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Two vortioxetine (VOT) salts with hydrochloride (VOT-HCl and VOT-0.5HCl) were prepared and structurally characterized. VOT-HCl features 1-dimensional P/M helical chains through N-H···Cl hydrogen bond interactions, whereas VOT-0.5HCl possesses a 1-dimensional zigzag structure in which 2 VOT molecules share a single proton through N···H+···N interactions. VOT-HCl converts into the monohydrate VOT-HCl·H2O after dissolution in water, whereas VOT-0.5HCl remains stable. The N 1s X-ray photoelectron spectroscopy analysis shows a characteristic binding energy peak at approximately 398.0 eV for VOT. The shift to high energy occurs at 400.3 eV for VOT-HCl and VOT-HBr, and at 399.7 eV for VOT-0.5HCl, which supports the salt formation by the degree of proton transfer and is confirmed by single-crystal X-ray analyses. The apparent equilibrium solubilities of VOT in water are significantly improved to 2.90 mg/mL (approximately a 32.0-fold increase over that of the free base) for VOT-HCl and to 0.59 mg/mL (approximately a 5.7-fold increase over that of the free base) for VOT-0.5HCl at 25°C.

A series of 1-(1'-hydroxyalkyl)rupestonic acid methyl esters were synthesized via the condensation of methyl rupestonate with various aldehydes in the presence of LDA. This mixed aldol reaction was highly stereoselective and all the new compounds were elucidated by detailed NMR and MS analyses. The absolute configurations of the newly formed stereocenters were further confirmed by X-ray crystallographic analysis of 3d, the results of which were found to be opposite to the prediction based on Zimmerman-Traxler's and Houk's models. All the compounds synthesized were then evaluated for their in vitro inhibitory activities against influenza A (H1N1 and H3N2) and B viruses. The data showed that 3p displayed the highest activity against influenza A H1N1 (IC50=0.69μg/mL) and H3N2 (IC50=0.69μg/mL) viruses, which were even better than Ribavirin and Oseltmivir. On the other hand, both 3c and 3o were found to show comparable activities with the reference drugs in inhibiting both influenza A and B viruses. Further studies will focus on reducing the cytotoxicity of the hits reported in this work.

We report here an original strategy to graft both electroactive conjugated oligomers and DNA probes on an electrode to make a label-free and reagentless DNA sensor. Oligomers of 5-amino-1,4-naphthoquinone are synthesized, then covalently immobilized on the electrode by electro-oxidation of their terminal amino group, along with aminophenylacetic acid. Then, short DNA probes modified by terminal amino group are chemically grafted via peptide bonds on the aminophenylacetic acid molecules. In this way, short DNA strands and electroactive oligomers, having barely the same length, are grafted close together and can interact. It is shown that hybridization with a complementary sequence, which changes the conformation of DNA strands, influences the redox kinetics of quinone groups and leads to a current increase, whereas no significant changes are observed for random or mismatch sequences.

Chemokine receptor CXCR4 is one of two principal coreceptors for the entry of HIV-1 into target cells. CXCR4 is known to form homodimers. We previously demonstrated that the amino terminus of viral macrophage protein II (vMIP-II) is the major determinant for CXCR4 recognition, and that V1 peptide derived from the N-terminus of vMIP-II (1-21 residues) showed significant CXCR4 binding. Interestingly, an all-d-amino acid analogue of V1 peptide, DV1 peptide, displayed an even higher binding affinity and strong antiviral activity in inhibiting the replication of CXCR4-dependent HIV-1 strains. In this study, we synthetically linked two DV1 peptides with the formation of a disulfide bond between the two cysteine residues present in the peptide sequence to generate a dimeric molecule potentially capable of interacting with two CXCR4 receptors. DV1 dimer exhibited enhanced binding affinity and antiviral activity compared with those of DV1 monomer. Ligand binding site mapping experiments showed that DV1 dimer overlaps with HIV-1 gp120 on CXCR4 binding sites, including several transmembrane (TM) residues located close to the extracellular side and the N-terminus of CXCR4. This finding was supported by the molecular modeling of CXCR4 dimer-DV1 dimer interaction based on the crystal structure of CXCR4, which showed that DV1 dimer is capable of interacting with the CXCR4 dimeric structure by allowing the N-terminus of each DV1 monomer to reach into the binding pocket of CXCR4 monomer. The development of this bivalent ligand provides a tool for further probing the functions of CXCR4 dimerization and studying CXCR4 heterodimerization with other receptors.

The replication of human immunodeficiency virus type 1 (HIV-1) can be profoundly inhibited by the natural ligands of two major HIV-1 coreceptors, CXCR4 and CCR5. Stromal cell-derived factor-1α (SDF-1α) is a natural ligand of CXCR4. We have recently developed a synthetic biology approach of using synthetically and modularly modified (SMM)-chemokines to dissect various aspects of the structure-function relationship of chemokines and their receptors. Here, we used this approach to design novel SMM-SDF-1α analogues containing unnatural N-methylated residues in the amino terminus to investigate whether the polypeptide main chain amide bonds in the N-terminus of SDF-1α play a role in SDF-1α signaling via CXCR4 and/or receptor internalization. The results show that SDF-1α analogues with a modified N-methylated main chain at position 2, 3, or 5 retain significant CXCR4 binding and yet completely lose signaling activities. Furthermore, a representative N-methylated analogue has been shown to be incapable of causing CXCR4 internalization. These results suggest that the ability of SDF-1α to activate CXCR4 signaling and internalization is dependent upon the main chain amide bonds in the N-terminus of SDF-1α. This study demonstrates the feasibility and value of applying a synthetic biology approach to chemically engineer natural proteins and peptide ligands as probes of important biological functions that are not addressed by other biological techniques.

