Abstract
Galactosylceramide (GalCer) is an alternative receptor allowing human immunodeficiency virus (HIV)-1 entry into CD4-negative cells of neural and colonic origin. Several lines of evidence suggest that this glycosphingolipid recognizes the V3 region of HIV-1 surface envelope glycoprotein gp120. Since the V3 loop plays a key role in the fusion process driven by HIV-1, we decided to synthesize soluble analogs of GalCer with the aim to develop a new class of anti-HIV-1 agents that could neutralize HIV-1 infection through masking of the V3 loop. We describe a short route, in three steps, for the synthesis of soluble analogs of GalCer, using unprotected lactose as the starting sugar. The analogs were prescreened in an assay based on the interaction between a V3 loop-derived synthetic peptide and [3H]suramin, a polysulfonyl compound displaying high affinity for the V3 loop. One of the soluble analogs, i.e. CA52(n15), strongly inhibited the binding of [3H]suramin to the V3 peptide, with an IC50 of 1.2 microM. This molecule was also able to inhibit [3H]suramin binding to recombinant gp120 with similar activity. Using a competition enzyme-linked immunosorbent assay with highly specific anti-gp120 monoclonal antibodies, the region recognized by CA52(n15) could be mapped to amino acids 318-323, which corresponds to the highly conserved consensus motif GPGRAF. Interestingly, the region recognized by suramin, i.e. IQRGP-R-F, was partially overlapping this motif. CA52(n15) was able to inhibit HIV-1-induced cell fusion as well as HIV-1 entry into both CD4(+) and CD4(-)/GalCer+ cells. A structure-activity relationship study showed that: (i) the antiviral activity of soluble analogs of GalCer correlates with V3 loop binding, and (ii) the hydrophobic moiety of the molecule plays an important role in this activity. Taken together, these data show that synthetic analogs of GalCer can inhibit HIV-1 entry into both CD4(-) and CD4(+) cells through masking of the V3 loop.
Highlights
Galactosylceramide (GalCer) is an alternative receptor allowing human immunodeficiency virus (HIV)-1 entry into CD4-negative cells of neural and colonic origin
The rationale for this strategy was based on previous observations suggesting that the GalCer receptor, putatively used by HIV-1 to infect neural cells [16, 17] and colon cells [18, 19], was recognized by the V3 domain of gp120: (i) anti-V3 antibodies block gp120 binding to GalCer [26]; (ii) these antibodies inhibit HIV-1 infection of CD4Ϫ/GalCerϩ human colon epithelial HT-29 cells [15, 26]; (iii) synthetic multimeric peptide constructs of the V3 consensus sequence (GPGRAF) bind to GalCer and prevent HIV-1 entry into HT-29 cells [24]; (iv) the V3 loop is a common genetic determinant controlling HIV-1 tropism for neural SKNMC cells [17, 28] and HT-29 cells [29], as demonstrated by using chimeric proviral clones
The V3 loop was mimicked by SPC3, a synthetic multibranched V3 peptide that has been recently characterized by our group as an inhibitor of HIV-1 infection in both CD4Ϫ and CD4ϩ cells [30]
Summary
Galactosylceramide (GalCer) is an alternative receptor allowing human immunodeficiency virus (HIV)-1 entry into CD4-negative cells of neural and colonic origin. We report here the synthesis and characterization of such soluble GalCer analogs that can block HIV-1-induced fusion as well as entry into both CD4Ϫ and CD4ϩ cells These analogs have been first evaluated for their ability to inhibit the binding of [3H]suramin to SPC3, a synthetic peptide displaying eight V3 consensus motifs (GPGRAF) radially branched on uncharged poly-Lys core matrix [15]. This prescreening assay proved useful to select those analogs that recognized the V3 loop, and a good correlation was found between the anti-HIV-1 activity of a given analog and its affinity for the V3 loop. These data show that synthetic soluble analogs of glycosphingolipids may represent a new class of anti-HIV-1 drugs that could be obtained at a large scale with a low cost
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