Carbohydrate-based peptidomimetics targeting neuropilin-1: Synthesis, molecular docking study and in vitro biological activities.

Neuropilin-1 (NRP-1), a transmembrane glycoprotein acting as a co-receptor of VEGF-A, is expressed by cancer and angiogenic endothelial cells and is involved in the angiogenesis process. Taking advantage of functionalities and stereodiversities of sugar derivatives, the design and the synthesis of carbohydrate based peptidomimetics are here described. One of these compounds (56) demonstrated inhibition of VEGF-A165 binding to NRP-1 (IC50=39µM) and specificity for NRP-1 over VEGF-R2. Biological evaluations were performed on human umbilical vein endothelial cells (HUVECs) through activation of downstream proteins (AKT and ERK phosphorylation), viability/proliferation assays and in vitro measurements of anti-angiogenic abilities.

Development of 6-[(18) F]fluoro-carbohydrate-based prosthetic groups and their conjugation to peptides via click chemistry.

This work describes the development of new 6-[(18) F]fluoro-carbohydrate-based prosthetic groups equipped with an azido arm that are able to participate in copper(I)-catalyzed cycloadditions for (18) F labeling of biomolecules under mild conditions. The radiolabeling in high radiochemical yields (up to 68 ± 6%) of these different prosthetic groups is presented. The flexibility of the azido arm introduced on the carbohydrate moieties allows efficient click reactions with different alkyne functionalized peptides such as gluthation or Arg-Gly-Asp derivatives in order to prepare glycopeptides. The radiosyntheses of (18) F-labeled glycopeptides proceed in high radiochemical yields (up to 76%) in an automated process with excellent radiochemical purity. The addition of a sugar moiety on peptides should enhance the bioavailability, pharmacokinetic, and in vivo clearance properties of these glycopeptides, compared with the unlabeled native peptide, and these properties are highly favorable for positron emission tomography imaging. A high uptake of (18) F-ß-gluco-c(RGDfC) is shown by positron emission tomography imaging in a subcutaneous abscess model in the rat, revealing the potential of this tracer to monitor integrin expression as a part of inflammation and/or angiogenesis processes.

Diastereoselective synthesis of new O-alkylated and C-branched inositols and their corresponding fluoro analogues.

Efficient routes were developed for the diastereoselective synthesis of new O-alkylated and C-branched inositols and their corresponding fluoro analogues. The key steps of the synthesis were the easy accessibility of different types of arms in term of configuration (myo and scyllo), the linking method and length, which could modulate the biological properties. These inositol derivatives, bearing an arm terminated either with a hydroxy group or a fluorine atom, could be interesting candidates for diastereoisomeric intermediates and biological evaluations, especially for PET imaging experiments.

PPARγ-inactive Δ2-troglitazone independently triggers ER stress and apoptosis in breast cancer cells.

Our aim was to better understand peroxisome proliferator-activated receptor gamma (PPARγ)-independent pathways involved in anti-cancer effects of thiazolidinediones (TZDs). We focused on Δ2-troglitazone (Δ2-TGZ), a PPARγ inactive TZD that affects breast cancer cell viability. Appearance of TUNEL positive cells, changes in mitochondrial membrane potential, cleavage of poly(ADP-ribose) polymerase (PARP)-1 and caspase-7 revealed that apoptosis occurred in both hormone-dependent MCF7 and hormone-independent MDA-MB-231 breast cancer cells after 24 and 48 h of treatment. A microarray study identified endoplasmic reticulum (ER) stress as an essential cellular function since many genes involved in ER stress were upregulated in MCF7 cells following Δ2-TGZ treatment. Δ2-TGZ-induced ER stress was further confirmed in MCF7 cells by phosphorylation of pancreatic endoplasmic reticulum kinase-like endoplasmic reticulum kinase (PERK) and its target eIF2α after 1.5 h, rapid increase in activating transcription factor (ATF) 3 mRNA levels, splicing of X-box binding protein 1 (XBP1) after 3 h, accumulation of binding immunogloblulin protein (BiP) and CCAAT-enhancer-binding protein homologous protein (CHOP) after 6 h. Immunofluorescence microscopy indicated that CHOP was relocalized to the nucleus of treated cells. Similarly, in MDA-MB-231 cells, overexpression of ATF3, splicing of XBP1, and accumulation of BiP and CHOP were observed following Δ2-TGZ treatment. In MCF7 cells, knock-down of CHOP or the inhibition of c-Jun N-terminal kinase (JNK) did not impair cleavage of PARP-1 and caspase-7. Altogether, our results show that ER stress is an early response of major types of breast cancer cells to Δ2-TGZ, prior to, but not causative of apoptosis.

