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In recent years, based on peptide models of HIV-1 RNA binding, NMR structures of Tat-responsive element-ligand complexes and aminoglycoside-RNA interactions, and HIV-1 Tat structure, we have designed and synthesized aminoglycoside-arginine conjugates (AACs) and aminoglycoside poly-arginine conjugates (APACs), to serve as Tat mimetics. These novel molecules inhibit HIV-1 infectivity with 50% effective concentration values in the low micromolar range, the most potent compounds being the hexa-arginine-neomycin B and nona-D-arginine-neomycin conjugates. Importantly, these compounds, in addition to acting as Tat antagonists, inhibit HIV-1 infectivity by blocking several steps in HIV-1 cell entry. The AACs and APACs inhibit HIV-1 cell entry by interacting with gp120 at the CD4-binding site, by interacting with CXCR4 at the binding site of the CXCR4 mAb 12G5, and apparently by interacting with transient structures of the ectodomain of gp41. In the current review, we discuss the mechanisms of anti-HIV-1 activities of these AACs, APACs and other aminoglycoside derivatives in detail. Targeting several key processes in the viral life cycle by the same compound not only may increase its antiviral efficacy, but more importantly, may reduce the capacity of the virus to develop resistance to the compound. AACs and APACs may thus serve as leading compounds for the development of multitargeting novel HIV-1 inhibitors.

We present the design, synthesis, anti-HIV-1 and mode of action of neomycin and neamine conjugated at specific sites to arginine 6- and 9-mers d- and l-arginine peptides (APACs). The d-APACs inhibit the infectivity of X4 HIV-1 strains by one or two orders of magnitude more potently than their respective l-APACs. d-arginine conjugates exhibit significantly higher affinity towards CXC chemokine receptor type 4 (CXCR4) than their l-arginine analogs, as determined by their inhibition of monoclonal anti-CXCR4 mAb 12G5 binding to cells and of stromal cell-derived factor 1α (SDF-1α)/CXCL12 induced cell migration. These results indicate that APACs inhibit X4 HIV-1 cell entry by interacting with CXCR4 residues common to glycoprotein 120 and monoclonal anti-CXCR4 mAb 12G5 binding. d-APACs readily concentrate in the nucleus, whereas the l-APACs do not. 9-mer-d-arginine analogues are more efficient inhibitors than the 6-mer-d-arginine conjugates and the neomycin-d-polymers are better inhibitors than their respective neamine conjugates. This and further structure–function studies of APACs may provide new target(s) and lead compound(s) of more potent HIV-1 cell entry inhibitors.

Aminoglycoside-arginine conjugates (AACs) inhibit HIV-1 replication and act as Tat antagonists. AACs compete with monoclonal antibody binding to CXCR4, compete with SDF-1alpha and HIV-1 gp120 cellular uptake, indicating that they interfere with initial steps of HIV-1 infection. We here present the selection of HIV-1 isolates resistant to hexa-arginine neomycin B conjugate (NeoR6), the most potent anti-HIV-1 AAC. We found in the NeoR6-resistant isolates the following mutations in gp120: I339T in the C3 region, S372L in the V4 region, and Q395K in the C4 region; and in gp41: S668R and F672Y in the 'heptad repeat' 2 (HR2) region. These findings strongly suggest that NeoR6 obstructs HIV-1 replication by interfering with the fusion step, dependent on both conformational changes in gp120 following CD4 and CXCR4 interaction, as well as by conformational changes in gp41 induced by HR1 and HR2 interaction. The AACs may thus represent a novel family of fusion inhibitors.

