SDHi fungicides: An example of mitotoxic pesticides targeting the succinate dehydrogenase complex.

Succinate dehydrogenase inhibitors (SDHi) are fungicides used to control the proliferation of pathogenic fungi in crops. Their mode of action is based on blocking the activity of succinate dehydrogenase (SDH), a universal enzyme expressed by all species harboring mitochondria. The SDH is involved in two interconnected metabolic processes for energy production: the transfer of electrons in the mitochondrial respiratory chain and the oxidation of succinate to fumarate in the Krebs cycle. In humans, inherited SDH deficiencies may cause major pathologies including encephalopathies and cancers. The cellular and molecular mechanisms related to such genetic inactivation have been well described in neuroendocrine tumors, in which it induces an oxidative stress, a pseudohypoxic phenotype, a metabolic, epigenetic and transcriptomic remodeling, and alterations in the migration and invasion capacities of cancer cells, in connection with the accumulation of succinate, an oncometabolite, substrate of the SDH. We will discuss recent studies reporting toxic effects of SDHi in non-target organisms and their implications for risk assessment of pesticides. Recent data show that the SDH structure is highly conserved during evolution and that SDHi can inhibit SDH activity in mitochondria of non-target species, including humans. These observations suggest that SDHi are not specific inhibitors of fungal SDH. We hypothesize that SDHi could have toxic effects in other species, including humans. Moreover, the analysis of regulatory assessment reports shows that most SDHi induce tumors in animals without evidence of genotoxicity. Thus, these substances could have a non-genotoxic mechanism of carcinogenicity that still needs to be fully characterized and that could be related to SDH inhibition. The use of pesticides targeting mitochondrial enzymes encoded by tumor suppressor genes raises questions on the risk assessment framework of mitotoxic pesticides. The issue of SDHi fungicides is therefore a textbook case that highlights the urgent need for changes in regulatory assessment.

Design, Synthesis, and Biological Evaluation of Covalent Inhibitors of Focal Adhesion Kinase (FAK) against Human Malignant Glioblastoma.

Human malignant glioblastoma (GBM) is a highly invasive and lethal brain tumor. Targeting of integrin downstream signaling mediators in GBM such as focal adhesion kinase (FAK) seems reasonable and recently demonstrated promising results in early clinical studies. Herein, we report the structure-guided development of a series of covalent inhibitors of FAK. These new compounds displayed highly potent inhibitory potency against FAK enzymatic activity with IC50 values in the nanomolar range. Several inhibitors retarded tumor cell growth as assessed by a cell viability assay in multiple human glioblastoma cell lines. They also significantly reduced the rate of U-87 cell migration and delayed the cell cycle progression by stopping cells in the G2/M phase. Furthermore, these inhibitors showed a potent decrease of autophosphorylation of FAK in glioblastoma cells and its downstream effectors Akt and Erk as well as nuclear factor-κB. These data demonstrated that these inhibitors may have the potential to offer a promising new targeted therapy for human glioblastomas.

A new AMPK activator, GSK773, corrects fatty acid oxidation and differentiation defect in CPT2-deficient myotubes.

Carnitine palmitoyl transferase 2 (CPT2) deficiency is one of the most common inherited fatty acid oxidation (FAO) defects and represents a prototypical mitochondrial metabolic myopathy. Recent studies have suggested a pivotal role of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle plasticity and mitochondrial homeostasis. Thus, we tested the potential of GSK773, a novel direct AMPK activator, to improve or correct FAO capacities in muscle cells from patients harboring various mutations. We used controls' and patients' myotubes and studied the parameters of FAO metabolism, of mitochondrial quantity and quality and of differentiation. We found that AMPK is constitutively activated in patients' myotubes, which exhibit both reduced FAO and impaired differentiation. GSK773 improves or corrects several metabolic hallmarks of CPT2 deficiency (deficient FAO flux and C16-acylcarnitine accumulation) by upregulating the expression of CPT2 protein. Beneficial effects of GSK773 are also likely due to stimulation of mitochondrial biogenesis and induction of mitochondrial fusion, by decreasing dynamin-related protein 1 and increasing mitofusin 2. GSK773 also induces a shift in myosin heavy chain isoforms toward the slow oxidative type and, therefore, fully corrects the differentiation process. We establish, through small interfering RNA knockdowns and pharmacological approaches, that these GSK773 effects are mediated through peroxisome proliferator-activated receptor gamma co-activator 1-alpha, reactive oxygen species and p38 mitogen-activated protein kinase, all key players of skeletal muscle plasticity. GSK773 recapitulates several important features of skeletal muscle adaptation to exercise. The results show that AMPK activation by GSK773 evokes the slow, oxidative myogenic program and triggers beneficial phenotypic adaptations in FAO-deficient myotubes. Thus, GSK773 might have therapeutic potential for correction of CPT2 deficiency.

