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At least 217 records · Page 12Linked to original sources

PubMed1998

Prediction of local structure in proteins using a library of sequence-structure motifs.

We describe a new method for local protein structure prediction based on a library of short sequence pattern that correlate strongly with protein three-dimensional structural elements. The library was generated using an automated method for finding correlations between protein sequence and local structure, and contains most previously described local sequence-structure correlations as well as new relationships, including a diverging type-II beta-turn, a frayed helix, and a proline-terminated helix. The query sequence is scanned for segments 7 to 19 residues in length that strongly match one of the 82 patterns in the library. Matching segments are assigned the three-dimensional structure characteristic of the corresponding sequence pattern, and backbone torsion angles for the entire query sequence are then predicted by piecing together mutually compatible segment predictions. In predictions of local structure in a test set of 55 proteins, about 50% of all residues, and 76% of residues covered by high-confidence predictions, were found in eight-residue segments within 1.4 A of their true structures. The predictions are complementary to traditional secondary structure predictions because they are considerably more specific in turn regions, and may contribute to ab initio tertiary structure prediction and fold recognition.

Algorithms↗

PubMed1997

Synthetic strategies in combinatorial chemistry.

Different synthetic strategies are required for the two kinds of libraries being developed for combinatoral chemistry. Preparation of a 'focused' library involves conversion of a known synthetic route to the solid-phase or automated format, whereas generation of a 'prospecting' library is driven more by availability of input materials and considerations of structural novelty. While both types can be identified over the history of combinatorial chemistry, elaborating syntheses of novel, nonoligomeric, 'drug-like' molecules for use in prospecting libraries is a relatively recent development.

Chemistry, Organic↗

PubMed1995

Synthetic chemical diversity: solid phase synthesis of libraries of C2 symmetric inhibitors of HIV protease containing diamino diol and diamino alcohol cores.

Solid phase synthesis of non-oligomeric organic compounds has been pursued for high-efficiency generation of large numbers of structurally diverse compounds for drug screening. Known as chemical diversity libraries or combinatorial libraries (when the synthesis is carried out in a combinatorial fashion), these compounds can be used for de novo discovery of drug leads or for expedient structure--activity relationship (SAR) studies. To expand the scope of solid phase synthesis beyond the capability of the traditional method of solid phase synthesis for peptides, a strategy was developed for bi-directional solid phase synthesis starting with diamino alcohol or diamino diol core structures. The strategy relies on using bifunctional linkers to modify the core structures, simultaneously protecting the hydroxyl group or the diol moiety of the core and providing a carboxyl group for attachment of the modified cores to a solid support. The two NH2 groups of the modified cores attached to the solid support were then deprotected and reacted with a wide variety of amine-reactive reagents (carboxylic acids, sulfonyl chlorides, isocyanates, chloroformates, etc.) to extend the molecule in both directions. This strategy was successfully applied to automated parallel synthesis of a library of C2 symmetric inhibitors of HIV protease containing the known symmetry-based diamino diol and diamino alcohol core structures, thus enabling expedient access of large numbers of analogs in this series. A library of over 300 discrete compounds was synthesized using this methodology in order to identify potent (IC50 < 100 nM) HIV protease inhibitors with reduced size. This paper describes the technical aspects of this technology.

Alcohols↗

PubMed1975

Computer-assisted cataloging: experiences at the UCLA Biomedical Library.

The computer-assisted procedures developed in the UCLA Biomedical Library Cataloging Division have been in effect for approximately three years. The system utilizes a Delta Data System cathode ray tube terminal and cassette attachment for on or off-line input of data. Products of the system include catalog card sets arranged in filing order, a monthly Recent Acquisitions List, and computer-generated book catalogs. Planning, personnel, and equipment requirements are discussed, and preliminary cost figures for various parts of the system are given. Potential applications of the automated system on a regional level and in terms of the library's future automation plans are considered.

California↗

PubMed1985

BACS: evolution of an integrated library system toward information management.