We have recently designed and synthesized aminoglycoside-arginine conjugates (AACs) as potential anti-HIV-1 agents. AACs exert a number of activities related to Tat antagonism. We here present a new set of AACs, conjugates of neomycin B, paromomycin, and neamine with different number of arginines (1-6), their (a) uptake by human T-cell lines, (b) antiviral activities, (c) competition with monoclonal antibody (mAb) 12G5 binding to CXCR4, (d) competition with stromal cell-derived factor-1 (SDF-1alpha) binding to CXCR4, and (e) competition with HIV-1 coat protein gp120 cell penetration. The appearance of mutations in HIV-1 gp120 gene in AACs resistant HIV-1 isolates, supports that AACs inhibit HIV-1 infectivity via interference of gp120-CXCR4 interaction. Our results point that the most potent AACs is the hexa-arginine-neomycin conjugate, the other multi-arginine-aminoglycoside conjugates are less active, and the mono-arginine conjugates display the lowest activity. Our studies demonstrate that, in addition to the core, the number of arginines attached to a specific aminoglycoside, are also important in the design of potent anti-HIV agents. The AACs play an important role, not only as HIV-1 RNA binders but also as inhibitors of viral entry into human cells.

A requisite for vaccines to confer protection against intracellular infections such as Human Immunodeficiency Virus or Mycobacterium tuberculosis is their capacity to induce Th1 immune responses. However, they may fail to do so in Africa and South East Asia, where most individuals have a dominant preexistent Th2 immune profile, due to persistent helminthic parasitic infections, which may undermine any Th1 response. It is well established that DNA vaccines induce strong Th1 biased immune responses against an encoded antigen, depending on the route and mode of immunization. Here, we demonstrate that intradermal immunization with plasmid DNA encoding β-gal (pCMV-LacZ) of Schistosoma-infected mice, with preexistent dominant Th2 immune background, induce a strong Th1 anti-β-gal response, as opposed to immunized with β-gal only. Importantly, the established protective Th2 immune response to schistosomes was not disrupted. These findings strongly support the possibility of using plasmid DNA as a Th1 inducing adjuvant when immunizing populations with a strong preexistent Th2 immune profile.

In recent publications, oligonucleotides joined by 2',5'-linkages were found to bind to complementary single-stranded RNA but to bind weakly, or not at all, to single-stranded DNA [e.g., P. A. Giannaris and M. J. Damha (1993) Nucleic Acids Res. 21, 4742-4749]. In this work, the biochemical and physicochemical properties of 2',5'-linked oligoribonucleotides containing mixed sequences of the four nucleobases (A, G, C, and U) were evaluated. CD spectra of RNA:2', 5'-RNA duplexes were compared with the spectra of DNA:DNA, RNA:RNA, and DNA:RNA duplexes of the same base sequence. The CD results indicated that the RNA:2',5'-RNA duplex structure more closely resembles the structure of the RNA:DNA hybrid, being more A-form than B-form in character. The melting temperature (Tm) values of the backbone-modified duplexes were compared with the Tm values of the unmodified duplexes. The order of thermal stability was RNA:RNADNA:DNA approximately RNA:DNA approximately DNA:RNARNA:2',5'-RNA2',5'-RNA:2',5'-RNADNA:2',5'-RNA (undetected). RNA:2',5'-RNA duplexes are not substrates of the enzyme RNase H (Escherichia coli, or HIV-1 reverse transcriptase), but they can inhibit the RNase H-mediated cleavage of a natural DNA:RNA substrate. Structural models that are consistent with the selective association properties of 2',5'-linked oligonucleotides are discussed.

Antisense oligonucleotides (AONs) targeted to the R-region near the 5'-LTR of HIV-1 genomic RNA inhibited both the synthesis of (-) strong stop DNA and the first template-switch reaction catalysed by HIV-1 reverse transcriptase (RT) in vitro. The 18 nucleotide (nt) AONs used were identical in sequence but differed in the sugar component of the 3'-terminal nucleotide, with either 2'-deoxy-D-ribose (DNA), 2'-deoxy-L-ribose (L), or arabinose (ARA) in this position. All three AONs hybridized to complementary 18 nt RNA (T(m) approximately 70 degrees C) and specifically interacted with the target RNA HIV-1 sequence at 37 degrees C. L was unable to serve as primer for RT-catalysed DNA polymerization, whereas priming from ARA was about 30% that noted with DNA. Each of the three AONs resulted in similar 85-95% decreases in the amount of full length (-) strong stop DNA and up to 75% decreases in the first template-switch reaction products formed by RT, implying that elongation of the AONs did not enhance the inhibitory activity in vitro. A concomitant increase in a truncated DNA product corresponding to polymerization termination at the 5'-end of the AON was noted, indicating that RT was unable to displace the AON. Interestingly, near maximal inhibition in vitro an AON:target RNA template ratio of 1:1 was noted. Our results confirm the validity of our in vitro system for the analysis of potential antisense oligonucleotide inhibitors, and suggest that antisense oligonucleotides directed to the R-region of HIV-1 RNA may be effective inhibitors of the initial stages of HIV-1 proviral DNA synthesis.

