Cyclin-dependent kinases (CDKs) and casein kinases 1 (CK1) are involved in the two key molecular features of Alzheimer's disease, production of amyloid-beta peptides (extracellular plaques) and hyper-phosphorylation of Tau (intracellular neurofibrillary tangles). A series of 2,6,9-trisubstituted purines, structurally related to the CDK inhibitor roscovitine, have been synthesized. They mainly differ by the substituent on the C-6 position. These compounds were screened for kinase inhibitory activities and antiproliferative effects. Several biaryl derivatives displayed potent inhibition of both CDKs and CK1. In particular, derivative 13a was a potent inhibitor of CDK1/cyclin B (IC 50: 220 nM), CDK5/p25 (IC 50: 80 nM), and CK1 (IC 50: 14 nM). Modeling of these molecules into the ATP-binding pocket of CK1delta provided a rationale for the increased selectivity toward this kinase. 13a was able to prevent the CK1-dependent production of amyloid-beta in a cell model. CDK/CK1 dual-specificity inhibitors may have important applications in Alzheimer's disease and cancers.

The development of CDC25 phosphatase inhibitors is an interesting approach toward new antitumor agents, as CDC25 play key roles in cell-cycle regulation and are overexpressed in numerous cancers. We previously reported a novel compound belonging to the thiazolopyrimidine family that inhibits CDC25 activity with an IC(50) value of 13 microM and displays cytotoxic properties against HeLa cells. Structural modifications were subsequently conducted on this new pharmacophore which led to a library of 45 thiazolopyrimidines. Regarding the in vitro effects, 14 compounds inhibit CDC25B with IC(50)<20 microM, with the most efficient inhibitor 44 improving the potency to 4.5 microM. Steady-state kinetics were performed and showed a mixed inhibition pattern for all tested compounds. Furthermore, 44 was able to revert the bypass of genotoxicity-induced G(2) arrest upon CDC25B overexpression, indicating that this compound targets the dual-specificity phosphatase in cultured cells. Finally, the cytotoxic activities of the compounds were determined against two human cancer cell lines. The results indicate that the prostatic LNCaP cell line is more sensitive to these derivatives than the pancreatic adenocarcinoma MiaPaCa-2 line. With its interesting enzymatic and cellular properties, compound 44 appears to be a promising CDC25B inhibitor for further development.

Grapevine trunk diseases: Eutypa dieback, esca and Botryosphaeria dieback, which incidence has increased recently, are associated with several symptoms finally leading to the plant death. In the absence of efficient treatments, these diseases are a major problem for the viticulture; however, the factors involved in disease progression are not still fully identified. In order to get a better understanding of Botryosphaeria dieback development in grapevine, we have investigated different factors involved in Botryosphaeriaceae fungi aggressiveness. We first evaluated the activity of the wood-degrading enzymes of different isolates of Neofusicoccum parvum and Diplodia seriata, two major fungi associated with Botryosphaeria dieback. We further examinated the ability of these fungi to metabolize major grapevine phytoalexins: resveratrol and δ-viniferin. Our results demonstrate that Botryosphaeriaceae were characterized by differential wood decay enzymatic activities and have the capacity to rapidly degrade stilbenes. N. parvum is able to degrade parietal polysaccharides, whereas D. seriata has a better capacity to degrade lignin. Growth of both fungi exhibited a low sensitivity to resveratrol, whereas δ-viniferin has a fungistatic effect, especially on N. parvum Bourgogne S-116. We further show that Botryosphaeriaceae are able to metabolize rapidly resveratrol and δ-viniferin. The best stilbene metabolizing activity was measured for D. seriata. In conclusion, the different Botryosphaeriaceae isolates are characterized by a specific aggressiveness repertory. Wood and phenolic compound decay enzymatic activities could enable Botryosphaeriaceae to bypass chemical and physical barriers of the grapevine plant. The specific signature of Botryosphaeriaceae aggressiveness factors could explain the importance of fungi complexes in synergistic activity in order to fully colonize the host.