'Click' glycosylation of peptides through cysteine propargylation and CuAAC.

'Click' glycosylation of cysteine-containing peptides were carried out in good yield by Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC). For that peptides were functionalized though direct propargylation of the cysteine residue allowing their use in CuAAC with suitable free or protected azido sugars of gluco, manno and galacto configuration. Among these free and protected glycopeptides a series of 'glycoRGD' peptides were obtained and submitted to in vitro platelet aggregation tests, showing that the pseudoglycosylation of the adhesion sequence lowers the IC50 value and thus could improve the in vivo pharmacokinetic properties.

Kinetic study of the rearrangement of deuterium-labeled 4'-O-methylnorbelladine in Leucojum aestivum shoot cultures by mass spectrometry. Influence of precursor feeding on amaryllidaceae alkaloid accumulation.

Alkaloids from plants of the family Amaryllidaceae have important pharmacological properties and can be regarded as derivatives of the common precursor 4'-O-methylnorbelladine (6) via intramolecular oxidative phenol coupling. Their biosynthetic pathway, particularly in Leucojum aestivum, has not yet been totally elucidated. Therefore, shoot cultures of this plant were subcultured in medium containing the labeled precursor 4'-O-methyl-d(3)-norbelladine (3) at various concentrations (0.05, 0.10, and 0.20 g/L) and were incubated for various periods of time (15, 30, and 40 days). The aim of this work was to study the influence of this precursor on both labeled and native alkaloid accumulation. Biotransformation into galanthamine (1) and lycorine (2) in shoot cultures was demonstrated using HPLC coupled to mass spectrometry. A maximal amount of 0.16% of 1 referred to the dry weight was obtained at day 15 in shoots fed with 0.10 g/L of precursor. In addition, a 20.5% dry weight of 2 was reached after 40 days of feeding with 0.20 g/L of precursor.

New troglitazone derivatives devoid of PPARγ agonist activity display an increased antiproliferative effect in both hormone-dependent and hormone-independent breast cancer cell lines.

Numerous recent studies indicate that most anticancer effects of PPARγ agonists like thiazolidinediones are the result of PPARγ-independent pathways. These conclusions were obtained by several approaches including the use of thiazolidinedione derivatives like Δ2-Troglitazone (Δ2-TGZ) that does not activate PPARγ. Since biotinylation has been proposed as a mechanism able to increase the specificity of drug delivery to cancer cells which could express a high level of vitamin receptor, a biotinylated derivative of Δ2-TGZ (bΔ2-TGZ) has been synthetized. In the present article, we have studied the in vitro effects of this molecule on both hormone-dependent (MCF-7) and hormone-independent (MDA-MB-231) breast cancer cells. In both cell lines, bΔ2-TGZ was more efficient than Δ2-TGZ to decrease cell viability. bΔ2-TGZ was also more potent than Δ2-TGZ to induce the proteasomal degradation of cyclin D1 in both cell lines and those of ERα in MCF-7 cells. However, in competition experiments, the presence of free biotin in the culture medium did not decrease the antiproliferative action of bΔ2-TGZ. Besides, other compounds that had no biotin but that were substituted at the same position of the phenolic group of the chromane moiety of Δ2-TGZ decreased cell viability similarly to bΔ2-TGZ. Hence, we concluded that the increased antiproliferative action of bΔ2-TGZ was not due to biotin itself but to the functionalization of the terminal hydroxyl group. This should be taken into account for the design of new thiazolidinedione derivatives able to affect not only hormone-dependent but also hormone-independent breast cancer cells in a PPARγ-independent pathway.