Molecularly imprinted polymers were coated on the surface of magnetic nanoparticles using an original and simple chemical strategy combining aryl diazonium salt chemistry and the iniferter method. This approach provides dispersed, highly soluble and BPA imprinted poly(methacrylic acid-co-ethylene glycoldimethacrylate) coated magnetic nanoparticles.

The natural ligands of two major human immunodeficiency virus type 1 (HIV-1) co-receptors, CXCR4 and CCR5, can profoundly inhibit the replication of HIV-1 that uses these co-receptors for entry into the target cells. It has been postulated that these natural chemokines inhibit HIV-1 infection by blocking common binding sites on CXCR4 or CCR5 that are required for HIV-1 envelope glycoprotein gp120 interaction with its co-receptor and/or by inducing receptor internalization. To investigate whether receptor internalization caused by stromal cell-derived factor (SDF)-1 α, a natural ligand of CXCR4, plays a role in its anti-HIV activity, we applied the SMM (synthetically and modularly modified)-chemokine approach to generate a functional probe of SDF-1 α that retains significant CXCR4 binding but does not induce CXCR4 internalization. The antiviral study of this functional probe analog versus wild-type SDF-1 α showed that, despite the significant CXCR4 binding activity, this probe analog displayed a complete loss of effect in causing CXCR4 internalization and greatly diminished antiviral activity. Interestingly, this new analog also showed a decreased number of overlapping binding sites with HIV-1 on CXCR4 transmembrane and extracellular domains. The correlation of the decrease in the anti-HIV activity with the loss of CXCR4 internalization observed with this probe molecule suggests that receptor internalization may play an important role in the anti-HIV activity of SDF-1 α and possibly other natural chemokines. This further implies that any modifications in SDF-1 α that result in a reduction or loss of internalization activity may result in analogs that are not suitable as effective HIV-1 inhibitors that target CXCR4, unless such modifications also result in improved CXCR4 interaction with increased number of overlapping binding sites with HIV-1, thus leading to more effective steric hindrance against HIV-1.

Rupestonic acid is the main active compound in Artemisia rupestris L, which mainly grows in Xinjiang of China. To find its active group, a series of novel rupestonic acid 3-carbonyl analogues were prepared. The structures of the compounds were confirmed by spectral and high resolution mass spectrum. All compounds were evaluated for antiviral activity against influenza A (H3N2 and H1N1) and B viruses in MDCK cell cultures. The compounds displayed a confined structure-activity relationship. Compound 13 with allyl group was the most potent inhibitor against influenza A/H1N1 virus with an IC50 value of 4.27 μM and a SI value of 27.04. Dihydrogen amide 3 possesses the good potency against influenza B virus with an IC50 value of 5.5 μM and a SI value of 13.

Chemokines and their receptors play important roles in normal physiological functions and the pathogeneses of a wide range of human diseases, including the entry of human immunodeficiency virus type 1 (HIV-1). However, the use of natural chemokines to probe receptor biology or to develop therapeutic drugs is limited by their lack of selectivity and the poor understanding of mechanisms in ligand-receptor recognition. We addressed these issues by combining chemical and structural biology in research into molecular recognition and inhibitor design. Specifically, the concepts of chemical biology were used to develop synthetically and modularly modified (SMM) chemokines that are unnatural and yet have properties improved over those of natural chemokines in terms of receptor selectivity, affinity, and the ability to explore receptor functions. This was followed by using structural biology to determine the structural basis for synthetically perturbed ligand-receptor selectivity. As a proof-of-principle for this combined chemical and structural-biology approach, we report a novel d -amino acid-containing SMM-chemokine designed based on the natural chemokine called viral macrophage inflammatory protein II (vMIP-II). The incorporation of unnatural d -amino acids enhanced the affinity of this molecule for CXCR4 but significantly diminished that for CCR5 or CCR2, thus yielding much more selective recognition of CXCR4 than wild-type vMIP-II. This d -amino acid-containing chemokine also showed more potent and specific inhibitory activity against HIV-1 entry via CXCR4 than natural chemokines. Furthermore, the high-resolution crystal structure of this d -amino acid-containing chemokine and a molecular-modeling study of its complex with CXCR4 provided the structure-based mechanism for the selective interaction between the ligand and chemokine receptors and the potent anti-HIV activity of d -amino acid-containing chemokines.

As the main coreceptors for human immunodeficiency virus type 1 (HIV-1) entry, CXCR4 and CCR5 play important roles in HIV-associated dementia (HAD). HIV-1 glycoprotein gp120 contributes to HAD by causing neuronal damage and death, either directly by triggering apoptotic pathways or indirectly by stimulating glial cells to release neurotoxins. Here, to understand the mechanism of CXCR4 or CCR5 signaling in neuronal apoptosis associated with HAD, we have applied synthetically and modularly modified (SMM)-chemokine analogs derived from natural stromal cell-derived factor-1alpha or viral macrophage inflammatory protein-II as chemical probes of the mechanism(s) whereby these SMM-chemokines prevent or promote neuronal apoptosis. We show that inherently neurotoxic natural ligands of CXCR4, such as stromal cell-derived factor-1alpha or viral macrophage inflammatory protein-II, can be modified to protect neurons from apoptosis induced by CXCR4-preferring gp120(IIIB), and that the inhibition of CCR5 by antagonist SMM-chemokines, unlike neuroprotective CCR5 natural ligands, leads to neurotoxicity by activating a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Furthermore, we discover distinct signaling pathways activated by different chemokine ligands that are either natural agonists or synthetic antagonists, thus demonstrating a chemical biology strategy of using chemically engineered inhibitors of chemokine receptors to study the signaling mechanism of neuronal apoptosis and survival.

