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At least 685 records · Page 38Linked to original sources
PubMed2004

Expression cloning and demonstration of Enterococcus faecalis lipoamidase (pyruvate dehydrogenase inactivase) as a Ser-Ser-Lys triad amidohydrolase.

Enterococcus faecalis lipoamidase was discovered almost 50 years ago (Reed, L. J., Koike, M., Levitch, M. E., and Leach, F. R. (1958) J. Biol. Chem. 232, 143-158) as an enzyme activity that cleaved lipoic acid from small lipoylated molecules and from pyruvate dehydrogenase thereby inactivating the enzyme. Although the partially purified enzyme was a key reagent in proving the crucial role of protein-bound lipoic acid in the reaction mechanism of the 2-oxoacid dehydrogenases, the identity of the lipoamidase protein and the encoding gene remained unknown. We report isolation of the lipoamidase gene by screening an expression library made in an unusual cosmid vector in which the copy number of the vector is readily varied from 1-2 to 40-80 in an appropriate Escherichia coli host. Although designed for manipulation of large genome segments, the vector was also ideally suited to isolation of the gene encoding the extremely toxic lipoamidase. The gene encoding lipoamidase was isolated by screening for expression in E. coli and proved to encode an unexpectedly large protein (80 kDa) that contained the sequence signature of the Ser-Ser-Lys triad amidohydrolase family. The hexa-histidine-tagged protein was expressed in E. coli and purified to near-homogeneity. The purified enzyme was found to cleave both small molecule lipoylated and biotinylated substrates as well as lipoic acid from two 2-oxoacid dehydrogenases and an isolated lipoylated lipoyl domain derived from the pyruvate dehydrogenase E2 subunit. Lipoamidase-mediated inactivation of the 2-oxoacid dehydrogenases was observed both in vivo and in vitro. Mutagenesis studies showed that the residues of the Ser-Ser-Lys triad were required for activity on both small molecule and protein substrates and confirmed that lipoamidase is a member of the Ser-Ser-Lys triad amidohydrolase family.

Amidohydrolases↗
PubMedDate not supplied

Small molecule shape-fingerprints.

The optimal overlap between two molecular structures is a useful measure of shape similarity. However, it usually requires significant computation. This work describes the design of shape-fingerprints: binary bit strings that encode molecular shape. Standard measures of similarity between two shape-fingerprints are shown to be an excellent surrogate for similarity based on volume overlap but several orders of magnitude faster to compute. Consequently, shape-fingerprints can be used for clustering of large data sets, evaluating the diversity of compound libraries, as descriptors in SAR and as a prescreen for exact shape comparison against large virtual databases. Our results show that a small set of shapes can be used to build these fingerprints and that this set can be applied universally.

Journal Article↗
PubMed2005

Recognition of proline-rich motifs by protein-protein-interaction domains.

Protein-protein interactions are essential in every aspect of cellular activity. Multiprotein complexes form and dissociate constantly in a specifically tuned manner, often by conserved mechanisms. Protein domains that bind proline-rich motifs (PRMs) are frequently involved in signaling events. The unique properties of proline provide a mechanism for highly discriminatory recognition without requiring high affinities. We present herein a detailed, quantitative assessment of the structural features that define the interfaces between PRM-binding domains and their target PRMs, and investigate the specificity of PRM recognition. Together with the analysis of peptide-library screens, this approach has allowed the identification of several highly conserved key interactions found in all complexes of PRM-binding domains. The inhibition of protein-protein interactions by using small-molecule agents is very challenging. Therefore, it is important to first pinpoint the critical interactions that must be considered in the design of inhibitors of PRM-binding domains.

Amino Acid Motifs↗
PubMed2006

Directed evolution of extradiol dioxygenase by a novel in vivo DNA shuffling.