We have recently developed a novel small animal model for HIV-1 infection (Ayash-Rashkovsky et al., http://www.fasebj.org/cgi/doi/10.1096/fj.04-3184fje; doi:10.1096/fj.04-3184fje). The mice were successfully infected with HIV-1 for 4-6 wk with different clades of either T- or M-tropic isolates. HIV-1 infection was accompanied by rapid loss of human CD4+ T cells, decrease in CD4/CD8 ratio, and increased T cell activation. HIV specific human humoral and cellular immune responses were observed in all HIV-1 infected animals. In the present study, HIV specific human immune responses, both humoral and cellular, were generated in noninfected Trimera mice, after their immunization with gp120-depleted HIV-1 antigen, presented by autologous human dendritic cells. Addition of CpG ODN to the antigen-pulsed DCs significantly enhanced (by 2- to 30-fold) the humoral and cellular HIV-1 specific immune responses. Only mice immunized with the HIV-1 immunogen and CpG were completely protected from infection with HIV-1 after challenge with high infection titers of the virus. This novel small animal model for HIV-1 infection may thus serve as an attractive platform for rapid testing of candidate HIV-1 vaccines and of adjuvants and may shorten the time needed for the development and final assessment of protective HIV-1 vaccines in human trials.

The K65R mutation in HIV-1 reverse transcriptase (RT) is associated with viral cross-resistance to 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and 2',3'-dideoxy-3'-thiacytidine. We have found that in vitro DNA synthesis by K65R RT is significantly more processive than that of wild type (wt) RT. Depending on the template/primer (T/P) used, the total incorporation of nucleotides under single processive cycle conditions was 20-50% higher with K65R RT than with wt RT. With heteropolymeric T/P, the total incorporation of dNMP by K65R and wt RT was similar under continuous DNA synthesis reaction conditions. However, under single processive cycle conditions, the rate of full-length polymerization product synthesis by K65R RT was about 2-fold higher than that by wt RT. We also found a decreased rate of T/P dissociation during K65R RT DNA synthesis, which is consistent with the increased processivity of the enzyme. We postulate that the increased processivity of the K65R RT may be a compensatory response to the decreased affinity of this mutant for certain dNTP substrates, allowing normal viral replication kinetics.

The synergism between the lytic activity of cytotoxin P4 isolated from the venom of Naja nigricollis nigricollis and various phospholipases (PLA) is demonstrated. The highest synergistic activity was obtained with one of the basic phospholipases found in the same venom. It was also shown between cytotoxin P4 and heterologous elapid and viperid snake phospholipases. Moreover, remarkable synergism was also demonstrated between cytotoxin P4 and bee venom PLA 2 or porcine pancreatic PLA 2 . However, the phospholipase fractions of Vipera palestinae and the bovine pancreas PLA 2 had no synergistic effect.

