Using IMAP technology to identify kinase inhibitors: comparison with a substrate depletion approach and analysis of the nature of false positives.

IMAP is a fluorescence polarisation-based assay method which can be applied to the measurement of protein kinase activity. Using a model serine/threonine kinase we found that IMAP generated a good assay window (Z' > 0.8), was very tolerant of DMSO, and was flexible with respect to sample processing (stopped reactions were stable over a period of several days). Using a set of six low molecular weight inhibitors of the kinase, we found a good correlation between IMAP and scintillation proximity assay (SPA) potency data. IMAP, which measures product accumulation, was compared in an HTS setting with a substrate depletion method (luminescence-based measurement of ATP concentration). There was a reasonable (approximately 50%) overlap in primary hits from a 17,000 compound set, but more apparent false positives were generated from the IMAP method. We followed up the compounds that showed activity in the IMAP method but not in the luminescence assay. Approximately 10% of these compounds displayed intrinsic fluorescence, suggesting that they were false actives by virtue of intrinsic spectroscopic properties. Compound activity by competition of phosphopeptide binding to IMAP beads can occur with high concentrations of chelating compounds, but did not occur with any of the false actives, suggesting that this form of interference is rare.

Transcriptional activation of the cloned Heliothis virescens (Lepidoptera) ecdysone receptor (HvEcR) by muristeroneA.

Ecdysteroids play an important role during insect development. We report here the isolation and characterisation of an Ecdysone receptor (EcR) homologue from Heliothis virescens (HvEcR) and present evidence supporting the HvEcR active role as an active component of the native insect receptor. Alignment of the deduced amino acid sequence of HvEcR with those of EcRs from other species confirmed its membership of this family and showed that it is closely related to the B1 isoform of Drosophila melanogaster. Northern blot analysis showed that two transcripts (6.0 and 6.5 kb) were recognised by a probe spanning the DNA and ligand binding domains of the HvEcR. Genomic Southern blots showed that the HvEcR is encoded by a single copy gene. Two lines of evidence towards the functional activity of the HvEcR are presented. In vitro transcribed and translated HvEcR showed specific binding to hsp27 and pall response elements in the presence of CfUSP. Stable expression of HvEcR in 293 cells induced reporter gene activity in the presence of muristeroneA in a dose dependant manner while dexamethasone failed to activate.

An RGS protein regulates the pheromone response in the fission yeast Schizosaccharomyces pombe.

The rate and extent of a cell's response to an extracellular stimulus is influenced by regulators that act on the intracellular signalling machinery. Although not directly involved in propagating the intracellular signal, regulators control the activity of the proteins that transmit the signals. To understand this aspect of cell signalling, we have studied the pheromone response pathway in the fission yeast Schizosaccharomyces pombe, a relatively simple signalling system in a genetically tractable organism. We demonstrate this approach by investigating the role of Rgs1, a member of the Regulator of G protein Signalling (RGS) family of proteins. The rgs1 gene was identified through the Sz. pombe genome sequencing project (accession number Q09777) and recognized as having similarity to RGS proteins [Tesmer et al. (1997) Cell 89: 251-261], but this is the first report concerning the activity of the protein. Strains lacking rgs1 (Deltargs1) are hypersensitive to pheromone stimulation and unable to conjugate with a mating partner. Inhibition of mating occurs at a relative late stage in the process as Deltargs1 strains exhibit pheromone-dependent transcription and form shmoos. Expression of SST2 (an RGS protein that regulates pheromone signalling in the budding yeast Saccharomyces cerevisiae) overcomes the hypersensitivity of the Deltargs1 strains but fails to rescue their mating defect.

A reporter gene assay for fungal sterol biosynthesis inhibitors.

Acetoacetyl-CoA thiolase (ACoAT) catalyses the condensation of two acetyl-CoA molecules, the first step in the sterol biosynthetic pathway. We constructed a yeast strain containing a fusion of the promoter of the Saccharomyces cerevisiae ACoAT gene to a reporter gene (Escherichia coli beta-galactosidase). Reporter gene activity in this strain can be induced by a variety of inhibitors of sterol biosynthesis. These results suggest that the ACoAT gene is feedback regulated at the transcriptional level by products of the sterol biosynthetic pathway. The reporter gene approach described here may be used to screen chemical collections for compounds which inhibit fungal sterol biosynthesis.

GAL4 fusion vectors for expression in yeast or mammalian cells.

