Inhibition of p56(lck) tyrosine kinase by isothiazolones

Lck encodes a 56-kDa protein-tyrosine kinase, predominantly expressed in T lymphocytes, crucial for initiating T cell antigen receptor (TCR) signal transduction pathways, culminating in T cell cytokine gene expression and effector functions. As a consequence of a high-throughput screen for selective, novel inhibitors of p56(lck), an isothiazolone compound was identified, methyl-3-(N-isothiazolone)-2-thiophenecarboxylate(A-125800), which inhibits p56(lck) kinase activity with IC50 = 1-7 microM. Under similar assay conditions, the isothiazolone compound was equipotent in blocking the ZAP-70 tyrosine kinase activity but was 50 to 100 times less potent against the catalytic activities of p38 MAP kinase and c-Jun N-terminal kinase 2alpha. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC50 = 35 microM) and blocked T cell proliferation in response to alloantigen (IC50 = 14 microM) and CD3/CD28-induced IL-2 secretion (IC50 = 2.2 microM) in primary T cell cultures. Inhibition of p56(lck )by A-125800 was dose- and time-dependent and was irreversible. A substitution of methylene for the sulfur atom in the isothiazolone ring of the compound completely abrogated the ability to inhibit p56(lck) kinase activity and TCR-dependent signal transduction. Incubation with thiols such as beta-ME or DTT also blocked the ability of the isothiazolone to inhibit p56(lck) kinase activity. LC/MS analysis established the covalent modification of p56(lck) at cysteine residues 378, 465, and 476. Together these data support an inhibitory mechanism, whereby cysteine -SH groups within the p56(lck) catalytic domain react with the isothiazolone ring, leading to ring opening and disulfide bond formation with the p56(lck) enzyme. Loss of p56(lck) activity due to -SH oxidation has been suggested to play a role in the pathology of AIDS. Consequently, a similar mechanism of sulfhydryl oxidation leading to p56(lck) inhibition, described in this report, may occur in the intact T cell and may underlie certain T cell pathologies.     

Abrogation of heparan sulfate synthesis in Drosophila disrupts the Wingless, Hedgehog and Decapentaplegic signaling pathways

Studies in Drosophila and vertebrate systems have demonstrated that heparan sulfate proteoglycans (HSPGs) play crucial roles in modulating growth factor signaling. We have isolated mutations in sister of tout velu (sotv), a gene that encodes a co-polymerase that synthesizes HSPG glycosaminoglycan (GAG) chains. Our phenotypic and biochemical analyses reveal that HS levels are dramatically reduced in the absence of Sotv or its partner co-polymerase Tout velu (Ttv), suggesting that both copolymerases are essential for GAG synthesis. Furthermore, we find that mutations in sotv and ttv impair Hh, Wg and Decapentaplegic (Dpp) signaling. This contrasts with previous studies that suggested loss of ttv compromises only Hh signaling. Our results may contribute to understanding the biological basis of hereditary multiple exostoses (HME), a disease associated with bone overgrowth that results from mutations in EXT1 and EXT2, the human orthologs of ttv and sotv.     

Aminoethoxyvinylglycine Effects on Late-Season Apple Fruit Maturation

Aminoethoxyvinylglycine (AVG) inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, and thus blocks ethylene synthesis. Preharvest foliar application of AVG to apple (Malus domestica Borkh.) fruit retards several key events of maturation including climacteric ethylene production, starch conversion to sugars, fruit softening, and abscission zone development. Although the impact of AVG on apple fruit maturation is well known, the biochemical basis of these effects is not well understood. The effects of AVG application on Redchief Delicious apple fruit maturation were studied. AVG applied four weeks prior to harvest significantly reduced internal ethylene levels, amylose degradation, and accumulation of sucrose, glucose, and sorbitol. Because AVG application coincidentally inhibited starch degradation and the increase in internal ethylene, we investigated the enzymatic basis of starch mobilization in apple fruit. Amylase activity was somewhat reduced in AVG-treated fruit. Amylase activity was less in AVG-treated fruit during the early stages of starch mobilization. Starch phosphorylase activity increased dramatically during the later stages of starch mobilization, but was not affected by AVG treatment. Soluble starch synthase activity was also unaffected by AVG treatment and remained constant throughout the eight-week harvest period. Moreover, AVG did not affect the levels of amylopectin, fructose, malate, ascorbate, citrate, or anthocyanin. These results suggest that apple fruit ripening has both ethylene-dependent and -independent processes occurring simultaneously.     

