The CBAs are an interesting new category of antiviral compounds that target the glycans on the envelope of viruses like HIV (for a review, see references 4
). However, since most CBAs are of a peptidic nature, which may cause problems such as high production costs, unfavorable pharmacokinetics, and possible immunological reactions, it is important to look for novel nonpeptidic low-molecular-weight compounds that behave as CBAs and prevent HIV infection. Recently, the antiviral properties of pradimicin A, an α-1,2-mannose-binding nonpeptidic semisynthetic antibiotic that binds to the glycans of HIV-1 gp120 and selects for glycan deletions in HIV-1 gp120, were described (12
In this study, we showed for the first time that a synthetic prototype compound within the structural class of the phthalocyanines, designated Alcian Blue, selectively inhibits retrovirus infection, presumably by binding to the glycans of the HIV-1 gp120 envelope. In contrast with the vast majority of CBAs that are endowed with anti-HIV activity, this compound is a synthetic nonpeptidic agent. Time-of-addition experiments clearly showed that AB acts on an antiviral target that is operative in an early stage of the viral life cycle (i.e., entry/fusion) but at a slightly later time point than DS8000, an attachment (adsorption) inhibitor of the HIV infection process. In fact, the time-of-addition study showed that AB acts at exactly the same time point as the CBAs HHA, UDA, and PRM-A. Our observation that AB dose dependently prevents syncytium formation (fusion) between persistently HIV-1-infected T cells and uninfected T lymphocytes is in agreement with the time-of-drug addition data, as well as with the mechanism of action of CBAs. This property may have the advantage that, if virus escaped the entry block afforded by AB, the transmission of virus from the virus-infected cell through cell-cell contact can still be blocked in the presence of the drug.
HHA shows specificity for internal (α-1,3 and α-1,6) mannoses, while PRM-A preferentially binds to α-1,2-mannose residues (7
). The glycoprotein gp120 of HIV is not only highly glycosylated, but also, ~ 33% of the N-glycans are high-mannose type (24
). This is remarkable, since human cells usually do not express high-mannose-type sugars on their glycoproteins and if they do, the high-mannose-type glycans never occur as dense on the glycoprotein as on gp120. In fact, the C-type mannose-binding lectin, which is part of the innate immune system, recognizes high-mannose-type sugars, initiating the lectin pathway (20
). However, whereas mannan reverses the anti-HIV activity of the CBAs HHA, GNA, and PRM-A (12
), high concentrations of mannan were not able to reverse the antiviral activity of AB (data not shown). Also, only 50% of the N-glycans deleted upon increasing concentrations of AB were high-mannose type. These data may indicate that AB may not preferentially recognize high-mannose-type glycans on HIV-1 gp120. The carbohydrate spectrum of AB still needs to be determined. In this respect, it is still unclear why AB tends to be more active against HIV-1 strains (EC50
, 3.7 to 9.3 μM) than HIV-2(ROD) (EC50
, 17 μM) or SIVMac251
, 12 μM). The fact that the HIV-1(IIIB
) and HIV-2(ROD) strains contain an equal amount of N-linked glycosylation sites may rule out that the differences in activity are simply due to the number (density) of N-glycans on the viral envelope. Glycan specificity, which has not been characterized for AB (38
), may play a more important role in its eventual antiviral efficacy, as well as the importance of the interaction of specific (glycosylated) envelope epitopes with the viral receptors. In fact, CBAs turned out to be potent inhibitors of Dengue virus entry and infection, whereas the viral E-envelope protein only contains two potential N-glycans (1
Besides mannose-binding lectin, other human mannose-binding lectins interact with gp120 of HIV. DC-SIGN, a lectin present on dendritic cells, functions in dendritic cell recognition and mediates uptake of pathogens, leading to antigen presentation to T cells (17
). Alike the CBAs HHA, GNA, and UDA (11
), AB was able to inhibit the binding of HIV-1 to DC-SIGN and the subsequent virus transmission to T cells. These observations indicate that CBAs such as Alcian Blue may not only have the potential to prevent cell-free HIV infection and syncytium formation between HIV-infected cells and noninfected cells but also the capture and subsequent transmission of the virus by DC-SIGN-expressing cells. These properties are important in view of a potential microbicidal application for this drug class.
