GFOGER Peptide Modifies the Protein Content of Extracellular Vesicles and Inhibits Vascular Calcification.

OBJECTIVE: Vascular calcification (VC) is an active process during which vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and release extracellular vesicles (EVs). In turn, the EVs serve as calcification foci via interaction with type 1 collagen (COL1). We recently showed that a specific, six-amino-acid repeat (GFOGER) in the sequence of COL1 was involved in the latter's interaction with integrins expressed on EVs. Our main objective was to test the GFOGER ability to inhibit VC. APPROACH: We synthesized the GFOGER peptide and tested its ability to inhibit the inorganic phosphate (Pi)-induced calcification of VSMCs and aortic rings. Using mass spectrometry, we studied GFOGER's effect on the protein composition of EVs released from Pi-treated VSMCs. RESULTS: Calcification of mouse VSMCs (MOVAS-1 cells), primary human VSMCs, and rat aortic rings was lower in the presence of GFOGER than with Pi alone (with relative decreases of 66, 58, and 91%, respectively; p < 0.001 for all) (no effect was observed with the scramble peptide GOERFG). A comparative proteomic analysis of EVs released from MOVAS-1 cells in the presence or absence of Pi highlighted significant differences in EVs' protein content. Interestingly, the expression of some of the EVs' proteins involved in the calcification process (such as osteogenic markers, TANK-binding kinase 1, and casein kinase II) was diminished in the presence of GFOGER peptide (data are available via ProteomeXchange with identifier PXD018169∗). The decrease of osteogenic marker expression observed in the presence of GFOGER was confirmed by q-RT-PCR analysis. CONCLUSION: GFOGER peptide reduces vascular calcification by modifying the protein content of the subsequently released EVs, in particular by decreasing osteogenicswitching in VSMCs.

Synthesis and Study of New Quinolineaminoethanols as Anti-Bacterial Drugs.

The lack of antibiotics with a novel mode of action associated with the spread of drug resistant bacteria make the fight against infectious diseases particularly challenging. A quinoline core is found in several anti-infectious drugs, such as mefloquine and bedaquiline. Two main objectives were set in this work. Firstly, we evaluated the anti-mycobacterial properties of the previous quinolines 3, which have been identified as good candidates against ESKAPEE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli) bacteria. Secondly, a new series 4 was designed and assessed against the same bacteria strains, taking the pair of enantiomers 3m/3n as the lead. More than twenty compounds 4 were prepared through a five-step asymmetric synthesis with good enantiomeric excesses (>90%). Interestingly, all compounds of series 3 were efficient on M. avium with MIC = 2-16 µg/mL, while series 4 was less active. Both series 3 and 4 were generally more active than mefloquine against the ESKAPEE bacteria. The quinolines 4 were either active against Gram-positive bacteria (MIC ≤ 4 µg/mL for 4c-4h and 4k/4l) or E. coli (MIC = 32-64 µg/mL for 4q-4v) according to the global lipophilicity of these compounds.

Synthesis, iron(III) complexation properties, molecular dynamics simulations and P. aeruginosa siderophore-like activity of two pyoverdine analogs.

P. aeruginosa ranks among the top five organisms causing nosocomial infections. Among the many novel strategies for developing new therapeutics against infection, targeting iron uptake mechanism seems promising as P. aeruginosa needs iron for its growth and survival. To scavenge iron, the bacterium produces siderophores possessing a very high affinity towards Fe(III) ions such as pyoverdines. In this work, we decided to study two pyoverdine analogs, aPvd2 and aPvd3, structurally close to the endogen pyoverdine. The pFe constants calculated with the values of formation showed a high affinity of aPvd3 towards Fe(III). Molecular dynamics calculations demonstrated that aPvd3-Fe forms with Fe(III) stable 1:1 complexes in water, whereas aPvd2 does not. Only aPvd3 is able to increase the bacterial growth and represents thus an alternative to pyoverdine for iron acquisition by the bacterium. The aPvd2-3 interaction studies with a lipid membrane indicated that they were unable to interact and to cross the plasma membrane of bacteria by passive diffusion. Consequently, the penetration of aPvd3 is ruled by a transport membrane protein. These results showed that aPvd3 may be used to inhibit pyoverdine uptake or to promote the accumulation and release of antibiotics into the cell following a Trojan horse strategy.

