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At least 703 records · Page 39Linked to original sources
PubMed1999

Phage antibodies against human dendritic cell subpopulations obtained by flow cytometry-based selection on freshly isolated cells.

In one application of phage display technology, large libraries of antibody fragments displayed on phage particles are used to select antibodies that bind to molecules expressed on the surface of eukaryotic cells. The advantage of this method is that antibodies can be selected against antigens in their native configuration, without the need to purify or express the antigen as a recombinant protein. Moreover, this approach may be used to search for novel membrane molecules expressed by subpopulations of cells that are difficult to address by conventional methods, e.g., small numbers of cells present in heterogeneous mixtures. It has been shown that the isolation of cell-bound phages is compatible with immunofluorescence staining and flow cytometric identification and sorting of cells based on multiparameter analysis. Here, we have employed a semi-synthetic phage display library of human single-chain Fv (scFv) antibody fragments in combination with flow cytometry to isolate antibodies against rare populations of precursor and mature dendritic cells (DCs) present in human peripheral blood. DCs are a phenotypically heterogeneous population of professional antigen presenting cells of bone marrow origin with complex and only partly understood developmental relationships and functions. We have isolated phage antibodies against subpopulations of blood DCs and analyzed the distribution of the target antigens. The results show that these phage antibodies are useful tools to further dissect relationships and function of DCs in healthy and diseased tissues.

Antibodies↗
PubMed2003

Discovery of an inhibitor of a transcription factor using small molecule microarrays and diversity-oriented synthesis.

Small molecule microarrays were screened to identify a small molecule ligand for Hap3p, a subunit of the yeast Hap2/3/4/5p transcription factor complex. The compound, named haptamide A, was determined to have a KD of 5.03 muM for binding to Hap3p using surface plasmon resonance analysis. Haptamide A also inhibited activation of a GDH1-lacZ reporter gene in a dose-dependent fashion. To explore structure-activity relationships, 11 derivatives of haptamide A were prepared using the same synthetic route that was developed for the original library synthesis. Analysis of dissociation constants and IC50 values for the reporter gene assay revealed a more potent inhibitor, haptamide B, with a KD of 330 nM. Whole-genome transcriptional profiling was used to compare effects of haptamide B with a hap3Delta yeast strain. Treatment with haptamide B, like the deletion mutant, reduced lactate-induced transcription of several genes from wild-type levels. Profiling the genetic "knockout" and the chemical genetic "knockdown" led to the identification of several genes that are regulated by Hap3p under nonfermentative conditions. These results demonstrate that a small molecule discovered using the small molecule microarray binding assay can permeate yeast cells and reach its target transcription factor protein in cells.

Amides↗
PubMed2006

Targeting the PAS-A domain of HIF-1alpha for development of small molecule inhibitors of HIF-1.

Hypoxia inducible factor-1 (HIF-1) is a master regulator of cellular adaptation to oxygen deprivation and activates transcription of genes involved in tumor metabolism, angiogenesis, invasion and metastasis, all of which are implicated in cancer progression. Several domains of HIF-1alpha mediate protein-protein interaction, which is essential for the formation of the active heterodimer with HIF-1beta. Targeting specific domains of HIF-1alpha might lead to the identification of more selective inhibitors. HIF-1alpha and HIF-1beta contain two Per-Arnt-Sim (PAS) domains, A and B, both of which appear to be important for heterodimer formation. In an attempt to identify small molecule inhibitors of the PAS-A domain of HIF-1 we expressed proteins containing amino acids 86-165 of HIF-1alpha and amino acids 159-240 of HIF-1beta fused to a His or FLAG tag, respectively. Expressed proteins retained functional activity as indicated by in vitro immunoprecipitation experiments and activation of luciferase expression in a mammalian two-hybrid system. Interestingly, over-expression of HIF-1alpha-PAS-A domain was sufficient to abrogate hypoxic induction of HIF-1-dependent luciferase expression, supporting its potential role as drug target. An ELISA based on the interaction between FLAG-HIF-1beta-PAS-A and HIF-1alpha-PAS-A-His was developed and used to screen libraries of synthetic compounds. NSC 50352 specifically inhibited PAS-A-dependent interaction between HIF-1alpha and HIF-1beta, but not the interaction mediated by unrelated domains. However, NSC 50352 was devoid of activity in cell-based assays. Our results provide proof-of-principle that the PAS-A domain of HIF-1alpha is a valid target for development of small molecule inhibitors.

