Identification of compounds that potentiate CREB signaling as possible enhancers of long-term memory.

Many studies have implicated the cAMP Response Element Binding (CREB) protein signaling pathway in long-term memory. To identify small molecule enhancers of CREB activation of gene expression, we screened approximately 73,000 compounds, each at 7-15 concentrations in a quantitative high-throughput screening (qHTS) format, for activity in cells by assaying CREB mediated beta-lactamase reporter gene expression. We identified 1,800 compounds that potentiated CREB mediated gene expression, with potencies as low as 16 nM, comprising 96 structural series. Mechanisms of action were systematically determined, and compounds that affect phosphodiesterase 4, protein kinase A, and cAMP production were identified, as well as compounds that affect CREB signaling via apparently unidentified mechanisms. qHTS followed by interrogation of pathway targets is an efficient paradigm for lead generation for chemical genomics and drug development.

Inhibition of morphine-induced cAMP overshoot: a cell-based assay model in a high-throughput format.

Opiates are not only potent analgesics but also drugs of abuse mainly because they produce euphoria. Chronic use of opiates results in the development of tolerance and dependence. Dr Marshall Nirenberg's group at the National Institutes of Health (NIH) was the first to use a cellular model system of Neuroblastoma × Glioma hybrid cells (NG108-15) to study morphine addiction. They showed that opiates affect adenylyl cyclase (AC) by two opposing mechanisms mediated by the opiate receptor. Although the cellular mechanisms that cause addiction are not yet completely understood, the most observed correlative biochemical adaptation is the upregulation of AC. This model also provides the opportunity to look for compounds which could dissociate the acute effect of opiates from the delayed response, upregulation of AC, and thus lead to the discovery of non-addictive drugs. To identify small molecule compounds that can inhibit morphine-induced cAMP overshoot, we have validated and optimized a cell-based assay in a high throughput format that measures cellular cAMP production after morphine withdrawal. The assay performed well in the 1536-well plate format. The LOPAC library of 1,280 compounds was screened in this assay on a quantitative high-throughput screening (qHTS) platform. A group of compounds that can inhibit morphine-induced cAMP overshoot were identified. The most potent compounds are eight naloxone-related compounds, including levallorphan tartrate, naloxonazine dihydrochloride, naloxone hydrochloride, naltrexone hydrochloride, and naltriben methanesulfonate. The qHTS approach we used in this study will be useful in identifying novel inhibitors of morphine induced addiction from a larger scale screening.

Historical review: Deciphering the genetic code--a personal account.

This is an autobiographical description of the events that led to the breaking of the genetic code and the subsequent race to decipher the code. The code was deciphered in two stages over a five-year period between 1961 and 1966. During the first stage, the base compositions of codons were deciphered by the directing cell-free protein synthesis with randomly ordered RNA preparations. During the second phase, the nucleotide sequences of RNA codons were deciphered by determining the species of aminoacyl-tRNA that bound to ribosomes in response to trinucleotides of known sequence. Views on general topics such as how to pick a research problem and competition versus collaboration also are discussed.

Identification of a potent new chemotype for the selective inhibition of PDE4.

A series of substituted 3,6-diphenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines were prepared and analyzed as inhibitors of phosphodiesterase 4 (PDE4). Synthesis, structure-activity relationships, and the selectivity of a highly potent analogue against related phosphodiesterase isoforms are presented.

A Cell-based beta-Lactamase Reporter Gene Assay for the CREB Signaling Pathway.