In the search for highly selective and potent cholinesterase inhibitors (AChEI) being able to improve oxidative injury, PMS777, a tetrahydrofuran derivative, was designed as a novel dual PAF and acetylcholinesterase inhibitor. The aim of this study was to investigate the modulatory effects of PMS777 and galanthamine, another AChEI, on the oxidative injury induced in neuronal cells. The SK-N-SH cells stimulated with LPS+IL-(1beta) were selected to investigate the direct inhibitory effect of PMS777 and galanthamine. LPS+IL-(1beta) induced oxidative injury as assessed by ROS production (29%), GSH depletion (11%) and loss of mitochondrial activity (22%). GSH depletion was never decreased by either drug. In contrast, ROS production and mitochondrial activity were totally prevented by addition of PMS777 but not galanthamine. PMS777 also inhibits butylcholinesterase and it shows selectivity for acetylcholinesterase. Thus, this PAF antagonist inaugurates a new type of AChEI, able to fight oxidative injury. Therefore, PMS777 could be of interest on patients with cognitive impairments and inflammatory damage, as in AD.

Starting from 4-tetradecyloxybenzamidine ( PMS815 ), a non-specific inhibitor of GI and GII PLA 2 s, we report in this work the discovery of the specificity through design, synthesis and structure–activity relationships studies of different kinds of PMS815 derivatives. The leading compound, 4,5-dihydro-3-(4-tetradecyloxybenzyl)-1,2,4-4 H -oxadiazol-5-one ( 9b , PMS1062 ) exhibits a micromolar IC 50 towards three group II PLA 2 s, while inactive towards four group I and one group III enzymes in two in vitro enzymatic assay conditions. It is also able to block the PLA 2 -II activities induced by LPS and IL-6 in HepG 2 cell line and no cytotoxicity is observed when PMS1062 is tested up to a concentration of 100 μM in two different cell lines (A549 and LLC-PK 1 ).

3-(4-Tetradecyloxybenzyl)-4H-1,2,4-oxadiazol-5-one (PMS1062 or I) can probably act as a PLA2-II inhibitor on the acute inflammation model, but its highly lipophilic character could prevent it from being biodistributed effectively. In this work, based on a molecular modeling study, we have proposed a model that may provide compounds retaining both anti-PLA2 activity and specificity while becoming active per os. Moreover, molecular dynamics and energy minimization enabled us to characterize the lowest-energy complexes of each derivative. Energy balances taking account of the conformational energy changes of both partners, along with the drug–protein interaction, were performed, and were further completed by single-point computations of the contributions of solvation/desolvation to the binding. The ordering of the resulting energy balances was found to be fully consistent with the experimentally ascertained ordering of affinities inferred from the IC50 values. The essential role of an indole group partaking in cation–π and hydrophobic interactions, together with the CaII-chelating oxadiazolone ring, was highlighted for the best binding compound. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Excessive levels of PAF and cells of macrophage lineage appear to play an important role in neuronal cell injury, inflammatory syndrome, and HIV replication in CNS resulting in AIDS dementia complex (ADC). The beneficial effects of PAF receptor antagonists are evident and give rise to expected therapeutic strategies for neurotrauma. Piperazine derivatives bearing a "cache-oreilles" (ear-muff) electronic distribution are able to inhibit in vitro PAF effects and, thus, could be used in pathologies where this mediator is involved. Therefore, their potential anti-HIV activity was investigated, and we find that (i) these PAF antagonists are effectively active in HIV-infected monocyte-derived macrophages (MDM) but there is no correlation between both anti-HIV and anti-PAF activities; (ii) the presence of a carbamate function (compounds 1a-d) is favorable to the antiviral activity; (iii) the lipophilicity of the substituent on the piperazinic cycle seems to be less important for the anti-PAF activity than for the antiviral one. Our leading compound, PMS 601 (compound 1a), presents a dual activity with IC(50) of 8 and 11 microM for anti-PAF and anti-HIV activity, respectively, without cytotoxic events at 1000 microM in MDM. Although its mode of action is not clearly defined, these data suggest that PMS 601, which displays no effect on acellular reverse transcriptase or protease tests, deserves further investigation in the treatment of HIV-1-associated dementia.

Combined inhibition of HIV-1 reverse transcriptase and protease has significantly improved the treatment of AIDS. Nevertheless, resistance to these drugs occurs rapidly because of viral mutations, emphasizing the importance of identifying novel retroviral targets to develop new drug combinations. The critical role played by the nucleocapsid protein NCp7 of HIV-1 at different steps of the retrovirus life cycle makes it an attractive target for the development of new antiviral agents. NCp7 contains two highly conserved zinc fingers and is characterized by a three-dimensional structure that cannot be modified without a complete loss of infectivity of mutated viruses. Based on these structural data, we report that RB 2121, a cyclic peptide designed to mimic several essential biological determinants of NCp7, displays antiviral activity by inhibiting HIV-1 replication in CEM-4 cells infected by HIV-1. In vitro , RB 2121 does not interfere with HIV-1 cell entry and viral enzymes but is able to inhibit the annealing activities of NCp7 by recognizing nucleic acids. Analysis of proviral DNA synthesis by means of PCR has shown that RB 2121 acts at an early step of the retrovirus life cycle by inducing a dose-dependent reduction in transcribed DNA levels through inhibition of NCp7–reverse transcriptase interaction. Because of its original mechanism of action, RB 2121 provides an interesting lead for the rational development of new anti-HIV-1 agents that could be associated advantageously with enzyme inhibitors to counteract rapid virus mutations and resistance problems observed in tritherapies.

A synthetic procedure for construction of the (+)-(3R,5R,6R)-5,6-dihydroxy-3,7-dimethyl-octanal and (−)-(3S,5S,6S)-5,6-dihydroxy-3,7-dimethyl-octanal derivatives, the intermediates for synthesis of the HIV-active didemnaketal analogue, was developed via a series of reactions from the natural (−)-menthone.

The orientation of chlorination and bromination of N, N-disubstituted benzamides in aqueous acetic acid is strongly influenced by the nature of the alkyl groups at the nitrogen atom. With large groups, the halogenation takes place fairly selectively at the ortho/para positions.

