Olivier Renaudet

Oxime ligation: a chemoselective click-type reaction for accessing multifunctional biomolecular constructs.

There is a growing need for biocompatible click reactions in order to prepare multifunctional conjugates, which are valuable molecules for innovative biomedical applications. In this context, we review the recent advances in the implementation of oxime ligation for the synthesis of multivalent or multicomponent systems. The value of these products is emphasized by their use in cell targeting, imaging, synthetic vaccines, and surface modifications.

Towards a Self‐Adjuvanting Multivalent B and T cell Epitope Containing Synthetic Glycolipopeptide Cancer Vaccine

Malignant tumor cells are characterized by the overexpression of altered glycoproteins or glycolipids resulting from the deregulation of glycosylation processes. The identification of these tumor-associated carbohydrate antigens (TACA) has largely contributed to the development of cancer diagnostic and immunotherapy. Particularly, TACA present strong antibody determinants (B cell epitopes) which are primarily targeted by tumor-specific antibodies (Abs). Although TACA are rightly considered as tremendous potential targets for cancer vaccines, their poor immunogenicity still hampers their use as therapeutic vaccines. To address this challenge, both careful rational design and robust chemical procedures should be considered to construct TACA-based vaccine prototypes capable of promoting a strong and selective Ab response against tumor cells. In the last decade, intensive research has focused on the development of molecularly defined TACA-based vaccine prototypes. These studies have clearly defined that not only the display of TACA, but also their nature and molecular formulation are crucial to improve immunity against tumors. First, a multivalent presentation of TACA, either on carrier protein (for example keyhole limpet hemocyanin) or on synthetic delivery systems containing CD4+ T helper (Th) cell epitope (for example multiple antigen glycopeptide) is required to elicit strong B cell responses and raise high affinity tumor-specific Abs. In addition, it was established that priming and sustaining of both Ab and CD8+ cytotoxic T cell (CTL) responses, the latter also crucial in cancer immunity, requires CD4+ Th cell help. This suggests that an ideal cancer vaccine formulation must incorporate B cell, CD4+ , and CD8+ T cell epitopes to ensure both humoral and cellular eradication of tumors. Finally, these synthetic multivalent vaccines should be delivered together with potent and safe external immunoadjuvants to ensure an early and strong immunity. To avoid the potential toxicity related to most of external adjuvants, especially often in immunocompromised cancer patients, recent reports have highlighted that palmitoyl-tailed B and T cell epitope peptides delivered in adjuvant-free saline are clinically safe, eliciting strong, long-lasting, and multivalent protective immunity. However, except for a few studies reporting the synthesis of up to three-component multivalent or “polytope” vaccines, 8] no molecular constructions have been designed so far on the basis of these overall structural features. This is presumably because of inherent difficulties, despite recent progresses in the synthesis, assembly, and formulation of oligosaccharide and glycoconjugate biomolecules. In this communication, we report for the first time on the design, synthesis, safety, immunogenicity, and protective efficacy of a prototype, molecularly defined, fully synthetic, self-adjuvanting multivalent glycolipopeptide (GLP) cancer vaccine. As illustrated on Figure 1, our GLP vaccine prototype associated four essential components displayed on a molecular delivery system: 1) a cluster of TACA B-cell epitope; 2) a CD4+ Th

Hepatocyte Targeting and Intracellular Copper Chelation by a Thiol-Containing Glycocyclopeptide

