Potent antiviral activity of carbohydrate-specific algal and leguminous lectins from the Brazilian biodiversity.

Brazil has one of the largest biodiversities in the world. The search for new natural products extracted from the Brazilian flora may lead to the discovery of novel drugs with potential to treat infectious and other diseases. Here, we have investigated 9 lectins extracted and purified from the Northeastern Brazilian flora, from both leguminous species: Canavalia brasiliensis (ConBr), C. maritima (ConM), Dioclea lasiocarpa (DLasiL) and D. sclerocarpa (DSclerL), and algae Amansia multifida (AML), Bryothamniom seaforthii (BSL), Hypnea musciformis (HML), Meristiella echinocarpa (MEL) and Solieria filiformis (SfL). They were exposed to a panel of 18 different viruses, including HIV and influenza viruses. Several lectins showed highly potent antiviral activity, often within the low nanomolar range. DSclerL and DLasiL exhibited EC50 values (effective concentration of lectin required to inhibit virus-induced cytopathicity by 50%) of 9 nM to 46 nM for HIV-1 and respiratory syncytial virus (RSV), respectively, DLasiL also inhibited feline corona virus at an EC50 of 5 nM, and DSclerL, ConBr and ConM showed remarkably low EC50 values ranging from 0.4 to 6 nM against influenza A virus strain H3N2 and influenza B virus. For HIV, evidence pointed to the blockage of entry of the virus into its target cells as the underlying mechanism of antiviral action of these lectins. Overall, the most promising lectins based on their EC50 values were DLasiL, DSclerL, ConBr, ConM, SfL and HML. These novel findings indicate that lectins from the Brazilian flora may provide novel antiviral compounds with therapeutic potential.

The Cellular Thioredoxin-1/Thioredoxin Reductase-1 Driven Oxidoreduction Represents a Chemotherapeutic Target for HIV-1 Entry Inhibition.

BACKGROUND: The entry of HIV into its host cell is an interesting target for chemotherapeutic intervention in the life-cycle of the virus. During entry, reduction of disulfide bridges in the viral envelope glycoprotein gp120 by cellular oxidoreductases is crucial. The cellular thioredoxin reductase-1 plays an important role in this oxidoreduction process by recycling electrons to thioredoxin-1. Therefore, thioredoxin reductase-1 inhibitors may inhibit gp120 reduction during HIV-1 entry. In this present study, tellurium-based thioredoxin reductase-1 inhibitors were investigated as potential inhibitors of HIV entry. RESULTS: The organotellurium compounds inhibited HIV-1 and HIV-2 replication in cell culture at low micromolar concentrations by targeting an early event in the viral infection cycle. Time-of-drug-addition studies pointed to virus entry as the drug target, more specifically: the organotellurium compound TE-2 showed a profile similar or close to that of the fusion inhibitor enfuvirtide (T-20). Surface plasmon resonance-based interaction studies revealed that the compounds do not directly interact with the HIV envelope glycoproteins gp120 and gp41, nor with soluble CD4, but instead, dose-dependently bind to thioredoxin reductase-1. By inhibiting the thioredoxin-1/thioredoxin reductase-1-directed oxidoreduction of gp120, the organotellurium compounds prevent conformational changes in the viral glycoprotein which are necessary during viral entry. CONCLUSION: Our findings revealed that thioredoxin-1/thioredoxin reductase-1 acts as a cellular target for the inhibition of HIV entry.

The role of cellular oxidoreductases in viral entry and virus infection-associated oxidative stress: potential therapeutic applications.

INTRODUCTION: Cellular oxidoreductases catalyze thiol/disulfide exchange reactions in susceptible proteins and contribute to the cellular defense against oxidative stress. Oxidoreductases and oxidative stress are also involved in viral infections. In this overview, different aspects of the role of cellular oxidoreductases and oxidative stress during viral infections are discussed from a chemotherapeutic viewpoint. AREAS COVERED: Entry of enveloped viruses into their target cells is triggered by the interaction of viral envelope glycoproteins with cellular (co)receptor(s) and depends on obligatory conformational changes in these viral envelope glycoproteins and/or cellular receptors. For some viruses, these conformational changes are mediated by cell surface-associated cellular oxidoreductases, which mediate disulfide bridge reductions in viral envelope glycoprotein(s). Therefore, targeting these oxidoreductases using oxidoreductase inhibitors might yield an interesting strategy to block viral entry of these viruses. Furthermore, since viral infections are often associated with systemic oxidative stress, contributing to disease progression, the enhancement of the cellular antioxidant defense systems might have potential as an adjuvant antiviral strategy, slowing down disease progression. EXPERT OPINION: Promising antiviral data were obtained for both strategies. However, potential pitfalls have also been identified for these strategies, indicating that it is important to carefully assess the benefits versus risks of these antiviral strategies.

