Determination of Gymnemic Acid I as a Protein Biosynthesis Inhibitor Using Chemical Proteomics.

The plant Gymnema sylvestre has been used widely in traditional medicine as a remedy for several diseases, and its leaf extract is known to contain a group of bioactive triterpene saponins belonging to the gymnemic acid class. Gymnemic acid I (1) is one of the main components among this group of secondary metabolites and is endowed with an interesting bioactivity profile. Since there is a lack of information about its specific biological targets, the full interactome of 1 was investigated through a quantitative chemical proteomic approach, based on stable-isotope dimethyl labeling. The ribosome complex was found to be the main partner of compound 1, and a full validation of the proteomics results was achieved by orthogonal approaches. Further biochemical and biological investigations revealed an inhibitory effect of 1 on the ribosome machinery.

Identification of cardiac glycoside molecules as inhibitors of c-Myc IRES-mediated translation.

Translation initiation is a fine-tuned process that plays a critical role in tumorigenesis. The use of small molecules that modulate mRNA translation provides tool compounds to explore the mechanism of translational initiation and to further validate protein synthesis as a potential pharmaceutical target for cancer therapeutics. This report describes the development and use of a click beetle, dual luciferase cell-based assay multiplexed with a measure of compound toxicity using resazurin to evaluate the differential effect of natural products on cap-dependent or internal ribosome entry site (IRES)-mediated translation initiation and cell viability. This screen identified a series of cardiac glycosides as inhibitors of IRES-mediated translation using, in particular, the oncogene mRNA c-Myc IRES. Treatment of c-Myc-dependent cancer cells with these compounds showed a decrease in c-Myc protein associated with a significant modulation of cell viability. These findings suggest that inhibition of IRES-mediated translation initiation may be a strategy to inhibit c-Myc-driven tumorigenesis.

Intact cell matrix-assisted laser desorption/ionization mass spectrometry as a tool to screen drugs in vivo for regulation of protein expression.

Here we demonstrate for the first time the application of intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) to study the regulation of protein expression. This technique can be extended to screen the drugs that inhibit protein synthesis in various diseases. We have used Escherichia coli cells expressing a recombinant glutathione-S-transferase (GST) gene under an arabinose-inducible promoter as a model system. Using ICM-MS analysis, we have detected a 28 kDa peak corresponding to the production of recombinant GST under the arabinose-induced condition. Furthermore, recombinant GST protein was purified by a single-step affinity purification using a glutathione Sepharose 4B affinity column from arabinose-induced E. coli cells. The purified GST protein was found to be a 28 kDa protein by MALDI analysis suggesting the arabinose-induced protein is indeed GST. The regulation of protein expression was studied using glucose as an alternative metabolite. The glucose-mediated regulation of the ara-operon was followed using the ICM-MS technique. All the results obtained from ICM-MS data were validated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The present technique can be extended for in vivo screening of drugs and it holds tremendous potential to discover novel drugs against specific protein expressions in different diseases.

Isolation of a putative ribosome inactivating protein from dried roots of Trichosanthes kirilowii used in Traditional Chinese Medicine.

A putative ribosome inactivating protein (RIP), named T 33, was isolated from dried roots of Trichosanthes kirilowii Maximovicz which are used in Traditional Chinese Medicine. After chromatographic enrichment a protein band with molecular mass of 33 kDa was found by SDS-PAGE. Partial amino acid sequences of this protein consisting of 8 - 33 amino acids reveal homology to highly conserved regions of different RIPs, but also indicate T 33 to be a novel type 2 RIP. Future heterologous expression of this protein will allow investigation of its biological activities in detail.

Heterogeneity of the ribosome-inactivating protein trichosanthin in Trichosanthes kirilowii tubers.

The ribosome-inactivating protein trichosanthin isolated from the tubers of Trichosanthes kirilowii, the Chinese drug Tianhuafen, has a molecular mass of approximately 26 kDa. We show here that T. kirilowii tubers also contain ribosome-inactivating proteins with a small extent of structural variation from and a larger molecular mass than trichosanthin.

Macrocyclic trichothecenes in Baccharis coridifolia plants and endophytes and Baccharis artemisioides plants.

Toxic disease in livestock caused by the shrubs Baccharis coridifolia and Baccharis artemisioides is very common in Argentina. The toxicity of Argentinian and Brazilian B. coridifolia plants and of Argentinian B. artemisioides was investigated. The toxicogenic capacity of 15 endothyte isolates of Ceratopicnidium baccharidicola from B. coridifolia was determined. Roridins and verrucarins were analyzed by thin-layer chromatography using a modified Jarvis method. One-hundred per cent of Argentinian B. coridifolia plants were positive for roridins (RA and RE) and verrucarins (VA and VJ), 16.2% for RD and 2.7% for RH. All of the Brazilian B. coridifolia plants were positive only for roridins. In B. artemisioides plants, RA, RE and RD were present in higher concentrations than VA and VJ, and all of them were more concentrated than in B. coridifolia. One-third of the endophyte isolates were toxicogenic for the same roridins and verrucarins, but in very low concentrations. This is the first report of macrocyclic trichothecenes in B. artemisioides, and a new report of B. coridifolia macrocyclic trichothecenes in Argentina.

Effects of dietary n-3 fatty acid supplementation versus thromboxane synthetase inhibition on gentamicin-induced nephrotoxicosis in healthy male dogs.

OBJECTIVE: To evaluate the protective effects of dietary n-3 fatty acid supplementation versus treatment with a thromboxane synthetase inhibitor (TXSI) in dogs given high-dose gentamicin. DESIGN: Clinicopathologic and renal histopathologic changes induced by gentamicin (10 mg/kg of body weight, IM, q 8 h, for 8 days) were compared in dogs fed an n-3 fatty acid-supplemented diet containing a fatty acid ratio of 5.7:1 (n-6:n-3), dogs treated with CGS 12970 (a specific TXSI given at 30 mg/kg, PO, q 8 h, beginning 2 days prior to gentamicin administration), and control dogs. The TXSI-treated and control dogs were fed a diet with a fatty acid ratio of 51.5:1 (n-6:n-3). Both diets were fed beginning 42 days prior to and during the 8-day course of gentamicin administration. ANIMALS: Eighteen 6-month-old male Beagles, 6 in each group. RESULTS: After 8 days of gentamicin administration, differences existed among groups. Compared with n-3-supplemented and control dogs. TXSI-treated dogs had higher creatinine clearance. Both TXSI-treated and n-3-supplemented dogs had higher urinary prostaglandin E2 and E3 (PGE2/3) and 6-keto prostaglandin F1a (PGF1a) excretion, compared with control dogs. Urinary thromboxane B2 (TXB2) excretion was higher in n-3-supplemented and control dogs, compared with TXSI-treated dogs. Urine PGE2/3-to-TXB2 and PGF(in)-to-TXB2, ratios were increased in TXSI-treated dogs, compared with n-3-supplemented and control dogs, and these ratios were increased in n-3-supplemented dogs, compared with control dogs. In addition, TXSI-treated and n-3-supplemented dogs had lower urinary protein excretion, compared with control dogs. Proximal tubular necrosis was less severe in TXSI-treated dogs, compared with control dogs. CONCLUSION: Treatment with CGS 12970 prior to and during gentamicin administration prevented increases in urinary TXB2 excretion and reduced nephrotoxicosis. CLINICAL RELEVANCE: Increased renal production/excretion of thromboxane is important in the pathogenesis of gentamicin-induced nephrotoxicosis.