Rémy Le Guével

Synthesis and kinase inhibitory activity of novel substituted indigoids

The bis-indole indigoids are a promising protein kinase inhibitor scaffold to be further evaluated against the numerous human diseases that imply abnormal regulation of kinases including neurodegenerative disorders. In an effort to identify new pharmacological inhibitors of disease-relevant protein kinases with increased potency and selectivity, we designed, synthesized new 5,7-disubstituted or 6-substituted bis-indole derivatives. On the basis of our previous synthetic work, 22 selected compounds were tested on CDK1/cyclin B, CDK5/p25, DYRK1A, CK1, and GSK-3alpha/beta kinases, five kinases involved in Alzheimers disease. Some of them were also evaluated for their cytotoxic and antiproliferative activities. 6-Nitro-3-N-oxime-indirubin and 5-amino-3-N-oxime-indirubin derivatives exhibited inhibitory activity in a submicromolar range against CDK1/cyclin B (0.18 and 0.1 microM, respectively), CK1 (0.6 microM and 0.13 microM) and GSK3 (0.04 microM and 0.36 microM).

Identification of small molecule regulators of the nuclear receptor HNF4α based on naphthofuran scaffolds

Nuclear receptors are ligand-activated transcription factors involved in all major physiological functions of complex organisms. In this respect, they are often described as drugable targets for a number of pathological states including hypercholesterolemia and atherosclerosis. HNF4alpha (NR2A1) is a recently deorphanized nuclear receptor which is bound in vivo by linoleic acid, although this natural ligand does not seem to promote transcriptional activation. In mouse, HNF4alpha is a major regulator of liver development and hepatic lipid metabolism and mutations in human have been linked to diabetes. Here, we have used a yeast one-hybrid system to identify small molecule activators of HNF4alpha in a library of synthetic compounds and found one hit bearing a methoxy group branched on a nitronaphthofuran backbone. A collection of molecules deriving from the discovered hit was generated and tested for activity toward HNF4alpha in yeast one-hybrid system. It was found that both the nitro group and a complete naphthofuran backbone were required for full activity of the compounds. Furthermore, adding a hydroxy group at position 7 of the minimal backbone led to the most active compound of the collection. Accordingly, a direct interaction of the hydroxylated compound with the ligand binding domain of HNF4alpha was detected by NMR and thermal denaturation assays. When used in mammalian cell culture systems, these compounds proved to be highly toxic, except when methylated on the furan ring. One such compound was able to modulate HNF4alpha-driven transcription in transfected HepG2C3A cells. These data indicate that HNF4alpha activity can be modulated by small molecules and suggest new routes for targeting the receptor in humans.

Comparative localization and functional activity of the main hepatobiliary transporters in HepaRG cells and primary human hepatocytes

The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various in vivo and in vitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HHs) are considered as the most suitable in vitro cell model but erratic availability and inter-donor functional variations limit their use. In this work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HHs in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular [bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1 (MDR1), and MDR3] or basolateral [Na(+)-taurocholate co-transporting polypeptide (NTCP) and MRP3] membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8- and 2.6-fold lower, than in SCHHs and CCHHs, respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHHs. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HHs and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis.

Estrogenic Potency of Benzophenone UV Filters in Breast Cancer Cells: Proliferative and Transcriptional Activity Substantiated by Docking Analysis

The results from recent studies show that some benzophenones (BPs) and their hydroxylated metabolites can function as weak estrogens (E2) in the environment. However, little is known about the structure-activity relationship of these molecules. We have examined the effects of exposure to ten different BPs on the proliferation of estrogen receptor (ER)-positive breast cancer cells and on the transcriptional activity of E2-target genes. We analyzed two genes that are tightly linked with estrogen-mediated proliferation, the CXCL12 and amphiregulin genes and two classical estrogen-responsive genes, the pS2 and progesterone receptor. Significant differences in the BPs efficiency to induce cell proliferation and endogenous E2-target gene expressions were observed. Using ERE-, Sp1-, AP1- and C3-reporter genes that contain different ER-binding sites in their promoter, we also showed significant differences in the BPs efficiency in activation of the ER transactivation. Together, our analyzes showed that the most active molecule is 4-hydroxy-BP. Docking analysis of the interaction of BPs in the ligand-binding pocket of ERα suggests that the minimum structural requirement for the estrogenic activity of BPs is a hydroxyl (OH) group in the phenyl A-ring that allows interaction with Glu-353, Arg-394 or Phe-404, which enhances the stability between BPs and ERα. Our modeling also indicates a loss of interaction between the OH groups of the phenyl B-ring and His-524. In addition, the presence of some OH groups in the phenyl B-ring can create repulsion forces, which may constrain helix 12 in an unfavorable position, explaining the differential estrogenic effects of BPs. These results, together with our analysis of BPs for their potency in activation of cell proliferation and ER-mediated transcription, report an improved understanding of the mechanism and structure–activity relationship of BPs.

Rho-kinase/myosin light chain kinase pathway plays a key role in the impairment of bile canaliculi dynamics induced by cholestatic drugs

Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injury; however, the mechanisms underlying such injuries are poorly understood. In this study of human HepaRG and primary hepatocytes, we found that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid (BA) efflux and require alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. Short exposure to 6 cholestatic compounds revealed that BC constriction and dilation were associated with disruptions in the ROCK/MLCK/myosin pathway. At the studied concentrations, cyclosporine A and chlorpromazine induced early ROCK activity, resulting in permanent MLC2 phosphorylation and BC constriction. However, fasudil reduced ROCK activity and caused rapid, substantial and permanent MLC2 dephosphorylation, leading to BC dilation. The remaining compounds (1-naphthyl isothiocyanate, deoxycholic acid and bosentan) caused BC dilation without modulating ROCK activity, although they were associated with a steady decrease in MLC2 phosphorylation via MLCK. These changes were associated with a common loss of BC contractions and failure of BA clearance. These results provide the first demonstration that cholestatic drugs alter BC dynamics by targeting the ROCK/MLCK pathway; in addition, they highlight new insights into the mechanisms underlying bile flow failure and can be used to identify new predictive biomarkers of drug-induced cholestasis.