Anti-HIV-1 activity of a tripodal receptor that recognizes mannose oligomers.

The glycoprotein gp120 of the HIV-1 viral envelope has a high content in mannose residues, particularly α-1,2-mannose oligomers. Compounds that interact with these high-mannose type glycans may disturb the interaction between gp120 and its (co)receptors and are considered potential anti-HIV agents. Previously, we demonstrated that a tripodal receptor (1), with a central scaffold of 1,3,5-triethylbenzene substituted with three 2,3,4-trihydroxybenzoyl groups, selectively recognizes α-1,2-mannose polysaccharides. Here we present additional studies to determine the anti-HIV-1 activity and the mechanism of antiviral activity of this compound. Our studies indicate that 1 shows anti-HIV-1 activity in the low micromolar range and has pronounced gp120 binding and HIV-1 integrase inhibitory capacity. However, gp120 binding rather than integrase inhibition seems to be the primary mechanism of antiviral activity of 1.

Tryptophan dendrimers that inhibit HIV replication, prevent virus entry and bind to the HIV envelope glycoproteins gp120 and gp41.

Dendrimers containing from 9 to 18 tryptophan residues at the peryphery have been efficiently synthesized and tested against HIV replication. These compounds inhibit an early step of the replicative cycle of HIV, presumably virus entry into its target cell. Our data suggest that HIV inhibition can be achieved by the preferred interaction of the compounds herein described with glycoproteins gp120 and gp41 of the HIV envelope preventing interaction between HIV and the (co)receptors present on the host cells. The results obtained so far indicate that 9 tryptophan residues on the periphery are sufficient for efficient gp120/gp41 binding and anti-HIV activity.

Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission.

The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.

The role of N-glycans of HIV-1 gp41 in virus infectivity and susceptibility to the suppressive effects of carbohydrate-binding agents.

BACKGROUND: Carbohydrate-binding agents (CBAs) are potent antiretroviral compounds that target the N-glycans on the HIV-1 envelope glycoproteins. The development of phenotypic resistance to CBAs by the virus is accompanied by the deletion of multiple N-linked glycans of the surface envelope glycoprotein gp120. Recently, also an N-glycan on the transmembrane envelope glycoprotein gp41 was shown to be deleted during CBA resistance development. RESULTS: We generated HIV-1 mutants lacking gp41 N-glycans and determined the influence of these glycan deletions on the viral phenotype (infectivity, CD4 binding, envelope glycoprotein incorporation in the viral particle and on the transfected cell, virus capture by DC-SIGN(+) cells and transmission of DC-SIGN-captured virions to CD4(+) T-lymphocytes) and on the phenotypic susceptibility of HIV-1 to a selection of CBAs. It was shown that some gp41 N-glycans are crucial for the infectivity of the virus. In particular, lack of an intact N616 glycosylation site was shown to result in the loss of viral infectivity of several (i.e. the X4-tropic IIIB and NL4.3 strains, and the X4/R5-tropic HE strain), but not all (i.e. the R5-tropic ADA strain) studied HIV-1 strains. In accordance, we found that the gp120 levels in the envelope of N616Q mutant gp41 strains NL4.3, IIIB and HE were severely decreased. In contrast, N616Q gp41 mutant HIV-1ADA contained gp120 levels similar to the gp120 levels in WT HIV-1ADA virus. Concomitantly deleting multiple gp41 N-glycans was often highly detrimental for viral infectivity. Using surface plasmon resonance technology we showed that CBAs have a pronounced affinity for both gp120 and gp41. However, the antiviral activity of CBAs is not dependent on the concomitant presence of all gp41 glycans. Single gp41 glycan deletions had no marked effects on CBA susceptibility, whereas some combinations of two to three gp41 glycan-deletions had a minor effect on CBA activity. CONCLUSIONS: We revealed the importance of some gp41 N-linked glycans, in particular the N616 glycan which was shown to be absolutely indispensable for the infectivity potential of several virus strains. In addition, we demonstrated that the deletion of up to three gp41 N-linked glycans only slightly affected CBA susceptibility.

Deletion of the highly conserved N-glycan at Asn260 of HIV-1 gp120 affects folding and lysosomal degradation of gp120, and results in loss of viral infectivity.