The Cyclic-AMP Response Element Binding (CREB) proteins comprise a family of transcription factors that stimulate or repress the expression of a wide variety of genes by binding to nucleotide sequences known as cAMP Response Elements (CREs). CREB-mediated transcription has been implicated in a wide variety of important physiological processes, including long-term memory, and enhancement of CREB signaling has been suggested as an attractive therapeutic strategy for human memory disorders. To identify small molecule compounds that enhance CREB pathway signaling, we have optimized and validated a cell-based beta-lactamase reporter gene CREB pathway assay in 1536-well plate format. The LOPAC library of 1280 compounds was screened in triplicate in this assay on a quantitative high throughput screening (qHTS) platform. A variety of compounds which affect known members of the CREB pathway were identified as active, including twelve known phosphodiesterase (PDE) inhibitors, and forskolin, a known activator of adenylate cyclase, thus validating the assay's performance. This qHTS platform assay will facilitate identification of novel small molecule CREB signaling enhancers, which will be useful for chemical genetic dissection of the CREB pathway and as starting points for potentially memory-enhancing therapeutics.

Silencing of genes in cultured Drosophila neurons by RNA interference.

Cultures of neuroblasts that generate abundant neurons were established from Drosophila embryos to study silencing of genes by RNA interference (RNAi). Cultured cells expressed ELAV, a marker of neurons, Futsch, a marker of neurites, and Synapsin, Synaptobrevin, and Synaptogamin, proteins involved in neurotransmitter secretion. Conditions were found for efficient transfection of cells with siRNAs for ELAV or the insulin-like receptor, which resulted in marked decreases in neurons that express ELAV and Futsch. Cells also were successfully transfected with long-chain Sox-Neuro dsRNA resulting in a 55% reduction of neurons expressing Futsch. The results suggest that this cultured neural cell system can be used to study RNAi-dependent silencing of genes involved in many kinds of neural functions.

RNA interference screen to identify genes required for Drosophila embryonic nervous system development.

RNA interference (RNAi) has been shown to be a powerful method to study the function of genes in vivo by silencing endogenous mRNA with double-stranded (ds) RNA. Previously, we performed in vivo RNAi screening and identified 43 Drosophila genes, including 18 novel genes required for the development of the embryonic nervous system. In the present study, 22 additional genes affecting embryonic nervous system development were found. Novel RNAi-induced phenotypes affecting nervous system development were found for 16 of the 22 genes. Seven of the genes have unknown functions. Other genes found encode transcription factors, a chromatin-remodeling protein, membrane receptors, signaling molecules, and proteins involved in cell adhesion, RNA binding, and ion transport. Human orthologs were identified for proteins encoded by 16 of the genes. The total number of dsRNAs that we have tested for an RNAi-induced phenotype affecting the embryonic nervous system, including our previous study, is 7,312, which corresponds to approximately 50% of the genes in the Drosophila genome.

Identification and analysis of vnd/NK-2 homeodomain binding sites in genomic DNA.

Vnd/NK-2 homeodomain affinity column chromatography was used to purify Drosophila DNA fragments bound by the vnd/NK-2 homeodomain. Sequencing the selected genomic DNA fragments led to the identification of 77 Drosophila DNA fragments that were grouped into 42 vnd/NK-2 homeodomain-binding loci. Most loci were within upstream or intronic regions, especially first introns. Nineteen of the Drosophila DNA fragments cloned correspond to one locus, termed Clone A, which is 312 bp in length and contains five vnd/NK-2 homeodomain core consensus binding sites, 5'-AAGTG, and is part of the first intron of the Beadex gene. We further analyzed the interactions between Clone A and vnd/NK-2 homeodomain protein by mobility-shift assay, DNase I footprinting, methylation interference, and ethylation interference. The DNase I footprinting analysis of Clone A with vnd/NK-2 homeodomain protein revealed three strong binding sites and one weak binding site between 15 and 130 bp of Clone A. We also analyzed binding of the vnd/NK-2 homeodomain to the 5'-flanking sequence of vnd/NK-2 genomic DNA. The DNase I footprinting result showed that there are two strong binding sites and five weak binding sites in the fragment between -385 and -675 bp from the transcription start site of the vnd/NK-2 gene.

Mapping activation and repression domains of the vnd/NK-2 homeodomain protein.