RecA-dependent homologous recombination in Escherichia coli is a very effective way to construct chimeras between two homologous genes. The disadvantage of in vivo method is a small library size of chimeric genes in comparison with in vitro DNA shuffling. In order to overcome the disadvantage, we have developed novel in vivo DNA shuffling methods with successive homologous recombinations. Linearized DNA molecules with two homologous genes were made with ligation rather than the conventional restriction enzyme cleavage between two genes. The three-way ligation of a vector and two homologous bphC genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenases or the two-way ligation of the donor bphC gene and an acceptor plasmid carrying the homologous bphC gene generated a variety of linearized DNA molecules. The homologous recombination between the genes on the linearized DNA molecules created the large chimeric bphC gene libraries in a recBC sbcA E. coli strain. After three rounds of recombinations, chimeric bphC genes with four-part gene fragments by triple-crossover were easily obtained. By employing a 96-well microtiter plate high-throughput screening, thermally stable chimeric 2,3-dihydroxybiphenyl 1,2-dioxygenases were selected from chimeric bphC gene libraries. This opens up a new way for directed evolution of proteins in vivo.

DNA Shuffling↗
PubMed2005

Size-specific ligands for RNA hairpin loops.

The targeting of one mRNA in the transcriptome requires small molecules that bind with substantial affinity and specificity. As such, compounds with specificity for individual RNA secondary structural motifs could be useful for targeting RNA. Described herein is the synthesis of a combinatorial library of 105 dimers of deoxystreptamine and the subsequent identification of compounds with specificity for specific RNA hairpin loop sizes, including tetraloops and octaloops. Such compounds will be useful for the perturbation of RNA function in vivo.

Base Sequence↗
PubMed2006

A cell-based ultra-high-throughput screening assay for identifying inhibitors of D-amino acid oxidase.

Enzymes are often considered less "druggable" targets than ligand-regulated proteins such as G-protein-coupled receptors, ion channels, or other hormone receptors. Reasons for this include cellular location (intracellular vs. cell surface), typically lower affinities for the binding of small molecules compared to ligand-specific receptors, and binding (catalytic) sites that are often charged or highly polar. A practical drawback to the discovery of compounds targeting enzymes is that screening of compound libraries is typically carried out in cell-free activity assays using purified protein in an inherently artificial environment. Cell-based assays, although often arduous to design for enzyme targets, are the preferred discovery tool for the screening of large compound libraries. The authors have recently described a novel cell-based approach to screening for inhibitors of a phosphatase enzyme and now report on the development and implementation of a homogeneous 3456-well plate assay for D-amino acid oxidase (DAO). Human DAO was stably expressed in Chinese hamster ovary (CHO) cells, and its activity was measured as the amount of hydrogen peroxide detected in the growth medium following feeding the cells with D-serine. In less than 12 weeks, the authors proved the concept in 96-and then 384-well formats, miniaturized the assay to the 3456-well (nanoplate) scale, and screened a library containing more than 1 million compounds. They have identified several cell-permeable inhibitors of DAO from this cell-based high-throughput screening, which provided the discovery program with a few novel and attractive lead structures.

Animals↗
PubMed2006

High-throughput screening for the discovery of inhibitors of fatty acid amide hydrolase using a microsome-based fluorescent assay.

Fatty acid amide hydrolase (FAAH) is a membrane-associated enzyme that catalyzes the hydrolysis of several endogenous bioactive lipids, including anandamide (AEA), N-palmitoylethanolamine (PEA), oleamide, and N-oleoylethanolamine (OEA). These fatty acid amides participate in many physiological activities such as analgesia, anxiety, sleep modulation, anti inflammatory responses, and appetite suppression. Because FAAH plays an essential role in controlling the tone and activity of these endogenous bioactive lipids, this enzyme has been implicated to be a drug target for the therapeutic management of pain, anxiety, and other disorders. In an effort to discover FAAH inhibitors, the authors have previously reported the development of a novel fluorescent assay using purified FAAH microsomes as an enzyme source and a fluorogenic substrate, arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA). Herein, the authors have adapted this assay to a high-throughput format and have screened a large library of small organic compounds, identifying a number of novel FAAH inhibitors. These data further verify that this fluorescent assay is sufficiently robust, efficient, and low-cost for the identification of FAAH inhibitory molecules and open this class of enzymes for therapeutic exploration.

