Sonia Arora

Identification of the Ubiquitin-Proteasome Pathway in the Regulation of the Stability of Eukaryotic Elongation Factor-2 Kinase

Eukaryotic elongation factor-2 kinase (eEF-2 kinase) is a highly conserved calcium/calmodulin-dependent enzyme involved in the regulation of protein translation and cell proliferation. Rapid changes in the activity and abundance of eEF-2 kinase have been observed on growth stimulation, and increased enzyme activity is characteristic of malignant cell growth. Yet the mechanism for controlling the turnover of this kinase is unknown. The ubiquitin-proteasome pathway regulates the degradation of many cellular proteins, including transcription factors, cell cycle regulators, and signal transduction proteins. Therefore, we determined whether the ubiquitin-proteasome pathway regulates the turnover of eEF-2 kinase. We found that eEF-2 kinase was a relatively short-lived protein with a half-life of less than 6 hours. eEF-2 kinase was ubiquitinated in vivo as determined by coimmunoprecipitation and polyubiquitin affinity matrix. Incubation of purified eEF-2 kinase with a source of ubiquitination enzymes (rabbit reticulocyte lysate), purified ubiquitin, and ATP revealed the presence of increasing molecular weight species of ubiquitinated eEF-2 kinase. Treatment of cells with MG132, a proteasome inhibitor, inhibited eEF-2 kinase degradation and induced the accumulation of polyubiquitinated forms of the enzyme, resulting in an increase in its half-life. These results suggest involvement of the proteasome in the turnover of the ubiquitinated kinase. Because eEF-2 kinase is chaperoned by heat shock protein 90 (Hsp90), we next determined if disruption of the Hsp90-eEF-2 kinase complex promoted degradation of the kinase. Treatment of cells with geldanamycin, an Hsp90 inhibitor, enhanced ubiquitination of eEF-2 kinase and decreased the half-life of the kinase to less than 2 hours. These results indicate that cellular levels of eEF-2 kinase are maintained by a balance between association with Hsp90 and degradation by the ubiquitin-proteasome pathway. In conclusion, these data show that the turnover of eEF-2 kinase is regulated by the ubiquitin-proteasome pathway and, therefore, modulating the ubiquitination of eEF-2 kinase might control the abundance of this enzyme and have implications in the treatment of certain forms of cancer.

Novel microtubule polymerization inhibitor with potent antiproliferative and antitumor activity.

Microtubule-stabilizing and microtubule-destabilizing agents are commonly used as anticancer agents. Although highly effective, success with these agents has been limited due to their relative insolubility, cumbersome synthesis/purification, toxic side effects, and development of multidrug resistance. Hence, the identification of improved agents that circumvent one or more of these problems is warranted. We recently described the rational design of a series of triazole-based compounds as antimitotic agents. Members of this N-substituted 1,2,4-triazole family of compounds exhibit potent tubulin polymerization inhibition and broad spectrum cellular cytotoxicity. Here, we extensively characterize the in vitro and in vivo effects of our lead compound from the series 1-methyl-5-(3-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-4-yl)-1H-indole, designated T115. We show that T115 competes with colchicine for its binding pocket in tubulin, produces robust inhibition of tubulin polymerization, and disrupts the microtubule network system inside the cells. In addition, T115 arrests human cancer cells in the G(2)-M phase of cell cycling, a hallmark of microtubule destabilizing drugs. T115 also inhibits cell viability of several cancer cell lines, including multidrug-resistant cell lines, in the low nanomolar range. No cytotoxicity was observed by T115 against normal human skin fibroblasts cell lines, and acute toxicity studies in normal nontumor-bearing mice indicated that T115 is well-tolerated in vivo (maximum total tolerated dose, 400 mg/kg). In a mouse xenograft model using human colorectal (HT-29) and prostate (PC3) cancer cells, T115 significantly inhibited tumor growth when administered i.p. Taken together, our results suggest that T115 is a potential drug candidate for cancer chemotherapy.

Application of Screening Methods, Shape Signatures and Engineered Biosensors in Early Drug Discovery Process

PurposeIn this study, two unreported estrogen antagonists were identified using a combination of computational screening and a simple bacterial estrogen sensor.MethodsMolecules here presented were initially part of a group obtained from a library of over a half million chemical compounds, using the Shape Signatures method. The structures within this group were then clustered and compared to known antagonists based on their physico-chemical parameters, and possible binding modes of the compounds to the Estrogen Receptor α (ERα) were analyzed. Finally, thirteen candidate compounds were purchased, and two of them were shown to behave as potential subtype-selective estrogen antagonists using a set of bacterial estrogen biosensors, which included sensors for ERα, ERβ, and a negative control thyroid hormone β biosensor. These activities were then analyzed using an ELISA assay against activated ERα in human MCF-7 cell extract.ResultsTwo new estrogen receptor antagonists were detected using in silico Shape Signatures method with an engineered subtype-selective bacterial estrogen biosensor and commercially available ELISA assay. Additional thyroid biosensor control experiments confirmed no compounds interacted with human thyroid receptor β.ConclusionsThis work demonstrates an effective combination of computational analysis and simple bacterial screens for rapid identification of potential hormone-like therapeutics.

Novel delta opioid receptor agonists exhibit differential stimulation of signaling pathways.

A novel family of 1,3,5-trisubstituted 1,2,4-triazoles was discovered as potent and selective ligands for the delta opioid receptor by rational design. Compound 5b exhibited low-nanomolar in vitro binding affinity (IC(50)=5.8 nM), excellent selectivity for the delta opioid receptor over the alternative mu and kappa opioid receptors, full agonist efficacy in receptor down-regulation and MAP kinase activation assays, and low-efficacy partial agonist activity in stimulation of GTPgammaS binding. The apparent discrepancy observed in these functional assays may stem from different signaling pathways involved in each case, as found previously for other G-protein coupled receptors. More biological studies are underway to better understand the differential stimulation of signaling pathways by these novel compounds.