Oligogalacturonic Acid Inhibits Vascular Calcification by Two Mechanisms: Inhibition of Vascular Smooth Muscle Cell Osteogenic Conversion and Interaction With Collagen.

OBJECTIVE: Cardiovascular diseases constitute the leading cause of mortality worldwide. Calcification of the vessel wall is associated with cardiovascular morbidity and mortality in patients having many diseases, including diabetes mellitus, atherosclerosis, and chronic kidney disease. Vascular calcification is actively regulated by inductive and inhibitory mechanisms (including vascular smooth muscle cell adaptation) and results from an active osteogenic process. During the calcification process, extracellular vesicles (also known as matrix vesicles) released by vascular smooth muscle cells interact with type I collagen and then act as nucleating foci for calcium crystallization. Our primary objective was to identify new, natural molecules that inhibit the vascular calcification process. APPROACH AND RESULTS: We have found that oligogalacturonic acids (obtained by the acid hydrolysis of polygalacturonic acid) reduce in vitro inorganic phosphate-induced calcification of vascular smooth muscle cells by 80% and inorganic phosphate-induced calcification of isolated rat aortic rings by 50%. A specific oligogalacturonic acid with a degree of polymerization of 8 (DP8) was found to inhibit the expression of osteogenic markers and, thus, prevent the conversion of vascular smooth muscle cells into osteoblast-like cells. We also evidenced in biochemical and immunofluorescence assays a direct interaction between matrix vesicles and type I collagen via the GFOGER sequence (where single letter amino acid nomenclature is used, O=hydroxyproline) thought to be involved in interactions with several pairs of integrins. CONCLUSIONS: DP8 inhibits vascular calcification development mainly by inhibition of osteogenic marker expression but also partly by masking the GFOGER sequence-thereby, preventing matrix vesicles from binding to type I collagen.

Wnt/ß-catenin signaling mediates osteoblast differentiation triggered by peptide-induced α5ß1 integrin priming in mesenchymal skeletal cells.

The α5ß1 integrin is a key fibronectin (FN) receptor that binds to RGD-containing peptides to mediate cell adhesion. We previously reported that α5ß1 integrin promotes osteogenic differentiation in mesenchymal skeletal cells (MSCs), but the underlying mechanisms are not fully understood. In this study, we determined the signaling mechanisms induced by α5ß1 integrin interaction with its high-affinity ligand CRRETAWAC in murine and human MSCs and in vivo. We show that cyclized CRRETAWAC fully displaced MSC adhesion to FN, whereas related peptides lacking the full RRET sequence produced a partial displacement, indicating that RRET acts as an RGD-like sequence that is required to antagonize FN-mediated cell adhesion. However, all peptides increased focal adhesion kinase phosphorylation, OSE2 transcriptional activity, osteoblast gene expression, and matrix mineralization in MSCs, indicating that peptide-induced α5ß1 integrin priming can promote osteogenic differentiation independently of the RRET sequence. Biochemical analyses showed that peptide-induced α5ß1 integrin priming transiently increased PI3K/Akt phosphorylation and promoted Wnt/ß-catenin transcriptional activity independently of RRET. Consistently, pharmacological inhibition of PI3K activity reduced osteoblast differentiation and abolished Wnt regulatory gene expression induced by α5ß1 integrin priming. In vivo, systemic delivery of cyclized GACRETAWACGA linked to (DSS)6 to allow delivery to bone-forming sites for 6 weeks increased serum osteocalcin levels and improved long bone mass and microarchitecture in SAMP-6 senescent osteopenic mice. The results support a mechanism whereby α5ß1 integrin priming by high-affinity ligands integrates Wnt/ß-catenin signaling to promote osteoblast differentiation independently of cell adhesion, which could be used to improve bone mass and microarchitecture in the aging skeleton.