Sugar-based peptidomimetics as potential inhibitors of the vascular endothelium growth factor binding to neuropilin-1.

Neuropilin-1 (NRP-1) is a co-receptor of VEGFR(165) and molecules interfering with VEGF(165) binding to NRP-1 seem to be promising candidates as new angiogenesis modulators. Based on the minimal four amino acid sequence of peptidic ligands known to bind NRP-1, we describe here the design, synthesis and biological evaluation of series of original sugar-based peptidomimetics using a C-glycosyl compound, derived from d-gulonolactone, as a scaffold, which was functionalized with side chains of the amino-acids arginine, and tryptophane or threonine. At 100 microM, all compounds exhibited a weak affinity for NRP-1, the most efficient being the bis-guanidinylated compound 32 (IC(50)=92 microM) which could be considered as a new NRP-1 non-peptidic ligand.

An efficient route to acyclic C-nucleosides and fused-ring analogues of uridine from exo-glycals.

Beta-amino esters prepared from activated exo-glycals are transformed into acyclic C-nucleoside with a C-4-substituted uracil derivative that can be cyclized under Mitsunobu conditions to provide a new family of fused-ring analogues of uridine nucleoside in which the N-1 nitrogen atom is embedded in an imino sugar ring. An analogue of uridine of D-ribo configuration is prepared.

Synthesis and solution conformation of homo-beta-peptides consisting of N-mannofuranosyl-3-ulosonic acids.

The synthesis and solution conformation of homo-oligomers of beta-aminoacids, beta-N-mannofuranosyl-3-ulosonic acids, have been studied by NMR, MD simulation, and circular dichroism. These oligomers feature a spirocyclic disubstitution and a N,O-acetal functionality at the beta-carbon of the backbone, an unprecedented situation in the realm of beta-peptides. Our study shows that tetramer 10 and hexamer 11 adopt a characteristic secondary structure. In the hexamer 11, NMR investigations coupled with MD simulations suggest the preference for a double C(8) turn forming conformation.

LCMS and GCMS for the screening of alkaloids in natural and in vitro extracts of Leucojum aestivum.

HPLC coupled to a mass spectrometer (MS) was used for the analysis of galanthamine and lycorine in natural extracts of Leucojum aestivum and in their in vitro cultures grown with a precursor (ACC), inhibitors (AgNO(3), STS), or an absorber (KMnO(4)) of ethylene. The maximum galanthamine (0.002%) and lycorine (0.02%) concentrations in tissue cultures were obtained in the presence of KMnO(4). GCMS was used to investigate underivatized alkaloid mixtures from L. aestivum. Seven alkaloids were identified in in vivo bulbs. KMnO(4) led to the highest diversity of alkaloids in tissue culture extracts.

Disruption of ERalpha signalling pathway by PPARgamma agonists: evidences of PPARgamma-independent events in two hormone-dependent breast cancer cell lines.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that can be activated by natural ligands such as 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ(2)) as well as synthetic drugs such as thiazolidinediones. The treatment of human breast cancer cell lines with PPARgamma agonists is known to have antiproliferative effects but the role of PPARgamma activation in the process remains unclear. In the present study, we investigated the effects of four PPARgamma agonists, Rosiglitazone (RGZ), Ciglitazone (CGZ), Troglitazone (TGZ) and the natural agonist 15d-PGJ(2), on estrogen receptor alpha (ERalpha) signalling pathway in two hormone-dependent breast cancer cell lines, MCF-7 and ZR-75-1. In both of them, TGZ, CGZ and 15d-PGJ(2) induced an inhibition of ERalpha signalling associated with the proteasomal degradation of ERalpha. ZR-75-1 cells were more sensitive than MCF-7 cells to these compounds. Treatments that induced ERalpha degradation inhibited cell proliferation after 24 h. In contrast, 24 h exposure to RGZ, the most potent activator of PPARgamma disrupted neither ERalpha signalling nor cell proliferation. 9-cis retinoic acid never potentiated the proteasomal degradation of ERalpha. PPARgamma antagonists (T0070907, BADGE and GW 9662) did not block the proteolysis of ERalpha in MCF-7 and ZR-75-1 cells treated with TGZ. ERalpha proteolysis still occurred in case of PPARgamma silencing as well as in case of treatment with the PPARgamma-inactive compound Delta2-TGZ, demonstrating a PPARgamma-independent mechanism. The use of thiazolidinedione derivatives able to trigger ERalpha degradation by a PPARgamma-independent pathway could be an interesting tool for breast cancer therapy.

