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At least 631 records · Page 35Linked to original sources
PubMed2004

Prevention of complement-mediated immune hemolysis by a small molecule compound.

Complement sensitization of red blood cells (RBCs) can result in transfusion reactions and hemolytic anemias. We hypothesized that manipulating the complement system using small organic molecules might prevent RBC destruction, thereby prolonging RBC survival in patients. Using a simple, rapid, large-scale hemolytic assay, we screened a 10,000 compound library, enriched in anti-inflammatory compounds at a final concentration of 25 microM, and identified a 549Da compound (C(34)H(24)N(6)O(2)) with a symmetrical structure containing two benzimidazole rings that, as compared to a known anti-complement molecule FUT-175, was more effective in reducing hemolysis by the classical pathway and had comparable anti-hemolytic activity against the alternative pathway. Furthermore, in a xenotransfusion mouse model, treatment of mice with 1.2mg/kg of the compound significantly prolonged the survival of transfused RBCs, reducing C3 deposition, but not the deposition of control IgG or IgM, for the first hour post-transfusion. These data suggest that further studies are warranted to determine if this compound has usefulness in a transfusion setting.

Anemia, Hemolytic↗
PubMed2005

Small molecules that delay S phase suppress a zebrafish bmyb mutant.

Bmyb is a ubiquitously expressed transcription factor involved in cellular proliferation and cancer. Loss of bmyb function in the zebrafish mutant crash&burn (crb) results in decreased cyclin B1 expression, mitotic arrest and genome instability. These phenotypic observations in crb mutants could be attributed to the decreased expression of cyclin B1, a cell-cycle regulatory protein that is responsible for driving cell progression from G2 through mitosis. To identify small molecules that interact with the bmyb pathway, we developed an embryo-based suppressor screening strategy. In 16 weeks we screened a diverse approximately 16,000 compound library, and discovered one previously unknown compound, persynthamide (psy, 1), that suppressed bmyb-dependent mitotic defects. Psy-treated embryos showed an S-phase delay, and knockdown of the cell-cycle checkpoint regulator ataxia telangiectasia--and Rad-related kinase (ATR) abrogated the suppression of crb. The DNA synthesis inhibitors aphidicolin (2) and hydroxyurea (3) also suppressed crb. S-phase inhibition upregulated cyclin B1 mRNA, promoting the progression of cells through mitosis. Our study demonstrates that chemical suppressor screening in zebrafish can identify compounds with cell-cycle activity and can be used to identify pathways that interact with specific cell-cycle phenotypes.

Adamantane↗
PubMed2002

Ribozyme-catalysed carbon-carbon bond formation.

Numerous RNA molecules with new catalytic properties have been isolated from synthetic combinatorial libraries. A broad range of chemical reactions is catalysed, and nucleic acids can accelerate bond formation between small organic substrates. This review focuses on carbon-carbon bond formation accelerated by in vitro selected ribozymes. Mechanistic investigations and structure-function relationships are discussed.

Base Sequence↗
PubMed2006

ES1, a new lung carcinoma antibody--an immunohistochemical study.

AIMS: To characterize a single domain antibody (sdAb), AFAI, obtained by panning a naive phage display library of single domain antibodies against the non-small cell lung carcinoma cell line A549. AFAI recognizes a variant form of carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6 or CEA6). METHODS AND RESULTS: Various normal tissues and 139 neoplastic lesions from lung and other organs were immunostained with ES1 antibody, a pentameric and highly avid form of AFAI. ES1 was immunoreactive with 34 of 35 non-squamous large cell lung carcinomas with staining intensities ranging from focal to extensive and from moderate to strong. Importantly, ES1 stained poorly differentiated lung adenocarcinomas and many undifferentiated large cell lung carcinomas that typically show negative immunoreactivity with an antibody specific for thyroid transcription factor-1. Non-lung adenocarcinomas showed more focal and weaker immunoreactivity than for lung adenocarcinoma. Other carcinomas showed negative or weak immunoreactivity and normal tissues showed no immunoreactivity. CONCLUSIONS: Compared with other antibodies used in the clinical evaluation of lung adenocarcinomas, ES1 is more lung carcinoma sensitive, more sensitive in immunostaining of poorly differentiated adenocarcinomas that are usually associated with distant metastasis and less immunoreactive with normal tissues.

