Determination of the Rituximab Binding Site to the CD20 Epitope Using SPOT Synthesis and Surface Plasmon Resonance Analyses.

Antibodies not only play a major role in clinical diagnostics and biopharmaceutical analysis but also are a class of drugs that are regularly used to treat numerous diseases. The identification of antibody-epitope binding sites is then of great interest to many emerging medical and bioanalytical applications, particularly to design monoclonal antibodies (mAb) mimics taking advantage of amino acid residues involved in the binding. Among relevant antibodies, the monoclonal antibody rituximab has received significant attention as it is exploited to treat several cancers including non-Hodgkin's lymphoma and chronic lymphocytic leukemia, as well as some autoimmune disorders such as rheumatoid arthritis. The binding of rituximab to the targeted cells occurs via the recognition of the CD20 epitope. A crystallographic study has shown that the binding area, named paratope, is located at the surface of rituximab. Combining the SPOT method and the complementary surface plasmon resonance technique allowed us to detect an extended recognition domain buried in the pocket of the rituximab Fab formed by four ß-sheets. More generally, the present study offers a comprehensive approach to identify antibody-epitope binding sites.

Fluorescent Peptide Biosensor for Probing CDK6 Kinase Activity in Lung Cancer Cell Extracts.

CDK6 kinase regulates cell-cycle progression in G1, together with CDK4, but has cell-, tissue- and developmentally distinct functions associated with transcription, angiogenesis and metabolism. Although CDK6 makes an attractive cancer biomarker and target, there are no means of assessing its activity in a complex environment. In this study, we describe the design, engineering and characterisation of a fluorescent peptide biosensor derived from 6-phosphofructokinase that reports on CDK6 kinase activity through sensitive changes in fluorescence intensity. This biosensor can report on CDK6 activity in a dose-dependent fashion, thereby enabling quantification of differences in kinase activity in complex and physiologically relevant environments. Further implementation of this biosensor in different lung and melanoma cell lines, as well as in mesothelioma cell lines derived from patients together with a CDK4 biosensor highlighted differences in kinase activity between CDK6 and CDK4 kinase. This work demonstrates the utility of these selective tools for monitoring two closely related kinases comparatively and simultaneously in the same samples, thereby offering attractive perspectives for diagnostic and therapeutic purposes.

Lanthanide DO3A-Complexes Bearing Peptide Substrates: The Effect of Peptidic Side Chains on Metal Coordination and Relaxivity.

Two DO3A-type ligands conjugated to substrates of urokinase (L3) and caspase-3 (L4) via a propyl-amide linker were synthesized and their lanthanide(III) (Ln3+) complexes studied. A model compound without peptide substrate (L2) and an amine derivative ligand mimicking the state after enzymatic cleavage (L1) were also prepared. Proton Nuclear Magnetic Relaxation Dispersion (NMRD) profiles recorded on the gadolinium(III) (Gd3+) complexes, complemented with the assessment of hydration numbers via luminescence lifetime measurements on the Eu3+ analogues, allowed us to characterize the lanthanide coordination sphere in the chelates. These data suggest that the potential donor groups of the peptide side chains (carboxylate, amine) interfere in metal coordination, leading to non-hydrated LnL3 and LnL4 complexes. Nevertheless, GdL3 and GdL4 retain a relatively high relaxivity due to an important second-sphere contribution generated by the strongly hydrophilic peptide chain. Weak PARACEST effects are detected for the amine-derivative EuL1 and NdL1 chelates. Unfortunately, the GdL3 and GdL4 complexes are not significantly converted by the enzymes. The lack of enzymatic recognition of these complexes can likely be explained by the participation of donor groups from the peptide side chain in metal coordination.

MCH-R1 Antagonist GPS18169, a Pseudopeptide, Is a Peripheral Anti-Obesity Agent in Mice.