Hypoxia Differentially Modulates the Genomic Stability of Clinical‐Grade ADSCs and BM‐MSCs in Long‐Term Culture

Long‐term cultures under hypoxic conditions have been demonstrated to maintain the phenotype of mesenchymal stromal/stem cells (MSCs) and to prevent the emergence of senescence. According to several studies, hypoxia has frequently been reported to drive genomic instability in cancer cells and in MSCs by hindering the DNA damage response and DNA repair. Thus, we evaluated the occurrence of DNA damage and repair events during the ex vivo expansion of clinical‐grade adipose‐derived stromal cells (ADSCs) and bone marrow (BM)‐derived MSCs cultured with platelet lysate under 21% (normoxia) or 1% (hypoxia) O2 conditions. Hypoxia did not impair cell survival after DNA damage, regardless of MSC origin. However, ADSCs, unlike BM‐MSCs, displayed altered γH2AX signaling and increased ubiquitylated γH2AX levels under hypoxic conditions, indicating an impaired resolution of DNA damage‐induced foci. Moreover, hypoxia specifically promoted BM‐MSC DNA integrity, with increased Ku80, TP53BP1, BRCA1, and RAD51 expression levels and more efficient nonhomologous end joining and homologous recombination repair. We further observed that hypoxia favored mtDNA stability and maintenance of differentiation potential after genotoxic stress. We conclude that long‐term cultures under 1% O2 were more suitable for BM‐MSCs as suggested by improved genomic stability compared with ADSCs. Stem Cells 2015;33:3608–3620

Synthesis and biological evaluation of 2,3-bis(het)aryl-4-azaindole derivatives as protein kinase inhibitors

The synthesis of several novel 4-azaindoles was carried out by novel Fischer reaction which offers as a main advantage, the synthesis of the bisfunctionalized 4-azaindolic building block in one step. The final compounds were evaluated on a panel of 5 kinases in order to evaluate their selectivity and on 7 cancer cell lines to determine their cytotoxic effects. RAF-1 and DYRK1A inhibitions were found, docking studies explain fully the results.

Synthesis and biological evaluation of novel oxophenylarcyriaflavins as potential anticancer agents.

We report the synthesis and biological evaluation of new oxophenylarcyriaflavins designed as potential anticancer agents. An efficient synthesis involving palladium-catalyzed Suzuki and Stille reactions is presented, without any indolic protective group. The central ring closure of the scaffold was performed through an electrophilic reaction on the position C-2 of the indole ring. The use of indole and 5-benzyloxyindole, along with substituted phenyl rings, generated three different scaffolds, which were successively exploited to modulate the structure. The cytotoxicity of the newly designed compounds on four cancer cell lines and activities against three kinases (CDK1, CDK5 and GSK3) were evaluated. Several compounds showed a marked cytotoxicity with IC(50) values in the sub-micromolar range, and induced important cell cycle perturbations, with a G2/M arrest. Some compounds revealed DNA binding properties and were found to inhibit topoisomerase-mediated DNA relaxation of supercoiled DNA, but these properties are not mandatory for a cytotoxic action. A novel lead compound () has been identified and warrants further investigations.

Synthesis and toxicity evaluation of hydrophobic ionic liquids for volatile organic compounds biodegradation in a two-phase partitioning bioreactor

Synthesis of several hydrophobic ionic liquids (ILs), which might be selected as good candidates for degradation of hydrophobic volatile organic compounds in a two-phase partitioning bioreactor (TPPB), were carried out. Several bioassays were also realized, such as toxicity evaluation on activated sludge and zebrafish, cytotoxicity, fluoride release in aqueous phase and biodegradability in order to verify their possible effects in case of discharge in the aquatic environment and/or human contact during industrial manipulation. The synthesized compounds consist of alkylimidazoliums, functionalized imidazoliums, isoqinoliniums, triazoliums, sulfoniums, pyrrolidiniums and morpholiniums and various counter-ions such as: PF6(-), NTf2(-) and NfO(-). Toxicity evaluation on activated sludge of each compound (5% v/v of IL) was assessed by using a glucose uptake inhibition test. Toxicity against zebrafish and cytotoxicity were evaluated by the ImPACCell platform of Rennes (France). Fluoride release in water was estimated by regular measurements using ion chromatography equipment. IL biodegradability was determined by measuring BOD28 of aqueous samples (compound concentration,1mM). All ILs tested were not biodegradable; while some of them were toxic toward activated sludge. Isoquinolinium ILs were toxic to human cancerous cell lines. Nevertheless no toxicity was found against zebrafish Danio rerio. Only one IL released fluoride after long-time agitation.

Synthesis and biological evaluation of new 5-benzylated 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indoles as PI3Kα inhibitors.

A series of novel 5-benzylated 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indoles was synthesized through a newly developed approach. All these compounds were evaluated against DYRK1A, CDK5 and PI3Kα and showed promising inhibitory activities against PI3Kα with most IC(50) values in the micromolar range. Among them, compound 18 was strongly considered as the most interesting compound with an IC(50) value of 0.091 μM. This series exhibited also significant anti-proliferative effects in various human cancer cell lines including those resulting in activation of the PI3K pathway.