Antibodies, Neoplasm↗
PubMed2004

Metabolic Diseases Drug Discovery-Strategic Research Institute's Third International World Summit. Dipeptidyl peptidase-IV inhibitors 26-27 July 2004, San Diego, CA, USA.

The majority of the presentations a the conference were on three highly sought-after targets for type 2 diabetes mellitus, namely PTP1B, PPARs and DPP-IV, reflecting the current focus and trend in the industry. A couple of novel targets were discussed, including the potential of myostatin as a type 2 diabetes mellitus target and a novel GPCR target. While small molecules were dominant, several biological-based approaches were covered: antibody therapeutics and oligonucleotide-based approaches (ASO and siRNA). In searching for small-molecule leads, structure-based rational design and focused combination chemistry appear to produce better results than a random high-throughput approach over the entire chemical library. The biggest challenges for diabetes and obesity drugs remain similar to those mentioned in previous meetings: increasing specificity to reduce side effects and maintaining long-term effect while maintaining or increasing efficacy. Due to the tremendous interest of the pharmaceutical industry in metabolic disease drug development, our knowledge of food intake and metabolism regulation has increased exponentially. Overall, the prospect of better drugs for, and better control of, type 2 diabetes mellitus and obesity is promising.

Animals↗
PubMed1997

A miniaturized arrayed assay format for detecting small molecule-protein interactions in cells.

BACKGROUND: Two complementary approaches to studying the cellular function of proteins involve alteration of function either by mutating protein-encoding genes or by binding a small molecule to the protein. A mutagen can generate millions of genetic mutations; correspondingly, split-pool synthesis can generate millions of unique ligands attached to individual beads. Genetic screening of mutations is relatively straightforward but, in contrast, split-pool synthesis presents a challenge to current methods of screening for compounds that alter protein function. The methods used to screen natural products are not feasible for large libraries composed of covalently immobilized compounds on synthesis beads. The sheer number of compounds synthesized by split-pool synthesis, and the small quantity of individual compound attached to each bead require assay miniaturization for efficient screening. RESULTS: We present a miniaturized cell-based technique for the screening of ligands prepared by split-pool synthesis. Spatially defined droplets with uniform volumes of approximately 50-150 nanoliters (depending on well dimensions) are arrayed on plastic devices prepared using a combination of photolithography and polymer molding. Using this microtechnology, approximately 6,500 assays using either yeast cells or mammalian tissue culture can be performed within the dimensions of a standard 10 cm petri dish. We demonstrate that the biological effect of a small molecule prepared by split-pool synthesis can be detected in this format following its photorelease from a bead. CONCLUSIONS: The miniaturized format described here allows uniformly sized nanodroplets to be arrayed on plastic devices. The design is amenable to a large number of biological assays and the spatially arrayed format ensures uniform and controlled ligand concentrations and should facilitate automation of assays. The screening method presented here provides an efficient means of rapidly screening large numbers of ligands made by split-pool synthesis in both yeast and mammalian cells.

Animals↗
PubMed1993

dlk, a putative mammalian homeotic gene differentially expressed in small cell lung carcinoma and neuroendocrine tumor cell line.

