The need for new therapeutic agents to fight the HIV pandemic is still actual. With the approval and successful use of enfuvirtide, the first fusion inhibitor targeting HIV entry, the field of HIV entry inhibitors is gaining increased attention with several new compounds targeting HIV coreceptor CCR5 in clinical trials (Esté & Telenti, 2007
). Other inhibitors of HIV entry that bind carbohydrate moieties on env glycoprotein such as cyanovirin (Dey et al., 2000
) and retrocyclin (Wang et al., 2003
) are also being investigated.
The HIV fusion reaction proceeds via binding of the gp120 subunit to receptor (CD4) and coreceptor (CXCR4/CCR5). This is followed by conformational changes in the fusion inducing gp41 subunit that mediates the fusion reaction (Gallo et al., 2003
). The involvement of these steps is well documented and targeted by drugs like the gp41 peptide enfuvirtide and coreceptor antagonists like AMD3100 and TAK779. However, other cofactors like membrane glycosphingolipids (GSLs) are known to pay a supporting role in HIV env-mediated fusion reaction (Rawat et al., 2005
). Indeed, specific glycolipids such as Gb3 and GM3 have been reported to act as cofactors for entry into CD4+
T cells (Fantini et al., 2000
). The binding of the V3 loop of gp120 to all of these glycolipids involves a common biochemical mechanism, i.e. a stacking interaction between the Phe residue in the GPGRAF core motif and the hydrophobic face of a terminal galactosyl ring in the glycolipid (Fantini, 2003
). This explains why synthetic analogs of GalCer could inhibit HIV-1 infection in CD4+
lymphocytes expressing Gb3 (Fantini et al., 2000
). Based on this premise, we had earlier evaluated the gp120 binding of analogs of GalCer with simple ceramide replacements. In the current study we have examined the interaction of these and related structures with a synthetic peptide corresponding to the glycolipid binding domain of the V3 loop of HIV-1 gp120, and tested their efficiency to inhibit HIV Env-mediated fusion and viral entry.
The very similar binding profiles of LAA-4
with respect to whole gp120 (previous study) (Augustin et al., 2006
), and the synthetic V3 peptide (current study), is consistent with the notion that these glycolipids interact specifically with the consensus sequence on gp120. As far as the relative binding of the different analogs to the V3 peptide is concerned, the generally higher binding of the O- and aza-C-glycosides compared to the C-glycosides suggests that the heteroatoms of the acetal or pseudo-acetal moiety might be involved in polar interactions with the receptor, or may be biasing a favored orientation of the sugar in relation to the hydrophobic chain (Taïeb et al., 2004
). The sharper increase in surface pressure with decreasing initial pressure that was observed for the linear chain O- and C- glycoside analogs could be an indication that the branched chain congeners arrange into more densely packed glycolipid monolayers such that insertion of the peptide into the corresponding monolayers occurs less readily compared to insertion into the monolayers formed from the linear analogs. The markedly higher binding of the aza-C- compared with the C- glycosides might be connected to changes in monolayer packing and/or additional receptor interactions that result from the replacement of the ring oxygen by an amino group (Augustin et al., 2006
While it appears likely that the difference in activity of the branched chain analogs in the peptide binding vs. cellular assays, is due to the poor water solubility of these compounds, any attempt to connect the activity of the linear chain analogs LAA-4, 5 and 6 to a similar molecular recognition process should be approached with caution. Such an analysis is complicated because of the absence of any clear structural information on the glycolipid receptor involved in the two assays, i.e. a monolayer vs. a monomer, micelle or a micelle aggregate. In addition, the possibility that receptors other than the V3 loop of gp120 may have an impact on the cellular effects should be considered. Not withstanding the molecular basis for interaction with gp120, the positive correlation between binding to the V3 peptide and inhibition of both cell-to-cell fusion and virus infection exhibited for LAA-4, -5 and -6 is consistent with the idea that antagonists to the glycolipid domain of the V3 loop may disrupt the attachment of HIV-1 to host cells.
The inhibition of both cell-to-cell fusion and virus infection suggests an inhibition of env function. We have also shown that the inhibition of viral entry was coreceptor-independent. The coreceptor-independent inhibition of viral entry mediated by LAA-4 and LAA-6 suggests that it is an early event common to a variety of HIV strains. This is further supported by the fact that inhibitory activity of the compounds is lost if added post viral entry. Our experiments using the TAS showed that these compounds rapidly loose activity within 2h of TAS induction. The loss of activity was faster than that for CD4 engagement suggesting a pre-CD4 binding step may be involved. Hence, we speculate that these glycoside analogs of GalCer bind to the virus and inhibit its adsorption on the target membrane at a pre-CD4 binding step. Although one of the compounds (LAA-5) was significantly toxic to the cells, LAA-4 and LAA-6 showed inhibition at non-toxic concentrations. This holds promise for further development of these compounds into potential antiviral agents.
Not surprising was the fact that these compounds also inhibited VSVG-pseudotyped virus infection. The mechanism of action of these compounds is likely to be similar to other antiviral agents like suramin and cyanovirin which we have shown here for the first time also inhibit VSVG pseudotype infection. This not only supports the broad antiviral activity of these compounds but also underscores the notion that interaction with cell surface carbohydrates is a common feature of a variety of enveloped viruses.
Although the compounds may not have HIV specific activity, the general antiviral activity makes them neverthelss useful. Compound LAA-5
which had the highest activity was also significantly toxic to the cells; however LAA-4
showed inhibition at non toxic concentrations holding promise for further development of these compounds into potential antiviral agents. Similar antiviral agents like cyanovirin (Chang & Bewley, 2002
; Barrientos et al., 2003
) retrocyclin (Wang et al., 2003
), and scytovirin (Xiong et al., 2006
) are proteins; inhibitors used in this study are small molecules that are not subject to the problems of immunogenicity that plague protein therapeutics. Other soluble analogues of GalCer have similarly been shown to inhibit HIV infection (Fantini et al., 1997
); however, our compounds are simpler versions of these early analogues with smaller side chains, better solubility and significantly enhanced activity. We provide a mechanistic approach to the antiviral activity of these compounds and hope to provide important leads for the further development of small-molecule inhibitors of HIV as well as other viruses.