Home About us Contact
|
Home About us Contact | ||
|
|
||
Library Screening (library + screening)
Selected AbstractsNew developments in small molecules targeting p53 pathways in anticancer therapyDRUG DEVELOPMENT RESEARCH, Issue 6 2008Chit Fang Cheok Abstract The tumor suppressor p53 is frequently inactivated in a wide variety of cancers and point mutations or deletions of the p53 gene are associated with poor prognosis in cancer. About half of all human tumors carry wildtype p53 but p53 wildtype functions are often suppressed by the overexpression of murine double minute 2 (MDM2), a negative regulator of p53. Restoration of p53 functions in tumor cells, therefore, represents an attractive strategy in combating cancer and has been the focus of intensive anticancer drug discovery. One strategy is to antagonize MDM2 functions and initial success was demonstrated in vitro and in xenograft tumor models using newly discovered small molecule inhibitors and antisense oligonucleotides. The new discovery of a compound targeting SirT1 (a member of the sirtuin family) in a p53-dependent reporter screen highlighted the importance of another negative regulator of p53 and offers an additional avenue for drug discovery and research on p53-activating therapeutics. Here, we discuss the developments of p53-activating small molecules and their potential use in combination therapy with established chemotherapeutics. These small molecules were discovered from chemical library screening using biochemical assays or cellular-based assays, and/or structure-based rational drug design strategies. Drug Dev Res 69:289,296, 2008. © 2008 Wiley-Liss, Inc. [source] Novel type III polyketide synthases from Aloe arborescensFEBS JOURNAL, Issue 8 2009Yuusuke Mizuuchi Aloe arborescens is a medicinal plant rich in aromatic polyketides, such as pharmaceutically important aloenin (hexaketide), aloesin (heptaketide) and barbaloin (octaketide). Three novel type III polyketide synthases (PKS3, PKS4 and PKS5) were cloned and sequenced from the aloe plant by cDNA library screening. The enzymes share 85,96% amino acid sequence identity with the previously reported pentaketide chromone synthase and octaketide synthase. Recombinant PKS4 and PKS5 expressed in Escherichia coli were functionally identical to octaketide synthase, catalyzing the sequential condensations of eight molecules of malonyl-CoA to produce octaketides SEK4/SEK4b. As in the case of octaketide synthase, the enzymes are possibly involved in the biosynthesis of the octaketide barbaloin. On the other hand, PKS3 is a multifunctional enzyme that produces a heptaketide aloesone (i.e. the aglycone of aloesin) as a major product from seven molecules of malonyl-CoA. In addition, PKS3 also afforded a hexaketide pyrone (i.e. the precursor of aloenin), a heptaketide 6-(2-acetyl-3,5-dihydroxybenzyl)-4-hydroxy-2-pyrone, a novel heptaketide 6-(2-(2,4-dihydroxy-6-methylphenyl)-2-oxoethyl)-4-hydroxy-2-pyrone and octaketides SEK4/SEK4b. This is the first demonstration of the enzymatic formation of the precursors of the pharmaceutically important aloesin and aloenin by a wild-type PKS obtained from A. arborescens. Interestingly, the aloesone-forming activity was maximum at 50 °C, and the novel heptaketide pyrone was non-enzymatically converted to aloesone. In PKS3, the active-site residue 207, which is crucial for controlling the polyketide chain length depending on the steric bulk of the side chain, is uniquely substituted with Ala. Site-directed mutagenesis demonstrated that the A207G mutant dominantly produced the octaketides SEK4/SEK4b, whereas the A207M mutant yielded a pentaketide 5,7-dihydroxy-2-methylchromone. [source] A trans -acting factor, isolated by the three-hybrid system, that influences alternative splicing of the amyloid precursor protein minigeneFEBS JOURNAL, Issue 13 2000Andrej Poleev Two clones were isolated in a three-hybrid screen of a rat fetal brain P5 cDNA library with an intronic splicing enhancer of the amyloid precursor protein (APP) gene as RNA bait. These clones represent the rat homologues of the previously described genes CUG-binding protein (CUG-BP) and Siah-binding protein (Siah-BP). Both interact in a sequence-specific manner with the RNA bait used for library screening as well as with the CUG repeat. In contrast, no interactions were observed in the three-hybrid assay with other baits tested. In two-hybrid assays, Siah-BP interacts with U2AF65 as well as with itself. EWS, an RGG-type RNA-binding protein associated with Ewing sarcoma, was identified as an interacting partner for the