Melanin-concentrating hormone (MCH) is a 19 amino acid long peptide found in the brain of animals, including fishes, batrachians, and mammals. MCH is implicated in appetite and/or energy homeostasis. Antagonists at its receptor (MCH-R1) could be major tools (or ultimately drugs) to understand the mechanism of MCH action and to fight the obesity syndrome that is a worldwide societal health problem. Ever since the deorphanisation of the MCH receptor, we cloned, expressed, and characterized the receptor MCH-R1 and started a vast medicinal chemistry program aiming at the discovery of such usable compounds. In the present final work, we describe GPS18169, a pseudopeptide antagonist at the MCH-R1 receptor with an affinity in the nanomolar range and a Ki for its antagonistic effect in the 20 picomolar range. Its metabolic stability is rather ameliorated compared to its initial parent compound, the antagonist S38151. We tested it in an in vivo experiment using high diet mice. GPS18169 was found to be active in limiting the accumulation of adipose tissues and, correlatively, we observed a normalization of the insulin level in the treated animals, while no change in food or water consumption was observed.

A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis.

Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a new general tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a heterobifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences ("helping hands") onto insoluble peptides. This tool is implemented in three simple and nearly quantitative steps: (i) on-resin incorporation of the linker at a Lys residue ε-amine, (ii) Fmoc-SPPS elongation of a desired solubilizing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful introduction and removal of a Lys6 helping hand is first demonstrated in two model systems (Ebola virus C20 peptide and the 70-residue ribosomal protein L31). It is then applied to the challenging chemical synthesis of the 97-residue co-chaperonin GroES, which contains a highly insoluble C-terminal segment that is rescued by a helping hand. Importantly, the Ddae linker can be cleaved in one pot following NCL or desulfurization. The purity, structure, and chaperone activity of synthetic l-GroES were validated with respect to a recombinant control. Additionally, the helping hand enabled synthesis of d-GroES, which was inactive in a heterochiral mixture with recombinant GroEL, providing additional insight into chaperone specificity. Ultimately, this simple, robust, and easy-to-use tool is expected to be broadly applicable for the synthesis of challenging peptides and proteins.

Towards the simplification of protein synthesis: iterative solid-supported ligations with concomitant purifications.

Please release me: a new linker for the temporary tagging of peptides at their N-terminus after solid-phase elongation, and its potential for capture/release purification is demonstrated. This concept is extended to a remarkably efficient self-purifying N-to-C iterative triazole ligation strategy, which is applied to the synthesis of a polypeptide having 160 residues, in a high purity without the need for chromatographic purification (orange blocks: peptide segments).

Caloxin-derived peptides for the inhibition of plasma membrane calcium ATPases.

Plasma membrane calcium ATPases (PMCAs) are a family of transmembrane proteins responsible for the extrusion of cytosolic Ca2+ to the extracellular milieu. They are important players of the calcium homeostasis possibly implicated in some important diseases. The reference inhibitors of PMCA extruding activity are on one hand ortho-vanadate (IC50 in the 30 mM range), and on the other a series of 12- to 20-mer peptides named caloxins (IC50 in the 100 µM scale). As for all integral membrane proteins, biochemistry and pharmacology are difficult to study on isolated and/or purified proteins. Using a series of reference blockers, we assessed a pharmacological window with which we could study the functionality of PMCAs in living cells. Using this system, we screened for alternative versions of caloxins, aiming at shortening the peptide backbone, introducing non-natural amino acids, and overall trying to get a glimpse at the structure-activity relationship between those new peptides and the protein in a cellular context. We describe a short series of equipotent 5-residue long analogues with IC50 in the low µM range.

RGD-cyclam conjugate: synthesis and potential application for positron emission tomography.

Cyclam and DOTA-containing positron emission tomography radiotracers were prepared by using a modular chemical strategy based on peptide synthesis and chemoselective ligations. These molecules encompass two functional domains, one a tumour 'homing' domain and the other a chelating ligand for copper allowing nuclear imaging of tumours.

Application of click-click chemistry to the synthesis of new multivalent RGD conjugates.

New multivalent RGD-containing macromolecules were designed by exploiting two orthogonal chemoselective ligations. They were next applied to a competitive cell adhesion assay and used for the non invasive optical imaging of tumour in small animals.