Amidohydrolases↗
PubMed2006

SuperMimic--fitting peptide mimetics into protein structures.

BACKGROUND: Various experimental techniques yield peptides that are biologically active but have unfavourable pharmacological properties. The design of structurally similar organic compounds, i.e. peptide mimetics, is a challenging field in medicinal chemistry. RESULTS: SuperMimic identifies compounds that mimic parts of a protein, or positions in proteins that are suitable for inserting mimetics. The application provides libraries that contain peptidomimetic building blocks on the one hand and protein structures on the other. The search for promising peptidomimetic linkers for a given peptide is based on the superposition of the peptide with several conformers of the mimetic. New synthetic elements or proteins can be imported and used for searching. CONCLUSION: We present a graphical user interface for finding peptide mimetics that can be inserted into a protein or for fitting small molecules into a protein. Using SuperMimic, promising locations in proteins for the insertion of mimetics can be found quickly and conveniently.

Algorithms↗
PubMed2003

The completed human genome: implications for chemical biology.

The recently completed human genome sequence represents an enormous opportunity to understand biology and accelerate the development of new therapeutics. However, it also presents equally large logistical, scientific and paradigmatic challenges to efficiently translate the enormous cache of sequence data into functional information that will be the precursor of new drug development. Small-molecule chemical biology applied on a genomic scale promises to speed this translation to novel therapeutics.

Animals↗
PubMed2003

Virtual screening to enrich hit lists from high-throughput screening: a case study on small-molecule inhibitors of angiogenin.

"Hit lists" generated by high-throughput screening (HTS) typically contain a large percentage of false positives, making follow-up assays necessary to distinguish active from inactive substances. Here we present a method for improving the accuracy of HTS hit lists by computationally based virtual screening (VS) of the corresponding chemical libraries and selecting hits by HTS/VS consensus. This approach was applied in a case study on the target-enzyme angiogenin, a potent inducer of angiogenesis. In conjunction with HTS of the National Cancer Institute Diversity Set and ChemBridge DIVERSet E (approximately 18,000 compounds total), VS was performed with two flexible library docking/scoring methods, DockVision/Ludi and GOLD. Analysis of the results reveals that dramatic enrichment of the HTS hit rate can be achieved by selecting compounds in consensus with one or both of the VS functions. For example, HTS hits ranked in the top 2% by GOLD included 42% of the true hits, but only 8% of the false positives; this represents a sixfold enrichment over the HTS hit rate. Notably, the HTS/VS method was effective in selecting out inhibitors with midmicromolar dissociation constants typical of leads commonly obtained in primary screens.

Binding Sites↗
PubMed2004

Regulating eukaryotic gene expression with aptamers.

Aptamers are RNA or DNA oligonucleotides identified within a randomly synthesized library, through an in vitro selection procedure. The selected candidates display a pre-determined property of interest with respect to a given target. Successful selection has been carried out against targets ranging from small (amino acids, antibiotics) to macro-molecules (proteins, nucleic acids). They generally show an affinity in the nanomolar range and a high specificity of target recognition. Interestingly, aptamers selected against purified targets in the test tube retain their properties within cells. RNA aptamers can be generated in situ from an appropriate DNA construct or delivered as nuclease-resistant oligonucleotide analogues. For example, aptamers recognizing RNA structure through loop-loop interactions modulate the trans-activation of in vitro transcription mediated by the TAR RNA element of human immunodeficiency virus type 1. Consequently, they constitute both exquisite tools for functional genomics analysis and promising prototypes of therapeutic agents. Natural aptameric motifs have been identified within mRNA sequences, which upon binding to a metabolite control the expression of the encoded gene, which is generally involved in the biosynthesis of this particular metabolite.

Amino Acid Motifs↗
PubMed1996

Isolation of a novel oncogene, NET1, from neuroepithelioma cells by expression cDNA cloning.