CUG-BP homologue in a two-hybrid assay for protein,protein interactions performed with various factors involved in RNA metabolism. Splicing assays performed by RT-PCR from cells cotransfected with certain cDNAs and an APP minigene, used as a reporter, indicate exclusion of exon 8 if the CUG-BP homologue is present. We conclude that clone AF169013 and its counterpart in human CUG-BP could be the trans -acting factors that interact with the splicing enhancer downstream of exon 8, and in this way influence alternative splicing of the APP minigene. [source] Mutational activation of the MAP3K8 protooncogene in lung cancerGENES, CHROMOSOMES AND CANCER, Issue 2 2004Adam Michael Clark The MAP3K8 protooncogene (Cot/Tpl-2) activates the MAP kinase, SAP kinase, and NF-,B signaling pathways. MAP3K8 mutations occur in the rat homologue, but activating mutations have yet to be identified in primary human tumors. We have identified MAP3K8 as a transforming gene from a human lung adenocarcinoma and characterized a 3, end mutation in the cDNA. In addition, we confirmed that the mutation occurs in the original lung tumor, and we screened a series of lung cancer cell lines to determine whether the MAP3K8 mutation is a common occurrence in lung tumorigenesis. The oncogene was isolated and identified with the NIH3T3 nude mouse tumorigenicity assay and cDNA library screening. The gene was analyzed by polymerase chain reaction (PCR), single-strand conformational polymorphism (SSCP), and 3,RACE for mutations. The mutation was localized to MAP3K8 exon 8 and confirmed in the primary tumor DNA. Both wild-type and mutant MAP3K8 cDNAs transformed NIH3T3 cells, but the transforming activity of the mutant was much greater than that of the wild type. PCR-SSCP screening of cell line cDNAs identified one silent polymorphism in cell line SK-LU-1. Although we were unable to find additional activating mutations, these data support a role for MAP3K8 activity in cellular transformation, but suggest that mutational activation of the gene is a rare event in lung cancer. © 2004 Wiley-Liss, Inc. [source] Novel epididymis-specific mRNAs downregulated by HE6/Gpr64 receptor gene disruptionMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2007Ben Davies Abstract Targeted disruption of the epididymis-specific HE6/Gpr64 receptor gene in mice led to male infertility. In order to characterize the phenotype at a molecular level, we compared the gene expression patterns of wild type (wt) versus knockout (KO) caput epididymides. The caput region of KO males, although morphologically normal, nevertheless showed an aberrant expression pattern. Combining micro array analysis, differential library screening, Northern blot analysis and quantitative RT-PCR, we found that the knockout of the HE6/Gpr64 receptor was mainly associated with the downregulation of genes specific to the initial segment. The list of KO downregulated transcripts comprised Enpp2/autotaxin, the lipocalins 8 and 9, the ,-defensin Defb42, cystatins 8 and 12, as well as the membrane proteins Adam (A Disintegrin And Metalloprotease) 28, claudin-10, EAAC1, and the novel Me9. Clusterin/ApoJ and osteopontin/Spp1 mRNAs, on the other hand, were upregulated in the KO tissues. The Me9 transcript was studied in further detail, and we report here a cluster of related epididymis-specific genes. Me9 is specifically expressed in the initial segment and is representative of a novel and highly conserved mammalian gene family. The family consists of single-exon genes only; intron-containing paralogs have not yet been ascertained. The cloned cDNA sequences predicted hydrophobic polytopic membrane proteins containing the DUF716 motif. Protein expression was shown in the rodent caput epididymidis but remained uncertain in primates. Mol. Reprod. Dev. 74: 539,553, 2007. © 2006 Wiley-Liss, Inc. [source] Quantitative specificity-based display library screening identifies determinants of antibody-epitope binding specificity,PROTEIN SCIENCE, Issue 9 2009Sejal S. Hall Abstract Despite the critical importance of molecular specificity in bimolecular systems, in vitro display technologies have been applied extensively for affinity maturation of peptides and antibodies without explicitly measuring the specificity of the desired interaction. We devised a general strategy to measure, screen, and evolve specificity of protein ligand interactions analogous to widely used affinity maturation strategies. The specificity of binding to target and nontarget antibodies labeled with spectrally distinct fluorophores was measured simultaneously in