Gastrin releasing peptide is mitogenic for mouse Swiss 3T3 fibroblasts and certain human small cell lung carcinoma (SCLC) cells but not for mouse Balb/c 3T3 fibroblasts. To identify new molecules associated with the gastrin releasing peptide-responsive phenotype, clones isolated from a differential cDNA library between Swiss and Balb/c 3T3 fibroblasts were used to screen for their expression in human SCLC cell lines. Using this approach, we have isolated and characterized human and mouse cDNA clones encoding a novel protein. This protein is a putative transmembrane protein belonging to the epidermal growth factor-like superfamily. In vitro transcription and translation studies detect a 42-kDa protein, in agreement with the size predicted from the translated cDNA sequence. This protein (termed Delta-like or dlk) is highly homologous to invertebrate homeotic proteins, including Delta, and Notch, the products of neurogenic loci involved in normal neural differentiation in Drosophila. dlk is expressed in tumors with neuroendocrine features, such as neuroblastoma, pheochromocytoma, and a subset of SCLC cell lines. However, its expression in normal tissues is restricted to the adrenal gland and placenta. These data suggest that dlk may be involved in neuroendocrine differentiation and, because of its cellular location and restricted expression in normal tissues, it may be a potential therapeutic target in neuroendocrine tumors, particularly SCLC.

3T3 Cells↗
PubMed1994

Novel bacterial P-type ATPases with histidine-rich heavy-metal-associated sequences.

Menkes disease and Wilson disease are human disorders of copper metabolism. It has recently been shown that both are due to mutations in P-type ATPase copper transport molecules. Related heavy metal transporting ATPases have been described in several strains of bacteria. In an effort to isolate other mammalian metal transporters, we screened a human small intestine library with probes homologous to conserved sequences in the known proteins. Two novel cDNAs were isolated, which encode new members of this family. Surprisingly, they were both of bacterial origin, most likely derived from E. coli sequences transduced during library construction.

Adenosine Triphosphatases↗
PubMed2003

High-throughput methods to detect dimerization of Bcl-2 family proteins.

Members of the Bcl-2 family are critical regulators of apoptosis. Antiapoptotic family proteins such as Bcl-2 and Bcl-x(L) function, at least in part, by binding proapoptotic members such as Bax and Bak and thereby preventing release of apoptotic proteins, including cytochrome c, from the mitochondria. "BH3-only" members of the family disrupt this interaction by binding, via their BH3 domain, to a hydrophobic pocket on the surface of the antiapoptotic members. Disruption of heterodimerizations by small-molecule inhibitors could be used to modulate cell death in both cancer (to increase apoptosis) and degenerative disorders (to decrease apoptosis), and assays are necessary to screen compound libraries. Fluorescence polarization and enzyme-linked immunosorbent assay-based methods to detect Bcl-2 protein interactions have been described. Here, two further methods that are rapid, "mix and read," homogeneous reactions, insensitive to compound autofluorescence, and amenable to high-throughput screening, are described: a scintillation proximity assay and a time-resolved fluorescence resonance energy transfer assay (HTRF). The assays are designed using tags such that different Bcl-2 family members or BH3 domain peptides can be readily applied to either format, as exemplified by the use here of histidine-tagged Bcl-x(L) and biotinylated BH3 peptides.

Amino Acid Sequence↗
PubMed1997

Identification of peroxisome proliferator-activated receptor ligands from a biased chemical library.

BACKGROUND: The peroxisome proliferator-activated receptors (PPARs) were cloned as orphan members of the nuclear receptor superfamily of transcription factors. The identification of subtype-selective ligands for PPARalpha and PPARgamma has led to the discovery of their roles in the regulation of lipid metabolism and glucose homeostasis. No subtype-selective PPARdelta ligands are available and the function of this subtype is currently unknown. RESULTS: A three-component library was designed in which one of the monomers was biased towards the PPARs and the other two monomers were chosen to add chemical diversity. Synthesis and screening of the library resulted in the identification of pools with activity on each of the PPAR subtypes. Deconvolution of the pools with the highest activity on PPARdelta led to the identification of GW 2433 as the first high-affinity PPARdelta ligand. [3H]GW 2433 is an effective radioligand for use in PPARdelta competition-binding assays. CONCLUSIONS: The synthesis of biased chemical libraries is an efficient approach to the identification of lead molecules for members of sequence-related receptor families. This approach is well suited to the discovery of small-molecule ligands for orphan receptors.