N-linked glycans covering the surface of the HIV-1 glycoprotein gp120 are of major importance for the correct folding of this glycoprotein. Of the, on average, 24 N-linked glycans present on gp120, the glycan at Asn260 was reported to be essential for the correct expression of gp120 and gp41 in the virus particle and deletion of the N260 glycan in gp120 heavily compromised virus infectivity. We show here that gp160 containing the N260Q mutation reaches the Golgi apparatus during biosynthesis. Using pulse-chase experiments with [35S] methionine/cysteine, we show that oxidative folding was slightly delayed in case of mutant N260Q gp160 and that CD4 binding was markedly compromised compared to wild-type gp160. In the search of compensatory mutations, we found a mutation in the V1/V2 loop of gp120 (S128N) that could partially restore the infectivity of mutant N260Q gp120 virus. However, the mutation S128N did not enhance any of the above-mentioned processes so its underlying compensatory mechanism must be a conformational effect that does not affect CD4 binding per se. Finally, we show that mutant N260Q gp160 was cleaved to gp120 and gp41 to a much lower extent than wild-type gp160, and that it was subject of lysosomal degradation to a higher extent than wild-type gp160 showing a prominent role of this process in the breakdown of N260-glycan-deleted gp160, which could not be counteracted by the S128N mutation. Moreover, at least part of the wild-type or mutant gp160 that is normally targeted for lysosomal degradation reached a conformation that enabled CD4 binding.

Exposure of HIV-1 to a combination of two carbohydrate-binding agents markedly delays drug resistance development and selects for virus strains with compromised fitness.

OBJECTIVES: We investigated the effect of combining two potent carbohydrate-binding agents (CBAs) with a complementary resistance profile (based on different N-glycan deletion selections in the HIV envelope glycoprotein gp120) on drug resistance development and viral fitness. METHODS: A long-term selection procedure was used to obtain virus strains resistant to the mannose-specific Hippeastrum hybrid agglutinin (HHA), the mannose-recognizing monoclonal antibody 2G12 and the combination of both compounds. The env genes of the mutant viruses were sequenced and the infectivity potential and phenotypic resistance profile of the virus strains were examined in CD4+ T lymphocyte C8166 cell cultures. RESULTS: The long-term exposure of HIV-1 to CBAs resulted in the selection of virus isolates containing deletions of several high-mannose-type N-glycans in their envelope. The generation of virus strains phenotypically resistant to the combination of both CBAs took markedly longer than the generation of virus strains resistant to the single compounds. The mutant HIV strains derived from the HHA/2G12 combination exposure showed much lower genotypic and phenotypic resistance than those isolated from the virus selection experiments with the single compounds. It was further shown that the CBA-resistant viruses had significantly decreased infectivities. CONCLUSIONS: The data revealed that CBAs are interesting anti-HIV drug candidates with an increased antiviral potential upon internal combination.

Inhibition of infection and transmission of HIV-1 and lack of significant impact on the vaginal commensal lactobacilli by carbohydrate-binding agents.

OBJECTIVES: A selection of carbohydrate-binding agents (CBAs) with different glycan specificities were evaluated for their inhibitory effect against HIV infection and transmission, and their interaction with vaginal commensal bacteria. METHODS: Several assays were used for the antiviral evaluation: (i) cell-free virus infection of human CD4+ T lymphocyte C8166 cells; (ii) syncytium formation in co-cultures of persistently HIV-1-infected HUT-78/HIV-1 and non-infected CD4+ SupT1 cells; (iii) DC-SIGN-directed capture of HIV-1 particles; and (iv) transmission of DC-SIGN-captured HIV-1 particles to uninfected CD4+ C8166 cells. CBAs were also examined for their interaction with vaginal commensal lactobacilli using several viability, proliferation and adhesion assays. RESULTS: The CBAs showed efficient inhibitory activity in the nanomolar to low-micromolar range against four events that play a crucial role in HIV-1 infection and transmission: cell-free virus infection, fusion between HIV-1-infected and non-infected cells, HIV-1 capture by DC-SIGN and transmission of DC-SIGN-captured virus to T cells. As candidate microbicides should not interfere with the normal human microbiota, we examined the effect of CBAs against Lactobacillus strains, including a variety of vaginal strains, a gastrointestinal strain and several non-human isolates. None of the CBAs included in our studies inhibited the growth of these bacteria in several media, affected their viability or had any significant impact on their adhesion to HeLa cell monolayers. CONCLUSIONS: The CBAs in this study were inhibitory to HIV-1 in several in vitro infection and transmission models, and may therefore qualify as potential microbicide candidates. The lack of significant impact on commensal vaginal lactobacilli is an important property of these CBAs in view of their potential microbicidal use.