Irreversible inhibition of glucose-6-phosphate dehydrogenase by the coenzyme A conjugate of ketoprofen: a key to oxidative stress induced by non-steroidal anti-inflammatory drugs?

Oxidative damage by non-steroidal anti-inflammatory drugs (NSAIDs) has been considered relevant to the occurrence of gastro-intestinal side-effects. In the case of chiral arylpropionate derivatives like ketoprofen (KPF), this mechanism has been evidenced for the R-enantiomer, especially when chiral inversion was observed, and lets us suppose the involvement of CoA conjugates. Glucose-6-phosphate dehydrogenase (G6PD) is the crucial enzyme to regenerate the GSH pool and maintain the intracellular redox potential. This enzyme is known to be down-regulated by palmitoyl-CoA thioester. We hypothesised then that G6PD is the target of carboxylic NSAIDs, via their CoA metabolites. We used molecular docking to localise a putative site in the human G6PD then we chose the Yeast orthologue, as the most suitable species to study experimentally the precise molecular interaction. KPF-CoA was effectively shown to bind covalently to the unique cysteine residue of the yeast enzyme. Binding was found to occur in the same site as palmitoyl-CoA. It was decreased in the presence of an allosteric inhibitor of G6PD, phospho(enol)pyruvate, and was not detected with G6PD of Leuconostoc mesenteroides, which does not possess the allosteric site. This site is distinct from the catalytic site, and probably allosteric, explaining the observed non-competitive inhibition of its activity by KPF-CoA. KPF-CoA was shown to induce the production of reactive oxygen species in Caco-2 cells, where its inhibition of G6PD activity was observed.

Spiro sugar-isoxazolidine scaffold as useful polyfunctional building block for peptidomimetics design.

Spiro sugar-isoxazolidines obtained by 1,3-dipolar cycloaddition of activated exo-glycals and nitrones were efficiently functionalized at two sites, i.e. C-4 and C-7, with arginine, arginine mimetics and guanidylated appendages. Two bicyclic sugar derivatives differing by the configuration at C-7 were chosen as model compounds. The small library of peptidomimetics was evaluated toward inhibition of VEGF-A165/neuropilin-1 binding. Unexpected cleavage of C3-C4 bond of isoxazolidine moiety was observed during hydrogenolysis and opened thus a new way toward hemiketal structures which could also find interesting applications as less constrained scaffold.

Fully automated production of sodium [(18)F]fluoride on AllInOne and miniAllInOne synthesizers.

A fully automated production of the imaging agent sodium [(18)F]fluoride ([(18)F]NaF) on two different modules commercialized by Trasis®, the AllInOne and the miniAllInOne, is reported. Both modules allow to prepare [(18)F]NaF in good radiochemical yield (around 97%) in less than 4min with the same specifications. Quality control of [(18)F]NaF produced by this way was performed according to the US and European Pharmacopeia monograph requirements.

Combining pharmacophore search, automated docking, and molecular dynamics simulations as a novel strategy for flexible docking. Proof of concept: docking of arginine-glycine-aspartic acid-like compounds into the alphavbeta3 binding site.