Hydrothermal carbonization (HTC) is considered as a promising technique for wastes conversion into carbon rich materials for various energetic, environmental and agricultural applications. In this work, the HTC of olive mill wastewater (OMWW) was investigated at different temperatures (180 -220°C) and both, the solid (i.e., hydrochars) and the final process liquid derived from the thermal conversion process were deeply analyzed. Results showed that the solid yield was affected by the temperature, i.e., decrease from 57% to 25% for temperatures of 180°C and 220°C, respectively. Furthermore, the hydrochars presented an increasing fixed carbon percentage with the increase of the carbonization temperature, suggesting that decarboxylation is the main reaction driving the HTC process. The decrease in the O/C ratio promoted an increase of the high heating value (HHV) by 32% for hydrochar prepared at 220°C. The process liquids were sampled and their organic contents were analyzed using GC-MS technique. Acids, alcohols, phenols and sugar derivatives were detected and their concentrations varied with carbonization temperatures. The assessment of the physico-chemical properties of the generated HTC by-products suggested the possible application of the hydrochars for energetic insights while the liquid fraction could be practical for in agricultural field.

CDC25 phosphatases play critical roles in cell cycle regulation and are attractive targets for anticancer therapies. Several small non-peptide molecules are known to inhibit CDC25, but many of them appear to form a covalent bond with the enzyme or act through oxidation of the thiolate group of the catalytic cysteine. Structure-based virtual ligand screening computations were performed with FRED, Surflex, and LigandFit, a compound collection of over 310,000 druglike molecules and the crystal structure of CDC25B in order to identify novel classes of ligands. In vitro experiments carried out on a selected list of 1500 molecules led to the discovery of 99 compounds able to inhibit CDC25B activity at 100 microM. Further docking computations were applied, allowing us to propose a binding mode for the most potent molecule (IC50 = 13 microM). Our best compounds represent promising new classes of CDC25 inhibitors that also exhibit antiproliferative properties.

Two practical and complementary methods are reported for the synthesis of trifluoromethylated alkynes. The first one, a mix-and-stir process, is based on the oxidative trifluoromethylation of readily available and bench-stable copper acetylides while the second one, which displays a broad substrate scope and has several advantages over existing procedures, is based on the oxidative copper-catalyzed direct trifluoromethylation of terminal alkynes. Both reactions provide user-friendly processes for the synthesis of trifluoromethylated acetylenes which can be easily obtained from readily available starting materials.

Botryosphaeria dieback, esca and Eutypa dieback are three economic major grapevine trunk diseases that cause severe yield reduction in vineyards worldwide. The frequency of disease symptoms has increased considerably over the past decade, and no efficient treatment is currently available to control these diseases. The different fungi associated with grapevine trunk diseases mainly induce necrotic wood and characteristic foliar symptoms. In this context, fungi virulence factors and host invasion are not well understood. We hypothesise that extracellular proteins produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with Botryosphaeria dieback, are virulence factors responsible for the pathogenicity. In our previous work, we demonstrated that the total extracellular compounds produced by N. parvum induced more necrosis on Chardonnay calli and triggered a different defence gene expression pattern than those produced by D. seriata. Furthermore, this aggressiveness was not clearly correlated with the production of mellein, a characteristic phytotoxin of Botryosphaeriaceae, in our in vitro calli model. To characterise other potential virulence factors and to understand the mechanisms of host invasion by the fungus, we evaluated the profile, quantity and the impact of extracellular proteins produced by these fungi on Vitis vinifera calli necrosis and defence gene expression. Our results reveal that, under the same conditions, N. parvum produces more extracellular proteins and in higher concentrations than D. seriata. With Vitis vinifera cv. Chardonnay cells, we showed that equivalent concentrations of proteins secreted by N. parvum were more aggressive than those of D. seriata in producing necrosis and that they clearly induced more grapevine defence genes.

Botryosphaeriaceae fungi are plant pathogens associated with Botryosphaeria dieback. To better understand the virulence factors of these fungi, we investigated the diversity of secreted proteins and extracellular enzyme activities involved in wood degradation and stilbene metabolization in Neofusicoccum parvum and Diplodia seriata, which are two major fungi associated with grapevine B. dieback. Regarding the analysis of proteins secreted by the two fungi, our study revealed that N. parvum, known to be more aggressive than D. seriata, was characterized by a higher quantity and diversity of secreted proteins, especially hydrolases and oxidoreductases that are likely involved in cell wall and lignin degradation. In addition, when fungi were grown with wood powder, the extracellular laccase and Mn peroxidase enzyme activities were significantly higher in D. seriata compared to N.parvum. Importantly, our work also showed that secreted Botryosphaeriaceae proteins produced after grapevine wood addition are able to rapidly metabolize the grapevine stilbenes. Overall, a higher diversity of resveratrol and piceatannol metabolization products was found with enzymes of N. parvum compared to D. seriata. This study emphasizes the diversity of secreted virulence factors found in B. dieback fungi and suggests that some resveratrol oligomers produced in grapevine wood after pathogen attack could be formed via pathogenic fungal oxidases.