We generated a cDNA expression library from a human neuroepithelioma cell line for detection of novel oncogenes by focus formation assay in NIH3T3 cells. A morphologically unique focus was identified upon transfection and the transforming plasmid was isolated. The transforming gene, designated NET1, encoded a predicted protein species of 54 kDa containing the Dbl-Homology (DH) motif. This motif is also present in other growth regulatory molecules including Bcr, Cdc24, Vav, Ras-Grf, Ect2, Ost, Tim and Tiam1, which have been implicated as regulators of small GTP-binding proteins. NIH3T3 cells transfected with NET1 expression plasmid showed altered growth properties in vitro and were tumorigenic when injected into nude mice. In addition, a 2.5 kb cDNA was isolated from a normal human cDNA library which represented the NET1 proto-oncogene contained a 5' extended open reading frame. The fact that the proto-oncogene failed to induce transformation in NIH3T3 cells suggested that the original NET1 oncogene was activated by 5'-truncation. The 3.0 and 2.4 kilobasepair (kb) transcripts of the NET1 gene was ubiquitously expressed in all tissues examined. Using fluorescence in situ hybridization, we localized the NET1 gene to the short arm of human chromosome 10 at band p15.

3T3 Cells↗
PubMed2006

Combining docking and molecular dynamic simulations in drug design.

A rational approach is needed to maximize the chances of finding new drugs, and to exploit the opportunities of potential new drug targets emerging from genomic and proteomic initiatives, and from the large libraries of small compounds now readily available through combinatorial chemistry. Despite a shaky early history, computer-aided drug design techniques can now be effective in reducing costs and speeding up drug discovery. This happy outcome results from development of more accurate and reliable algorithms, use of more thoughtfully planned strategies to apply them, and greatly increased computer power to allow studies with the necessary reliability to be performed. Our review focuses on applications and protocols, with the main emphasis on critical analysis of recent studies where docking calculations and molecular dynamics (MD) simulations were combined to dock small molecules into protein receptors. We highlight successes to demonstrate what is possible now, but also point out drawbacks and future directions. The review is structured to lead the reader from the simpler to more compute-intensive methods. Thus, while inexpensive and fast docking algorithms can be used to scan large compound libraries and reduce their size, more accurate but expensive MD simulations can be applied when a few selected ligand candidates remain. MD simulations can be used: during the preparation of the protein receptor before docking, to optimize its structure and account for protein flexibility; for the refinement of docked complexes, to include solvent effects and account for induced fit; to calculate binding free energies, to provide an accurate ranking of the potential ligands; and in the latest developments, during the docking process itself to find the binding site and correctly dock the ligand a priori.

Computer Simulation↗
PubMed2006

Insights into synergistic interactions in binary mixtures of chemical permeation enhancers for transdermal drug delivery.

Chemical permeation enhancers (CPEs) are known to increase skin permeability to therapeutic drugs. Single chemicals, however, offer limited enhancements of skin permeability. Mixtures of chemicals can overcome this limitation owing to their synergistic interactions. However, identification of potent mixtures of chemicals requires screening of a large number of formulations. Discovery of CPE mixtures can be significantly accelerated by identifying patterns that occur in the existing data on CPEs. In this study, we systematically mine through a huge database on skin permeabilizing effect of over 4000 binary formulations generated by high throughput screening and extract general principles that govern the effect of binary combinations of chemicals on skin's barrier properties. Potencies and synergies of these formulations are analyzed to identify the role played by the formulation composition and chemistry. The analysis reveals several intuitive but some largely non-intuitive trends. For example, formulations made from enhancer mixtures are most potent when participating moieties are present in nearly equal fractions. Methyl pyrrolidone, a small molecule, is particularly effective in forming potent and synergistic enhancer formulations, and zwitterionic surfactants are more likely to feature in potent enhancers. Simple but invaluable rules like these will provide guiding principles for designing libraries to further speed up the formulation discovery process.

Administration, Cutaneous↗
PubMed1997

Minimized proteins.

The design of bioactive small molecules for interaction at larger protein-protein interfaces remains a challenge. Recent progress towards minimizing proteins into significantly smaller polypeptides has been achieved via both rational design processes and selection from vast combinatorial libraries. Such 'mini-proteins' represent a potential intermediate step toward the development of drugs targeted to protein-protein interfaces.