The Drosophila Lissencephaly1 (DLis1) gene is required for nuclear migration

Nuclear movement is critical for several developmental processes in eukaryotes. Drosophila oogenesis provides a paradigmatic example in which localization of the nucleus generates a source of cellular asymmetry that is used in patterning both the anterior-posterior and the dorsal-ventral axes of the oocyte. In this study we show that mutations in the Drosophila Lissencephaly1 (DLis1) gene result in partial ventralization of the eggshell. DLis1 mutations affect the localization of gurken mRNA and protein in the oocyte. These defects are correlated with incorrect positioning of the oocyte nucleus, suggesting that DLis1 is required for nuclear migration. DLis1 shows significant sequence conservation across the evolutionary spectrum. Fungal cognates of DLis1 are involved in nuclear migration while homologs in humans and mice are implicated in neuronal migration. DLis1 shows genetic interactions with the Glued and Dynein heavy chain subunits of the dynein/dynactin complex, supporting the idea that the Lis1 family of proteins plays a role in microtubule motor-based nuclear motility.     

Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists

Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.     

Kinesin-1 tail autoregulation and microtubule-binding regions function in saltatory transport but not ooplasmic streaming

The N-terminal head domain of kinesin heavy chain (Khc) is well known for generating force for transport along microtubules in cytoplasmic organization processes during metazoan development, but the functions of the C-terminal tail are not clear. To address this, we studied the effects of tail mutations on mitochondria transport, determinant mRNA localization and cytoplasmic streaming in Drosophila. Our results show that two biochemically defined elements of the tail - the ATP-independent microtubule-binding sequence and the IAK autoinhibitory motif - are essential for development and viability. Both elements have positive functions in the axonal transport of mitochondria and determinant mRNA localization in oocytes, processes that are accomplished by biased saltatory movement of individual cargoes. Surprisingly, there were no indications that the IAK autoinhibitory motif acts as a general downregulator of Kinesin-1 in those processes. Time-lapse imaging indicated that neither tail region is needed for fast cytoplasmic streaming in oocytes, which is a non-saltatory bulk transport process driven solely by Kinesin-1. Thus, the Khc tail is not constitutively required for Kinesin-1 activation, force transduction or linkage to cargo. It might instead be crucial for more subtle elements of motor control and coordination in the stop-and-go movements of biased saltatory transport.     

Application of a high-content multiparameter cytotoxicity assay to prioritize compounds based on toxicity potential in humans

Prioritization of compounds based on human hepatotoxicity potential is currently a key unmet need in drug discovery, as it can become a major problem for several lead compounds in later stages of the drug discovery pipeline. The authors report the validation and implementation of a high-content multiparametric cytotoxicity assay based on simultaneous measurement of 8 key cell health indicators associated with nuclear morphology, plasma membrane integrity, mitochondrial function, and cell proliferation. Compounds are prioritized by (a) computing an in vitro safety margin using the minimum cytotoxic concentration (IC(20)) across all 8 indicators and cell-based efficacy data and (b) using the minimal cytotoxic concentration alone to take into account concentration of drug in tissues. Feasibility data using selected compounds, including quinolone antibiotics, thiazolidinediones, and statins, suggest the viability of this approach. To increase overall throughput of compound prioritization, the authors have identified the higher throughput, plate reader-based CyQUANT assay that is similar to the high-content screening (HCS) assay in sensitivity of measuring inhibition of cell proliferation. It is expected that the phenotypic output from the multiparametric HCS assay in combination with other highly sensitive approaches, such as microarray-based expression analysis of toxic signatures, will contribute to a better understanding and predictivity of human hepatotoxicity potential.