Another important similarity with the CBAs such as HHA, UDA, or PRM-A is the selection of mutant virus strains that predominantly show deletions in the glycans on HIV gp120. Interestingly, an accumulation of four N
-glycan deletions occurred under exposure of HIV-1 to escalating AB concentrations (Fig. ). These four N-glycosylation site positions in gp120 have also been shown to be deleted under HHA, GNA, and/or UDA pressure (5
). While the plant lectins HHA and GNA showed a clear preference for deleting high-mannose-type N-glycans, this preference was not so (statistically) clear for AB, since only two out of four deleted glycans were high-mannose types. The selection of the AB-resistant virus strain containing four deletions in N-linked glycans of gp120 took up to 80 subcultivations in the presence of escalating drug concentrations. Similar long selection times have been observed for other CBAs as well, including GNA and HHA (65 to 70 passages) (2
) and UDA (90 passages) (3
), whereas somewhat shorter selection times were required for PRM-A (15 to 20 passages) (10
). However, in contrast with the AB-resistant virus strains, no additional positively charged amino acid changes have been observed in their gp120/gp41 envelope for the virus strains resistant to HHA, GNA, UDA, and PRM-A.
Also noteworthy is the observation that none of the CBAs tested showed cross-resistance toward the AB-resistant HIV-1(IIIB) isolate 7 (Fig. ). A possible explanation for this observation could be that HHA, GNA, UDA, and PRM-A need a higher number of high-mannose-type glycan deletions in gp120 to become phenotypically resistant. The lack of cross-resistance against the other CBAs is also in line with our previous findings that many CBAs have a high genetic barrier, requiring multiple (≥4) glycan deletions in HIV-1 gp120 before phenotypic resistance becomes measurable. Alike the CBAs, AB is also endowed with a relatively high genetic barrier. It took up to 70 passages (~245 days) for the first N-glycan mutations to appear and up to 80 passages (~ 280 days) for HIV-1 to become moderately (~ 10-fold) resistant to AB (Fig. ). In this respect, AB closely mimics the antiviral mechanism of action and resistance profile of the plant and prokaryotic lectins.
The cationic phthalocyanine derivative Alcian Blue has been used to quantify glycosaminoglycans by its ability to form complexes with such carbohydrates (39
). It was also previously shown by Vzorov et al. (36
) to inhibit HIV in cell culture. Many other structurally related porphyrins, like the metalloporphyrin-ellipticine complexes (15
) and the negatively charged carboxyphenyl porphyrine derivatives (14
), have been described as potent inhibitors of HIV infection. It is clear that the class of porphyrins, either cationic, neutral, or anionic, may show promise as potential anti-HIV drug leads for systemic as well as microbicidal applications. However, it seems likely that cationic and anionic derivatives, although they both block virus entry by interacting with gp120, may inhibit HIV infectivity by different mechanisms. The mutational pattern observed under escalating Alcian Blue concentrations provided evidence that this compound blocks viral infection by interacting with the envelope glycans. Such a mechanism of action is very interesting because it provokes a unique resistance profile by forcing the virus to delete its glycans on its envelope. By doing this, it is speculated that AB triggers the immune system to produce a neutralizing antibody or cellular immune response against the uncovered immunogenic epitopes and, thus, AB may represent a unique prototype agent for further exploration as a potential antiviral agent. However, not all mutations in the envelope of HIV-1 occurred in N-glycosylation motifs (Table ). Although most of the mutations in the envelope resulted in changes to an identical or homologous amino acid, five mutations led to the creation of positive charges by creating a lysine or histidine residue in the envelope. It may be of particular importance to notice that three out of the five created positive charges in the HIV-1 envelope occurred in gp41. The significance of these observations is currently still unclear. It cannot be excluded that the appearance of the positively charged amino acids may afford a repulsing effect against the (positively charged) AB molecules. Surface plasmon resonance interaction studies of AB with immobilized gp120 and gp41 to clarify this issue failed due to a significant level of aspecific binding of AB to the envelope-coated chip (B. Hoorelbeke, personal communication). Thus, although Alcian Blue undoubtedly targets the glycans on the envelope of HIV-1, this may not be its sole mode of antiviral activity. It cannot be excluded that CBAs such as AB also interact with glycoproteins that are present on the cell surface and which play a direct or indirect role in the entry process of HIV. In this respect, we have observed an interaction of AB with one of the coreceptors of HIV (i.e., CCR5) by fluorescence-activated cell sorter analysis, but the significance of this observation in the eventual antiviral efficacy of AB is currently unclear and will be the subject of further studies.
More than 25 years after the discovery of HIV and AIDS, the search for an efficient vaccine is still ongoing and is more technically challenging than ever expected. Microbicides are chemical agents that can be used intravaginally by women. The ideal microbicide should be easy to use, active against most HIV strains, and safe, but also economically affordable for people in the developing world (8
). In this respect, CBAs are especially promising, since they have a high genetic barrier and enable “neutralization” of a broad variety of HIV clades (5
). Alcian Blue, belonging to the chemical class of phthalocyanines, opens the way to the exploration of other (cationic) phthalocyanines in the development of novel antiviral drugs targeting the glycans on the envelope glycoproteins of HIV. It has the same interesting properties as the CBAs HHA, UDA, and PRM-A and, thus, AB can be considered a prototype compound in the development of new CBA microbicides of a nonpeptidic nature.