We describe two sets of vectors, one for yeast (pY1, pY2 and pY3) and one for mammalian cells (pM1, pM2, and pM3), that simplify the production of fusion proteins containing the DNA-binding domain of GAL4. This protein fragment, consisting of GAL4 amino acid (aa) residues 1-147, binds to a specific 17-bp nucleotide sequence, but is incapable of activating transcription unless fused to a protein that can contribute an activating function. Genetic strategies exploiting this property of GAL4 (aa 1-147) have been developed to characterize transcription factor functional domains, protein-protein interactions, and site-specific proteolysis. The vectors we describe allow fusion to the C terminus of GAL4 (aa 1-147) in any reading frame, and thus facilitate these experimental strategies.

Structure and methylation of the human calcitonin/alpha-CGRP gene.

We report a detailed analysis of the human calcitonin/alpha-CGRP gene locus. About 39kb of DNA containing the gene has been mapped and a common Pvu II RFLP identified downstream of the gene. DNA sequence analysis revealed an extensive CpG island containing several rare restriction enzyme sites at the 5' end of the gene. The structure of this island is unusual in that it contains two distinct CpG-rich regions, one located around exon 1 and the other about 1.5kb further upstream. Msp I sites within both CpG-rich regions were found to be unmethylated, regardless of whether the calcitonin/alpha-CGRP gene was being expressed. However, a correlation was found between demethylation of Msp I sites in intron 2, downstream of the CpG island, and calcitonin/alpha-CGRP gene expression. DNA sequence analysis also revealed the presence of several binding sites for constitutive and regulatory transcription factors in the promoter of the gene. These results suggest that both unmethylated CpG islands and specific demethylation of internal sequences may play a role in the activation of calcitonin/alpha-CGRP gene transcription.

Association of parathyroid tumors in multiple endocrine neoplasia type 1 with loss of alleles on chromosome 11.

Familial multiple endocrine neoplasia type 1 (MEN-1) is an autosomal dominant disorder characterized by the combined occurrence of tumors of the parathyroid glands, the pancreas, and the pituitary gland. Pancreatic tumors have previously been shown to be associated with the loss of alleles on chromosome 11; we therefore looked for similar genetic alterations in specimens of parathyroid tumors, which are the most common feature of MEN-1. We obtained parathyroid tumors and peripheral-blood leukocytes from six patients with MEN-1; 18 cloned human DNA sequences from chromosome 11 were then used to identify restriction-fragment-length polymorphisms. A loss of heterozygosity was detected in parathyroid tumors from three of the six patients with MEN-1; this finding demonstrated that allelic deletions on chromosome 11 are involved in the monoclonal development of parathyroid tumors in patients with MEN-1. In addition, studies of three affected families (with 17 affected members and 51 unaffected members) established linkage with the oncogene INT2 (peak lod score, 3.30, at 0 percent recombination); the MEN-1 gene was thus mapped to the pericentromeric region of the long arm of chromosome 11 (11q13). Our location of the MEN-1 gene at 11q13 is close to the location previously reported. We conclude that a single inherited locus on chromosome 11, band q13, causes MEN-1 and that the monoclonal development of parathyroid and pancreatic tumors in patients with MEN-1 involves similar allelic deletions on chromosome 11.

Expression and function of the human calcitonin/alpha-CGRP gene in health and disease.

Studies on the structure and expression of the rat and human calcitonin gene provide a remarkable example by which a combination of molecular, immunocytochemical and pharmacological techniques have led to the identification of a novel gene product, the calcitonin gene-related peptide, a peptide of unexpected tissue distribution and biological activity. Future studies at the molecular level will provide insight into post-transcriptional mechanisms by which a single gene can give rise to discrete mRNAs in a tissue-specific manner. The isolation and characterization of the CGRP receptor, and hence the site(s) and mechanism(s) of action of the CGRP family within the vasculature, will also add significantly to our understanding of molecular mechanisms involved in the modulation of cardiovascular function in man. Furthermore, the role and mechanism of action of the CGRP peptide family in the central and peripheral nervous systems remains to be elucidated. It is also apparent that measurement of circulating plasma levels of CGRP may be of diagnostic and prognostic value, providing information concerning the onset and progression of lung and thyroid carcinoma. Our analysis of the structure and expression of the calcitonin/alpha-CGRP gene also demonstrates, for the first time, the molecular basis of large calcitonin secretion by lung carcinoma cells. Whether the expression of this gene in lung carcinoma cell-lines can truly be described as 'ectopic' is however questionable, in the light of recent immunocytochemical evidence which demonstrates that the lung is a major source of CGRP producing cells in the rat (see Springall et al., 1984).