A label-free electrochemical immunosensor was developed by electropolymerization of N-(3-(4-(2-(4-hydroxyphenyl)propan-2-yl)phenoxy)propyl) 3-(5-hydroxy-1,4-dihydro-1,4-dioxonaphthalen-2(3)-yl)propionamide (JugBPA). By combination with an antibody directed to bisphenol A (αBPA), this conducting polymer-based biosensor can detect BPA directly with a limit of detection of 2pgmL(-1). Square wave voltammetry shows that the polymer film presents a current decrease upon anti-BPA binding and an opposite current increase upon BPA addition in solution. This electrochemical immunosensor (E-assay) also shows high selectivity towards closely related compounds (bisphenol A dimethacrylate, and dibutyl phthalate). The E-assay concept described here could be a promising tool for simple, low-cost and reagentless on-site environmental monitoring.

Two series of rupestonic acid derivatives, (1-substituted-1H-1,2,3-triazol-4-yl)methyl 2-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate and N-(1-substituted-1H-1,2,3-triazol-4-yl)methyl 2-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide were easily and efficiently synthesized via click chemistry. These compounds were tested for their in vitro activities against various strains of influenza A virus (H1N1, oseltamivir resistant H1N1, H3N2) and influenza B virus. The results showed that nine compounds were active against the H1N1 strain of influenza A virus and among them the best one 14a, was as active as the reference drugs, Oseltamivir and Ribavirin. Some of them were also active on the Oseltamivir resistant H1N1 strain. In regards to influenza B virus, twenty-one compounds over thirty were active and seven of them 7b, 8b, 9b, 10a, 11b, 12b, 13b showed better activity than Ribavirin. The structure-activity relationship of these compounds is discussed on the basis of each type of the viruses studied. Furthermore, four best representative compounds 7b, 10a, 12b and 14a were evaluated in a plaque assay experiment using MDCK cells and RBV as control compound and the results showed that 7b, 10a and 12b were better than RBV in inhibiting plaque formation, in good accordance with their anti-influenza B activities.

The three-dimensional structure of peptides encompassing the two zinc-saturated finger motifs of the nucleocapsid protein NCp7 of HIV-1 has been reported by several groups. Whereas the folded structures of the finger motifs were in good agreement, discrepancies existed concerning their spatial relationship since the fingers were found either close to each other [Morellet, N., Jullian, N., De Rocquigny, H., Maigret, B., Darlix, J. L., & Roques, B. P. (1992) Embo J. 11, 3059-3065] or independently folded [Omichinski, J. G., Clore, G. M., Sakaguchi, K., Appella, E., & Gronenborn, A. M. (1991) FEBS Lett. 292, 25-30, Summers, M. F., Henderson, L. E., Chance, M. R., Bess, J. W., Jr., South, T. L., Blake, P. R., Sagi, I., Perez-Alvarado, G., Sowder, R.C., III, Hare, D.R., & Arthur, L. O. (1992) Protein Sci. 1, 563-574]. As in the interacting finger model, Phe16 in the NH2-terminal finger and Trp37 in the COOH-terminal finger were found to be spatially close, the fluorescence properties of the aromatic residues at positions 16 and 37 in the wild-type and two conservatively substituted (12-53) NCp7 peptides were investigated and compared with those of three negative control derivatives where the finger motifs were not in close contact. Direct distance measurements by Tyr-Trp fluorescence resonance energy transfer of the former derivatives yielded a 7-12 A interchromophore distance range which is clearly inconsistent with the 12.5-18 A range measured for the negative controls and thus a random orientation of the zinc finger motifs.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of ionizing radiation, used in post-harvest treatment of fruit and vegetables. were investigated on cultured apple cells (Pyrus malus L. cv. Royal Red) on a short-term period. Irradiation (2 kGy) induced an increase of passive ion effluxes from cells and a decrease of cell capacity to regulate external pH. These alterations are likely due to effects on plasma membrane structure and function and were further investigated by studying the effects of irradiation on plasma membrane H+-ATPase activity. Plasma membrane-enriched vesicles were prepared and the H+-ATPase activity was characterized. Irradiation of the vesicles induced a dose dependent inhibition of H+-ATPase activity. The loss of enzyme activity was immediate, even at low doses (0.5 kGy), and was not reversed by the addition of 2mM dithiothreitol. This inhibition may be the result of an irreversible oxidation of enzyme sulfhydryl moieties and/or the result of changes induced within the lipid bilayer affecting the membrane-enzyme interactions. Further analysis of the H+-ATPase activity was carried out on vesicles obtained from irradiated cells confirming the previous results. In vivo recovery of activity was not observed within 5 h following the treatment, thus explaining the decrease of cell capacity to regulate external pH. This rapid irreversible inhibition of the plasma membrane H+-ATPase must be considered as one of the most important primary biochemical events occurring in irradiated plant material.

A series of polyphenolic curcumin analogs were synthesized and their inhibitory effects on mushroom tyrosinase and the inhibition of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical formation were evaluated. The results indictated that the analogs possessing m-diphenols and o-diphenols exhibited more potent inhibitory activity on tyrosinase than reference compound rojic acid, and that the analogs with o-diphenols exhibited more potent inhibitory activity of DPPH free-radical formation than reference compound vitamin C. The inhibition kinetics, analyzed by Lineweaver-Burk plots, revealed that compounds B(2) and C(2) bearing o-diphenols were non-competitive inhibitors, while compounds B(11) and C(11) bearing m-diphenols were competitive inhibitors. In particular, representative compounds C(2) and B(11) showed no side effects at a dose of 2,000 mg/kg in a preliminary evaluation of acute toxicity in mice. These results suggest that such polyphenolic curcumin analogs might serve as lead compounds for further design of new potential tyrosinase inhibitors.