Antineoplastic Agents↗
PubMed2005

Screening a library of potential prion therapeutics against cellular prion proteins and insights into their mode of biological activities by surface plasmon resonance.

The conversion of cellular prion protein (PrP(C)) to the protease resistant isoform (PrP(Sc)) is considered essential for the progression of transmissible spongiform encephalopathies (TSEs). A potential therapeutic strategy for preventing the accumulation of PrP(Sc) is to stabilize PrP(C) through the direct binding of a small molecule to make conversion less energetically favourable. Using surface plasmon resonance (SPR)-based technology we have developed a procedure, based on direct binding, for the screening of small molecules against PrP(C) immobilized on a sensor chip. In this paper we report some problems associated with the immobilization of PrP(C) onto the sensor surface for conducting drug screening and how these problems were overcome. We demonstrated that the conformational change of PrP(C) on the chip surface leads to increased exposure of the C-terminal which was observed by the increase in quinacrine binding over time, and loss of heparin binding to the N-terminal. In addition, we also report the results of the successful screening of a library of 47 compounds of known activity in cell line or cell free conversion studies for direct binding to three forms of PrP(C) (huPrP(C), t-huPrP(C) and moPrP(C)). These results show the usefulness of this technique for the identification of PrP(C) binding ligands and to gain some insight as to their potential mode of action.

Biosensing Techniques↗
PubMed2000

Anticalins versus antibodies: made-to-order binding proteins for small molecules.

Engineering proteins to bind small molecules presents a challenge as daunting as drug discovery, for both hinge upon our understanding of receptor-ligand molecular recognition. However, powerful techniques from combinatorial molecular biology can be used to rapidly select artificial receptors. While traditionally researchers have relied upon antibody technologies as a source of new binding proteins, the lipocalin scaffold has recently emerged as an adaptable receptor for small molecule binding. 'Anticalins', engineered lipocalin variants, offer some advantages over traditional antibody technology and illuminate features of molecular recognition between receptors and small molecule ligands.

Amino Acid Sequence↗
PubMed2004

[Screening and identification of human telomerase reverse transcriptase epitopes from random phage display peptide library].

OBJECTIVE: To identify and characterize the epitopes of human telomerase reverse transcriptase (hTERT). METHODS: A random phage displayed dodecapeptide library was screened with anti-hTERT antibody. The selected phage clones were identified by sandwich enzyme-linked immunosorbent assay, anti-hTERT antibody blocking assay and competitive inhibition assay. RESULTS: After 3 rounds of screening, 13 of 24 randomly selected phage clones were identified as positive clones that could specifically bind to anti-hTERT antibody but not to normal mouse IgG. Amino acid sequences deduced from DNA sequences showed 11 different sequences with high percentage of histone and hydrophilic amino acids. CONCLUSION: Eleven epitopes have been obtained that can effectively mimic hTERT, which will be useful for the research of small-molecule inhibitor targeting at hTERT.

Base Sequence↗
PubMed2004

A three-hybrid approach to scanning the proteome for targets of small molecule kinase inhibitors.

In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors. Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography. The Y3H system described here may prove generally useful in the discovery of candidate drug targets.

Adenine↗
PubMed2004

Discovery of a small molecule that inhibits the interaction of anthrax edema factor with its cellular activator, calmodulin.

