Synthesis of Novel Glycolipids That Bind HIV-1 Gp120
Synthesis of novel glycolipids that bind HIV-1 Gp120 — …
Synthesis of novel glycolipids that bind hiv 1 gp120
AB - As part of a research effort to design and prepare high affinity ligands for the galactosyl ceramide (GalCer) binding site on the HIV cell surface glycoprotein, gp120, several GalCer analogues have been prepared and characterized. The molecular design of analogues permits independent variations of the carbohydrate, the length of a hydrophilic spacer between the ligand and the lipid, and the composition of the hydrophobic lipid chains. Five different galactosyl analogues were synthesized having hydrophilic spacers of tri-, tetra-, and penta-ethylene glycol separating the carbohydrate from the lipid region which has either oleoyl or stearoyl lipid chains. The synthetic design allows for a convergent synthesis of the three components of the glycolipid conjugate. The structural characterization includes the proton and carbon chemical shifts, which were assigned after analysis of 1D and 2D NMR spectra.
The interaction between HIV gp120 and galactose-containing cell surface glycolipids such as GalCer or Gb3 are known to facilitate HIV binding to both CD4+ as well as CD4− cells. In an effort to develop small molecule HIV-1 entry inhibitors with improved solubility and efficacy, we have synthesized a series of C glycoside analogs of GalCer and tested their anti HIV-1 activity. The analogs were tested for gp120 binding using a HIV-1 (IIIB) V3-loop specific peptide. Two of the six analogs that interfered with gp120 binding also inhibited HIV Env-mediated cell-to-cell fusion and viral entry in the absence of any significant cytotoxicity. Analogues with two side chains did not show inhibition of fusion and/or infection under identical conditions. The inhibition of virus infection seen by these compounds was not coreceptor dependent, as they inhibited CXCR4, CCR5 as well as dual tropic viruses. These compounds showed inhibition of HIV entry at early steps in viral infection since the compounds were inactive if added post viral entry. Temperature-arrested state experiments showed that the compounds act at the level of virus attachment to the cells likely at a pre CD4 engagement step. These compounds also showed inhibition of VSV glycoprotein-pseudotyped virus. The results presented here show that the glycoside derivatives of GalCer with simple side chains may serve as a novel class of small molecule HIV-1 entry inhibitors that would be active against a number of HIV isolates as well as other enveloped viruses.
novel glycolipids that bind HIV-1 Gp120.
N2 - As part of a research effort to design and prepare high affinity ligands for the galactosyl ceramide (GalCer) binding site on the HIV cell surface glycoprotein, gp120, several GalCer analogues have been prepared and characterized. The molecular design of analogues permits independent variations of the carbohydrate, the length of a hydrophilic spacer between the ligand and the lipid, and the composition of the hydrophobic lipid chains. Five different galactosyl analogues were synthesized having hydrophilic spacers of tri-, tetra-, and penta-ethylene glycol separating the carbohydrate from the lipid region which has either oleoyl or stearoyl lipid chains. The synthetic design allows for a convergent synthesis of the three components of the glycolipid conjugate. The structural characterization includes the proton and carbon chemical shifts, which were assigned after analysis of 1D and 2D NMR spectra.
The isolation of highly potent antibodies that recognize a broad diversity of HIV-1 clades has opened up tremendous opportunities for enhancing our understanding of how to neutralize HIV-1. Crystal structures of antibodies have been determined that recognize different epitopes including the gp41 membrane proximal region, the gp120 receptor binding site and clusters of high mannose glycans on gp120. New human monoclonal antibodies (PG and PGT families) have been discovered recently that potently neutralize 70-90% of HIV-1 isolates across all clades. These exciting new antibodies were derived from direct functional screening of B cells from IAVI protocol G donors (Theraclone/Monogram) and are unusually potent. These antibodies have unique features and bind to novel epitopes that include the glycan shield as well as segments of gp120. Structural characterization of these broadly neutralizing antibodies, as well as identification of their glycan epitopes on HIV-1 Env, provide tremendous insights for neutralization of HIV-1 and can now be used for structure-assisted vaccine design.
Synthesis of Novel Glycolipids That Bind HIV-1 Gp120.
To further characterize the mechanism of action for these compounds, a temperature-arrested state experiment was conducted. HIV Env-mediated fusion is quite complex and involves several intermediate steps and conformational changes before the fusion reaction is completed (). Binding of HIV gp120 to CD4 induces conformational changes in gp120 that result in exposure of the coreceptor (CXCR4/CCR5) binding site. Once gp120 binds to the coreceptor, the HIV-1 gp41 six helix bundle forms resulting in the fusion of viral and cellular membranes. Incubating the virus with cells at lower than optimal temperature for fusion can arrest the fusion process at the TAS step that involves binding of gp120 to CD4 and to variable levels the coreceptor (CXCR4/CCR5) in the absence of fusion (). We used the TAS model to study the step at which the glycoside analogs inhibit HIV fusion. As seen in , the compounds LAA-4 and -6 lost significant activity during the 2h TAS. This was followed by loss of Leu3A activity, indicating CD4 engagement and to some degree loss of AMD3100 activity, suggesting CXCR4 recruitment. However, C34 did not loose any activity, suggesting that gp41 structural changes do not occur during TAS, which is consistent with previous reports (). The loss of activity was more significant for LAA-4 and LAA-6 than for Leu3A suggesting that the compounds inhibit a pre-CD4 step likely involving HIV gp120 binding to cell surface glycolipids or glycoproteins.