A new electropolymerizable monomer, [N-(6-(4-hydroxy-6-isopropylamino-1,3,5-triazin-2-ylamino)hexyl) 5-hydroxy-1,4-naphthoquinone-3-propionamide], has been designed for use in a label-free electrochemical immunosensor when polymerized on an electrode and coupled with a monoclonal anti-atrazine antibody. This monomer contains three functional groups: hydroxyl group for electropolymerization, quinone group for its transduction capability, and hydroxyatrazine as bioreceptor element. Square wave voltammetry shows that the polymer film, poly[N-(6-(4-hydroxy-6-isopropylamino-1,3,5-triazin-2-ylamino)hexyl) 5-hydroxy-1,4-naphthoquinone-3-propionamide], presents negative current change following anti-atrazine antibody complexation and positive current change after atrazine addition in solution. This constitutes a direct, label-free and signal-on electrochemical immunosensor, with a very low detection limit of ca. 1 pM, i.e. 0.2 ng L(-1), one of the lowest reported for such immunosensors. This is far lower than the detection limit required by the European Union directives for drinkable water and food samples (0.1 μg L(-1)). The strategy described has great promise for the development of simple, cost-effective and reagentless on-site environmental monitors.

The cell differentiation-inducing effect of 2-N,N-diethylaminocarbonyloxymethyl-1-diphenylmethyl-4-(3,4,5-trimethoxybenzoyl) piperazine, hydrochloride (PMS-1077) was determined in human leukaemic HL-60 cells with profiling of cell proliferation, analysis of cell cycling, characterization of expression of various CD molecules and determination of phagocytotic activity of differentiated HL-60 cells. After treatment with PMS-1077, HL-60 cells exhibited a decreased cell viability during which cell cycle was arrested in G₀-/G₁-phase. Flow cytometric analysis showed CD11b and CD14 were up-regulated, whereas CD15 was unaffected. Together with the finding that PMS-1077-treated HL-60 cells exhibited activities of differentiation by examining their ability of phagocytosing latex beads, an antiproliferative effect and a differentiation-inducing role were determined for PMS-1077 in HL-60 cells.

A synthetic procedure for construction of the (+)-(3R,5R,6R)-5,6-dihydroxy-3,7-dimethyl-octanal and (−)-(3S,5S,6S)-5,6-dihydroxy-3,7-dimethyl-octanal derivatives, the intermediates for synthesis of the HIV-active didemnaketal analogue, was developed via a series of reactions from the natural (−)-menthone.

1-Benzoyl-2-alkyl piperazines are strong inhibitors of Group I and II secreted PLA2s. An improvement of their activity was obtained by replacing the amide function by a sulfamide and by introduction of electrodonor substituents on the para position of the benzenesulfonyl moiety. Neither the position on one of the carbon of the piperazine ring nor the absolute configuration of this carbon have an effect on the affinity for one or the other group of PLA2, but the lipophilicity remains for these series an essential parameter. In addition structure–activity relationships allow new hypothesis on interaction of these piperazine derivatives with the catalytic site of PLA2s.

The neuroregulatory activities of PMS-601, a platelet activating factor antagonist, were investigated in laboratory and animal models of HIV-1 encephalitis (HIVE). For the former, PMS-601 reduced monocyte-derived macrophage pro-inflammatory factors, multinucleated giant cell (MGC) formation, and neuronal loss independent of antiretroviral responses. PMS-601 treatment of HIVE severe combined immunodeficient mice showed reduced microgliosis, MGC formation and neurodegeneration. These observations support the further development of PMS-601 as an adjunctive therapy for HIV-1 associated neurocognitive disorders.

Piperazine is one of the heterocycles which are associated with diverse pharmacological activities. 2-N,N-Diethylaminocarbonyloxymethyl-1-diphenylmethyl-4-(3,4,5-trimethoxybenzoyl) piperazine hydrochloride (PMS-1077) is a trisubstituted piperazine which contains a trimethoxybenzene ring and a benzhydrylpiperazine fragment, both of which can induce cell proliferation regression by different mechanisms. We have therefore examined the effects of PMS-1077 on Human Burkitt's lymphoma cells (Raji). The viability of Raji cells was determined by MTT assay and also assessed by trypan blue dye exclusion method. The results demonstrate that PMS-1077 can suppress the proliferation of Raji cells in a dose- and timedependent manner, while inhibit colony formation ability of Raji cells merely in a dose-dependent manner in vitro. Meanwhile, morphological changes were observed using fluorescence microscope. Flow cytometric analysis through PI stains showed that PMS-1077 blocked the growth of Raji cells in the G(0)/G(1) period, and induced apoptosis of Raji cells after 48 h of incubation. Cell apoptosis induced by PMS-1077 was further confirmed by staining with Annexin-V FITC and PI. Preliminary study by molecular docking suggests that PMS-1077 may inhibit tubulin polymerization. More experiments are in progress in our laboratory to reveal the mode of action of PMS-1077 in the induction of apoptosis of Raji cells.

Amyloid-beta (Abeta) plays a central role in the neuroinflammation and cholinergic neuronal apoptosis in Alzheimer's disease, and thus has been considered as a main determinant of this disease. In the previous study, we reported that PMS777, a novel bis-interacting ligand for acetylcholinesterase (AChE) inhibition and platelet-activating factor (PAF) receptor antagonism, could significantly attenuate PAF-induced neurotoxicity. Continuing our efforts, we further investigated the protective effect of PMS777 on Abeta-induced neuronal apoptosis in vitro and neuroinflammation in vivo. PMS777 (1-100 microM) was found to inhibit Abeta-induced human neuroblastoma SH-SY5Y cell apoptosis in a concentration-dependent manner. Concurrently, PMS777 increased ratio of bcl-2 to bax mRNA, and inhibited both mRNA expression and activity of caspase-3 in SH-SY5Y cells after the exposure with Abeta. In vivo experimental study demonstrated that PMS777 could attenuate Abeta-induced microglial and astrocytic activation in the rat hippocampus after systemic administration. These results suggest that PMS777 potently protects against Abeta-induced neuronal apoptosis and neuroinflammation, and warrants further investigations in connection with its potential value in the treatment of Alzheimer's disease.

