Synthesis and characterisation of chitosan-graft-poly(OEGMA) copolymers prepared by ATRP

Two synthetic routes, “grafting-from” and “grafting-to” the chitosan backbone, were investigated to prepare chitosan-graft-poly(OEGMA) copolymers by ATRP. The copolymers were characterised by ¹H NMR and FT-IR spectroscopy, elemental analyses and GPC. Estimates of the degree of grafting were quantitatively found from integration of the ¹H NMR spectra. The GPC data indicated that the hydrodynamic volumes of the polymers were significantly altered by the incorporation of poly(OEGMA). A comparison of the two synthetic methods showed differences in the ratio of synthetic and natural polymers present in the resulting combs. The “grafting-to” synthetic route was preferable as the polymers were of higher purity and more defined.     

Synthesis and biological evaluation of novel 5-alkyl-2-arylthio-6-((3,4-dihydroquinolin-1(2H)-yl)methyl)pyrimidin-4(3H)-ones as potent non-nucleoside HIV-1 reverse transcriptase inhibitors

A series of novel S-DABO analogues of 5-alkyl-2-arylthio-6-((3,4-dihydroquinolin-1(2H)-yl)methyl)pyrimidin-4(3H)-ones were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were compounds 6c1,6c6, and 6b1 (EC(50)=0.24 ± 0.05, 0.38 ± 0.13, 0.39 ± 0.05 μM, respectively), which possess improved or similar HIV-1 inhibitory activity compared with nevirapine (NVP) (EC(50)=0.21 μM) and delavirdine (DLV) (EC(50)=0.32 μM). None of these compounds were active against HIV-2 replication. Furthermore, enzyme inhibitory assays were performed with selected derivatives against HIV-1 wtRT, confirming that the main target of these compounds is the HIV-1 RT and these new S-DABOs are acting as NNRTIs. The preliminary structure-activity relationship (SAR) of these new congeners is discussed briefly and rationalized by docking studies.     

Thermoprecipitation of lysozyme from egg white using copolymers of N-isopropylacrylamide and acidic monomers

Thermoprecipitation of lysozyme from egg white was demonstrated using copolymers of N-isopropylacrylamide with acrylic acid, methacrylic acid, 2-acryloylamido-2-methylpropane-sulfonic acid and itaconic acid, respectively. Polymers synthesized using molar feed ratio of N-isopropylacrylamide:acidic monomers of 98:2 exhibited lower critical solution temperatures in the range of 33--35 degrees C. These polymers exhibited electrostatic interactions with lysozyme and inhibited its bacteriolytic activity. The concentration of acidic groups required to attain 50% relative inhibition of lysozyme by the polymers, was 10(4)--10(5) times lower than that required for the corresponding monomers. This was attributed to the multimeric nature of polymer-lysozyme binding. More than 90% lysozyme activity was recovered from egg white. Polymers exhibited reusability up to at least 16 cycles with retention of >85% recovery of specific activity from aqueous solution. In contrast, copolymer comprising natural inhibitor of lysozyme i.e. poly (N-isopropylacrylamide-co-O-acryloyl N-acetylglucosamine) lost 50% recovery of specific activity. Thermoprecipitation using these copolymers, which enables very high recovery of lysozyme from egg white, would be advantageous over pH sensitive polymers, which generally exhibit lower recovery.     

Synthesis and carbonic anhydrase inhibitory properties of novel 4-(2-aminoethyl)benzenesulfonamide-dipeptide conjugates

Thirty novel sulfonamide derivatives incorporating dipeptide were synthesized by facile acylation through benzotriazole mediated reactions and their structures were identified by ¹H NMR, ¹³C NMR, MS and FT-IR spectroscopic techniques and elemental analysis. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against four human (h) isoforms, hCA I, hCA II, hCA IV and hCA XII. Most of the synthesized compounds showed excellent in vitro carbonic anhydrase inhibitory properties comparable to those of the clinically used drug acetazolamide (AAZ). The new unprotected dipeptide-sulfonamide conjugates showed very effective inhibitory activity, in the low nanomolar range against II and XII, being less effective as hCA I and IV inhibitors. Four of the thirty compounds also showed strong inhibitory activity against hCA XII compared to AAZ.     

