Pascal Sonnet

New aromatase inhibitors. Synthesis and biological activity of aryl-substituted pyrrolizine and indolizine derivatives

We report herein the design and the synthesis of some aryl-substituted pyrrolizine and indolizine derivatives, on the basis of a hypothetical pharmacophore structure designed to fit the catalytic site of the human cytochrome P450 aromatase. The in vitro biological evaluation of these compounds allowed us to point out two new potent non-steroidal aromatase inhibitors, MR 20494 and MR 20492, with IC50 values in the range of 0.1 microM.

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.

Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors

Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML.

New ferrocenic pyrrolo[1,2-a]quinoxaline derivatives: Synthesis, and in vitro antimalarial activity – Part II

Following our search for antimalarial compounds, novel series of ferrocenyl-substituted pyrrolo[1,2-a]quinoxalines 1-2 were synthesized from ferrocene-carboxaldehyde and tested for their in vitro activity upon the erythrocytic development of Plasmodium falciparum strains with different chloroquine-resistance status. The ferrocenic pyrrolo[1,2-a]quinoxalines 1-2 were prepared in 6 or 9 steps through a Barton-Zard reaction. Promising pharmacological results against FcB1, K1 and F32 strains were obtained with ferrocenyl pyrrolo[1,2-a]quinoxalines 1j-l linked by a bis-(3-aminopropyl)piperazine linker substituted by a nitrobenzyl moiety.

Synthesis and antiplasmodial activity of betulinic acid and ursolic acid analogues.

More than 40% of the World population is at risk of contracting malaria, which affects primarily poor populations in tropical and subtropical areas. Antimalarial pharmacotherapy has utilised plant-derived products such as quinine and artemisinin as well as their derivatives. However, worldwide use of these antimalarials has caused the spread of resistant parasites, resulting in increased malaria morbidity and mortality. Considering that the literature has demonstrated the antimalarial potential of triterpenes, specially betulinic acid (1) and ursolic acid (2), this study investigated the antimalarial activity against P. falciparum chloroquine-sensitive 3D7 strain of some new derivatives of 1 and 2 with modifications at C-3 and C-28. The antiplasmodial study employed flow cytometry and spectrofluorimetric analyses using YOYO-1, dihydroethidium and Fluo4/AM for staining. Among the six analogues obtained, compounds 1c and 2c showed excellent activity (IC50 = 220 and 175 nM, respectively) while 1a and b demonstrated good activity (IC50 = 4 and 5 μM, respectively). After cytotoxicity evaluation against HEK293T cells, 1a was not toxic, while 1c and 2c showed IC50 of 4 μM and a selectivity index (SI) value of 18 and 23, respectively. Moreover, compound 2c, which presents the best antiplasmodial activity, is involved in the calcium-regulated pathway(s).

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. J. Cell. Biochem. 113: 3029–3038, 2012.

Wnt/β-catenin signaling mediates osteoblast differentiation triggered by peptide-induced α5β1 integrin priming in mesenchymal skeletal cells.

Background: The mechanisms whereby α5β1 integrin triggers osteogenesis are poorly understood. Results: CRRETAWAC-mediated α5β1 integrin priming promotes osteoblast differentiation via PI3K/Wnt/β-catenin signaling independently of the RGD-like REET sequence. Systemic delivery of the α5β1 integrin-priming peptide improved long bone microarchitecture in senescent osteopenic mice. Conclusion: Wnt signaling mediates osteoblast differentiation induced by peptide-mediated α5β1 integrin priming. Significance: A novel mechanism underlying α5β1 integrin-mediated osteoblast differentiation is provided. The α5β1 integrin is a key fibronectin (FN) receptor that binds to RGD-containing peptides to mediate cell adhesion. We previously reported that α5β1 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 α5β1 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 α5β1 integrin priming can promote osteogenic differentiation independently of the RRET sequence. Biochemical analyses showed that peptide-induced α5β1 integrin priming transiently increased PI3K/Akt phosphorylation and promoted Wnt/β-catenin transcriptional activity independently of RRET. Consistently, pharmacological inhibition of PI3K activity reduced osteoblast differentiation and abolished Wnt regulatory gene expression induced by α5β1 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 α5β1 integrin priming by high-affinity ligands integrates Wnt/β-catenin signaling to promote osteoblast differentiation independently of cell adhesion, which could be used to improve bone mass and microarchitecture in the aging skeleton.

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/β‐catenin signaling in murine osteoblasts. We showed that truncated N‐cadherin constructs that impair N‐cadherin‐LRP5/6 interactions promote Wnt/β‐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/β‐catenin signaling in osteoblasts. We also show that the peptide‐mediated disruption of N‐cadherin‐LRP5/6 interaction increases Wnt/β‐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/β‐catenin signaling that can be used for promoting osteoblast function and bone formation.

Evidence for new non-steroidal human aromatase inhibitors and comparison with equine aromatase inhibition for an understanding of the mammalian active site

Abstract We developed a new comparative model in order to better understand the structure-function relationships of the active site in human aromatase. Thus, we undertook the comparative inhibition of human and equine aromatases with new compounds. In fact, equine aromatase represents the only easy and available mammalian membrane-bound enzyme model, besides the human one, which is biochemically purified, well characterized and cloned. During the course of our work concerning the synthesis and screening of new drugs on human and equine aromatases, we identified two new indane derivatives which inhibited the human enzyme (IC 50 = 3.5 μM and 5.9 μM) strongly and selectively while they were much less active on the equine one (IC 50 > 10 μM). The hitherto known aromatase inhibitors, such as 4-hydroxyandrostenedione (4-OHA) and some other indane-related derivatives, are equally efficient on both human and equine enzymes. In this work, using a theoretical 3D model of aromatase, we have explained the human selectivity of the new described compounds as due to the specific differences between the primary structure of both active sites in human and equine enzymes. These results could allow synthesis of a new family of compounds that are much more potent and selective aromatase inhibitors.

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 IC50 ranging from 5 to 12 μM. As expected, most of the compounds showed a significant level of cytotoxicity against monocytes (IC50 = 2-50 μM). 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.