N-cadherin/wnt interaction controls bone marrow mesenchymal cell fate and bone mass during aging.

Age-related bone loss is characterized by reduced osteoblastogenesis and excessive bone marrow adipogenesis. The mechanisms governing bone marrow mesenchymal stromal cell (BMSC) differentiation into adipocytes or osteoblasts during aging are unknown. We show here that overexpressing N-cadherin (Cadh2) in osteoblasts increased BMSC adipocyte differentiation and reduced osteoblast differentiation in young transgenic (Tg) mice whereas this phenotype was fully reversed with aging. The reversed phenotype with age was associated with enhanced Wnt5a and Wnt10b expression in osteoblasts and a concomitant increase in BMSC osteogenic differentiation. Consistent with this mechanism, conditioned media from young wild type osteoblasts inhibited adipogenesis and promoted osteoblast differentiation in BMSC from old Cadh2 Tg mice, and this response was abolished by Wnt5a and Wnt10b silencing. Transplantation of BMSC from old Cadh2 Tg mice into young Tg recipients increased Wnt5a and Wnt10b expression and rescued BMSC osteogenic differentiation. In senescent osteopenic mice, blocking the CADH2-Wnt interaction using an antagonist peptide increased Wnt5a and Wnt10b expression, bone formation, and bone mass. The data indicate that Cadh2/Wnt interaction in osteoblasts regulates BMSC lineage determination, bone formation, and bone mass and suggest a therapeutic target for promoting bone formation in the aging skeleton.

Absolute Configuration and Antimalarial Activity of erythro-Mefloquine Enantiomers.

Mefloquine (MQ), an antimalarial drug, is used as a racemate of (-)- and (+)-enantiomers, which display biological differences. The question concerning their exact configuration remains a matter of debate. The absolute configuration of the two MQ enantiomers as well as their biological activity has been established, thus confirming the importance of the stereochemistry in the design of MQ analogues that have fewer adverse side effects.

Peptide-based activation of alpha5 integrin for promoting osteogenesis.

Promoting osteoblastogenesis remains a major challenge in disorders characterized by defective bone formation. We recently showed that the alpha 5 integrin subunit (ITGA5) is critically involved in human mesenchymal cell osteoblast differentiation. In this study, we determined the potential of pharmacological ITGA5 activation by a synthetic cyclic peptide (GA-CRRETAWAC-GA) on murine osteoblast differentiation and function in vitro and bone formation in vivo. Peptide-mediated activation of ITGA5 in murine C3H10T1/2 mesenchymal cells resulted in the generation of the integrin-mediated cell signals FAK and ERK1/2-MAPKs. In vitro, peptide-based activation of ITGA5 protected from cell apoptosis but did not affect cell adhesion or replication, while it enhanced the expression of the osteoblast marker genes Runx2 and type I collagen and increased extracellular matrix (ECM) mineralization as also found with bone morphogenetic protein-2 (BMP2), a standard bone anabolic factor. When injected on adult mouse cranial bone for 3 weeks, the peptide-mediated activation of ITGA5 increased bone thickness by twofold, an effect also induced by BMP2. Histomorphometric analysis showed that this anabolic effect resulted from decreased cell apoptosis and increased bone forming surfaces and bone formation rate (BFR). We conclude that pharmacological activation of ITGA5 in mesenchymal cells is effective in promoting de novo bone formation as a result of increased osteoprogenitor cell differentiation into osteoblasts and increased cell protection from apoptosis. This peptide-based approach could be used therapeutically to promote the osteogenic capacity of osteoblast progenitor cells and to induce de novo bone formation in conditions where osteoblastogenesis is compromised.

Peptide-based mediated disruption of N-cadherin-LRP5/6 interaction promotes Wnt signaling and bone formation.

