Bioluminescent kinase strips: A novel approach to targeted and flexible kinase inhibitor profiling.

In addition to target efficacy, drug safety is a major requirement during the drug discovery process and is influenced by target specificity. Therefore, it is imperative that every new drug candidate be profiled against various liability panels that include protein kinases. Here, an effective methodology to streamline kinase inhibitor profiling is described. An accessible standardized profiling system for 112 protein kinases covering all branches of the kinome was developed. This approach consists of creating different sets of kinases and their corresponding substrates in multi-tube strips. The kinase stocks are pre-standardized for optimal kinase activity and used for inhibitor profiling using a bioluminescent ADP detection assay. We show that these strips can routinely generate inhibitor selectivity profiles for small or broad kinase family panels. Lipid kinases were also assembled in strip format and profiled together with protein kinases. We identified two specific PI3K inhibitors that have off-target effects on CK2 that were not reported before and would have been missed if compounds were not profiled against lipid and protein kinases simultaneously. To validate the accuracy of the data generated by this method, we confirmed that the inhibition potencies observed are consistent with published values produced by more complex technologies such as radioactivity assays.

Ethylene-regulated gene expression in tomato fruit: characterization of novel ethylene-responsive and ripening-related genes isolated by differential display

Differential display was used to isolate early ethylene-regulated genes from late immature green tomato fruit in order to obtain a broader understanding of the molecular basis by which ethylene coordinates the ripening process. Nineteen novel ethylene-responsive (ER) cDNA clones were isolated that fell into three classes: (i) ethylene up-regulated (ii) ethylene down-regulated, and (iii) transiently induced. Expression analysis revealed that ethylene-dependent changes in mRNA accumulation occurred rapidly (15 min) for most of the ER clones. The predicted proteins encoded by the ER genes are putatively involved in processes as diverse as primary metabolism, hormone signalling and stress responses. Although a number of the isolated ER clones correspond to genes already documented in other species, their responsiveness to ethylene is described here for the first time. Among the ER clones sharing high homology with regulatory genes, ER43, a putative GTP-binding protein, and ER50, a CTR1-like clone, are potentially involved in signal transduction. ER24 is homologous to the multi-protein bridging factor MBF1 involved in transcriptional activation, and finally, two clones are homologous to genes involved in post-transcriptional regulation: ER49, a putative translational elongation factor, and ER68, a mRNA helicase-like gene. Six ER clones correspond to as yet unidentified genes. The expression studies indicated that all the ER genes are ripening-regulated, and, depending on the clone, show changes in transcript accumulation either at the breaker, turning, or red stage. Analysis of transcript accumulation in different organs indicated a strong bias towards expression in the fruit for many of the clones. The potential roles for some of the ER clones in propagating the ethylene response and regulating fruit ripening are discussed.

Expression and characterization of three tomato 1-aminocyclopropane-1-carboxylate oxidase cDNAs in yeast.

Heterologous expression in yeast has previously shown that the tomato cDNA LE-ACO1 encodes a functional 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) protein [Hamilton, A. J., Bouzayen, M. & Grierson, D. (1991) Proc. Natl Acad. Sci. USA 88, 7434-7437]. In the present work, full-length cDNAs encoding the two other members of the tomato ACO family (LE-ACO2 and LE-ACO3) were isolated and expressed in Saccharomyces cerevisiae. Analysis of the predicted amino acid sequences showed that the ACO1 and ACO3 proteins are highly similar (95%) while ACO2 is more divergent (89%). Yeast strains transformed with each of the three cDNAs were able to convert exogenous ACC to ethylene, the ACO1 strain exhibiting the highest activity in vivo and the ACO3 and ACO2 strains reaching 65% and 45% of ACO1 maximum activity, respectively. None of the ACO activities expressed in yeast required addition of ascorbate in vivo. ACO activities assayed in vitro revealed no significant differences between the three isoforms with regards to optimum temperature (29 degrees C), optimum pH (6.8-7.2), absolute dependence for ascorbate, Fe2+ and carbon dioxide, and inhibition by iron-chelating agents (1,10-phenanthroline and EDTA), Co2+ and free-radical scavengers (n-propyl gallate). However, differences were detected in the apparent Km values for ACC, the pI and the specific activity. The biochemical features that might explain the differences between the isoenzyme activities are discussed.

ER5, a tomato cDNA encoding an ethylene-responsive LEA-like protein: characterization and expression in response to drought, ABA and wounding.

We report the isolation by differential display of a novel tomato ethylene-responsive cDNA, designated ER5. RT-PCR analysis of ER5 expression revealed an early (15 min) and transient induction by ethylene in tomato fruit, leaves and roots. ER5 mRNA accumulated during 2 h of ethylene treatment and thereafter underwent a dramatic decline leading to undetectable expression after 5 h of treatment. The full-length cDNA clone of 748 bp was obtained and DNA sequence analysis showed strong homologies to members of the atypical hydrophobic group of the LEA protein family. The predicted amino acid sequence shows 67%, 64%, 64%, and 61% sequence identity with the tomato Lemmi9, soybean D95-4, cotton Lea14-A, and resurrection plant pcC27-45 gene products, respectively. As with the other members of this group, ER5 encodes a predominantly hydrophobic protein. Prolonged drought stress stimulates ER5 expression in leaves and roots, while ABA induction of this ethylene-responsive clone is confined to the leaves. The use of 1-MCP, an inhibitor of ethylene action, indicates that the drought induction of ER5 is ethylene-mediated in tomato roots. Finally, wounding stimulates ER5 mRNA accumulation in leaves and roots. Among the Lea gene family this novel clone is the first to display an ethylene-regulated expression.