Multiple antiviral activities of cyanovirin-N: blocking of human immunodeficiency virus type 1 gp120 interaction with CD4 and coreceptor and inhibition of diverse enveloped viruses

Cyanovirin-N (CV-N) is a cyanobacterial protein with potent neutralizing activity against human immunodeficiency virus (HIV). CV-N has been shown to bind HIV type 1 (HIV-1) gp120 with high affinity; moreover, it blocks the envelope glycoprotein-mediated membrane fusion reaction associated with HIV-1 entry. However, the inhibitory mechanism(s) remains unclear. In this study, we show that CV-N blocked binding of gp120 to cell-associated CD4. Consistent with this, pretreatment of gp120 with CV-N inhibited soluble CD4 (sCD4)-dependent binding of gp120 to cell-associated CCR5. To investigate possible effects of CV-N at post-CD4 binding steps, we used an assay that measures sCD4 activation of the HIV-1 envelope glycoprotein for fusion with CCR5-expressing cells. CV-N displayed equivalently potent inhibitory effects when added before or after sCD4 activation, suggesting that CV-N also has blocking action at the level of gp120 interaction with coreceptor. This effect was shown not to be due to CV-N-induced coreceptor down-modulation after the CD4 binding step. The multiple activities against the HIV-1 envelope glycoprotein prompted us to examine other enveloped viruses. CV-N potently blocked infection by feline immunodeficiency virus, which utilizes the chemokine receptor CXCR4 as an entry receptor but is CD4 independent. CV-N also inhibited fusion and/or infection by human herpesvirus 6 and measles virus but not by vaccinia virus. Thus, CV-N has broad-spectrum antiviral activity, both for multiple steps in the HIV entry mechanism and for diverse enveloped viruses. This broad specificity has implications for potential clinical utility of CV-N.     

Synthesis of acyl thiourea derivatives of chitosan and their antimicrobial activities in vitro

Three different acyl thiourea derivatives of chitosan (CS) were synthesized and their structures were characterized by FT-IR spectroscopy and elemental analysis. The antimicrobial behaviors of CS and its derivatives against four species of bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Sarcina) and four crop-threatening pathogenic fungi (Alternaria solani, Fusarium oxysporum f. sp. vasinfectum, Colletotrichum gloeosporioides (Penz.) Saec, and Phyllisticta zingiberi) were investigated. The results indicated that the antimicrobial activities of the acyl thiourea derivatives are much better than that of the parent CS. The minimum value of MIC and MBC of the derivatives against E. coli was 15.62 and 62.49 microg/mL, respectively. All of the acyl thiourea derivatives had a significant inhibitory effect on the fungi in concentrations of 50-500 microg/mL; the maximum inhibitory index was 66.67%. The antifungal activities of the chloracetyl thiourea derivatives of CS are noticeably higher than the acetyl and benzoyl thiourea derivatives. The degree of grafting of the acyl thiourea group in the derivatives was related to antifungal activity; higher substitution resulted in stronger antifungal activity.     

Synthesis, biological evaluation, and molecular docking studies of 2,5-substituted-1,4-benzoquinone as novel urease inhibitors

A series of 2,5-substituted-1,4-benzoquinone (1-6) were prepared and structurally characterized by elemental analysis, IR spectra, (1)H and (13)C NMR spectra, and single crystal X-ray determination. The urease inhibitory activities of the compounds against H. pylori urease were studied. Among the compounds, 2,5-bis(2-morpholin-4-ylethylamino)-[1,4]benzoquinone (2) shows the most effective activity with IC(50) value of 27.30±2.17μM. Docking simulation was performed to insert compound 2 into the crystal structure of H. pylori urease at the active site to determine the probable binding mode. As a result, compound 2 may be used as a potential urease inhibitor.     

New organotropic compounds. Synthesis,characterization and reactivity of Pt(II) and Au(III) complexes with bile acids: DNA interactions and 'in vitro' anticancer activity

Based on the ability of bile acids to vectorialize the cytostatic activity of other agents, we have designed and synthesized a new series of platinum and gold complexes. These compounds were studied and characterized by elemental analysis, FT-IR, FAB(+)/MS, 1H, 13C and 195Pt NMR, UV-Vis spectroscopy and conductivity measurements in solution, among other techniques. Kinetic studies carried out in aqueous solution and in the presence of different NaCl concentrations: 4 mM (similar to cytoplasmic concentration), 150 mM (similar to plasmatic concentration). The effects on the electrophoretic mobility of the pUC18 plasmid, the DNA denaturation temperature, and ethidium bromide (EtBr) binding to DNA were studied. The complexes are able to inter-react with DNA to inhibit DNA synthesis and hence, to reduce cell proliferation. The complexes were evaluated for in vitro cytostatic activity against human colon adenocarcinoma, mouse hepatoma, human hepatoma, mouse leukemia, etc. The antitumor effect of some of the compounds prepared was similar to that of cisplatin. However, other compounds had lower cytostatic activity. This different behavior can be accounted for by the structure/activity relationship (SAR), although other factors, such as uptake and the different kinetic behavior in solution, may be responsible for these differences.     