Wnt signaling plays an important role in skeletal biology and diseases. In osteoblasts, we recently showed that the cell-cell adhesion molecule N-cadherin interacts with the Wnt coreceptors LRP5/6 to regulate osteogenesis. In this study we investigated whether targeting the intracellular domain of N-cadherin that interacts with LRP5/6 may promote Wnt signaling and bone formation. By investigating the molecular interactions between the Wnt coreceptors LRP5/6 and N-cadherin, we identified specific LRP5/6- and N-cadherin-interacting intracellular domains that impact Wnt/ß-catenin signaling in murine osteoblasts. We showed that truncated N-cadherin constructs that impair N-cadherin-LRP5/6 interactions promote Wnt/ß-catenin signaling and osteoblast differentiation. Based on this finding, we developed a peptide-based approach targeting N-cadherin-LRP5 interaction for promoting Wnt signaling and osteoblast function. We found that a competitor peptide containing the 28 last amino acids of LRP5 disrupts LRP5/6-N-cadherin interaction and thereby enhances Wnt/ß-catenin signaling in osteoblasts. We also show that the peptide-mediated disruption of N-cadherin-LRP5/6 interaction increases Wnt/ß-catenin signaling and osteoblast function in vitro and promotes calvaria bone formation in vivo. The targeted competitor peptide-based strategy reported here may provide a novel approach to stimulate Wnt/ß-catenin signaling that can be used for promoting osteoblast function and bone formation.

Circular dichroism studies of type III collagen mimetic peptides with anti- or pro-aggregant activities on human platelets.

We report the synthesis of collagen related peptides containing the peptide sequence Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys. The anti-thrombotic activity effects of different glycine mutations in this sequence were studied in regard with their different adopted conformations. The biological results could be correlated to the glycine propensity to adopt a more stable polyproline II helix conformation. The incorporation of these sequences in "collagen-like" alpha-triple-helix peptides shows a pro-thrombotic activity compared to a scrambled negative control peptide which possesses no significant activity.

Synthesis and preliminary evaluation of new ursolic and oleanolic acids derivatives as antileishmanial agents.

A series of new ursolic and oleanolic acids derivatives was synthesized via ursolic or oleanolic acids, previously extracted from South American Ilex species. These new compounds were tested for in vitro antiparasitic activity on Leishmania amazonensis and Leishmania infantum strains. Some of these compounds showed activity against the promastigote forms of L. amazonensis or L. infantum, with IC(50) ranging from 5 to 12 microM. As expected, most of the compounds showed a significant level of cytotoxicity against monocytes (IC(50) = 2-50 microM). From a structure-activity relationships point of view, these pharmacological results enlightened mainly the importance of an acetylation at position 3 of the oleanolic acid skeleton in the activity against the L. amazonensis strain, and of a bis-(3-aminopropyl)piperazine moiety on the carboxylic function of ursolic acid against the L. infantum strain.

Evaluation of ursolic acid isolated from Ilex paraguariensis and derivatives on aromatase inhibition.

The inhibitory potency of ursolic acid extracted from Ilex paraguariensis, a plant used in South American population for a tea preparation known as maté, and its derivatives to inhibit aromatase activity was assessed and compared to a phytoestrogen apigenin and a steroidal aromatase inhibitor 4-hyroxyandrostenedione (4-OHA). Among all compounds tested only ursolic acid 1 showed an efficient and dose-dependent aromatase inhibition with IC50 value of 32 microM as did apigenin (IC50=10 microM), whereas IC50 value of 4-OHA was 0.8 microM. Our results show that the incorporation of a metallocene moiety into the ursolic acid derivatives decreases the aromatase inhibition. Moreover, comparison of the structure/inhibitory potency relationship of compounds indicates that the presence of cycle A and the configuration of C3-OH and C17-COOH seems to be more favourable to recognize the active site of aromatase and to block its activity.

Pharmacomodulation on the 3-acetylursolic acid skeleton: Design, synthesis, and biological evaluation of novel N-{3-[4-(3-aminopropyl)piperazinyl]propyl}-3-O-acetylursolamide derivatives as antimalarial agents.