Binding, Competitive↗
PubMed2007

Characterization of a potent and selective small-molecule inhibitor of the PIM1 kinase.

The pim-1 kinase is a true oncogene that has been implicated in the development of leukemias, lymphomas, and prostate cancer, and is the target of drug development programs. We have used experimental approaches to identify a selective, cell-permeable, small-molecule inhibitor of the pim-1 kinase to foster basic and translational studies of the enzyme. We used an ELISA-based kinase assay to screen a diversity library of potential kinase inhibitors. The flavonol quercetagetin (3,3',4',5,6,7-hydroxyflavone) was identified as a moderately potent, ATP-competitive inhibitor (IC(50), 0.34 micromol/L). Resolution of the crystal structure of PIM1 in complex with quercetagetin or two other flavonoids revealed a spectrum of binding poses and hydrogen-bonding patterns in spite of strong similarity of the ligands. Quercetagetin was a highly selective inhibitor of PIM1 compared with PIM2 and seven other serine-threonine kinases. Quercetagetin was able to inhibit PIM1 activity in intact RWPE2 prostate cancer cells in a dose-dependent manner (ED(50), 5.5 micromol/L). RWPE2 cells treated with quercetagetin showed pronounced growth inhibition at inhibitor concentrations that blocked PIM1 kinase activity. Furthermore, the ability of quercetagetin to inhibit the growth of other prostate epithelial cell lines varied in proportion to their levels of PIM1 protein. Quercetagetin can function as a moderately potent and selective, cell-permeable inhibitor of the pim-1 kinase, and may be useful for proof-of-concept studies to support the development of clinically useful PIM1 inhibitors.

Chromones↗
PubMed2006

Interactive tools for risk reduction and efficiency improvements in medicinal chemistry.

There are many decisions and risks associated with the design and development of new pharmaceutical agents. To help improve decision-making, and reduce the associated risks--prior to synthesis, we have developed interactive web-browser tools for: (i) tracking, searching, clustering and categorizing (by reactive moieties) chemical reactants, (ii) interactively assessing risks, either synthetic--based on prior experience, absorption following oral administration--based on rules of 5, or diversity, and (iii) a complete architecture for enumerating, analyzing, submitting and plating large combinatorial or small biased libraries. We believe the implementation of this highly interactive system has given our scientists a competitive advantage by maintaining their focus on the lowest risk, highest quality molecules throughout the research process.

Administration, Oral↗
PubMed2005

Small molecule tgf-beta mimetics as potential neuroprotective factors.

Neurodegenerative and dementing illnesses are becoming an increasing social and economical burden as the number of older people continues to grow in industrialized countries. Current knowledge of the processes leading to these diseases is still limited, and very few effective treatments are available. Because neurodegeneration is associated with an activation of injury and innate immune responses in the brain, drugs that could mimic the beneficial aspects of this response are potential therapeutic candidates. The cytokine transforming growth factor (TGF)-beta1 is an organizer of the brain's response to injury and is known to be neuroprotective. Previous studies from our lab also showed that TGF-beta1 can reduce accumulation of beta-amyloid peptide (Abeta), which appears to be central to Alzheimer's disease (AD) pathogenesis, and we therefore initiated a search for small molecule chemical compounds that could mimic this effect. We report here the identification of several such TGF-beta mimetics detected in an in vitro screen of a library with 5000 chemically diverse compounds. If active in vivo, these mimetics could be developed into candidates for the treatment of neurodegeneration.

Alzheimer Disease↗
PubMed2005

In vitro selection of DNA aptamers binding ethanolamine.