Enhancement of Human Immunodeficiency Virus Type 1 (HIV-1) Infection via Increased Membrane Fluidity by a Cationic Polymer

Cationic polymers are known to have potent activity against bacteria, but their effects on viral activity have been little studied. We investigated the effect of one such polymer, polyethyleneimine (PEI), on HIV-1 infection. Although virus-cell binding was significantly inhibited by PEI, HIV-1 infection in human T-cell lines such as MT-4 and MOLT-4 was accelerated conversely when the drug treatment was carried out, after the virus had attached to the cells or PEI was simultaneously added to the virus and cell culture system. This paradoxical effect of PEI on HIV-1 infection was examined using HIV-1 chronically infected cells (MOLT-4/HIV-1). Dissociation of the glycoprotein gp120 (as revealed by exposure of transmembrane protein gp41) from MOLT-4/HIV-1 cells and the resultant fusion of these cells was shown to be induced by the addition of PEI. Accordingly, it was suggested that the binding inhibition of HIV-1 to CD4-positive cells by PEI was due to the shedding of gp120 from HIV-1 particles, and this PEI rather promoted membrane fusion between the virus and cells leading to the enhancement of HIV-1 infection. Similarly, dissociation of gp120 from MOLT-4/HIV-1 was also induced by sCD4. The effect of these reagents on changes in membrane fluidity was evaluated by polarization (p) measurements, and it was observed that the acceleration of membrane fluidity occurred only in the PEI system. Therefore, it is likely that PEI accelerates HIV-1 infection by facilitating virus entry into the host cells through an increase in membrane fluidity.     

Mediation of human immunodeficiency virus type 1 binding by interaction of cell surface heparan sulfate proteoglycans with the V3 region of envelope gp120-gp41.

The mechanism of heparan sulfate (HS)-mediated human immunodeficiency virus type 1 (HIV-1) binding to and infection of T cells was investigated with a clone (H9h) of the T-cell line H9 selected on the basis of its high level of cell surface CD4 expression. Semiquantitative PCR analysis revealed that enzymatic removal of cell surface HS by heparitinase resulted in a reduction of the amount of HIV-1 DNA present in H9h cells 4 h after exposure to virus. Assays of the binding of recombinant envelope proteins to H9h cells demonstrated a structural requirement for an oligomeric form of gp120/gp41 for HS-dependent binding to the cell surface. The ability of the HIV-1 envelope to bind simultaneously to HS and CD4 was shown by immunoprecipitation of HS with either antienvelope or anti-CD4 antibodies from 35SO4(2-)-labeled H9h cells that had been incubated with soluble gp140. Soluble HS blocked the binding of monoclonal antibodies that recognize the V3 and C4 domains of the envelope protein to the surface of H9 cells chronically infected with HIV-1IIIB. The V3 domain was shown to be the major site of envelope-HS interaction by examining the effects of both antienvelope monoclonal antibodies and heparitinase on the binding of soluble gp140 to H9h cells.     

Inhibition of HIV-1 proviral DNA synthesis and RNA accumulation by mismatched dsRNA

The antiviral activity of mismatched dsRNA of the form poly(I):poly(C12-U)n (Ampligen) against the human immunodeficiency virus type 1 (HIV-1) was investigated by RNA-RNA and RNA-DNA hybridizations. Mismatched dsRNA delayed the appearance of newly transcribed HIV-1 RNA as detected by liquid dot-blot hybridization in cultures of H9 T-lymphoblastoid cells following virus challenge. The appearance of proviral DNA as detected by Southern hybridization following virus challenge in H9 cells was also delayed. Mismatched dsRNA had no effect in syncytium inhibition assays performed by fusing MT-2 cells with H9/HTLV-IIIB cells. These results suggest that the in vitro anti-HIV-1 activity of mismatched dsRNA occurs, at least in part, at an early stage in the viral replication cycle following initial gp120-CD4 binding.     

Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120.

Monoclonal antibodies (MAbs) were obtained by immunizing mice with synthetic peptides corresponding to the third variable (V3) or the third conserved (C3) domain of the external envelope protein (gp120) of human immunodeficiency virus type 2 (HIV-2ROD). One MAb, designated B2C, which was raised against V3 peptide NKI26, bound to the surface of HIV-2-infected cells but not to their uninfected counterparts. B2C was capable of neutralizing cell-free and cell-associated virus infection in an isolate-specific fashion. The antibody-binding epitope was mapped to a 6-amino-acid peptide in the V3 variable domain which had the core sequence His-Tyr-Gln. Two MAbs, 2H1B and 2F19C, which were raised against the C3 peptide TND27 reacted with gp120 of HIV-2ROD in a Western immunoblot assay. The C3 epitopes recognized by these two MAbs appeared inaccessible because of their poor reactivity in a surface immunofluorescence assay. Although partial inhibition of syncytium formation was observed in the presence of the anti-C3 MAbs, their neutralizing activity appeared weak. Finally, the effects of these MAbs against CD4-gp120 binding were assessed. Partial inhibition of CD4-gp120 binding was observed in the presence of high concentrations of B2C. On the other hand, no inhibition of CD4-gp120 binding was observed in the presence of anti-C3 MAbs. Since complete neutralization could be achieved at a concentration corresponding to that of partial binding inhibition by B2C, some different mechanisms may be involved in the B2C-mediated neutralization. These results, taken together, indicated that analogous to the function of the V3 region of HIV-1, the V3 region of HIV-2ROD contained at least a type-specific fusion-inhibiting neutralizing epitope. In this respect, the V3 sequence of HIV-2 may be a useful target in an animal model for HIV vaccine development.     

