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Development of a binding assay between the HIV-1 envelope protein (gp120) and coreceptors CCR5/CXCR4 by Surface Plasmon Resonance : Screening and optimization of viral entry inhibitors

Abstract : It is well-established that cell-associated Heparan Sulphate (HS) binds the V3 loop of gp120 of HIV-1 thus aiding in viral infectivity. However, a variety of soluble polyanions have antiviral properties once conjugated to CD4 and a CD4-conjugated HS (mCD4-HS12), showed nM activity against HIV-1 in vitro. Due to the structural complexity of HS, screening differently sulphated oligosaccharides to improve the molecule's activity would be too cumbersome, thus in order to obtain a more specific, higher affinity and easier to produce moiety, collaborators synthesized HS mimetic peptides. We aimed to screen these peptides and other anionic molecules for their capacity to inhibit HIV-1 entry. To this end we set-up a platform whereby solubilised CCR5 and CXCR4 were immobilized on biosensors (biacore) and used to screen for molecules that inhibited gp120-CD4 binding to the coreceptors. To control the solubilization process, CXCL12, the natural ligand of CXCR4, was injected over the immobilized CXCR4. The affinities of CXCL12 isoforms (α and γ) for CXCR4 were calculated within the ranges of values that have been previously described with different techniques, thus proving the functionality of our system and enabling us to investigate the binding mechanisms of these two isoforms with CXCR4 and their regulation by HS. The system was subsequently used to screen the inhibitory capacity of the HS mimetic peptides. Each peptide, [S(XDXS)n], contained amino acids that mimic the hydroxyl, carboxyl and sulphate groups on HS chains. The peptide containing sulphotyrosine residues, when conjugated to mCD4 (mCD4-P3YSO3), displayed nM IC50 for simultaneously inhibiting gp120 binding to HS, CD4, antibody, coreceptors and HIV-1 infection in vitro. This is the first bivalent entry inhibitor that targets both R5 and X4 viruses and the concept of a HSmimetic peptide lends itself to structural-functional analysis of HS chains binding to proteins, a novel technique in this field.
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https://tel.archives-ouvertes.fr/tel-00721774
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Submitted on : Monday, July 30, 2012 - 12:12:48 PM
Last modification on : Tuesday, October 6, 2020 - 4:12:09 PM
Long-term archiving on: : Wednesday, October 31, 2012 - 3:22:46 AM

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Bridgette Janine Connell. Development of a binding assay between the HIV-1 envelope protein (gp120) and coreceptors CCR5/CXCR4 by Surface Plasmon Resonance : Screening and optimization of viral entry inhibitors. Agricultural sciences. Université de Grenoble, 2012. English. ⟨NNT : 2012GRENV013⟩. ⟨tel-00721774⟩

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