Metal overload plays an important role in several diseases or intoxications, like in Wilsons disease, a major genetic disorder of copper metabolism in humans. To efficiently and selectively decrease copper concentration in the liver that is highly damaged, chelators should be targeted at the hepatocytes. In the present work, we synthesized a molecule able to both lower intracellular copper, namely Cu(I), and target hepatocytes, combining within the same structure a chelating unit and a carbohydrate recognition element. A cyclodecapeptide scaffold displaying a controlled conformation with two independent faces was chosen to introduce both units. One face displays a cluster of carbohydrates to ensure an efficient recognition of the asialoglycoprotein receptors, expressed on the surface of hepatocytes. The second face is devoted to metal ion complexation thanks to the thiolate functions of two cysteine side-chains. To obtain a chelator that is active only once inside the cells, the two thiol functions were oxidized in a disulfide bridge to afford the glycopeptide P(3). Two simple cyclodecapeptides modeling the reduced and complexing form of P(3) in cells proved a high affinity for Cu(I) and a high selectivity with respect to Zn(II). As expected, P(3) becomes an efficient Cu(I) chelator in the presence of glutathione that mimics the intracellular reducing environment. Finally, cellular uptake and ability to lower intracellular copper were demonstrated in hepatic cell lines, in particular in WIF-B9, making P(3) a good candidate to fight copper overload in the liver.

A green fluorescent chemosensor for amino acids provides a versatile high-throughput screening (HTS) assay for proteases

The water soluble fluorescein-based ligand 1 forms a non-fluorescent complex with Cu(2+). This complex serves as a fluorescent sensor for amino acids in the 10(-3) M concentration range. Since the signal response is very fast, the sensor can be used to detect the hydrolytic activity of various proteases (trypsin, chymotrypsin, subtilisin) on bovine serum albumin as a whole protein substrate, and more generally to follow reactions releasing or removing free amino acids, in real time.

Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction

X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2beta which contacts at the centre of the CK2alpha/CK2beta interface dominates affinity. The results indicate that a double mutation in CK2beta of amino acids Tyr188 and Phe190, which are complementary and fill up a hydrophobic pocket of CK2alpha, is the most disruptive to CK2alpha binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr188-Phe190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2beta-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference.

Antitumor activity of a self-adjuvanting glyco-lipopeptide vaccine bearing B cell, CD4+ and CD8+ T cell epitopes

Molecularly defined synthetic vaccines capable of inducing both antibodies and cellular anti-tumor immune responses, in a manner compatible with human delivery, are limited. Few molecules achieve this target without utilizing external immuno-adjuvants. In this study, we explored a self-adjuvanting glyco-lipopeptide (GLP) as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. A prototype B and T cell epitope-based GLP molecule was constructed by synthesizing a chimeric peptide made of a CD8+ T cell epitope, from ovalbumin (OVA257–264) and an universal CD4+ T helper (Th) epitope (PADRE). The resulting CTL–Th peptide backbones was coupled to a carbohydrate B cell epitope based on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules at C-terminal. The N terminus of the resulting glycopeptides (GP) was then linked to a palmitic acid moiety (PAM), obviating the need for potentially toxic external immuno-adjuvants. The final prototype OVA-GLP molecule, delivered in adjuvant-free PBS, in mice induced: (1) robust RAFT-specific IgG/IgM that recognized tumor cell lines; (2) local and systemic OVA257–264-specific IFN-γ producing CD8+ T cells; (3) PADRE-specific CD4+ T cells; (4) OVA-GLP vaccination elicited a reduction of tumor size in mice inoculated with syngeneic murine MO5 carcinoma cells and a protection from lethal carcinoma cell challenge; (5) finally, OVA-GLP immunization significantly inhibited the growth of pre-established MO5 tumors. Our results suggest self-adjuvanting glyco-lipopeptide molecules as a platform for B Cell, CD4+, and CD8+ T cell epitopes-based immunotherapeutic cancer vaccines.

Multivalent glyco(cyclo)peptides

Because of the importance of carbohydrate-protein interactions in biological processes, the development of glycoclusters and glycodendrimers capable of mimicking the multivalent display of carbohydrates at the cell surface has become a major field of research over the last decade. Among the large variety of scaffolds that are now available, peptides and cyclopeptides are widely used for the multivalent presentation of glycans. This review will provide an overview of the most recent advances in the preparation and utilization of linear glycopeptides and glycocyclopeptides in glycobiology.