A novel and highly efficient flexible docking approach is presented where the conformations (internal degrees of freedom) and orientations (external degrees of freedom) of the ligands are successively considered. This hybrid method takes advantage of the synergistic effects of structure-based and ligand-based drug design techniques. Preliminary antagonist-derived pharmacophore determination provides the postulated bioactive conformation. Subsequent docking of this pharmacophore to the receptor crystal structure results in a postulated pharmacophore/receptor binding mode. Pharmacophore-oriented docking of antagonists is subsequently achieved by matching ligand interacting groups with pharmacophore points. Molecular dynamics in water refines the proposed complexes. To validate the method, arginine-glycine-aspartic acid (RGD) containing peptides, pseudopeptides, and RGD-like antagonists were docked to the crystal structure of alphavbeta3 holoprotein and apoprotein. The proposed directed docking was found to be more accurate, faster, and less biased with respect to the protein structure (holo and apoprotein) than DOCK, Autodock, and FlexX docking methods. The successful docking of an antagonist recently cocrystallized with the receptor to both apo and holoprotein is particularly appealing. The results summarized in this report illustrated the efficiency of our light CoMFA/rigid body docking hybrid method.

Elucidation of the mechanism of inhibition of cyclooxygenases by acyl-coenzyme A and acylglucuronic conjugates of ketoprofen.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the cyclooxygenase (COX) isoforms which accounts for their clinical effects. The differential inhibition of COX-1 and COX-2 is not sufficient to explain the absence of a correlation between in vitro and in vivo effects, especially for 2-aryl-propionates, thus indicating the participation of metabolites. Conjugates to glucuronic acid and to coenzyme-A are mainly produced, and have been shown to be chemically reactive. Therefore, we studied the interaction of the ketoprofen metabolites with the COX enzymes. After incubation with bovine pulmonary artery endothelial cells (BPAEC), COX-1 was inhibited stereoselectively by S-ketoprofen acylglucuronide, and more significantly by CoA-thioester. After washing-out the medium, COX-1 activity was essentially recovered, indicating a reversible inhibition. In LPS-stimulated J774.2 cells, COX activity (mainly inducible COX-2) was inhibited reversibly and stereospecifically by S-ketoprofen glucuronide, whereas it disappeared totally and was not recovered after incubation with CoA-thioester. Correspondingly, inhibition of purified COX-2 with this compound was observed to be rapid and irreversible. Using an anti-ketoprofen antibody, COX immunoprecipitated from cells exhibited adduct formation for COX-2 but not for COX-1. This was observed after incubation with CoA-thioester, and, surprisingly, also with glucuronide. Molecular docking gave support to explain this discrepancy: the glucuronide was found to establish a strong interaction with Y115 located in the membrane binding domain, whereas the thioester was preferentially bound to the active site of the enzyme. Overall, our results suggest a contribution of CoA-thioester metabolites of carboxylic NSAIDs to their pharmacological action by irreversibly and selectively inhibiting COX-2.

Vitronectin receptor alpha(V)beta(3) integrin antagonists: chemical and structural requirements for activity and selectivity.

Alpha(V)beta(3) integrin, a cell surface protein, has been targeted by a variety of natural and synthetic antagonists in the search for potential cancer and osteoporosis drug candidates. This review discusses chemical and structural requirements for activity and selectivity deduced from SAR studies and draws a tentative picture of the pharmacophore.

From D-glucuronic acid to L-iduronic acid derivatives via a radical tandem decarboxylation-cyclization.

A synthesis to L-iduronic derivatives, major components of heparin derived pentasaccharides was accomplished by formal inversion of configuration at C-5 of a D-glucuronic acid derivative through radical formation at C-5 using Barton decarboxylation followed by intramolecular radical addition on an acetylenic tether at O-4 giving exclusively a bicyclic sugar of L-ido configuration. Oxidation and ring opening of this bicyclic sugar led to a L-iduronate. This method opens the way to short syntheses of pentasaccharidic moiety of Idraparinux and congeners.

Optimization of troglitazone derivatives as potent anti-proliferative agents: towards more active and less toxic compounds.

Δ2-Troglitazone derivatives were shown to exhibit anti-proliferative activity in a PPARγ-independent manner. We prepared various compounds in order to increase their potency and decrease their toxicity towards non-malignant primary cultured hepatocytes. Many compounds induced viabilities less than 20% at 10 µM on various cancer cell lines. Furthermore, five of them showed hepatocyte viability of 80% or more at 200 µM. In addition, compounds 17 and 18 exhibited promising maximum tolerated doses on a murine model, enabling future investigations.