Glycoside analogs of β-galactosylceramide, a novel class of small molecule antiviral agents that inhibit HIV-1 entry

doi:10.1016/j.antiviral.2008.04.004

Antiviral Research

Volume 80, Issue 1, October 2008, Pages 54-61

https://doi.org/10.1016/j.antiviral.2008.04.004 Get rights and content

Abstract

The interaction between HIV gp120 and galactose-containing cell surface glycolipids such as GalCer or Gb₃ is 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. Analogs 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.

Introduction

HIV gains entry into cells via binding of Env glycoprotein to CD4 and a chemokine receptor (CXCR4/CCR5). Besides the use of these well-defined receptor and coreceptor other cofactors involved in HIV-1 entry have been suggested (Rawat et al., 2006). A variety of glycosphingolipids (GSL) have been shown to regulate Env-mediated fusion including galactosyl ceramide (GalCer) and a monoganglioside GM3 (Fantini et al., 2000, Hammache et al., 1998). More specifically GalCer has been shown to be a cofactor in HIV Env binding and infection of CD4⁺ cells (Fantini et al., 1997).

The binding of HIV gp120 to cell surface expressed GalCer maps to the V3 loop region (Fantini et al., 1997, Bhat et al., 1993). The V3 loop of HIV, though highly variable, has a few conserved basic residues. This region has therefore been shown to bind a variety of anionic compounds such as polysulfonated albumin (Kuipers et al., 1996), suramin (Yahi et al., 1994) and heparin sulfate (Rider et al., 1994). A number of soluble analogs of GalCer as well as other sphingolipids have been shown to inhibit HIV infection (Mahfoud et al., 2002, Villard et al., 2002, Lund et al., 2006).

With a view towards new anti HIV agents we have previously examined the gp120 binding of both a C-glycoside (LAA-4) and an aza-C-glycoside (LAA-5), analogs of GalCer in which one or other of the acetal oxygens is replaced with a methylene or amino group and the ceramide residue substituted with a simple hydrocarbon chain (Augustin et al., 2006). The binding of both LAA-4 and LAA-5 was found to be similar to that of GalCer (Tanahashi et al., 1997). In the present study we have widened the range of structures to include analogs with O-glycoside residues and branched-chain ceramide substitutes and examined their binding to a synthetic peptide corresponding to the glycolipid binding domain of the V3 loop of HIV-1 gp120. The inhibition of HIV-1 Env glycoprotein-mediated fusion and infection was also investigated. Binding to the HIV-1 peptide was found to vary with both the structure of the glycoside or pseudoglycoside linkage and the ceramide replacement, and selected analogs showed a potent inhibition of both HIV Env glycoprotein-mediated cell-to-cell fusion as well as viral infection. Analysis of intermediate steps in the fusion process revealed that the inhibition is at the level of viral entry and is independent of coreceptor use by the virus.

Section snippets

C-glycoside analogs of GalCer

The syntheses of O-glycosides LAA-3 (Tanahashi et al., 1997) and LAA-6, C-glycoside LAA-4 and aza-C-glycoside LAA-5 (Augustin et al., 2006) have been previously described (Nehete et al., 1993). The new analogs LAA-1 and LAA-2 were prepared by modification of these procedures.

Structure and binding of glycoside analogs of GalCer

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 (Fig. 1). 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

Discussion

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 and 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.,

Acknowledgements

We are grateful to the NIH AIDS Research and Reference Reagent Program, for supplying valuable reagents, Eric Freed for pNLluc and pNLAD8 and Barry O’Keefe for providing cyanovirin-N. This investigation was supported by grants R01 GM57865 and SCORE S06 GM60654 from the National Institute of General Medical Sciences of the National Institutes of Health (NIH). “Research Centers in Minority Institutions” award RR-03037 from the National Center for Research Resources of the NIH, which supports the

References (28)