A series of new piperazine derivatives of ursolic acid was synthesized and tested against Plasmodium falciparum strains. They were also tested on their cytotoxicity effects upon MRC-5 cells. Seven new piperazinyl analogues showed significant activity in the nanomolar range (IC(50)=78-167nM) against Plasmodium falciparum CQ-resistant strain FcB1. A possible mechanism of interaction implicating binding of these compounds to beta-hematin was supported by in vitro tests. Moreover, the importance of the hydrophilic framework attached at the terminal nitrogen atom of the bis-(3-aminopropyl)piperazine joined to the triterpene ring was also explored through molecular dynamic simulations.

Type III collagen mimetic peptides designed with anti- or pro-aggregant activities on human platelets.

We report the synthesis of collagen related peptides containing the peptide sequence Lys-Hyp-Gly-Glu-Hyp-Gly-Pro-Lys. The alpha-triple helix peptides behave as type III collagen analogues supporting platelet aggregation, while the homotrimer which does not exhibit a triple-helical conformation inhibits type III collagen-induced human platelet aggregation. The incorporation of the octapeptide sequence in type III collagen mimetic peptides may lead to the loss of the anti-thrombotic activity for a pro-thrombotic one.

Antithrombotic effect of the type III collagen-related octapeptide (KOGEOGPK) in the mouse.

Platelet adhesion to subendothelial types I and III collagens exposed upon vascular injury plays a crucial role in hemostasis and thrombosis. We previously identified a KOGEOGPK sequence (O for hydroxyproline) within type III collagen interacting with platelets, and demonstrated a strong inhibitory effect of the KOGEOGPK peptide on human platelet interactions with type III collagen in vitro. In the present study, we tested the antithrombotic effect of KOGEOGPK in vivo. In a mouse model of pulmonary thromboembolism induced by intravenous injection of type III collagen and epinephrine, prior administration of 80 mg/kg KOGEOGPK reduced by 50% the size of thrombi embolized in lungs, compared to vehicle-treated mice (p<0.0001). In a mouse model of photochemically induced lesion of caecum venules and arterioles, intravenous injection of 80 mg/kg KOGEOGPK decreased by 76% the occurrence of arteriole occlusion 45 min after vascular injury (p<0.05). A moderate antithrombotic effect of KOGEOGPK was also observed in the injured venules. In addition, intracardiac injection of KOGEOGPK had no effect on the tail bleeding time. These findings demonstrate a substantial contribution of platelet interactions with the type III collagen-related KOGEOGPK sequence in thrombus formation in vivo with preferential involvement in arterial thrombosis.

Further evidence that the CCK2 receptor is coupled to two transduction pathways using site-directed mutagenesis.

A heterogeneity of CCK2 receptors has been reported which could correspond to different states of coupling to G proteins and/or association with different second messenger systems. To investigate these hypotheses, the wild-type CCK2 receptor and three mutants F347A, D100N and K333M/K334T/R335L, expected to modify the coupling of the G protein with the third intracellular loop of the receptor, were transfected into Cos-7 cells and their binding and signalling properties were evaluated using the natural ligand CCK8. Activation of wild-type as well as F347A, D100N or K333M/K334T/R335L CCK2 receptors by this ligand led to a similar arachidonic acid release which was blocked by pertussis toxin and the phospholipase A2 inhibitor, mepacrine. Nevertheless, in contrast to the wild-type CCK2 receptor, addition of CCK8 to cells transfected with the F347A or K333M/K334T/R335L mutants did not result in the production of inositol phosphates while the maximum increase in this second messenger formation was reduced by 30% with the D100N mutant. Taken together, these results suggest that the CCK2 receptor is coupled to two G proteins and that Phe347 and the cluster of basic residues K333/K334/R335 probably play a key role in Gq protein stimulation leading to inositol phosphate production but not in activation of the G protein coupled to phospholipase A2. These data bring additional support at the molecular level to the existence of different affinity states of CCK2 receptors suggested from the results of binding assays and behavioural studies.