Skin aging is associated with the loss of the structural collagens and the elastin fiber components that form the extracellular matrix (ECM). It is associated with reduced transforming growth factor-β (TGF-β), angiogenesis and increased oxidative stress. Copper has been incorporated into cosmetics for anti-skin aging. This research investigated the mechanism for the anti-skin aging effect copper ions, from cuprous oxide powders. Dermal fibroblasts were exposed to copper and examined for expression (protein and/or promoter levels) of types I, III, V collagen, heat shock protein-47 (HSP-47), elastin, fibrillin-1, and fibrillin-2, TGF-β1, vascular endothelial growth factor (VEGF), and in addition for membrane damage and lipid peroxidation. The direct antioxidant activity of copper was also determined. The research indicates that copper's anti-skin aging and skin regeneration potential is through its stimulation of ECM proteins, TGF-β1, VEGF, and inhibition of oxidative stress effects at physiological concentrations; and supports its use in cosmetics.

Aminoglycoside-arginine conjugates (AACs) are multi-target HIV-1 inhibitors. The most potent AAC is neomycin hexa-arginine conjugate, NeoR6. We here demonstrate that NeoR6 interacts with CXCR4 without affecting CXCL12-CXCR4 ordinary chemotaxis activity or loss of CXCR4 cell surface expression. Importantly, NeoR6 alone does not affect cell migration, indicating that NeoR6 interacts with CXCR4 at a distinct site that is important for HIV-1 entry and mAb 12G5 binding, but not to CXCL12 binding or signaling sites. This is further supported by our modeling studies, showing that NeoR6 and CXCL12 bind to two distinct sites on CXCR4, in contrast with other CXCR4 inhibitors, e.g. T140 and AMD3100. This complementary utilization of chemical, biology, and computation analysis provides a powerful approach for designing anti-HIV-1 drugs without interfering with the natural function of CXCL12/CXCR4 binding.

The generation of small animal models, which preserve the ability for the generation of primary and memory immune responses of the engrafted human immune cells and in which a robust HIV-1 infection may occur, may enable the rapid screening, development and evaluation of HIV-1 protective vaccines and adjuvants. This manuscript reviews the existing mouse HIV-1 models used to study virologic, immunologic and pathogenic aspects of HIV-1 infection and disease and discusses their limitations and advantages, especially in the context of vaccine development, with special focus on the recently developed Trimera-HIV-1 animal model.

Copper ions, either alone or in copper complexes, have been used to disinfect liquids, solids and human tissue for centuries. Today copper is used as a water purifier, algaecide, fungicide, nematocide, molluscicide as well as an anti-bacterial and anti-fouling agent. Copper also displays potent anti-viral activity. This article reviews (i) the biocidal properties of copper; (ii) the possible mechanisms by which copper is toxic to microorganisms; and (iii) the systems by which many microorganisms resist high concentrations of heavy metals, with an emphasis on copper.

Lethally irradiated normal BALB/c mice, reconstituted with murine SCID bone marrow and engrafted with human PBMC (Trimera mice), were used to establish a novel murine model for HIV-1 infection. The Trimera mice were successfully infected with different clades and primary isolates of T- and M-tropic HIV-1, with the infection persisting in the animals for 4-6 wk. Rapid loss of the human CD4+ T cells, decrease in CD4/CD8 ratio, and increased T cell activation accompanied the viral infection. All HIV-1 infected animals were able to generate both primary and secondary immune responses, including HIV specific human humoral and cellular responses. In addition to testing the efficacy of new antiviral compounds, this new murine HIV-1 model may be used for studying host-virus interactions and, most importantly, for screening and developing potential HIV-1 protective vaccines and adjuvants (Ayash-Rashkovsky et al., http://www.fasebj.org/cgi/doi/10.1096/fj.04-3185fje; doi:10.1096/fj.04-3185fje.).

Millions of individuals in developing countries are infected with helminths and other chronic infectious diseases, such as HIV-1, which lead to persistent immune activation and unbalanced immune state. We have suggested that the capacity of chronically immune activated individuals to protect themselves, cope with infections, and mount protective immunity following vaccination, is highly impaired. Here we examined the expression of toll-like receptor 9 (TLR9), as an essential component in the recognition of immunostimulating bacterial CpG-DNA motifs, in different subsets of human peripheral blood mononuclear cells (PBMC) obtained from chronically immune activated and non-activated individuals. TLR9 expression was correlated to immune cell activation and was upregulated following phytohemagglutinin or anti-CD3 activation. PBMC obtained from chronically immune activated individuals had a different overall pattern of TLR9 expression, including reduced upregulation of this receptor following additional immune activation, and diminished responsiveness to CpG-DNA stimulation, in comparison to non-activated individuals. These differences may partly account for the reduced capacity of chronically immune activated individuals to mount effective immune responses and strengthen the notion that the host immune background should be considered in the design and trial of potential adjuvants and vaccines.