The evolution of the Washington University School of Medicine BACS integrated library system toward information management functions is outlined. The creation of a machine-readable database and its extension through telecommunications have consequences that reach beyond the functions of the library as we have perceived them. It is argued that libraries are flexible institutions that, with automation, are likely to enlarge rather than to diminish.

Catalogs, Library↗

PubMed2005

Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor.

Small-molecule kinase inhibitors are predominantly discovered in pure protein assays. We have discovered an inhibitor of Rho-kinase (ROCK) through an image-based, high-throughput screen of cell monolayer wound healing. Using automated microscopy, we screened a library of approximately 16,000 compounds finding many that affected cell migration or cell morphology as well as compounds that blocked mitotic progression. We tested approximately 200 compounds in a series of subassays and chose one, 3-(4-pyridyl)indole (Rockout), for more detailed characterization. Rockout inhibits blebbing and causes dissolution of actin stress fibers, phenocopying Rho-kinase inhibitors. Testing Rho-kinase activity in vitro, Rockout inhibits with an IC50 of 25 microM ( approximately 5-fold less potent than Y-27632) but has a similar specificity profile. We also profile the wound healing assay with a library of compounds with known bioactivities, revealing multiple pathways involved in the biology.

Animals↗

PubMed2002

Universal solid-phase approach for the immobilization, derivatization, and resin-to-resin transfer reactions of boronic acids.

Boronic acid-containing molecules are employed in a broad range of biological, medicinal, and synthetic applications. These compounds, however, tend to be difficult to handle by solution-phase methods. Herein, this problem is addressed with the development of the first general solid-phase approach for the derivatization of functionalized boronic acids. This approach is based on the use of a diethanolamine resin anchor that facilitates boronic acid immobilization by avoiding the need for exhaustive removal of water in the esterification process. The immobilization of a wide variety of boronic acids onto N,N-diethanolaminomethyl polystyrene (DEAM-PS, 1) can be performed within minutes by simple stirring in anhydrous solvents at room temperature. Evidence for the formation of a bicyclic diethanolamine boronate with putative N-B coordination was shown by (1)H NMR analysis of DEAM-PS-supported p-tolylboronic acid. The hydrolytic cleavage of the same model boronic acid from the DEAM-PS resin was studied by UV spectroscopy. Hydrolysis and attachment were shown to occur under a rapidly attained equilibrium, and a large excess of water (>32 equiv) is required to effect a practically quantitative release of boronic acids from DEAM-PS. Despite their relative sensitivity to water and alcohols, DEAM-PS-bound arylboronic acids functionalized with a formyl, a bromomethyl, a carboxyl, or an amino group can be transformed in good to excellent yields into a wide variety of amines, amides, anilides, and ureas, respectively. Ugi multicomponent reactions on DEAM-PS-supported aminobenzeneboronic acids, derivatization of multifunctional arylboronic acids, and sequential reactions can also be carried out efficiently. These new DEAM-PS-supported arylboronic acids can be employed directly into resin-to-resin transfer reactions (RRTR). This type of multiresin process helps eliminate time-consuming cleavage and transfer operations, thereby considerably simplifying the outlook of combinatorial library synthesis by manual or automated means. This concept was illustrated by a set of optimized procedures for the Suzuki cross-coupling and the borono-Mannich reactions.

Boronic Acids↗

PubMed2006

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM): A novel method for biomolecular screening.

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a new separation-based affinity method. It has kinetic capabilities exceeding those of surface plasmon resonance (SPR) and does not require immobilization of molecules on the surface. Another distinctive feature of NECEEM is that--if it is combined with an advanced method for the mixing solutions inside a capillary, termed transverse diffusion of laminar flow profiles (TDLFP)--it requires only nanoliter volumes of solutions. The proven applications of NECEEM to biomolecular screening include 1) measuring kinetic and thermodynamic parameters of protein-ligand interactions, 2) quantitative affinity analyses of proteins and hybridization analyses of DNA and RNA, and 3) selection of binding ligands from combinatorial libraries. NECEEM is easy to automate and parallelize. Because of its simplicity and analytical power, NECEEM has the potential to become a workhorse in studies of biomolecular interactions. The author reviews theoretical bases of NECEEM and its applications to biomolecular screening.

