Identification of Phosphate-Containing Compounds as New Inhibitors of 14-3-3/c-Abl Protein-Protein Interaction.

The 14-3-3/c-Abl protein-protein interaction (PPI) is related to carcinogenesis and in particular to pathogenesis of chronic myeloid leukemia (CML). Previous studies have demonstrated that molecules able to disrupt this interaction improve the nuclear translocation of c-Abl, inducing apoptosis in leukemia cells. Through an X-ray crystallography screening program, we have identified two phosphate-containing compounds, inosine monophosphate (IMP) and pyridoxal phosphate (PLP), as binders of human 14-3-3σ, by targeting the protein amphipathic groove. Interestingly, they also act as weak inhibitors of the 14-3-3/c-Abl PPI, demonstrated by NMR, SPR, and FP data. A 37-compound library of PLP and IMP analogues was investigated using a FP assay, leading to the identification of three further molecules acting as weak inhibitors of the 14-3-3/c-Abl complex formation. The antiproliferative activity of IMP, PLP, and the three derivatives was tested against K-562 cells, showing that the parent compounds had the most pronounced effect on tumor cells. PLP and IMP were also effective in promoting the c-Abl nuclear translocation in c-Abl overexpressing cells. Further, these compounds demonstrated low cytotoxicity on human Hs27 fibroblasts. In conclusion, our data suggest that 14-3-3σ targeting compounds represent promising hits for further development of drugs against c-Abl-dependent cancers.

Toxicological effects of pet food ingredients on canine bone marrow-derived mesenchymal stem cells and enterocyte-like cells.

We developed an in vitro method to assess pet food ingredients safety. Canine bone marrow-derived mesenchymal stem cells (BMSC) were differentiated into enterocyte-like cells (ELC) to assess toxicity in cells representing similar patterns of exposure in vivo. The toxicological profile of clove leave oil, eugenol, guanosine monophosphate (GMP), GMP + inosine monophosphate, sorbose, ginger root extract, cinnamon bark oil, cinnamaldehyde, thyme oil, thymol and citric acid was assessed in BMSC and ELC. The LC50 for GMP + inosine monophosphate was 59.42 ± 0.90 and 56.7 ± 3.5 mg ml(-1) for BMSC and ELC; 56.84 ± 0.95 and 53.66 ± 1.36 mg ml(-1) for GMP; 0.02 ± 0.001 and 1.25 ± 0.47 mg ml(-1) for citric acid; 0.077 ± 0.002 and 0.037 ± 0.01 mg ml(-1) for cinnamaldehyde; 0.002 ± 0.0001 and 0.002 ± 0.0008 mg ml(-1) for thymol; 0.080 ± 0.003 and 0.059 ± 0.001 mg ml(-1) for thyme oil; 0.111 ± 0.002 and 0.054 ± 0.01 mg ml(-1) for cinnamon bark oil; 0.119 ± 0.0004 and 0.099 ± 0.011 mg ml(-1) for clove leave oil; 0.04 ± 0.001 and 0.028 ± 0.002 mg ml(-1) for eugenol; 2.80 ± 0.11 and 1.75 ± 0.51 mg ml(-1) for ginger root extract; > 200 and 116.78 ± 7.35 mg ml(-1) for sorbose. Lemon grass oil was evaluated at 0.003-0.9 in BMSC and .03-0.9 mg ml(-1) in ELC and its mechanistic effect was investigated. The gene toxicology studies showed regulation of 61% genes in CYP450 pathway, 37% in cholestasis and 33% in immunotoxicity pathways for BMSC. For ELC, 80% for heat shock response, 69% for beta-oxidation and 65% for mitochondrial energy metabolism. In conclusion, these studies provide a baseline against which differential toxicity of dietary feed ingredients can be assessed in vitro for direct effects on canine cells and demonstrate differential toxicity in differentiated cells that represent gastrointestinal epithelial cells.

In vitro safety assessment of food ingredients in canine renal proximal tubule cells.

In vitro models are useful tools to initially assess the toxicological safety hazards of food ingredients. Toxicities of cinnamaldehyde (CINA), cinnamon bark oil, lemongrass oil (LGO), thymol, thyme oil (TO), clove leaf oil, eugenol, ginger root extract (GRE), citric acid, guanosine monophosphate, inosine monophosphate and sorbose (SORB) were assessed in canine renal proximal tubule cells (CPTC) using viability assay and renal injury markers. At LC50, CINA was the most toxic (0.012mg/ml), while SORB the least toxic (>100mg/ml). Toxicities (LC50) of positive controls were as follows: 4-aminophenol (0.15mg/ml in CPTC and 0.083mg/ml in human PTC), neomycin (28.6mg/ml in CPTC and 27.1mg/ml in human PTC). XYL displayed lowest cytotoxic potency (LC50=82.7mg/ml in CPTC). In vivo renal injury markers in CPTC were not significantly different from controls. The LGO toxicity mechanism was analyzed using qPCR and electron microscopy. Out of 370 genes, 57 genes (15.4%) were significantly up (34, 9.1%) or down (23, 6.2%) regulated, with the most upregulated gene gsta3 (∼200-fold) and the most affected pathway being oxidative stress. LGO induced damage of mitochondria, phospholipid accumulation and lack of a brush border. Viability assays along with mechanistic studies in the CPTC model may serve as a valuable in vitro toxicity screening tool.

The biological activity in mammals and insects of the nucleosidic fraction from the spider Parawixia bistriata.

Previous work has shown that the crude venom of Parawixia bistriata induces convulsive seizures in rats after intracerebroventricular injection. In this work, the isolation of a bioactive fraction with ultraviolet absorption characteristics of nucleic acid and trace protein or amino acid content is described. NMR analysis demonstrated that the major component of the active fraction is the nucleoside inosine. An analogue of this component (inosine 5'-monophosphate) induced a delayed paralysis effect in termites.

Potentiation of immune responses in mice by a new inosine derivative--methyl inosine monophosphate (MIMP).

Inosine 5'-methyl monophosphate (MIMP) is a new immunomodulator designed to improve upon the activity of other thymomimetic purines. In Balb/c mice, MIMP was assessed for toxicity and activity on immune responses. The lethal dose for half the mice (LD50) exceeded 500 mg/kg of body weight by both the parenteral and oral routes. At doses of 1-100 mg/kg, the mice showed no visible untoward effects. The antibody response of splenocytes to sheep erythrocytes (SRBC) was measured by IgM plaque-forming cells (PFC) in soft agar under optimal conditions of immunization and challenge. MIMP (1-100 mg/kg) was given by both the intraperitoneal and oral routes (gavage) at the time of SRBC injection and 4 days thereafter. The PFC response was found to be significantly augmented. The maximum effect (approximately 2x) was observed at 50 and 100 mg/kg, via intraperitoneal (i.p.) and oral routes, respectively. Increases (maximally 1.5x) in the responses of splenic lymphocytes to mitogen stimulation with phytohemagglutinin (PHA) and concanavalin A (Con A) were observed under similar conditions of MIMP treatment. SRBC-induced delayed-hypersensitivity (DTH) was also measured under optimal conditions. By both i.p. and oral routes, enhancement of DTH response was produced by the lower doses of MIMP (0.01-1 mg/kg). Again, a second peak of optimum stimulation of DTH response was produced by 50 mg/kg of MIMP when administered by both routes. The effect was observed mainly on the sensitization rather than on the expression phase. MIMP qualifies as an effective immunopotentiator in normal mice.