Chronic immune activation is associated with impaired signal transduction. Since such activation is commonly found during HIV-1 infection, we studied cellular responses to non-specific T-cell receptor stimulation of PBMC obtained from 20 HIV-1 non-infected individuals and 23 highly or partially immune activated HIV-1 infected individuals. PBMC proliferation and ERK-1/2 phosphorylation following anti-CD3 stimulation, and constitutive levels of Cbl-b, were determined. Increased levels of Cbl-b, decreased proliferation, and lower ERK-1/2 phosphorylation were found in PBMC of highly immune activated HIV-1 infected individuals. The elevated expression of Cbl-b and impaired phosphorylation of ERK-1/2 associated with immune activation probably contribute to the attenuated proliferative and cellular responses characteristic of HIV-1 infection. Therefore, targeting immune negative modulators, such as Cbl-b, may serve as a novel approach for controlling HIV-1 disease progression.

HIV-1 transactivating protein Tat is essential for virus replication and progression of HIV disease. HIV-1 Tat stimulates transactivation by binding to HIV-1 transactivator responsive element (TAR) RNA, and while secreted extracellularly, it acts as an immunosuppressor, an activator of quiescent T-cells for productive HIV-1 infection, and by binding to CXC chemokine receptor type 4 (CXCR4) as a chemokine analogue. Here we present a novel HIV-1 Tat antagonist, a neomycin B-hexaarginine conjugate (NeoR), which inhibits Tat transactivation and antagonizes Tat extracellular activities, such as increased viral production, induction of CXCR4 expression, suppression of CD3-activated proliferation of lymphocytes, and upregulation of the CD8 receptor. Moreover, Tat inhibits binding of fluoresceine isothiocyanate (FITC)-labeled NeoR to human peripheral blood mononuclear cells (PBMC), indicating that Tat and NeoR bind to the same cellular target. This is further substantiated by the finding that NeoR competes with the binding of monoclonal Abs to CXCR4. Furthermore, NeoR suppresses HIV-1 binding to cells. Importantly, NeoR accumulates in the cell nuclei and inhibits the replication of M- and T-tropic HIV-1 laboratory isolates (EC(50) = 0.8-5.3 microM). A putative model structure for the TAR-NeoR complex, which complies with available experimental data, is presented. We conclude that NeoR is a multitarget HIV-1 inhibitor; the structure, and molecular modeling and dynamics, suggest its binding to TAR RNA. NeoR inhibits HIV-1 binding to cells, partially by blocking the CXCR4 HIV-1 coreceptor, and it antagonizes Tat functions. NeoR is therefore an attractive lead compound, capable of interfering with different stages of HIV infection and AIDS pathogenesis.

By using a Sendai virus-human erythrocyte model, this work found that virus-infected cells were 10-fold more susceptible to lysis in two of five examined cobra venoms. Four cytotoxins were isolated from the venom of the cobra Naja nigricollis that also showed 10-fold higher cytotoxicity toward virus-infected cells than to untreated cells. As selective destruction of virus-infected cells is of immense importance in clinical practice, this work demonstrates the potential of cobra cytotoxins to serve as leading compounds for the generation of derivatives or fractions with high cytotoxic specificity toward virus-infected cells.