HIV-1 replication in Langerhans and interstitial dendritic cells is inhibited by neutralizing and Fc-mediated inhibitory antibodies

Langerhans cells (LCs) and interstitial dendritic cells (IDCs) may be among the first human immunodeficiency virus type 1 (HIV-1) targets after sexual transmission. We generated cells of these types by differentiation of purified CD34(+) cord blood cells. After in vitro infection with R5-tropic strains, we obtained similar percentages of infected cells for both dendritic cell (DC) subsets. Moreover, LC infection was not increased by blockage of langerin by antilangerin. These results indicate that, under our experimental conditions, there was no evidence of any preference of HIV replication in LCs versus IDCs. The inhibitory activity of HIV-1-specific IgAs and IgGs against HIV-1 replication in LCs and IDCs was analyzed. We found that neutralizing antibodies inhibit HIV-1 infection of both DC subsets. Interestingly, HIV-1 was inhibited more efficiently by the IgGs than the corresponding IgA, due to an Fcγ receptor-dependent mechanism. Moreover, nonneutralizing inhibitory IgGs were able to inhibit infection of both LCs and IDCs. These results underline the importance of HIV-1 inhibition by the binding of the Fc part of IgGs to Fcγ receptors and suggest that the induction of neutralizing and nonneutralizing inhibitory IgGs in addition to neutralizing IgAs at mucosal sites may contribute to protection against sexual transmission of HIV-1.     

HIV-1 protease inhibitors containing an N-hydroxyamino acid core structure

Two series of peptidomimetics containing an N-hydroxyamino acid core structure were prepared by mixed solution solid-phase synthesis and tested for inhibitory activity against the human immunodeficiency virus (HIV-1) protease (Pr) and the virus in cell culture. In general, N-hydroxy Gly containing pseudopeptides displayed modest HIV Pr inhibition (IC50 > or = 930 nM). In the N-hydroxy Phe derivatives, Fmoc-Phe-psi[CO-N(OH)]-Phe-Pro-NHtBu was the best inhibitor of the series (IC50 = 144nM) showing satisfactory inhibition of HIV replication in cell culture (ED50 = 98 nM) and remarkable stability against cell culture and plasma enzymes.     

The synthesis and biological evaluation of novel bridging nucleoside analogues.

Novel bridging nucleoside analogues were prepared by cycloaddition reactions between pyranose glycals and barbiturate-derived, reactive thionoimides in modest yields. In all of the reactions conducted, the major cycloadducts obtained were the bottom faced adducts resulting from endo addition to the glycal. The adducts were stable to a variety of acidic reaction conditions and several of the compounds showed moderate activities against HIV-1 in primary human lymphocytes. One compound displayed anti-herpes simplex virus type-1 activity in Vero cells. Cytotoxicity measurements were also obtained.     

Synthesis and in vitro cytotoxicity of novel lipophilic (diamine)platinum(II) complexes of salicylate derivatives

Novel lipophilic (diamine)platinum(II) complexes of salicylate derivatives as the leaving groups were synthesized and characterized by elemental analysis, FAB(+)-MS, FT-IR, and (1)H NMR spectroscopy. Most of the resulting platinum complexes had high solubility in organic solvents such as ethanol, acetone, and ether, and had right partition coefficient suited to be encapsulated in liposomes. The pertinent complexes were evaluated for their in vitro cytotoxicity against A549 human lung carcinoma and SGC-7901 human gastric carcinoma cell lines. They showed better cytotoxic activity than carboplatin and oxaliplatin.     

Design,synthesis, docking studies and biological evaluation of novel chalcone derivatives as potential histone deacetylase inhibitors

A group of novel chalcone derivatives comprising hydroxamic acid or 2-aminobenzamide group as zinc binding groups (ZBG) were synthesized. The structure of the prepared compounds was fully characterized by IR, NMR and elemental microanalyses. Most of the tested compounds displayed strong to moderate HDAC inhibitory activity. Some of these compounds showed potent anti-proliferative activity against human HepG2, MCF-7 and HCT-116 cell lines. In particular, compounds 4a and 4b exhibited significant anti-proliferative activity against the three cell lines compared to SAHA as reference drug and displayed promising profile as anti-tumor candidates. The results indicated that these chalcone derivatives could serve as a promising lead compounds for further optimization as antitumor agents.