Evidence indicates that galactosyl ceramide (GalCer) and its 3′-sulfated derivative, sulfatide (SGalCer), may act as alternate coreceptors for human immunodeficiency virus type 1 (HIV-1) in CD4− cells. Glycosphingolipids (GSLs) may also be necessary for fusion of HIV-1 and host cell membranes. Using an enzyme-linked immunosorbent assay to determine which GSL was the best ligand for both recombinant and virus-associated gp120, we found that SGalCer was the best ligand for each rgp120 and HIV-1 isolate tested. Therefore, novel multivalent glycodendrimers, which mimic the carbohydrate clustering reportedly found in lipid rafts, were synthesized based on the carbohydrate moiety of SGalCer. Here we describe the synthesis of a polysulfated galactose functionalized, fifth generation DAB dendrimer (PS Gal 64mer), containing on average two sulfate groups per galactose residue. Its ability to inhibit HIV-1 infection of cultured indicator cells was compared to that of dextran sulfate (DxS), a known, potent, binding inhibitor of HIV-1. The results indicate that the PS Gal 64mer inhibited infection by the HIV-1 isolates tested as well as DxS.
Jacobsen, Jacquelyn Gervay-Hague, and David F
by the synthesis and testing ..
LaBell, Neil E
February 14th, 2006 - Volume 20 - Issue 3
New Catanionic Glycolipids
to HIV and inhibition of GSL synthesis ..
Non-natural glycosphingolipids and structurally simpler analogues bind HIV-1 ..
Case Essays: CHARACTERIZATION polymer synthesis an …
A synthetic V3 loop peptide, RIQRGPGRAFVTIGK, corresponding to the R15K peptide, from gp120 of HIV-1 IIIb isolate was used, instead of the recombinant gp120 molecule, for binding studies. This synthetic peptide was originally designed as an inhibitor of HIV-1 infection in vitro () and was later shown to bind to monomolecular films of GalCer, Gb3 and GM3, and therefore, could be used as a model for the glycolipid binding domain of gp120 (). To validate this hypothesis and also to allow for more accurate comparison of the data, the binding of LAA-4 and LAA-5 was re-evaluated against the synthetic peptide. Indeed, the values for the critical pressure of insertion for GalCer, LAA-4 and LAA-5 with respect to the synthetic peptide (ca 22, 22, 30 mN/m respectively), were very close to those determined for the binding with gp120. GlcCer showed no insertion at all, regardless of the initial pressure of the film, and was used as a negative control.
WRITING methodology research paper
In this study, we characterized the adherence of several viral isolates with different cellular tropisms [HIV-1 IIIB (X4), MN (X4), and Ba-L (R5)], and the corresponding rgp120s (gp120 IIIB, MN, and Ba-L) to GSLs immobilized on polyvinyl chloride wells. The results of the binding studies led us to hypothesize that a multivalent sulfated galactose-derivatized dendrimer might be an effective antagonist of HIV-1 binding. Here we describe the synthesis of a polysulfated galactose functionalized glycodendrimer (PS Gal 64mer) that contained on average two sulfate groups per galactose residue. The ability of the PS Gal 64mer to inhibit HIV-1 infection of cultured indicator cell lines was compared to that of dextran sulfate (DxS), a known potent inhibitor of HIV-1 (X4) infectivity. The results indicated that (i) of the GSLs tested, immobilized SGalCer was the best ligand for all of the virions and rgp120s studied; (ii) monosulfated galactose saccharides were unable to inhibit rgp120 adherence to immobilized sulfatide; and (iii) the PS Gal 64mer was as effective an inhibitor of HIV-1 infectivity or better than DxS for the isolates tested.
core shell nanoparticles synthesis;
Our interest has been in GalCer analogs that are amenable to development as therapeutic agents. In this context we have synthesized hydrolytically stable analogs with simple hydrocarbon chains as replacements for the ceramide residue (). Accordingly, the gp120 binding of C-glycoside LAA-4 and aza-C-glycoside LAA-5 with a linear hydrocarbon chain as a ceramide substitute were previously examined in a monolayer assay. The relative binding was determined by measuring the change in surface pressure of the glycolipid monolayer, on exposure to an aqueous solution of recombinant gp120 (). The critical pressure of insertion was in the range of 24 mN/m for both GalCer and the C-glycoside LAA-4, and 28.5 mN/m for the aza-C-glycoside LAA-5. This data suggests that gp120 exhibits similar binding to monolayers of GalCer and LAA-4, and noticeably higher affinity for the monolayer formed from LAA-5 (). In order to develop a wider structure activity relationship, we extended this study to LAA-6, the O-glycoside corresponding to LAA-4 and LAA-5, and the series of C-, aza-C- and O-glycosides with a branched chain hydrocarbon as the ceramide substitute ().
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