The nucleocapsid protein (NC) plays seminal roles in HIV replication, thus representing a major drug target. NC functions rely on its two zinc-fingers and flanking basic residues. Zinc ejectors inhibit NC functions, but with limited specificity. New classes of molecules competing with NC or its viral nucleic acid and enzyme partners are reviewed here.

Inflammatory injury and induction of oxidative stress have been implicated as causative factors in neurodegenerative diseases such as Alzheimer's disease (AD). Using LPS-stimulated RAW 264.7 macrophages as a model of inflammatory injury, LPS was found to stimulate ROS production (159%), GSH depletion (15%) and loss of mitochondrial activity (32%) as well as TNFalpha release (40%), and NO production (13.7 times), all parameters involved in AD. PMS777, a tetrahydrofuran derivative, designed as a dual PAF and acetylcholinesterase inhibitor, was found to decrease ROS (up to 32%) and NO production (up to 5 times), TNFalpha release (33%). PMS777 also prevents loss of mitochondrial activity, and GSH depletion. In contrast, tacrine was found to decrease ROS production (57% up to 102%) and TNFalpha level (up to 30%). It decreases NO release only at the highest concentrations without preventing loss of mitochondrial activity and GSH depletion. In this study, we show that PMS777 is strongly anti-inflammatory against LPS-induced responses in RAW 264.7. Differential effects between PMS777 and tacrine could be attributed to the anti-PAF activity of PMS777 which was able to fight inflammatory events and oxidative injury whereas tacrine only minimizes them. PMS777 could open a new approach in the treatment of AD.

(1) HIV-1 and viral proteins-evoked chronic brain inflammation, which is characterized by microglial activation, is the pivotal neuropathogenesis of HIV-1-associated dementia (HAD). Platelet-activating factor (PAF), mainly released from activated microglia and acts as a high potent inflammatory mediator and a neurotoxin, is indicated to be a principle initiator of neuroinflammation, neuronal dysfunction, and apoptosis related to HAD. Thus, bis-interacting ligands of acetylcholinesterase (AChE) inhibition and PAF receptor antagonism would be of great interest in the therapeutic potential of HAD not only for improvement of cognitive performance, but also for disease-modifying. (2). We have previously reported that a novel tetrahydrofuran-derived bis-interacting ligand PMS777 had satisfying potencies for PAF receptor blockade and AChE inhibition, and markedly improved cholinergic dysfunction-induced cognitive impairment in mice. Continuing with our research, we further investigated the neuroprotective activities of PMS777 on PAF-triggered neuronal injury in human neuroblastoma SH-SY5Y cells. (3) The bis-interacting ligand PMS777 (10 muM) obviously alleviated PAF-induced cell apoptosis in SH-SY5Y cells. Pretreatment with PMS777 also markedly inhibited intracellular Ca(2+) overload, down-regulation of anti-apoptotic bcl-2 mRNA, stimulation of pro-apoptotic bax mRNA expression and activation of caspase-3 pathway. Also, PMS777 could fine-tune pro-inflammatory cyclooxygenase-2 (cox-2) mRNA expression in PAF-treated cells. (4) These results suggest that PMS777 possesses a neuroprotective profile via anti-apoptotic/inflammatory signaling and warrant further investigations in connection with the potential value of this compound in HAD treatment.

Secreted phospholipases A2 (sPLA2s) have been reported to play an important role in various inflammatory conditions and thus represent an attractive therapeutic target. Previous SAR studies from our laboratory have revealed certain important features of our recently discovered specific hGIIA sPLA2 inhibitors, and we report here the synthesis and biological activities of glycerol-containing derivatives of our lead compound III (Figure 1). Efficient and selective synthesis methods have been developed to make glycerol trisubstituted by different groups on desired positions. In terms of biological activities, the best compounds (A3, A6, and A15) are more active than III (Figure 1), as potent as Me-Indoxam, an sPLA2s inhibitor of reference, against hGIIA, hGV, and hGX sPLA2s and at least 10 times less active toward the GIB enzymes in two in vitro assay systems. By synthesis of enantiopure (S)-A6, we demonstrated that no important improvement of the inhibitory potency could be achieved by this approach. Furthermore, the results show that the global lipophilicity is likely responsible for the anti-PLA2 activity and two oxadiazolone moieties seem too big to be accommodated by the active site of the hGIIA enzyme.

Based on the positions of the first two conservative cys-teine residues, chemokines are classified into four sub-families: CXC, CC, CX3C, and C. As a viral chemokine,vMIP-II shows a broad spectrum of binding to CXC, CC,and CX3C chemokine receptors. In particular, vMIP-IIbinds to both CXCR4 and CCR5, which are the only twomajor coreceptors of human immunodeficiency virusType 1 (HIV-1). Although most mammalian chemokinesare agonists to their corresponding receptors, vMIP-II isin most cases an antagonist to its receptors includingCXCR4 and CCR5.

Stepwise solid phase synthesis using the Fmoc chemistry is reported for a panel of 71-residue and novel unnatural chemokine analogs derived from vMIP-II. This demonstrates the feasibility of using this synthetic method to generate de novo designed protein ligand molecules to study the biology and pharmacology of chemokine receptors.