Affinity Labels↗

PubMed2004

A step ahead: combining protein purification and correct folding selection.

The success of recombinant protein expression seems unpredictable and even good yields of soluble proteins do not guarantee the correct folding. The search for soluble constructs can be performed by exploiting libraries and speeded up by automation, but these approaches are money and time consuming and the tags used for affinity purification can mask the real stability of the target proteins. The ideal purification protocol would include the structure quality control. A recent paper commented in this article describes a phage-display method to screen for antibodies that are able to re-fold after heat-denaturation and can be selectively affinity-purified only if monodispersed. It turned out that the proteins with high recovery performance after heat-shock were also suitable for efficient recombinant expression.

Editorial↗

PubMed2004

The NLM Indexing Initiative's Medical Text Indexer.

The Medical Text Indexer (MTI) is a program for producing MeSH indexing recommendations. It is the major product of NLM's Indexing Initiative and has been used in both semi-automated and fully automated indexing environments at the Library since mid 2002. We report here on an experiment conducted with MEDLINE indexers to evaluate MTI's performance and to generate ideas for its improvement as a tool for user-assisted indexing. We also discuss some filtering techniques developed to improve MTI's accuracy for use primarily in automatically producing the indexing for several abstracts collections.

Abstracting and Indexing↗

PubMed1991

Comparison of traditional gas chromatography (GC), headspace GC, and the microbial identification library GC system for the identification of Clostridium difficile.

Three gas chromatography (GC) methods were compared for the identification of 52 clinical Clostridium difficile isolates, as well as 17 non-C. difficile Clostridium isolates. Headspace GC and Microbial Identification System (MIS) GC, an automated system which utilizes a software library developed at the Virginia Polytechnic Institute to identify organisms based on the fatty acids extracted from the bacterial cell wall, were compared against the reference method of traditional GC. Headspace GC and MIS were of approximately equivalent accuracy in identifying the 52 C. difficile isolates (52 of 52 versus 51 of 52, respectively). However, 7 of 52 organisms required repeated sample preparation before an identification was achieved by the MIS method. Both systems effectively differentiated C. difficile from non-C. difficile clostridia, although the MIS method correctly identified only 9 of 17. We conclude that the headspace GC system is an accurate method of C. difficile identification, which requires only one-fifth of the sample preparation time of MIS GC and one-half of the sample preparation time of traditional GC.

Bacteriological Techniques↗

PubMed2005

Construction, evaluation and refinement of a large human antibody phage library based on the IgD and IgM variable gene repertoire.

The ability to isolate antibodies against any antigen of interest has become increasingly important as antibodies have proved their utility both in antigen detection, quantification and as specific in vivo targeting agents. To this end, we have constructed a large antibody phage library in the single chain Fv (scFv) phagemid format based on the naive human variable (V) gene repertoire dictated by IgD and IgM. Optimizing each step of the library construction has resulted in a highly diverse and functional library, as assessed by sequencing analysis, large-scale automated expression analysis and antigen screening. Furthermore, the versatile format of the library, which comprises 14 separate sub-libraries, adds considerably flexibility with respect to which part of the antibody repertoire that is to be probed. This versatility has been further exploited to generate a refined antibody library, which exhibits one of the highest prokaryotic expression levels reported to date for a naive repertoire. The construction of the refined library was based on the functional purification of expressed V genes in the context of the protein L interaction with correctly folded V genes of the kappa light chain family. Antigen screening of this library indicated that the functional purification improved the ability to retrieve antigen specific antibodies, but at the cost of potential loss of diversity in the isolated repertoire.

Amino Acid Sequence↗

PubMed1996

Characterization of benzodiazepine "combinatorial" chemical libraries by on-line immunoaffinity extraction, coupled column HPLC-ion spray mass spectrometry-tandem mass spectrometry.