Structure-Based Design, Synthesis, and Characterization of the First Irreversible Inhibitor of Focal Adhesion Kinase.

Focal Adhesion Kinase signaling pathway and its functions have been involved in the development and aggressiveness of tumor malignancy, it then presents a promising cancer therapeutic target. Several reversible FAK inhibitors have been developed and are being conducted in clinical trials. On the other hand, irreversible covalent inhibitors would bring many desirable pharmacological features including high potency and increased duration of action. Herein we report the structure-guided development of the first highly potent and irreversible inhibitor of the FAK kinase. This inhibitor showed a very potent decrease of autophosphorylation of FAK in squamous cell carcinoma. A cocrystal structure of the FAK kinase domain in complex with this compound revealed the inhibitor binding mode within the ATP binding site and confirmed the covalent linkage between the targeted Cys427 of the protein and the inhibitor.

Aryl hydrocarbon receptor upregulates IL-1ß expression in hCMEC/D3 human cerebral microvascular endothelial cells after TCDD exposure.

The AhR is a cytosolic ligand-dependent transcription factor activated by both endogenous and exogenous chemicals. It can regulate expression of many target genes including some inflammatory cytokines and chemokines. To date AhR implication in the regulation of inflammatory cytokines and chemokines at human cerebral endothelium has not been addressed. In the present study, we investigated whether AhR could regulate the expression of two pro-inflammatory cytokines and one chemokine i.e. IL-1ß, IL-6 and IL-8 in the hCMEC/D3 human cerebral microvascular endothelial cell line. Exposure to TCDD increased IL-1ß mRNA expression levels in hCMEC/D3. By using small interfering RNA against AhR we demonstrated that TCDD effects on IL-1ß expression were mediated through AhR activation. Regarding IL-6 and IL-8, TCDD exposure had little or no effect on their mRNA levels in hCMEC/D3. In conclusion, our findings suggest that AhR-mediated IL-1ß regulation in cerebral endothelium could induce BBB breakdown and contribute to the pathogenesis of a variety of neurologic disorders.

Exposure to metal oxide nanoparticles administered at occupationally relevant doses induces pulmonary effects in mice.

In spite of the great promises that the development of nanotechnologies can offer, concerns regarding potential adverse health effects of occupational exposure to nanoparticle (NP) is raised. We recently identified metal oxide NP in lung tissue sections of welders, located inside macrophages infiltrated in fibrous regions. This suggests a role of these NP in the lung alterations observed in welders. We therefore designed a study aimed to investigate the pulmonary effects, in mice, of repeated exposure to NP administered at occupationally relevant doses. We therefore chose four metal oxide NPs representative of those found in the welder's lungs: Fe2O3, Fe3O4, MnFe2O4 and CrOOH. These NPs were administered weekly for up to 3 months at two different doses: 5 µg, chosen as occupationally relevant to welding activity, and 50 µg, chosen as occupationally relevant to the context of an NP-manufacturing facility. Our results show that 3 month-repeated exposures to 5 µg NP induced limited pulmonary effects, characterized by the development of a mild peribronchiolar fibrosis observed for MnFe2O4 and CrOOH NP only. This fibrotic event was further extended in terms of intensity and localization after the repeated administration of 50 µg NP: all but Fe2O3 NP induced the development of peribronchiolar, perivascular and alveolar fibrosis, together with an interstitial inflammation. Our data demonstrate for the first time a potential risk for respiratory health posed by repeated exposure to NP at occupationally relevant doses. Given these results, the development of occupational exposure limits (OELs) specifically dedicated to NP exposure might therefore be an important issue to address.

Design, synthesis, and evaluation of novel imidazo[1,2-a][1,3,5]triazines and their derivatives as focal adhesion kinase inhibitors with antitumor activity.

A series of triazinic inhibitors of focal adhesion kinase (FAK) have been recently shown to exert antiangiogenic activity against HUVEC cells and anticancer efficacy against several cancer cell lines. We report herein that we further explored the heterocyclic core of these inhibitors by a fused imidazole ring with the triazine to provide imidazo[1,2-a][1,3,5]triazines. Importantly, these new compounds displayed 10(-7)-10(-8) M IC50 values, and the best inhibitor showed IC50 value of 50 nM against FAK enzymatic activity. Several inhibitors potently inhibited the proliferation of a panel of cancer cell lines expressing high levels of FAK. Apoptosis analysis in U87-MG and HCT-116 cell lines suggested that these compounds delayed cell cycle progression by arresting cells in the G2/M phase of the cell cycle, retarding cell growth. Further investigation demonstrated that these compounds strongly inhibited cell-matrix adhesion, migration, and invasion of U87-MG cells.