Human immunodeficiency virus type 1 (HIV-1) uses a chemokine receptor, usually CXCR4 or CCR5, for entry into the target cells. Here, we used a chemical biology approach to demonstrate that binding and signaling domains in CXCR4 are possibly distinct and separate, as the new analogue, D(1-10)-vMIP-II-(9-68)-SDF-1alpha (RCP222), could not activate CXCR4 despite the fact that its binding activity was comparable to that of stromal cell-derived factor (SDF)-1alpha, the only natural ligand of CXCR4.

The chemokine receptor CXCR4 plays an important role as the receptor for the normal physiological function of stromal cell-derived factor 1α (SDF-1α) and the coreceptor for the entry of human immunodeficiency virus type 1 (HIV-1) into the cell. In a recent work (S. Tian et al., J. Virol.79:12667-12673, 2005), we found that many residues throughout CXCR4 transmembrane (TM) and extracellular loop 2 domains are specifically involved in interaction with HIV-1 gp120, as most of these sites did not play a role in either SDF-1α binding or signaling. These results provided direct experimental evidence for the distinct functional sites on CXCR4 for HIV-1 and the normal ligand SDF-1α. To further understand the CXCR4-ligand interaction and to develop new CXCR4 inhibitors to block HIV-1 entry, we have recently generated a new family of unnatural chemokines, termed synthetically and modularly modified (SMM) chemokines, derived from the native sequence of SDF-1α or viral macrophage inflammatory protein II (vMIP-II). These SMM chemokines contain various de novo-designed sequence replacements and substitutions byd-amino acids and display more enhanced CXCR4 selectivity, binding affinities, and/or anti-HIV activities than natural chemokines. Using these novel CXCR4-targeting SMM chemokines as receptor probes, we conducted ligand binding site mapping experiments on a panel of site-directed mutants of CXCR4. Here, we provide the first experimental evidence demonstrating that SMM chemokines interact with many residues on CXCR4 TM and extracellular domains that are important for HIV-1 entry, but not SDF-1α binding or signaling. The preferential overlapping in the CXCR4 binding residues of SMM chemokines with HIV-1 over SDF-1α illustrates a mechanism for the potent HIV-1 inhibition by these SMM chemokines. The discovery of distinct functional sites or conformational states influenced by these receptor sites mediating different functions of the natural ligand versus the viral or synthetic ligands has important implications for drug discovery, since the sites shared by SMM chemokines and HIV-1 but not by SDF-1α can be targeted for the development of selective HIV-1 inhibitors devoid of interference with normal SDF-1α function.

Acetylcholinesterase inhibitors are commonly used as cognitive enhancers for dementia in aged people. Among them, tacrine (THA) but not galanthamine, was shown to exhibit hepatotoxicity which reduces its clinical use. PMS777, both a PAF antagonist and a new potent acetylcholinesterase inhibitor was recently demonstrated to reverse scopolamine-induced amnesia in mice without toxicity. In the present study, the effects of THA, galanthamine and PMS777 were compared in HepG2 cells on the oxidative parameters involved in the reported hepatotoxicity of THA. THA (or = 10 microM) induced an oxidative stress as shown by elevated ROS and MDA production and by a decrease in GSH level. Moreover, mitochondrial membrane potential and redox status were decreased. At low concentrations (or =10 microM), there was no significant disturbance. None of the oxidative stress markers was affected by PMS777 up to the maximum concentration tested and it is suggested that PMS777 is not cytotoxic for HepG2 cells. Galanthamine was also without cytotoxicity. Our results suggest that the toxic effect of THA above 10 microM may be caused by drug-induced mitochondrial energization impairment and destabilisation of membrane phospholipids associated with an oxidative stress. In contrast by preventing these dysfunctions, PMS777 could be safer than THA.

The HIV-1 central nervous system infection leads to the onset of neurological impairments called AIDS dementia complex (ADC). PAF plays an important role in this pathology, as it is an HIV-1-induced neurotoxin produced by infected or activated macrophages and microglia, in the brain. We previously reported that PAF-antagonists bearing a trisubstituted piperazine presented in vitro anti-HIV-1 activity in human macrophages. To improve the pharmacological activities of our lead compound, 1a, we modified its carbamate function and evaluated both its antiretroviral and anti-PAF activities. One carbamate derivative (10c) demonstrated a similar antiviral activity but a higher anti-PAF potency, whereas 4a, with an ureide function, presents an increased antiviral activity and can be considered as a pure antiretroviral drug, as it does not present PAF-antagonism. Moreover, we measured the ability of 1a to cross the blood-brain barrier, using the in situ mouse brain perfusion method and its plasmatic concentrations after iv and po administration. The transport parameter measured (K(in)) proves that 1a is able to cross this biological barrier, but a pharmacokinetic study reveals its weak bioavailability in rats.

We have recently reported the discovery of a series of specific inhibitors of human group IIA phospholipase A2 (hGIIA PLA2) to display promising in vitro and in vivo properties. Here we describe the influence of different structural modifications on the specificity and potency against hGIIA PLA2 versus porcine group IB PLA2. The SAR results, as well as the log P and pKa values of oxadiazolone determined in this work, provide important information towards the comprehension of the mode of action of this kind of compounds.

Starting from 4-tetradecyloxybenzamidine (PMS815), a non-specific inhibitor of GI and GII PLA2s, we report in this work the discovery of the specificity through design, synthesis and structure-activity relationships studies of different kinds of PMS815 derivatives. The leading compound, 4,5-dihydro-3-(4-tetradecyloxybenzyl)-1,2,4-4H-oxadiazol-5-one (9b, PMS1062) exhibits a micromolar IC50 towards three group II PLA2s, while inactive towards four group I and one group III enzymes in two in vitro enzymatic assay conditions. It is also able to block the PLA2-II activities induced by LPS and IL-6 in HepG2 cell line and no cytotoxicity is observed when PMS1062 is tested up to a concentration of 100 microM in two different cell lines (A549 and LLC-PK1).