One-pot approach to well-defined biomolecular assemblies by orthogonal chemoselective ligations.

Click-click cyclopeptides: Well-defined biomolecular assemblies are synthesized using orthogonal oxime bond formation and copper(I)-mediated alkyne-azide cycloaddition reactions in a stepwise or in a one-pot approach. To illustrate this strategy, regioselective ligation of biologically relevant peptides onto a cyclopeptidic scaffold was performed.

Imaging of extracellular cathepsin S activity by a selective near infrared fluorescence substrate-based probe.

We designed a near-infrared fluorescent substrate-based probe (SBP), termed MG101, for monitoring extracellular cathepsin S (CatS) activity. We conceived a fused peptide hairpin loop-structure, combining a CatS recognition domain, an electrostatic zipper (with complementary charges of a polyanionic (D-Glu)5 segment and a polycationic (D-Arg)5 motif, as well as a N and C terminal Förster resonance energy transfer pair (donor: AlexaFluor680; quencher: BHQ3) to facilitate activity-dependent imaging. MG101 showed excellent stability since no fluorescence release corresponding to a self-dequenching was observed in the presence of either 2 M NaCl or after incubation at a broad range of pH (2.2-8.2). Cathepsins B, D, G, H, and K, neutrophil elastase and proteinase 3 did not cleave MG101, while CatS, and to a lesser extent CatL, hydrolysed MG101 at pH 5.5. However MG101 was fully selective for CatS at pH 7.4 (kcat/Km = 140,000 M-1 s-1) and sensitive to low concentration of CatS (<1 nM). The selectivity of MG101 was successfully endorsed ex vivo, as it was hydrolysed in cell lysates derived from wild-type but not knockout CatS murine spleen. Furthermore, application of the SBP probe with confocal microscopy confirmed the secretion of active CatS from THP-1 macrophages, which could be abrogated by pharmacological CatS inhibitors. Taken together, present data highlight MG101 as a novel near-infrared fluorescent SBP for the visualization of extracellular active CatS from macrophages and other cell types.

VHH characterization. Comparison of recombinant with chemically synthesized anti-HER2 VHH.

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody-like protein, using an anti-HER2 VHH as a model. The total chemical synthesis of the 125 amino-acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its "antigen." Using this combination of orthogonal techniques, it was possible to show that the three VHHs-whether synthetic or recombinant ones-were properly and similarly folded and recognized the "antigen" HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH-drug conjugates.

Combining triazole ligation and enzymatic glycosylation on solid phase simplifies the synthesis of very long glycoprotein analogues.

The solid-phase chemical assembly of a protein through iterative chemoselective ligation of unprotected peptide segments can be followed with chemical and/or enzymatic transformations of the resulting immobilized protein, the latter steps thus benefitting from the advantages provided by the solid support. We demonstrate here the usefulness of this strategy for the chemo-enzymatic synthesis of glycoprotein analogues. A linker was specifically designed for application to the synthesis of O-glycoproteins: this new linker is readily cleaved under mild aqueous conditions compatible with very sensitive glycosidic bonds, but is remarkably stable under a wide range of chemical and biochemical conditions. It was utilized for solid-supported N-to-C peptidomimetic triazole ligation followed by enzymatic glycosylation, ultimately leading to a very large MUC1-derived glycoprotein containing 160 amino acid residues, 24 α-GalNAc moieties linked to Ser and Thr, and 3 triazoles as peptide bond mimetics.

Rational design of triazololipopeptides analogs of kisspeptin inducing a long-lasting increase of gonadotropins.