A transient transfection assay using Drosophila S2 tissue culture cells and WT and mutant Drosophila vnd/NK-2 homeobox cDNAs was used to localize repression and activation domains of vnd/NK-2 homeodomain protein. A repression domain was identified near the N terminus of vnd/NK-2 homeodomain protein (amino acid residues 154-193), which contains many hydrophobic amino acid residues. The major determinants of the repression domain were shown to be amino acid residues F155, W158, I161, L162, L163, and W166. Truncated protein consisting of the N-terminal repression domain and the DNA-binding homeodomain repressed transcription as efficiently as WT vnd/NK-2 protein. An activation domain was identified between the tinman domain and the homeodomain (amino acid residues 277-543), which consists of a glutamine-rich subdomain and two acidic subdomains. No effect was detected of the tinman domain or the NK-2-specific domain on either activation or repression of a beta-galactosidase reporter gene.

Sequence-specific DNA binding by the vnd/NK-2 homeodomain of Drosophila.

The ventral nervous system defective (vnd)/NK-2 homeodomain and some flanking amino acid residues were expressed in Escherichia coli, purified to homogeneity, and the protein was covalently coupled to Sepharose. Oligodeoxynucleotides that contained 16-bp random sequences were purified by vnd/NK-2 affinity column chromatography, cloned, and sequenced. The consensus nucleotide sequence of the vnd/NK-2 homeodomain binding site was shown to be T(T/C)AAGTG(G/C). The apparent equilibrium dissociation constant (K(D)) of the vnd/NK-2 homeodomain for the consensus sequence is 1.9 x 10(-10) M. In addition, results of competition between oligodeoxynucleotides for binding to the vnd/NK-2 homeodomain and determination of the apparent K(D) values of oligodeoxynucleotides that differ from the consensus sequence by only a single base pair demonstrate that the four central nucleotides, AAGT, in this sequence play a major role in determining the affinity of binding.

A functional genomic screen for cardiogenic genes using RNA interference in developing Drosophila embryos.

Identifying genetic components is an essential step toward understanding complex developmental processes. The primitive heart of the fruit fly, the dorsal vessel, which is a hemolymph-pumping organ, has provided a unique model system to identify cardiogenic genes and to further our understanding of the molecular mechanisms of cardiogenesis. Using RNA interference in developing Drosophila embryos, we performed a genomewide search for cardiogenic genes. Through analyses of the >5800 genes that cover approximately 40% of all predicted Drosophila genes, we identified a variety of genes encoding transcription factors and cell signaling proteins required for different steps during heart development. Analysis of mutant heart phenotypes and identified genes suggests that the Drosophila heart tube is segmentally patterned, like axial patterning, but assembled with regional modules. One of the identified genes, simjang, was further characterized. In the simjang mutant embryo, we found that within each segment a subset of cardial cells is missing. Interestingly, the simjang gene encodes a protein that is a component of the chromatin remodeling complex recruited by methyl-CpG-DNA binding proteins, suggesting that epigenetic information is crucial for specifying cardiac precursors. Together, these studies not only identify key regulators but also reveal mechanisms underlying heart development.

Regulatory DNA required for vnd/NK-2 homeobox gene expression pattern in neuroblasts.

Vnd/NK-2 protein was detected in 11 neuroblasts per hemisegment in Drosophila embryos, 9 medial and 2 intermediate neuroblasts. Fragments of DNA from the 5'-flanking region of the vnd/NK-2 gene were inserted upstream of an enhancerless betagalactosidase gene in a P-element and used to generate transgenic fly lines. Antibodies directed against Vnd/NK-2 and beta-galactosidase proteins then were used in double-label experiments to correlate the expression of beta-galactosidase and Vnd/NK-2 proteins in identified neuroblasts. DNA region A, which corresponds to the -4.0 to -2.8-kb fragment of DNA from the 5'-flanking region of the vnd/NK-2 gene was shown to contain one or more strong enhancers required for expression of the vnd/NK-2 gene in ten neuroblasts. DNA region B (-5.3 to -4.0 kb) contains moderately strong enhancers for vnd/NK-2 gene expression in four neuroblasts. Hypothesized DNA region C, whose location was not identified, contains one or more enhancers that activate vnd/NK-2 gene expression only in one neuroblast. These results show that nucleotide sequences in at least three regions of DNA regulate the expression of the vnd/NK-2 gene, that the vnd/NK-2 gene can be activated in different ways in different neuroblasts, and that the pattern of vnd/NK-2 gene expression in neuroblasts of the ventral nerve cord is the sum of partial patterns.

Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells.

Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes.

Distortion of the three-dimensional structure of the vnd/NK-2 homeodomain bound to DNA induced by an embryonically lethal A35T point mutation.

The three-dimensional solution structure obtained by NMR of the A35T mutant vnd/NK-2 homeodomain bound to the vnd/NK-2 consensus 16 bp DNA sequence was determined. This mutation to threonine from alanine in position 35 in helix II of the vnd/NK-2 homeodomain is associated with early embryonic lethality in Drosophila melanogaster. Although the unbound mutant protein is not structured, in the DNA-bound state it adopts the three-helix fold characteristic of all known homeodomains, but with alterations relative to the structure of the wild-type analogue. These structural modifications occur, and are accompanied by a 50-fold reduction in the DNA binding affinity, even though most of the protein-DNA interactions originally seen for the wild-type homeodomain are found likewise in the threonine analogue. Alterations include torsional angle changes in the loop between helix I and helix II, and in the turn between helix II and helix III, as well as in a distortion of the usual antiparallel orientation of helix I with respect to helix II. The alteration of the position of leucine 40 in the A35T mutant is proposed to explain the observed 1.27 ppm upfield shift of the corresponding amide proton resonance relative to the value observed for the wild-type analogue. A detailed comparison of the structures of the mutant A35T and wild-type vnd/NK-2 homeodomains bound to the cognate DNA is presented. The consequences of the structural alteration of the DNA-bound A35T mutant vnd/NK-2 protein may constitute the basis of the observed early embryonic lethality.

Mutations that affect the ability of the vnd/NK-2 homeoprotein to regulate gene expression: transgenic alterations and tertiary structure.

The importance in downstream target regulation of tertiary structure and DNA binding specificity of the protein encoded by the vndNK-2 homeobox gene is analyzed. The ectopic expression patterns of WT and four mutant vndNK-2 genes are analyzed together with expression of two downstream target genes, ind and msh, which are down-regulated by vndNK-2. Three mutants are deletions of conserved regions (i.e., tinman motif, acidic motif, and NK-2 box), and the fourth, Y54M vndNK-2, corresponds to a single amino acid residue replacement in the homeodomain. Of the four ectopically expressed mutant genes examined, only the Y54M mutation inactivates the ability of the vndNK-2 homeodomain protein to repress ind and msh. The acidic motif deletion mutant slightly reduced the ability of the protein to repress ind and msh. By contrast, both tinman and NK-2 box deletion mutants behaved as functional vndNK-2 genes in their ability to repress ind and msh. The NMR-determined tertiary structures of the Y54M vndNK-2 homeodomain, both free and bound to DNA, are compared with the WT analog. The only structural difference observed for the mutant homeodomain is in the complex with DNA and involved closer interaction of the methionine-54 with A2, rather than with C3 of the (-) strand of the DNA. This subtle change in the homeodomain-DNA complex resulted in modifications of binding affinities to DNA. These changes resulting from a single amino acid residue replacement constitute the molecular basis for the phenotypic alterations observed on ectopic expression of the Y54M vndNK-2 gene during embryogenesis.

Genes required for Drosophila nervous system development identified by RNA interference.

RNA interference was used to screen 3,314 Drosophila double-stranded RNAs, corresponding to approximately 25% of Drosophila genes, for genes that affect the development of the embryonic nervous system. RNA-interference-mediated gene silencing in Drosophila embryos resulted in loss-of-function mutant phenotypes for 43 genes, which is 1.3% of the genes that were screened. We found 18 genes that were not known previously to affect the development of the nervous system. The functions of some of the genes are unknown. Other genes encode protein kinases, transcription factors, and signaling proteins, as well as proteins with other functions.