Human group IIA secretory phospholipase A2 (hGIIA sPLA2) is reported to be involved in inflammation, since its expression level is enhanced under various inflammatory conditions. In this work, we report the total chemical synthesis of this enzyme (124 amino acids) by solid-phase method. The identity of the protein, in denatured or folded (7 disulphide bonds) forms, was confirmed by electrospray MS. Synthetic sPLA2 possesses the same circular dichroism spectrum, enzymic activity in hydrolysing different phospholipid substrates, and inhibitory effect in thrombin formation from prothrombinase complex as the recombinant sPLA2. Furthermore, LY311727, a reported specific hGIIA sPLA2 inhibitor, is able to inhibit the synthetic and the recombinant enzymes with the same efficiency. This study demonstrates that chemically continuous solid phase synthesis is an alternative and less time-consuming approach to producing small, structurally folded and fully active proteins of up to 124 amino acids, such as hGIIA sPLA2. Moreover, this technique provides more flexibility in analogue synthesis to elucidate their physiological functions and pathological effects.

Chemokine receptor CXCR4 plays an important role in the immune system and the cellular entry of human immunodeficiency virus type 1 (HIV-1). To probe the stereospecificity of the CXCR4-ligand interface, d-amino acid peptides derived from natural chemokines, viral macrophage inflammatory protein II (vMIP-II) and stromal cell-derived factor-1alpha (SDF-1alpha), were synthesized and found to compete with (125)I-SDF-1alpha and monoclonal antibody 12G5 binding to CXCR4 with potency and selectivity comparable with or higher than their l-peptide counterparts. This was surprising because of the profoundly different side chain topologies between d- and l-enantiomers, which circular dichroism spectroscopy showed adopt mirror image conformations. Further direct binding experiments using d-peptide labeled with fluorescein (designated as FAM-DV1) demonstrated that d- and l-peptides shared similar or at least overlapping binding site(s) on the CXCR4 receptor. Structure-activity analyses of related peptide analogs of mixed chiralities or containing alanine replacements revealed specific residues at the N-terminal half of the peptides as key binding determinants. Acting as CXCR4 antagonists and with much higher biological stability than l-counterparts, the d-peptides showed significant activity in inhibiting the replication of CXCR4-dependent HIV-1 strains. These results show the remarkable stereochemical flexibility of the CXCR4-peptide interface. Further studies to understand the mechanism of this unusual feature of the CXCR4 binding surface might aid the development of novel CXCR4-binding molecules like the d-peptides that have high affinity and stability.

1-Benzoyl-2-alkyl piperazines are strong inhibitors of Group I and II secreted PLA2s. An improvement of their activity was obtained by replacing the amide function by a sulfamide and by introduction of electrodonor substituents on the para position of the benzenesulfonyl moiety. Neither the position on one of the carbon of the piperazine ring nor the absolute configuration of this carbon have an effect on the affinity for one or the other group of PLA2, but the lipophilicity remains for these series an essential parameter. In addition structure–activity relationships allow new hypothesis on interaction of these piperazine derivatives with the catalytic site of PLA2s.

The HIV-1 NCp7 contains two spatially close zinc fingers, required for the production of infectious particles. To investigate in more detail the function of the zinc finger domain, monoclonal antibodies were generated with a cyclic analog of the NCp7 proximal zinc finger. This analog was shown to bind zinc ions and to preserve the highly folded structure of the native peptide (Dong C-Z et al.: J Am Chem Soc 1995;117:2726-2731). We report here two monoclonal antibodies (2B10 and 4D3), which are the first monoclonal antibodies directed against CCHC NCp7 zinc fingers. Dot-blot experiments revealed that a few nanograms of synthetic NCp7 can be detected on a nitrocellulose membrane. Whereas 2B10 appears specific for an epitope located in sequence 19-27 of NCp7, 4D3 appears to be structurally specific. Immunocomplex affinities were evaluated, using BIAcore technology, to be up to 1 and 10 nM, respectively, for 2B10 and 4D3 in 100 mM NaCl. These antibodies were able to recognize NCp7 in the Gag polyprotein precursor and were shown to immunoprecipitate NCp7 from a cell supernatant. Moreover, NCp7-Vpr interaction mediated by the zinc fingers is inhibited by 2B10, emphasizing the role of these domains in the protein-protein complex. These results indicate that 2B10 and 4D3 behave as useful tools for studying both NC protein functions during the course of virion morphogenesis and the role played by its zinc finger domain at various steps in the retroviral life cycle.

Our previous studies have demonstrated that PMS1077, a platelet-activating factor (PAF) antagonist, could induce apoptosis of Raji cells. However, the mechanism of action has not yet been determined. The nuclear transcription factor-kappa B (NF-κB) signaling pathway plays a critical role in tumor cell survival, proliferation, invasion, metastasis, and angiogenesis, so we determined the effects of PMS1077 and its structural analogs on tumor necrosis factor-α (TNF-α) induced activation of NF-κB signaling. In this study, we found that PMS1077 inhibited TNF-α induced expression of the NF-κB regulated reporter gene in a dose dependent manner. Western blot assay indicated that PMS1077 suppressed the TNF-α induced inhibitor of κB-α (IκB-α) phosphorylation, IκB-α degradation, and p65 phosphorylation. PMS1077 consistently blocked TNF-α induced p65 nuclear translocation as demonstrated in the immunofluorescence assay used. Docking studies by molecular modeling predicted that PMS1077 might interact directly with the IκB kinase-β (IKK-β) subunit. These results suggested that PMS1077 might suppress the activation of NF-κB by targeting IKK-β involved in the NF-κB signaling pathway. Finally, we showed that PMS1077 sensitized cells to TNF-α induced apoptosis by suppressing the expression of NF-κB regulated anti-apoptotic genes. Our results reveal a novel function of PMS1077 on the NF-κB signaling pathway and imply that PMS1077 can be considered as an anti-tumor lead compound.