protein mixtures via multiparameter flow cytometry, thereby enabling screening for high target antibody specificity. Isolated antibody specific ligands exhibited varying specificity, revealing critical amino acid determinants for target recognition and nontarget avoidance in complex mixtures. Molecular specificity in the mixture was further enhanced by quantitative directed evolution, yielding a family of epitopes exhibiting improved specificities equivalent, or superior to, the native peptide antigen to which the antibody was raised. Specificity screening simultaneously favored affinity, yielding ligands with three-fold improved affinity relative to the parent epitope. Quantitative specificity screening will be useful to screen, evolve, and characterize the specificity of protein and peptide interactions for molecular recognition applications. [source] Comparison of PERV genomic locations between Asian and European pigsANIMAL GENETICS, Issue 1 2010W. Y. Jung Summary Xenotransplantation from pigs provides a possible solution to the shortage of human organs for allotransplantation. Porcine endogenous retroviruses (PERVs) are a possible obstacle to using porcine organs in addition to the immunological barriers. Three main types of PERVs (A, B and C) have been previously investigated in diverse pig breeds. To examine the copy numbers of PERVs and their genomic locations in the Korean native pig genome, we screened a BAC (Bacterial Artificial Chromosome) library with PERV-specific protease primers for initial recognition of PERV-positive clones and three sets of envelope-specific primers for the identification of PERV types. A total of 45 PERV-positive clones, nine PERV-A and 36 PERV-B, have been identified from the library screening and the BAC contigs were constructed using the primers designed from BAC end sequences (BESs). These primers were also used for SCH (Somatic Cell Hybrid) and RH (Radiation Hybrid) mapping of the PERV-positive clones. The results indicate that 45 PERV-positive BAC clones belong to nine contigs and a singleton. SCH and IMpRH (INRA-Minnesota Porcine Radiation Hybrid) mapping results indicated that there are at least eight separate PERV genomic locations, consisting of three PERV-A and five PERV-B. One contig could not be mapped, and two contigs are closely located on SSC7. Southern blotting indicates there may be up to 15 additional sites. Further investigation of these clones will contribute to a general strategy to generate PERV-free lines of pigs suitable for xenotransplantation. [source] RNA helicase encoded by melanoma differentiation,associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis: Association with rapidly progressive interstitial lung disease,ARTHRITIS & RHEUMATISM, Issue 7 2009Shinji Sato Objective To identify the autoantigen recognized by the autoantibody that is associated with clinically amyopathic dermatomyositis (C-ADM) and rapidly progressive interstitial lung disease (ILD). Methods An anti,CADM-140 antibody,positive prototype serum sample was used to screen a HeLa cell,derived complementary DNA (cDNA) library. Selected cDNA clones were further evaluated by immunoprecipitation of their in vitro,transcribed and in vitro,translated products using anti,CADM-140 antibody,positive and anti-CADM-140 antibody,negative sera. The lysates of COS-7 cells transfected with the putative antigen were similarly tested. An enzyme-linked immunosorbent assay (ELISA) to detect the anti,CADM-140 antibody was established using a recombinant CADM-140 antigen, and its specificity and sensitivity for C-ADM and rapidly progressive ILD were assessed in 294 patients with various connective tissue diseases. Results By cDNA library screening and immunoprecipitation of in vitro,transcribed and in vitro,translated products, we obtained a cDNA clone encoding melanoma differentiation,associated gene 5 (MDA-5). The anti,CADM-140 antibodies in patients' sera specifically reacted with MDA-5 protein expressed in cells transfected with full-length MDA-5 cDNA, confirming the identity of MDA-5 as the CADM-140 autoantigen. The ELISA, using recombinant MDA-5 protein as the antigen, showed an analytical sensitivity of 85% and analytical specificity of 100%, in comparison with the "gold standard" immunoprecipitation assay, and was useful for identifying patients with C-ADM and/or rapidly progressive ILD. Conclusion Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD. Moreover, our ELISA using recombinant MDA-5 protein makes detection of the anti,CADM-140 antibody routinely available. [source] | ||||||||||||||||