The multiple mutations associated with high-level AZT resistance (D67N, K70R, T215F, K219Q) arise in two separate subdomains of the viral reverse transcriptase (RT), suggesting that these mutations may contribute differently to overall resistance. We compared wild-type RT with the D67N/K70R/T215F/K219Q, D67N/K70R, and T215F/K219Q mutant enzymes. The D67N/K70R/T215F/K219Q mutant showed increased DNA polymerase processivity; this resulted from decreased template/primer dissociation from RT, and was due to the T215F/K219Q mutations. The D67N/K70R/T215F/K219Q mutant was less sensitive to AZTTP (IC50 approximately 300 nM) than wt RT (IC50 approximately 100 nM) in the presence of 0.5 mM pyrophosphate. This change in pyrophosphate-mediated sensitivity of the mutant enzyme was selective for AZTTP, since similar Km values for TTP and inhibition by ddCTP and ddGTP were noted with wt and mutant RT in the absence or in the presence of pyrophosphate. The D67N/K70R/T215F/K219Q mutant showed an increased rate of pyrophosphorolysis (the reverse reaction of DNA synthesis) of chain-terminated DNA; this enhanced pyrophosphorolysis was due to the D67N/K70R mutations. However, the processivity of pyrophosphorolysis was similar for the wild-type and mutant enzymes. We propose that HIV-1 resistance to AZT results from the selectively decreased binding of AZTTP and the increased pyrophosphorolytic cleavage of chain-terminated viral DNA by the mutant RT at physiological pyrophosphate levels, resulting in a net decrease in chain termination. The increased processivity of viral DNA synthesis may be important to enable facile HIV replication in the presence of AZT, by compensating for the increased reverse reaction rate.

The thiocarboxanilide nonnucleoside inhibitor (NNI) UC781 inhibited HIV-1 reverse transcriptase (RT) DNA polymerase activity at a 1:1 molar ratio of inhibitor to enzyme. Inhibition was linear uncompetitive with respect to template/primer (T/P) and mixed noncompetitive with respect to deoxynucleoside triphosphate (dNTP), typical of NNI. When the RT-T/P binary complex was incubated with UC781 and then separated from unbound inhibitor, recovery of enzyme activity was slow, with only about 60% activity recovered after 25 min. The inactivation of the RT-T/P complex was prevented by the presence of a large excess of UC84, another carboxanilide NNI that interacts with this RT mechanistic form. UC781 protected the RT-T/P-dNTP ternary complex from irreversible inactivation by a photoactivatable azido analog of nevirapine, implying that UC781 binds to the NNI pocket of this RT mechanistic form. UC781 did not photoprotect either the free enzyme or the RT-T/P binary complex; however, protein fluorescence quenching studies indicated that UC781 interacted with all RT mechanistic forms, with the order of affinity being RT-T/P-dNTP ternary complexRT-T/P binary complexfree RT. Reaction progress curve analysis showed that the binding of UC781 to RT is rapid (k(on) approximately 1.7 x 10(6) M(-1) s(-1)), but that dissociation is slow (k(off) approximately 1.6 x 10(-3) s(-1)). UC781 is therefore a rapid tight-binding inhibitor of HIV-1 RT, the first NNI to demonstrate this property.

HIV-1 reverse transcriptase (RT) is multifunctional, with RNA-dependent DNA polymerase (RDDP), DNA-dependent DNA polymerase (DDDP), and ribonuclease H (RNase H) activities. N-(4-tert-Butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH) inhibited both the polymerase and the RNase H activities of HIV-1 RT in vitro. IC50 values for inhibition of RDDP were 0.8-3.4 microM, depending on the template/primer (T/P) used in the assay. The IC50 for DDDP inhibition was about 12 microM, while that for inhibition of RNase H was 3.5 microM. EC50 for inhibition of HIV-1 replication in cord blood mononuclear cells was 1.5 microM. BBNH inhibition of RNase H in vitro was time-dependent, whereas inhibition of RT polymerase activities was immediate. BBNH was a linear mixed-type inhibitor of RT RDDP activity with respect to both T/P and to dNTP, whereas BBNH inhibition of RT RNase H activity was linear competitive. Protection experiments using an azidonevirapine photolabel showed that BBNH binds to the non-nucleoside RT inhibitor (NNRTI) binding pocket. Importantly, the compound inhibited recombinant RT containing mutations associated with high-level resistance to other NNRTI. While BBNH did not inhibit the DNA polymerase activities of other retroviral reverse transcriptases and DNA polymerases, the compound inhibited Escherichia coli RNase HI and the RNase H activity of murine leukemia virus RT. BBNH also inhibited HIV-1 RT RNase H in the presence of high concentrations of other non-nucleoside inhibitors with higher affinities for the NNRTI binding pocket, and of RT in which the NNRTI binding pocket had been irreversibly blocked by the azidonevirapine photolabel. We conclude that BBNH may therefore bind to two sites on HIV-1 RT. One site is the polymerase non-nucleoside inhibitor binding site and the second may be located in the RNase H domain. BBNH is therefore a promising lead compound for the development of multisite inhibitors of HIV-1 RT.