The catalytic efficiency of adenylyl cyclase activity of edema factor (EF) from Bacillus anthracis is enhanced by approximately 1000-fold upon its binding to mammalian protein calmodulin (CaM). A tandem cell-based and protein binding-based screen of a 10,000 member library identified a molecule that inhibits the EF-CaM interaction and therefore the adenylyl cyclase activity. A combination of fluorescence spectroscopy and photolabeling studies showed that the molecule targets the CaM binding region of EF. A series of related compounds were synthesized and evaluated to identify one compound, 4-[4-(4-nitrophenyl)-thiazolylamino]-benzenesulfonamide, that maintained activity against EF but showed minimal toxicity to two cultured cell lines. This compound represents an important reagent to study the role of EF in anthrax pathology and may represent a drug lead against anthrax infection.

Adenylyl Cyclase Inhibitors↗
PubMed2004

Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity.

Apoptosis resistance commonly occurs in cancers, preventing activation of Caspase family cell death proteases. XIAP is an endogenous inhibitor of Caspases overexpressed in many cancers. We developed an enzyme derepression assay, based on overcoming XIAP-mediated suppression of Caspase-3, and screened mixture-based combinatorial chemical libraries for compounds that reversed XIAP-mediated inhibition of Caspase-3, identifying a class of polyphenylureas with XIAP-inhibitory activity. These compounds, but not inactive structural analogs, stimulated increases in Caspase activity, directly induced apoptosis of many types of tumor cell lines in culture, and sensitized cancer cells to chemotherapeutic drugs. Active compounds also suppressed growth of established tumors in xenograft models in mice, while displaying little toxicity to normal tissues. These findings validate IAPs as targets for cancer drug discovery.

Animals↗
PubMedDate not supplied

Strategy for discovering chemical inhibitors of human cyclophilin a: focused library design, virtual screening, chemical synthesis and bioassay.

The discovery of cyclophilin A (CypA) inhibitor is now of special interest in the treatment of immunological disorders. In this work, using a strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and bioassay, a series of novel small molecular CypA inhibitors have been discovered. First, using the fragments taken from our previously discovered CypA inhibitors (Bioorg. Med. Chem. 2006, 14, 2209-2224) as building blocks, we designed a focused combinatorial library containing 255 molecules employing the LD1.0 program (J. Comb. Chem. 2005, 7, 398-406) developed by us. Sixteen compounds (1a-e, 2a-b, 3a-b, and 4a-g) were selected by using virtual screening against the X-ray crystal structure of CypA as well as druglike analysis for further synthesis and bioassay. All these sixteen molecules are CypA binders with binding affinities (K(D) values) ranging from 0.076 to 41.0 microM, and five of them (4a, 4c, and 4e-g) are potent CypA inhibitors with PPIase inhibitory activities (IC(50) values) of 0.25-6.43 microM. The hit rates for binders and inhibitors are as high as 100% and 31.25%, respectively. Remarkably, both the binding affinity and inhibitory activity of the most potent compound increase approximately 10 times than that of the most active compound discovered previously. The high hit rate and the high potency of the new CypA inhibitors demonstrated the efficiency of the strategy for focused library design and screening. In addition, the novel chemical entities reported in this study could be leads for discovering new therapies against the CypA pathway.

Binding Sites↗
PubMed2002

New tools and concepts for modern organic synthesis.

The increasing need to efficiently assemble small molecules as potential modulators of therapeutic targets that are emerging from genomics and proteomics is driving the development of novel technologies for small-molecule synthesis. Here, we describe some of the general applications and approaches to synthesis using one such technology--solid-supported reagents--that has been shown to significantly improve productivity in the generation of combinatorial libraries and complex target molecules.

Organic Chemicals↗
PubMed2006

Opposite effects of rho family GTPases on engulfment of apoptotic cells by macrophages.