L.A. Augustin et al.
C-Glycoside analogues of beta-galactosylceramide with a simple ceramide substitute: synthesis and binding to HIV-1 gp120
Bioorg. Med. Chem.
(2006)
L.G. Barrientos et al.
Cyanovirin-N binds to the viral surface glycoprotein, GP1, 2 and inhibits infectivity of Ebola virus
Antiviral Res.
(2003)
L.C. Chang et al.
Potent inhibition of HIV-1 fusion by cyanovirin-N requires only a single high affinity carbohydrate binding site: characterization of low affinity carbohydrate binding site knockout mutants
J. Mol. Biol.
(2002)
J.A. Esté et al.
HIV entry inhibitors
Lancet
(2007)
J. Fantini et al.
Synthetic soluble analogs of galactosylceramide (GalCer) bind to the V3 domain of HIV-1 gp120 and inhibit HIV-1-induced fusion and entry
J. Biol. Chem.
(1997)
S.A. Gallo et al.
The HIV Env-mediated fusion reaction
Biochim. Biophys. Acta
(2003)
N. Garmy et al.
Interaction of cholesterol with sphingosine: physicochemical characterization and impact on intestinal absorption
J. Lipid Res.
(2005)
D. Hammache et al.
Specific interaction of HIV-1 and HIV-2 surface envelope glycoproteins with monolayers of galactosylceramide and ganglioside GM3
J. Biol. Chem.
(1998)
R. Mahfoud et al.
A novel soluble analog of the HIV-1 fusion cofactor, globotriaosylceramide (Gb(3)), eliminates the cholesterol requirement for high affinity gp120/Gb(3) interaction
J. Lipid Res.
(2002)
D. Marsh
Lateral pressure in membranes
Biochim. Biophys. Acta
(1996)

P.N. Nehete et al.

A post-CD4-binding step involving interaction of the V3 region of viral gp120 with host cell surface glycosphingolipids is common to entry and infection by diverse HIV-1 strains

Antiviral Res.

(2002)

N. Taïeb et al.

Rafts and related glycosphingolipid-enriched microdomains in the intestinal epithelium: bacterial targets linked to nutrient absorption

Adv. Drug Deliv. Rev.

(2004)

C. Xiong et al.

Overexpression and purification of scytovirin, a potent, novel anti-HIV protein from the cultured cyanobacterium Scytonema varium

Protein Expr. Purif.

(2006)

N. Yahi et al.

Suramin inhibits binding of the V3 region of HIV-1 envelope glycoprotein gp120 to galactosylceramide, the receptor for HIV-1 gp120 on human colon epithelial cells

J. Biol. Chem.

(1994)

Cited by (34)

