Rosario Díaz

Third target of rapamycin complex negatively regulates development of quiescence in Trypanosoma brucei

African trypanosomes are protozoan parasites transmitted by a tsetse fly vector to a mammalian host. The life cycle includes highly proliferative forms and quiescent forms, the latter being adapted to host transmission. The signaling pathways controlling the developmental switch between the two forms remain unknown. Trypanosoma brucei contains two target of rapamycin (TOR) kinases, TbTOR1 and TbTOR2, and two TOR complexes, TbTORC1 and TbTORC2. Surprisingly, two additional TOR kinases are encoded in the T. brucei genome. We report that TbTOR4 associates with an Armadillo domain-containing protein (TbArmtor), a major vault protein, and LST8 to form a unique TOR complex, TbTORC4. Depletion of TbTOR4 caused irreversible differentiation of the parasite into the quiescent form. AMP and hydrolysable analogs of cAMP inhibited TbTOR4 expression and induced the stumpy quiescent form. Our results reveal unexpected complexity in TOR signaling and show that TbTORC4 negatively regulates differentiation of the proliferative form into the quiescent form.

Identification and characterization of hundreds of potent and selective inhibitors of Trypanosoma brucei growth from a kinase-targeted library screening campaign.

In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs.

Establishment of a structure-activity relationship of 1H-imidazo[4,5-c]quinoline-based kinase inhibitor NVP-BEZ235 as a lead for African sleeping sickness.

Compound NVP-BEZ235 (1) is a potent inhibitor of human phospoinositide-3-kinases and mammalian target of rapamycin (mTOR) that also showed high inhibitory potency against Trypanosoma brucei cultures. With an eye toward using 1 as a starting point for anti-trypanosomal drug discovery, we report efforts to reduce host cell toxicity, to improve the physicochemical properties, and to improve the selectivity profile over human kinases. In this work, we have developed structure–activity relationships for analogues of 1 and have prepared analogues of 1 with improved solubility properties and good predicted central nervous system exposure. In this way, we have identified 4e, 9, 16e, and 16g as the most promising leads to date. We also report cell phenotype and phospholipidomic studies that suggest that these compounds exert their anti-trypanosomal effects, at least in part, by inhibition of lipid kinases.

The effects of the arbuscular mycorrhizal fungus Glomus deserticola on growth of tomato plants grown in the presence of olive mill residues modified by treatment with saprophytic fungi.

Olive oil extraction generates large amounts of olive mill residues (DOR) which may be used as fertilizer. The influence of arbuscular mycorrhizal (AM) on the phytotoxicity of dry olive residue (DOR) transformed with saprophytic fungi was studied. Aqueous extraction of DOR gave an (ADOR) fraction and an exhausted (SDOR) fraction, both of which had less phytotoxicity for tomato than the original DOR. The saprophytic fungiTrametes versicolor andPycnoporus cinnabarinus further decreased the phytotoxicity of ADOR and SDOR on tomato. The decrease of phenols concentration and the differences in the level of laccase activity caused by these fungi suggest did not account fully for the reduced phytoxicity but the fact that the higher hydrolytic enzyme activity ofP. cinnabarinus, paralleled the decrease of phytotoxicity, indicates that these enzymes seem to be involved. The AM fungusGlomus deserticola increased or exacerbated the beneficial effect of SDOR incubated with saprophytic fungi, in terms of dry weight of tomato plants. The percentage of root length colonized byG. deserticola strongly decreased in presence of DOR, but the level of mycorrhization was higher in presence of ADOR or SDOR. Our results suggest that the combination of aqueous extraction and incubation with saprophytic fungi will open the way for the use of olive oil extraction residues as organic amendment in agricultural soils.

Effect of DOR Incubated with Saprobe Fungi on Hydrolytic Enzymes Activities and Chemical Properties of Rhizospheric Soil of Lettuce

The dry olive residue (DOR), a by-product of the olive mill industry, is produce in large quantities in Mediterranean countries. The phytotoxic and antimicrobial properties of this residue adversely impact on soil qualities. The objective of this study was to investigate the evolution of soil enzymes activities (acid phosphatase, β-glucosidase, protease and urease) and chemical properties (pH, phenols, total organic carbon (TOC) and soluble carbohydrates) after the agronomic application on lettuce of either un-treated DOR or DOR incubated with inmobilized Panus tigrinus or Fusarium lateritium. A decrease of total phenols and phytotoxicity on lettuce plants in presence of treated DOR related to the un-treated residue was detected. We observed a decrease of all hydrolytic enzymes, except protease, after the soil incubation with un-treated DOR for 15 days. It appears that the high concentration of phenolics compounds inhibited the activities of these enzymes. However, the microbial activity was stimulated by the addition of DOR incubated with the saprobe fungi as indicated by the increase of soil enzyme activities detected. The protease activity was always higher than the control soil irrespective of the type of amendant. The TOC and phenols of rhizospheric soil of lettuce increased after 15 and 30 days of agronomic application of untreated or treated residue and a general decrease of the soluble carbohydrates contents was found after 30 days of soil incubation. The DOR detoxified by saprobe fungi has been seen to have a positive effect to restore the loss rhizospheric soil functionality detected after the addition of un-treated residue.