The efficient engulfment of apoptotic cells by professional or nonprofessional phagocytes is critical to maintain mammalian homeostasis. To identify molecules involved in the engulfment of apoptotic cells, we established a retrovirus-based expression cloning system coupled with the engulfment assay. By screening a cDNA library of a mouse macrophage cell line, we identified two small GTPase family members (RhoG and Rab5) that enhanced the engulfment of apoptotic cells. By examining other small GTPase family members, we found that Rac1 enhanced the engulfment of apoptotic cells, whereas RhoA inhibited the process. Accordingly, the expression of a dominant-negative form of RhoG or Rac1 in primary macrophage cultures severely reduced the ability of the macrophages to engulf apoptotic cells, and a dominant-negative form of RhoA enhanced the process. These results indicated that the efficient engulfment of apoptotic cells requires the concerted action of small GTPase family members. We demonstrated previously that NIH3T3 cells expressing the alphav beta3 integrin efficiently engulf apoptotic cells in the presence of milk fat globule epidermal growth factor 8 via a phosphatidylserine-dependent mechanism. The dominant-negative form of RhoG or Rac1 inhibited this process, which suggested RhoG and Rac1 are also involved in the integrin-mediated engulfment.

Animals↗
PubMed2003

Development of peptide mimotopes of lipooligosaccharide from nontypeable Haemophilus influenzae as vaccine candidates.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of otitis media in children and lower respiratory tract diseases in adults. So far there is no effective vaccine against NTHi. A major surface-exposed component of NTHi, lipooligosaccharide (LOS), is a virulence factor as well as a potential protective Ag. LOS is too toxic to be administered in humans. However, detoxified LOS is a T cell-independent small molecule and is poorly immunogenic in vivo, so we converted LOS into a nontoxic T cell-dependent Ag through the use of peptides that mimic the LOS by screening a phage-display peptide library with a rabbit Ab specific for NTHi LOS. Fifty-six phage clones were found to share LOS mimicry molecules. Among them, 22 clones were subjected to DNA sequencing, and four consensus sequences were identified as NMMRFTSQPPNN, NMMNYIMDPRTH, NMMKYISPPIFL, and NMMRFTELSTPS. Three of the four synthetic peptides showed strong binding reactivity to the rabbit anti-LOS Ab and also a mouse bactericidal monoclonal anti-LOS Ab in vitro, and elicited specific serum anti-LOS Abs in rabbits (27- to 81-fold) after conjugation with keyhole limpet hemocyanin. Passive immunization with the rabbit antisera resulted in a significantly enhanced pulmonary bacterial clearance in a mouse model. The enhanced bacterial clearance was eliminated if the rabbit serum was preabsorbed with NTHi LOS. These data indicate that the peptide mimotopes of LOS that we have identified might be potential components of peptide vaccines against NTHi.

Animals↗
PubMed2003

Natural product-like and other biologically active heterocyclic libraries using solid-phase techniques in the post-genomic era.

High-throughput technologies allow the selection of new biological targets for drug discovery in the post-genomic era. These tools increase the need of new methods to rapidly obtain potent small molecules and natural products to discover new lead structures. In particular, the solid-phase synthesis offers a great potential to obtain large compound sets.

Combinatorial Chemistry Techniques↗
PubMed1998

In vitro biological characterization and antiangiogenic effects of PD 166866, a selective inhibitor of the FGF-1 receptor tyrosine kinase.