Amino Acid Sequence↗
PubMed2006

Identification of a polymeric Ig receptor binding phage-displayed peptide that exploits epithelial transcytosis without dimeric IgA competition.

The polymeric Ig receptor (pIgR), also called membrane secretory component (SC), mediates epithelial transcytosis of polymeric immunoglobulins (pIgs). J Chain-containing polymeric IgA (pIgA) and pentameric IgM bind pIgR at the basolateral epithelial surface. After transcytosis, the extracellular portion of the pIgR is cleaved at the apical side, either complexed with pIgs as bound SC or unoccupied as free SC. This transport pathway may be exploited to target bioactive molecules to the mucosal surface. To identify small peptide motifs with specific affinity to human pIgR, we used purified free SC and selection from randomized, cysteine-flanked 6- and 9-mer phage-display libraries. One of the selected phages, called C9A, displaying the peptide CVVWMGFQQVC, showed binding both to human free SC and SC complexed with pIgs. However, the pneumococcal surface protein SpsA (Streptococcus pneumoniae secretory IgA-binding protein), which binds human SC at a site distinct from the pIg binding site, competed with the C9A phage for binding to SC. The C9A phage showed greatly increased transport through polarized Madin-Darby canine kidney cells transfected with human pIgR. This transport was not affected by pIgA nor did it inhibit pIgR-mediated pIgA transcytosis. A free peptide of identical amino acid sequence as that displayed by the C9A phage inhibited phage interaction with SC. This implied that the C9A peptide sequence may be exploited for pIgR-mediated epithelial transport without interfering with secretory immunity.

Animals↗
PubMed2006

Discovery of a novel shp2 protein tyrosine phosphatase inhibitor.

Shp2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. It is involved in growth factorinduced activation of mitogen-activated protein (MAP) kinases Erk1 and Erk2 (Erk1/2) and has been implicated in the pathogenicity of the oncogenic bacterium Helicobacter pylori. Moreover, gain-of-function Shp2 mutations have been found in childhood leukemias and Noonan syndrome. Thus, small molecule Shp2 PTP inhibitors are much needed reagents for evaluation of Shp2 as a therapeutic target and for chemical biology studies of Shp2 function. By screening the National Cancer Institute (NCI) Diversity Set chemical library, we identified 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877) as a potent Shp2 PTP inhibitor. Molecular modeling and site-directed mutagenesis studies suggested that NSC-87877 binds to the catalytic cleft of Shp2 PTP. NSC-87877 cross-inhibited Shp1 in vitro, but it was selective for Shp2 over other PTPs (PTP1B, HePTP, DEP1, CD45, and LAR). It is noteworthy that NSC-87877 inhibited epidermal growth factor (EGF)-induced activation of Shp2 PTP, Ras, and Erk1/2 in cell cultures but did not block EGF-induced Gab1 tyrosine phosphorylation or Gab1-Shp2 association. Furthermore, NSC-87877 inhibited Erk1/2 activation by a Gab1-Shp2 chimera but did not affect the Shp2-independent Erk1/2 activation by phorbol 12-myristate 13-acetate. These results identified NSC-87877 as the first PTP inhibitor capable of inhibiting Shp2 PTP in cell cultures without a detectable off-target effect. Our study also provides the first pharmacological evidence that Shp2 mediates EGF-induced Erk1/2 MAP kinase activation.

Adaptor Proteins, Signal Transducing↗
PubMed2002

A role for the protease falcipain 1 in host cell invasion by the human malaria parasite.

Cysteine proteases of Plasmodium falciparum are required for survival of the malaria parasite, yet their specific cellular functions remain unclear. We used a chemical proteomic screen with a small-molecule probe to characterize the predominant cysteine proteases throughout the parasite life cycle. Only one protease, falcipain 1, was active during the invasive merozoite stage. Falcipain 1-specific inhibitors, identified by screening of chemical libraries, blocked parasite invasion of host erythrocytes, yet had no effect on normal parasite processes such as hemoglobin degradation. These results demonstrate a specific role for falcipain 1 in host cell invasion and establish a potential new target for antimalarial therapeutics.

Animals↗