The role of lipids in retroviral replication
2018, Retrovirus-Cell Interactions
Retroviruses interact with cellular membranes at various stages of their replication cycle, including the complex processes of membrane fusion during virus entry, and membrane fission late in the replication cycle. For most retroviruses, both entry and egress take place at the plasma membrane of the host cell. Relative to the other components of the retrovirus particle (proteins and nucleic acids), the role of viral lipids is poorly understood. However, recent studies have revealed important functions for lipids and membrane microdomains in virus–membrane interactions for a number of retroviruses. In this chapter, we provide an overview of our current understanding of the role that lipids and membrane microdomains play in retroviral replication.
Rapid screening and identification of active ingredients in licorice extract interacting with V3 loop region of HIV-1 gp120 using ACE and CE-MS
2015, Journal of Pharmaceutical and Biomedical Analysis
The binding of envelope protein gp120 to glycosphingolipids is very important during the human immunodeficiency virus entering into the host cell. This step occurs in the V3 loop region in particularly. The conserved core sequence of V3 loop in gp120 was named R15K. Anti-HIV drug targeting to R15K would avoid the drug-resistance caused by HIV-1 genetic diversity. Here, for the first time, affinity capillary electrophoresis (ACE) and capillary electrophoresis-mass spectrometry (CE-MS) were used for establishing a simple, rapid and effective method of screening the licorice extract for biological activity (anti-HIV), which avoided the complicated isolation and purification process. R15K, 3′-sialyllactose (the positive control), and d-galactose (the negative control) were used for the development and validation of ACE method. After the interaction between licorice extract and R15K was confirmed by ACE, the relative active ingredients were isolated by SPE and their structures were determined by CE-ESI-MS online. In this research, two mixtures from licorice extract were found to be active. Furthermore, glycyrrhizin and licorice saponin G2 were verified as the main ingredients that significantly interacted with R15K via CE-MS and LC–MS. The results of quantitative assays showed that the active mixture contained glycyrrhizin of 74.23% and licorice saponin G2 of 9.52%. Calculated by Scatchard analysis method, glycyrrhizin/R15K complex had the highest binding constant (1.69 ± 0.08) × 10⁷ L/mol among 27 compounds isolated from licorice extract. The anti-HIV activity of glycyrrhizin was further confirmed by bioactive experiment of cellular level. This strategy might provide a high throughput screening and identifying platform for seeking HIV-1 inhibitors in natural products.
Continuous lipase-catalyzed production of pseudo-ceramides in a packed-bed bioreactor
2014, Journal of Molecular Catalysis B: Enzymatic
Ceramides are spingolipid compounds that are very attractive as active components in both the pharmaceutical and the cosmetic industries. In this study, the synthesis of ceramide analogs, the so-called pseudo-ceramides, was carried out using for the first time a two-step continuous enzymatic process with immobilized Candida antarctica lipase B (Novozym^® 435) in a packed-bed bioreactor. The first step involved the selective N-acylation of 3-amino-1,2-propanediol using stearic acid as the first acyl donor (i). This was followed by the selective O-acylation of the N-stearyl 3-amino-1,2-propanediol synthesized in the first step, with myristic acid as the second acyl donor, to produce a N,O-diacyl 3-amino-1,2-propanediol-type pseudo-ceramide, namely 1-O-myristyl,3-N-stearyl 3-amino-1,2-propanediol (ii). The process was first optimized by evaluating the influences of three factors: feed flow rate, quantity of biocatalyst and substrate concentration. Under optimal conditions an amide synthesis yield of 92% and a satisfying production rate of almost 3.15 mmol h⁻¹ g_biocatalyst⁻¹ (1128 mg h⁻¹ g_biocatalyst⁻¹) were obtained. The second step, N-acyl 3-amino-1,2-propanediol O-acylation, was similarly optimized and in addition the effect of the substrate molar ratio was studied. Thus, an optimal pseudo-ceramide synthesis yield of 54% and a production rate of 0.46 mmol h⁻¹ g_biocatalyst⁻¹ (261 mg h⁻¹ g_biocatalyst⁻¹) were reached at a 1:3 ratio of amide to fatty acid. In addition, it was demonstrated that this two-step process has great potential for the production of N,O-diacyl 3-amino-1,2-propanediol-type pseudo-ceramides on an industrial scale. It was shown in particular that Novozym^® 435 could be used for more than 3 weeks without a drop in the yield during the first step of 3-amino-1,2-propanediol N-acylation, proving that this biocatalyst is very stable under these operational conditions. This factor would greatly reduce the need for biocatalyst replacement and significantly lower the associated cost.
The control of Novozym<sup>®</sup> 435 chemoselectivity and specificity by the solvents in acylation reactions of amino-alcohols
2013, Journal of Molecular Catalysis B: Enzymatic
Citation Excerpt :
Acylated amino-alcohols such as ceramides, glucamides and amino-acid derivatives have found important applications, such as in pharmaceutical and cosmetic industries, as potential anti-viral or anti-tumour drugs [1–3], anti-oxidant stabilizers [4] and as active ingredients in hair and skin care products [5].