We have identified aptamers (synthetic oligonucleotides) binding to the very small molecule ethanolamine with high affinity down to the low nanomolar range. These aptamers were selected for their ability to bind to ethanolamine immobilised on magnetic beads, from an 96mer library of initially about 1 x 10(16) randomised ssDNA molecules. The dissociation constants of these aptamers range between K(D)=6 and K(D)=19 nmol L(-1). The aim of the development of ethanolamine aptamers is their use for the detection of this substance in clinical and environmental analysis. Ethanolamine is associated with several diseases. Moreover, ethanolamine and its derivatives di- and tri-ethanolamine are used in chemical and cosmetic industries. The use of biosensors with ethanolamine aptamer as new molecular recognition element could be an innovative method for an easy and fast detection of ethanolamine.

Aptamers, Nucleotide↗
PubMed2002

From combinatorial chemistry to chemical microarray.

Combinatorial chemistry was first applied to the generation of peptide arrays in 1984. Since then, the field of combinatorial chemistry has evolved rapidly into a new discipline. There is a great need for the development of methods to examine the proteome functionally at a global level. Using many of the techniques and instruments developed for DNA microarrays, chemical microarray methods have advanced significantly in the past three years. High-density chemical microarrays can now be synthesized in situ on glass slides or be printed through covalent linkage or non-specific adsorption to the surface of the solid-support with fully automatic arrayers. Microfabrication methods enable one to generate arrays of microsensors at the end of optical fibers or arrays of microwells on a flat surface. In conjunction with the one-bead one-compound combinatorial library method, chemical microarrays have proven to be very useful in lead identification and optimization. High-throughput protein expression systems, robust high-density protein, peptide and small-molecule microarray systems, and automatic mass spectrometers are critical tools for the field of functional proteomics.

Combinatorial Chemistry Techniques↗
PubMed1982

Cloning and characterization of cDNA copies of the 7S RNAs of HeLa cells.

We have cloned cDNA copies of in vitro adenylated 7S RNA of HeLa cells. The most representative clones in the library contain DNA fragments copied from the 7SL and 7SK small RNAs. The two classes of recombinants share no homology. The 7SL RNA contains at the 5' end of the molecule sequences homologous to the Alu sequence family. Hybridization to human genomic DNA shows that the 7SL and 7SK clones are homologous to two different families of repetitive sequences.

Cloning, Molecular↗
PubMedDate not supplied

Simple method for cDNA amplification starting from small amount of total RNA.

We describe a novel simple PCR-based technique for construction of cDNA libraries starting from the small samples of cells or tissues. This technique is based on the insertion of inverted terminal repeats (ITR) into amplified cDNA which causes a part of molecules to generate "pan"-type structures at each cycle of PCR amplification. This allows to avoid generation of the primer dimmer and makes possible the regulation of an average length of amplified sequences varying in concentration of primers.

Animals↗
PubMed2006

REPLACE: a strategy for iterative design of cyclin-binding groove inhibitors.

We describe a drug-design strategy termed REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) in which nonpeptidic surrogates for specific determinants of known peptide ligands are identified in silico by using a core peptide-bound protein structure as a design anchor. In the REPLACE application example, we present the effective replacement of two critical binding motifs in a lead protein-protein interaction inhibitor pentapeptide with more druglike phenyltriazole and diphenyl ether groups. These were identified through docking of fragment libraries into the volume of the cyclin-binding groove of CDK2/cyclin A vacated through truncation of the inhibitor peptide-binding determinants. Proof of concept for this strategy was obtained through the generation of potent peptide-small-molecule hybrids and by the confirmation of inhibitor-binding modes in X-ray crystal structures. This method therefore allows nonpeptide fragments to be identified without the requirement for a high-sensitivity binding assay and should be generally applicable in replacing amino acids as individual residues or groups in peptide inhibitors to generate pharmaceutically acceptable lead molecules.

Amino Acid Substitution↗