Linear and Branched Glyco-Lipopeptide Vaccines Follow Distinct Cross-Presentation Pathways and Generate Different Magnitudes of Antitumor Immunity

Background Glyco-lipopeptides, a form of lipid-tailed glyco-peptide, are currently under intense investigation as B- and T-cell based vaccine immunotherapy for many cancers. However, the cellular and molecular mechanisms of glyco-lipopeptides (GLPs) immunogenicity and the position of the lipid moiety on immunogenicity and protective efficacy of GLPs remain to be determined. Methods/Principal Findings We have constructed two structural analogues of HER-2 glyco-lipopeptide (HER-GLP) by synthesizing a chimeric peptide made of one universal CD4+ epitope (PADRE) and one HER-2 CD8+ T-cell epitope (HER420–429). The C-terminal end of the resulting CD4–CD8 chimeric peptide was coupled to a tumor carbohydrate B-cell epitope, based on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules. The resulting HER glyco-peptide (HER-GP) was then linked to a palmitic acid moiety, attached either at the N-terminal end (linear HER-GLP-1) or in the middle between the CD4+ and CD8+ T cell epitopes (branched HER-GLP-2). We have investigated the uptake, processing and cross-presentation pathways of the two HER-GLP vaccine constructs, and assessed whether the position of linkage of the lipid moiety would affect the B- and T-cell immunogenicity and protective efficacy. Immunization of mice revealed that the linear HER-GLP-1 induced a stronger and longer lasting HER420–429-specific IFN-γ producing CD8+ T cell response, while the branched HER-GLP-2 induced a stronger tumor-specific IgG response. The linear HER-GLP-1 was taken up easily by dendritic cells (DCs), induced stronger DCs maturation and produced a potent TLR- 2-dependent T-cell activation. The linear and branched HER-GLP molecules appeared to follow two different cross-presentation pathways. While regression of established tumors was induced by both linear HER-GLP-1 and branched HER-GLP-2, the inhibition of tumor growth was significantly higher in HER-GLP-1 immunized mice (p<0.005). Significance These findings have important implications for the development of effective GLP based immunotherapeutic strategies against cancers.

Expedient synthesis of aminooxylated-carbohydrates for chemoselective access of glycoconjugates

Abstract Herein, we describe an efficient preparation of various biologically important carbohydrate motifs bearing an aminooxy group at the anomeric position. These nucleophilic sugar analogues represent useful intermediates for the chemoselective preparation of glycoconjugates. The key glycosylation step involves the coupling of fluoro-activated protected sugar and N -hydroxyphthalimide in the presence of BF 3 ·Et 2 O. Final deprotection and cleavage of the phthalimide moiety with methylhydrazine afforded new Glc-β-ONH 2 3 , GalNAc-β-ONH 2 9 , Glc-α-ONH 2 14 , Gal-α-ONH 2 17 and Man-α-ONH 2 20 derivatives with good yields. Compared to the literature results, the preparation of Gal-β-ONH 2 6 , GalNAc-α-ONH 2 11 and Lac-β-ONH 2 23 proved to be more efficient.

On-bead synthesis and binding assay of chemoselectively template-assembled multivalent neoglycopeptides

The investigation of recognition events between carbohydrates and proteins, especially the control of how spatial factors and binding avidity are correlated in, remains a great interest for glycomics. Therefore, the development of efficient methods for the rapid evaluation of new ligands such as multivalent glycoconjugates is essential for diverse diagnostic or therapeutic applications. In this paper we describe the synthesis of chemoselectively-assembled multivalent neoglycopeptides and the subsequent recognition assay on a solid support. Aminooxylated carbohydrates (betaLac-ONH(2) 4, alphaGalNAc-ONH(2) 9 and alphaMan-ONH(2) 13) have been prepared as carbohydrate-based recognition elements and assembled as clusters onto a cyclopeptidic scaffold by an oxime-based strategy in solid phase. Further binding tests between lectins and beads of resin derivatized with neoglycopeptides displaying clustered lactoses, N-acetylgalactoses and mannoses (18-20) have shown specific recognition and enhanced affinity through multivalent interactions, suggesting that the local density of carbohydrate-based ligands at the bead surface is crucial to improve the interaction of proteins of weak binding affinity. This solid phase strategy involving both molecular assembly and biological screening provides a rapid and efficient tool for various applications in glycomics.