The current work describes the differential behaviour of Novozym^® 435 (immobilized Candida antarctica lipase B) in O-acylation and N-acylation catalysis of bifunctional amino-alcohols acyl acceptors. We performed acylation experiments on three amino-alcohols (alaninol, 4-amino-1-pentanol and 6-amino-1-hexanol) using myristic acid as an acyl donor. Two organic solvents (tert-amyl alcohol and n-hexane) and one ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate: Bmim [PF₆]) were used to determine the effect of the solvent. The influence of the amino-alcohol carbon chain length between the alcohol and amino groups on chemoselectivity C (k_{cat,app O-acylation}/k_{cat,app N-acylation}) was highlighted. N-acylation is improved using alaninol, a short chain amino-alcohol (no mono-O-acylation in tert-amyl alcohol and C = 0.12 in n-hexane) whereas O-acylation is improved using 4-amino-1-pentanol and 6-amino-1-hexanol which are amino-alcohols with longer chain (C = 10.5 in tert-amyl alcohol and C = 539 in n-hexane). On the other hand, the production of the acylated amino-alcohols after 96 h of reaction was shown to be strongly affected by the solvent nature and the amino-alcohol structure: starting from alaninol as an acyl acceptor, the yield of amide synthesis reaches up to 98% in tert-amyl alcohol using 0.7 equivalents of myristic acid while the yield of amido-ester synthesis reaches up to 88% in Bmim [PF₆] using 1.75 equivalents of myristic acid.
The effect of substrate structure on the chemoselectivity of Candida antarctica lipase B-catalyzed acylation of amino-alcohols
2013, Journal of Molecular Catalysis B: Enzymatic
The selective acylation of multifunctional compounds exhibiting both alcohol and amino groups gives interesting products with many applications in food, cosmetic and pharmaceutical industries, but it is real challenge. The current work describes the different behavior shown by Candida antarctica lipase B (Novozym 435) when catalyzing the O-acylation and N-acylation of bifunctional acyl acceptors. The acylation of three amino-alcohols (alaninol, 4-amino-1-pentanol and 6-amino-1-hexanol) was studied using myristic acid as an acyl donor. To achieve this, a structure-reactivity study was performed in tert-amyl alcohol as a solvent, comparing the three amino-alcohols as acyl acceptors and a series of structurally related amines, namely (R)-sec-butylamine, 1-methoxy-2-propylamine and 1,2-diaminopropane. These substrates were designed to investigate the effect of the group located in β-position of the amino group on the acyl acceptor: the more nucleophilic the group, the more the apparent maximal velocity (V_max,app) of N-acylation increases. Moreover, the crucial role of the carbon chain length between the alcohol and amino groups on the chemoselectivity was also demonstrated. The chemoselectivity for the N-acylation was improved when the carbon chain included two carbons (alaninol) whereas the chemoselectivity for the O-acylation was improved when the carbon chain included four carbons or more (4-amino-1-pentanol and 6-amino-1-hexanol).
These results provided new insights for the selective synthesis of amides or esters produced from the acylation of bifunctional substrates.
Synthesis, gp120 binding and anti-HIV activity of fatty acid esters of 1,1-linked disaccharides
2011, Bioorganic and Medicinal Chemistry
Citation Excerpt :
In this vein, GSL analogues have attracted interest as potential inhibitors of HIV infectivity.2,7–10 We have previously synthesized and tested the gp120 binding and antiviral activity of GalCer analogues with simple ceramide substitutes, against both X4 and R5 tropic virus.11 Some of the lead compounds, represented by 2, bound gp120 similar to GalCer and showed significant activity against HIV envelope glycoprotein (Env)-mediated fusion.
Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of β-galactosylceramide (GalCer), a set of mono- and di-saccharide fatty acid esters were designed as GalCer mimetics and their binding to the V3 loop peptide of HIV-1 and anti-HIV activity evaluated. 1,1-linked Gal-Man and Glu-Man disaccharides with an ester on the Man subunit bound the V3 loop peptide and inhibited HIV infectivity in single round infection assays with the TZM-bl cell line. IC₅₀’s were in the 50 μM range with no toxicity to the cells at concentrations up to 200 μM. These compounds appear to inhibit virus entry at early steps in viral infection since they were inactive if added post viral entry. Although these compounds were found to bind to the V3 loop peptide of gp120, it is not clear that this interaction is responsible for their anti-HIV activity because the relative binding affinity of closely related analogues did not correlate with their antiviral behavior. The low cytotoxicity of these 1,1-linked disaccharide fatty acid esters, combined with the easy accessibility to structurally diverse analogues make these molecules attractive leads for new topical anti-viral agents.

View all citing articles on Scopus

View full text

Published by Elsevier B.V.

Glycoside analogs of β-galactosylceramide, a novel class of small molecule antiviral agents that inhibit HIV-1 entry

Abstract

Introduction

Section snippets

C-glycoside analogs of GalCer

Structure and binding of glycoside analogs of GalCer

Discussion

Acknowledgements

Bioorg. Med. Chem.

Antiviral Res.

J. Mol. Biol.

Lancet

J. Biol. Chem.

Biochim. Biophys. Acta

J. Lipid Res.

J. Biol. Chem.

J. Lipid Res.

Biochim. Biophys. Acta

Antiviral Res.

Adv. Drug Deliv. Rev.

Protein Expr. Purif.

J. Biol. Chem.