the association rate. This latter mutation also caused a severe reduction in the catalytic efficiency of the RT. These results provide a correlation between the efficiency of nucleotide utilization by RT and its resistance to PBO inhibition.

The present study examines the molecular mechanisms by which a member of a novel series of pyrrolo-1,5-benzoxazepines, PBOX-21, induces G1 arrest in 1321N1 cells. PBOX-21-induced G1 arrest is preceded by both a decrease in CDK2 kinase activity, which is critical for the G1/S transition, and a downregulation in cyclin D 3 protein expression levels, suggesting that these two events may be crucially involved in the mediation of the cell cycle arrest. The decrease in CDK2 activity may be due to an observed decrease in CDK2 protein levels following PBOX-21 treatment. Coinciding with the arrest is a reduction in the activity of CDK4, due to either the observed PBOX-21 induced downregulation in CDK4 expression, or a reduction in complex formation between cyclin D 3 –CDK4 leading to a decrease in the levels of active cyclin D 3 –CDK4 complexes with kinase activity. The level of CDK6 activity was also seen to be reduced following PBOX-21 treatment, also possibly due to a reduction in complex formation with cyclin D 3 . However, this reduction in CDK6 kinase activity was not seen until after PBOX-21-induced G1 arrest has reached its maximum, and therefore may be viewed as a consequence of, and a method of maintaining the PBOX-21-induced arrest, rather than a cause. Also in parallel with the G1 arrest elicited by PBOX-21 is an upregulation in the universal CDK inhibitor, p21. Furthermore, the retinoblastoma protein (Rb), a substrate of CDK2 and CDK6, whose phosphorylation is necessary for cell cycle progression, becomes hypophosphorylated. These results indicate that PBOX-21 exerts its growth inhibitory effects through the modulation of the expression and activity of several key G1 regulatory proteins.

Effector caspases-3, -6 and -7 are responsible for producing the morphological features associated with apoptosis, such as DNA fragmentation. The present study demonstrates that a member of a novel series of pyrrolo-1,5-benzoxazepines, PBOX-6, induces apoptosis in MCF-7 cells, which lack caspase-3. Apoptosis was accompanied by DNA fragmentation and the activation of caspase-7, but not caspases-3 and -6. Inhibition of caspase-7 activity reduced the extent of apoptosis induced, indicating that activation of caspase-7 is involved in the mechanism by which PBOX-6 induces apoptosis in MCF-7 cells. This study suggests that caspase-3 is not necessarily essential for DNA fragmentation and the morphological changes associated with apoptosis.

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.

In the present study, we have synthesized and tested novel pyridopyrrolo- and pyrrolobenzoxazepine derivatives, as novel and selective peripheral type benzodiazepine receptor (PBR) ligands, and their ability to modulate steroid biosynthesis has been investigated. A subset of new ligands bind the PBR (rat brain and testis) with picomolar affinity, representing the most potent ligands that have been identified to date, and elicited effects on endogenous rate of steroidogenesis in MA10 Leydig cells, having similar potency and effect as PK11195. Several compounds, differently substituted at C-7, were used as molecular yardsticks to probe the spatial dimension of the lipophilic pocket L4 in the receptor binding site.

(S)-CPW399 (2b) is a novel, potent, and subtype-selective AMPA receptor full agonist that, unlike (S)-willardiine and related compounds, in mouse cerebellar granule cells, stimulated an increase in [Ca(2+)](i), and induced neuronal cell death in a time- and concentration-dependent manner. Compound 2b appears to be a weakly desensitizing, full agonist at AMPA receptors and therefore represents a new pharmacological tool to investigate the role of AMPA receptors in excitotoxicity and their molecular mechanisms of desensitization.

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.

Pyrrolobenzoxazepinone (PBO) derivatives represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTs) whose prototype is (+/-)-6-ethyl-6-phenylpyrrolo[2,1-d][1,5]benzoxazepin-7(6H)- one (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis and biological evaluation of novel derivatives and analogues of 6 featuring a meta-substituted phenyl or a 2-thienyl ring at C-6 and a pyridine system in place of the fused-benzene ring to yield pyrrolopyridooxazepinones (PPOs). Compared with the lead 6 and nevirapine, several of the synthesized compounds (PBOs 13a-d and PPOs 13i-k) displayed higher inhibitory activity against wild-type RT and clinically relevant mutant RTs containing the single amino acid substitutions L100I, K103N, V106A, Y181I, and Y188L. The most potent inhibitors were further evaluated for in vitro antiviral activity on lymphocytes and monocyte-macrophages, for cytotoxicity on a panel of cell lines, and for potential synergistic antiviral activity with AZT. Pharmacokinetic studies performed on 13b, 13c, and 13i showed that these compounds achieve high concentrations in the brain. The results of the biological and pharmacokinetic experiments suggest a potential clinical utility of analogues such as 13b-d, 13i, and 13j, in combination with nucleoside RT inhibitors, against strains of HIV-1 bearing those mutations that confer resistance to known NNRTI.