New potent and selective KISS1R agonists were designed using a combination of rational chemical modifications of the endogenous neuropeptide kisspeptin 10 (KP10). Improved resistance to degradation and presumably reduced renal clearance were obtained by introducing a 1,4-disubstituted 1,2,3-triazole as a proteolysis-resistant amide mimic and a serum albumin-binding motif, respectively. These triazololipopeptides are highly potent full agonists of KISS1R and are >100 selective over the closely related NPFF1R. When injected in ewes with a quiescent reproductive system, the best compound of our series induced a much prolonged increase of luteinizing hormone release compared to KP10 and increased follicle-stimulating hormone plasma concentration. Hence, this KISS1R agonist is a new valuable pharmacological tool to explore the potential of KP system in reproduction control. Furthermore, it represents the first step to develop drugs treating reproductive system disorders due to a reduced activity of the hypothalamo-pituitary-gonadal axis such as delayed puberty, hypothalamic amenorrhea, and hypogonadotropic hypogonadism.

Preparation of peptide and other biomolecular conjugates through chemoselective ligations.

The synthesis of molecular conjugates through chemoselective ligations represents a very convenient strategy to prepare complex macromolecules with diverse functional elements. Herein, we describe chemical methods based on the preparation of chemoselectively addressable peptides allowing successive oxime ligations and/or alkyne-azide cycloaddition ("click") reactions of various biomolecules. This modular synthetic approach can be applied to a broad range of purposes.

Redox strategy for reversible attachment of biomolecules using bifunctional linkers.

Soft attachment of streptavidin to ß-cyclodextrin-modified pegylated SAMs was efficiently performed in a reversible and repetitive way via orthogonal bifunctional linkers involving streptavidin-biotin recognition and redox-driven multivalent host-guest (ß-cyclodextrin-ferrocene) interactions.

Noninvasive visualization and quantification of tumor αVß3 integrin expression using a novel positron emission tomography probe, 64Cu-cyclam-RAFT-c(-RGDfK-)4.

INTRODUCTION: The α(V)ß(3) integrin is a well-known transmembrane receptor involved in tumor invasion, angiogenesis and metastasis. Our aim was to evaluate a novel positron emission tomography (PET) probe, (64)Cu-cyclam-RAFT-c(-RGDfK-)(4), for noninvasive visualization and quantification of α(V)ß(3) integrin expression. METHODS: RAFT-c(-RGDfK-)(4), a tetrameric cyclic Arg-Gly-Asp (RGD)-based peptide, was conjugated with a bifunctional chelator, 1,4,8,11-tetraazacyclotetradecane (cyclam), radiolabeled with the positron emitter (64)Cu and evaluated in vitro by cell binding and competitive inhibition assays and in vivo by biodistribution and receptor blocking studies, and PET imaging. The following cell lines, human embryonic kidney HEK293(ß(1)) [α(V)ß(3)-negative] and HEK293(ß(3)) [α(V)ß(3)-overexpressing] and human glioblastoma U87MG [naturally expressing α(V)ß(3)], together with their subcutaneous xenografts in athymic nude mice, were used for the present study. The expression levels of α(V)ß(3) on these cell lines and tumor xenografts were analyzed by flow cytometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography, respectively. RESULTS: (64)Cu-cyclam-RAFT-c(-RGDfK-)(4) demonstrated the in vitro and in vivo specificity for the α(V)ß(3) integrin and displayed rapid blood clearance, predominantly renal excretion and low uptake in nontumor tissues. Tumor uptake of (64)Cu-cyclam-RAFT-c(-RGDfK-)(4) (3 h postinjection) in HEK293(ß(3)) (high levels of α(V)ß(3)), U87MG (moderate levels of α(V)ß(3)) and HEK293(ß(1)) (undetectable levels of α(V)ß(3)) tumors was 9.35%±1.19%, 3.46%±0.45% and 1.18%±0.30% injected dose per gram, respectively, with a strong and positive correlation with the tumor α(V)ß(3) expression levels (correlation coefficient=0.967; P<.0001). Positron emission tomographic images showed that α(V)ß(3)-positive tumors were clearly visualized with high tumor-to-background contrast, and agreed well with the biodistribution results. CONCLUSION: (64)Cu-cyclam-RAFT-c(-RGDfK-)(4) exhibits potential for noninvasively quantifying α(V)ß(3) expression.