Through direct synthetic efforts, we discovered a small molecule that is a nanomolar inhibitor of the human fibroblast growth factor-1 receptor (FGFR) tyrosine kinase. PD 166866, a member of a new structural class of tyrosine kinase inhibitors, the 6-aryl-pyrido[2,3-d]pyrimidines, was identified by screening a compound library with assays that measure protein tyrosine kinase activity. PD 166866 inhibited human full-length FGFR-1 tyrosine kinase with an IC50 value of 52.4 +/- 0.1 nM and was further characterized as an ATP competitive inhibitor of the FGFR-1. In contrast, PD 166866 had no effect on c-Src, platelet-derived growth factor receptor-beta, epidermal growth factor receptor or insulin receptor tyrosine kinases or on mitogen-activated protein kinase, protein kinase C and CDK4 at concentrations as high as 50 microM. PD 166866 was a potent inhibitor of basic fibroblast growth factor (bFGF)-mediated receptor autophosphorylation in NIH 3T3 cells expressing endogenous FGFR-1 and in L6 cells overexpressing the human FGFR-1 tyrosine kinase, confirming a tyrosine kinase-mediated mechanism. PD 166866 also inhibited bFGF-induced tyrosine phosphorylation of the 44- and 42-kDa (ERK 1/2) mitogen-activated protein kinase isoforms in L6 cells, presumably via inhibition of bFGF-stimulated FGFR-1 tyrosine kinase activation. PD 166866 did not inhibit platelet-derived growth factor, epidermal growth factor or insulin-stimulated receptor autophosphorylation in vascular smooth muscle, A431 or NIHIR cells, respectively, further supporting its specificity for the FGFR-1. In addition, daily exposure of PD 166866 to L6 cells at concentrations from 1 to 100 nM resulted in a concentration-related inhibition of bFGF-stimulated cell growth for 8 consecutive days with an IC50 value of 24 nM. In contrast, PD 166866 had little effect on platelet-derived growth factor-BB-stimulated growth of L6 cells or serum-stimulated vascular smooth muscle cell proliferation. Finally, PD 166866 was found to be a potent inhibitor of microvessel outgrowth (angiogenesis) from cultured artery fragments of human placenta. These results highlight the discovery of PD 166866, a new nanomolar potent and selective small molecule inhibitor of the FGFR-1 tyrosine kinase with potential use as antiproliferative/antiangiogenic agent for such therapeutic targets as tumor growth and neovascularization of atherosclerotic plaques.

3T3 Cells↗
PubMed2006

Quantitative measurement of selectin-ligand interactions: assays to identify a sweet pill in a library of carbohydrates.

Soluble oligosaccharides and glycoproteins can inhibit leukocyte adhesion during a range of vascular ailments including inflammation, thrombosis, and cancer metastasis. The design of such molecules in many cases is based on the structure of naturally occurring selectin ligands. In this case, synthetic selectin-ligand mimetics act as competitive inhibitors of cell adhesion. In an alternate approach, cell-permeable, small-molecule oligosaccharides have been shown to alter the metabolic pathways that lead to the biosynthesis of functional selectin-ligands. The addition of such molecules results in glycoproteins that are defective in their ability to bind selectins. Quantitative in vitro testing of the efficacy of the above inhibition strategies ideally requires the application of assays that mimic the in vivo physiological milieu in terms of the valency of selectin and selectin-ligands, the physiological fluid-flow conditions, and the use of blood cells. Assays that are performed in small volumes are preferable when the quantity of available inhibitor is scarce. Finally, the measurements must account for the rapid on- and off-rates of selectin-mediated binding interactions. This chapter addresses these issues by presenting methods to measure selectin function in enzyme-linked immunosorbent assay and flow cytometry-based static assays, cell-adhesion assays performed under shear flow in cone-plate viscometers, and Biacore surface plasmon resonance measurements of molecular-binding kinetics. Examples are presented where such methods are applied to measure the ability of simple oligosaccharides based on sialyl Lewis-X and complex molecules with the core-2 structure to block selectin function. Such methods may be extended to identify potent selectin antagonists in a library of carbohydrates.

Combinatorial Chemistry Techniques↗
PubMed1996

Identification of human OGR1, a novel G protein-coupled receptor that maps to chromosome 14.

We describe the cloning of a novel G protein-coupled receptor, termed ovarian cancer G protein-coupled receptor 1 (OGR1), from an ovarian cancer cell line that maps to chromosome 14q31. The predicted open reading frame of OGR1 encodes a protein of 365 amino acids. OGR1 is expressed as a single 3.0-kb transcript in several tissues, including spleen, testis, small intestine, peripheral blood leukocytes, brain, heart, lung, placenta, and kidney, with no detectable OGR1 expression in thymus, prostate, ovary, colon, liver, skeletal muscle, or pancreas. OGR1 shares strongest homology (49-54%) with the human orphan receptor GPR4. The structural features of OGR1 suggest that the ligand for OGR1 is likely to be a small peptide or a small bioactive molecule.

Amino Acid Sequence↗