A potent antihemorrhagic factor (BaSAH1) was isolated from the serum of the snake Bothrops asper by ammonium sulfate precipitation at 40-60%, Sephacryl S-200 and Sephadex G-50 gel filtration, DEAE-Sepharose, and hydrophobic Phenyl-Sepharose chromatography. The purified protein showed one band with an isoelectric point of 5.2 and a molecular weight of 66 kDa. 4 micrograms of the purified factor BaSAH were needed to neutralize the hemorrhagic dose of B. asper whole venom compared to 60 micrograms of the clinically used horse polyvalent immunoglobulins. Moreover, 0.35 microgram of BaSAH were sufficient to achieve complete neutralization of the main hemorrhagic toxin (BaH1), with a molar ratio of 2:1. The antihemorrhagic activity was stable between pH 1.5-9 and up to 60 degrees C but lost activity completely after 30 min of heating at 70 degrees C. BaSAH did not digest the hemorrhagic toxin BaH1 or formed a precipitin line with it, nor with the whole venom. Both ELISA experiments and chromatography of BaSAH after incubation with the 125I-labeled hemorrhagic toxin BaH1 demonstrated that the mechanism of the neutralization involves a formation of an inactive soluble complex between the natural antihemorrhagin and the main hemorrhagin of B. asper venom.

The carboxanilides UC84 and UC38 are nonnucleoside inhibitors of both the RNA-dependent and DNA-dependent DNA polymerase activities of HIV-1 reverse transcriptase (RT). We have previously shown that UC84 and UC38 bind to the same site as nevirapine but interact with different RT mechanistic forms, with UC84 preferentially binding to the RT-primer/template complex and UC38 binding only to the RT-primer/template-dNTP ternary complex [Fletcher, R. S., et al. (1995) Biochemistry 34, 4346-4353]. Here we demonstrate that combinations of UC84 and UC38 inhibit RT DNA polymerase activity in vitro in a synergistic manner. This synergy was noted primarily in reactions containing high concentrations of primer/template and Km levels of dNTP substrate and was independent of both primer/template identity and the molar ratio of UC84:UC38. Combination indices were in the range of 0.4-0.6, indicating substantial synergy in the inhibition of RT activity. More importantly, combinations of UC84 and UC38 also showed a high degree of synergy in inhibiting HIV-1 replication in both MT-4 and cord blood mononuclear cells. We believe this to be the first example of synergistic inhibition of HIV-1 RT by combinations of structurally related nonnucleoside inhibitors.

A metalloproteinase, named BaP1, was purified to homogeneity from the venom of Bothrops asper (Pacific region) of Costa Rica by ion-exchange chromatography on CM-Sephadex and gel filtration on Sephacryl S-200. The enzyme has a mol. wt of 24,000 and contains few Cys and high numbers of Asp, Leu, Ser and Glu. BaP1 hydrolyzes casein, hide powder azure and fibrinogen, having an optimal pH of 8.0. It rapidly digests the A alpha-chain of fibrinogen and, later on, the B beta-chain, leaving the gamma-chain unaffected. Chelating agents (EDTA and 1,10-phenanthroline) inhibited proteolytic activity, whereas 2-mercaptoethanol and soybean trypsin inhibitor did not affect this activity. BaP1 has a weak hemorrhagic activity, with a minimum hemorrhagic dose of 20 micrograms; this activity was inhibited by EDTA and was abolished after incubation at 60 degrees C. In addition, BaP1 induces edema and a mild myotoxic effect, lacking coagulant, defibrinating and lethal effects. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

The main natural antihemorrhagic factor (NtAH), which inhibits the hemorrhagic activity of Bothrops asper snake venom, was isolated from the serum of the non-poisonous water snake Natrix tessellata by ammonium sulfate precipitation at 35-55%, Sephadex G-75 gel filtration, ion exchange chromatography on DEAE-Sepharose and CM-Sepharose and hydrophobic Phenyl-Sepharose chromatography. The purified protein showed one band with an isoelectric point of 4.5 and a molecular mass of about 880 kDa. The antihemorrhagic activity was stable between pH 5.5-11.7 and up to 50 degrees C, but lost activity after 20 min at 60 degrees C. It did not form a precipitin line with the main hemorrhagin of Bothrops asper snake venom (BaH1), nor with the whole venom, which suggests that the antihemorrhagic factor is not an immunoglobulin. The mechanism of neutralization by the isolated antihemorrhagic factor NtAH did not include digestion of the hemorrhagic toxin BaH1. Chromatography of NtAH with active 125I-labeled BaH1 toxin as well as ELISA experiments demonstrated that the mechanism of neutralization involves formation of an inactive soluble complex between the natural NtAH of the non-poisonous water snake and the main hemorrhagin of Bothrops asper venom. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Three hemorrhagic factors (BaH1, BH2 and BH3) were isolated from the venom of Bothrops asper by gel filtration on Sephacryl S-200, DEAE-Sepharose chromatography, metal chelate affinity chromatography and hydrophobic interaction chromatography. They contain 55% of the total hemorrhagic activity of the whole venom when they are mixed, but lose almost half of the activity if they are separated, indicating a synergism between the three. The main hemorrhagin is BaH1 (Bothrops asper hemorrhagin 1); the other two are weak hemorrhagins but contribute to the synergism. They are acidic proteins with a pI of 4.5, 5.2 and 5; their mol. wt is 64,000, 26,000 and 55,000 respectively. The minimal hemorrhagic dose (MHD) of BaH1, BH2 and BH3 is 0.18, 2 and 1.6 micrograms, with a specific activity 55, 5 and 6.25 higher than that of the whole venom. The hemorrhagic activity of all three factors was inhibited by EDTA and ortho-phenathroline, indicating that the hemorrhagic activity is metal dependent. Phosphoramidon, soybean trypsin inhibitor, PMSF, pepstatin and aprotinin did not affect the hemorrhagic activity of the isolated factors. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Bacitracin and the membrane-impermeant thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) are agents known to inhibit protein disulfide isomerase (PDI), a cell-surface protein critical in HIV-1 entry therefore they are fusion inhibitors (FI). Here we investigated the possibility that Bacitracin and or DTNB might have other antiviral activities besides FI. By means of residual activity assays, we found that both compounds showed antiviral activity only to viruses T-tropic HIV-1 strain. Cell-based fusion assays showed inhibition on HeLa-CD4-LTR-β-gal (CD4) and HL2/3 cells treated with Bacitracin, and DTNB with the latest compound we observed fusion inhibition on both cells but strikingly in HL2/3 cells (expressing Env) indicating a possible activity on both, the cell membrane and the viral envelope. A time-of-addition experiment showed that both compounds act on HIV entry inhibition but DTNB also acts at late stages of the viral cycle. Lastly, we also found evidence of long-lasting host cell protection in vitro by DTNB, an important pharmacodynamic parameter for a topical microbicide against virus infection, hours after the extracellular drug was removed; this protection was not rendered by Bacitracin. These drugs proved to be leading compounds for further studies against HIV showing antiviral characteristics of interest.