To characterize combinatorial chemical libraries of small drug compounds, an automated column switching system incorporating an immunoaffinity extraction (IAE) column and two reversed-phase HPLC columns was coupled to a triple-quadrupole mass spectrometer. A Protein G column and antibodies to benzodiazepines were used to screen library components. A pH change in the mobile phase eluted the benzodiazepine-antibody complexes onto a C-18 restricted access media (RAM) column, thereby separating the selected benzodiazepines from the antibody. In a final step, backflushing the RAM column eluted the benzodiazepines onto a C-8 analytical reversed-phase column for separation before detection and preliminary structural characterization using ion spray mass spectrometry (MS) and tandem mass spectrometry (MS/ MS). A known 19-component library and an unknown 20-component library were analyzed. Full-scan IAE/LC/ LC/MS and IAE/LC/LC/MS/MS chromatograms suggested the feasibility of this combination of techniques, although the antibodies used were not highly specific. Inspection of MS/MS spectra of components in the unknown library compared to the MS/MS spectrum of a known standard (chlordiazepoxide) identified a subclass of benzodiazepines. Productions of the known standard and an unknown benzodiazepine were successively captured and fragmented (MSn experiments) using an iontrap mass spectrometer off-line, which confirmed that the unknown was an analogue of chlordiazepoxide.

Benzodiazepines↗

PubMedDate not supplied

Efficient split synthesis for targeted libraries.

We propose a new approach for fabricating more sophisticated combinatorial chemistry libraries via split synthesis and evaluate its potential through extensive simulation. Our algorithmically intensive method promises to reduce the time and materials costs of synthesizing libraries which are (1) too large to synthesize economically by sequential or parallel synthesis, (2) too long or irregular for conventional split synthesis generation techniques, and (3) not used in sufficient quantity to justify the setup costs of array makers. It also encourages the design of more focused and interesting libraries than are typically constructed using split synthesis. Our algorithms automate the design of efficient synthesis procedures for motif-based libraries which are too complex to design by hand. Our software allows the user to select the most desirable tradeoff between minimizing the number of steps in the synthesis process and containing the combinatorial explosion of the number of compounds synthesized.

Algorithms↗

PubMed1999

Automation of yeast two-hybrid screening.

We have developed an automated format for screening yeast two-hybrid libraries for protein-protein interactions. The format consists of a liquid array in which pooled library subsets of yeast, expressing up to 1000 different cDNAs, are mated to a yeast strain of the opposite mating type, expressing a protein of interest. Interactors are detected by a liquid assay for beta-galacsidase following prototrophic selection. The method is demonstrated by the detection of interactions between two encoded yeast RNA polymerase subunits in simulated libraries of varied complexity. To demonstrate its utility for large scale screening of complex cDNA libraries, two nuclear receptor ligand-binding domains were screened through two cDNA libraries arrayed in pooled subsets. Screening these libraries yielded clones which had previously been identified in traditional yeast two hybrid screens, as well as several new putative interacting proteins. The formatting of the cDNA library into pooled subsets lends itself to functional subtraction of the promiscuous positive class of interactor from the library. Also, the liquid arrayed format enables electronic handling of the data derived from interaction screening, which, together with the automated handling of samples, should promote large-scale proteome analysis.

Automation↗

PubMed2004

POCOman: new system for quantifying posterior capsule opacification.

PURPOSE: To describe a new method of measuring posterior capsule opacification (PCO) and intraocular lens (IOL) rotation and report the validation of the method. SETTING: Ophthalmology Department, St. Thomas' Hospital, and Medical Imaging, Department of Physics, King's College, London, United Kingdom. METHOD: A new interactive software program, POCOman, was developed for the semiobjective assessment of PCO. Digital images of the posterior capsule, which can be acquired by any technique, are analyzed by the observer to determine the percentage area of PCO and assign a severity score. The system was validated by comparing it to clinical slitlamp evaluation of PCO and automated POCO system analysis using a library of 100 images taken from archives. The software also measures sequential IOL rotation for the evaluation of toric IOLs. RESULTS: An image could be analyzed in approximately 2 minutes. The results of the POCOman system correlated well with the results of the automated POCO system and clinical evaluation. CONCLUSIONS: The POCOman is an effective, user-friendly system for quantifying PCO. It can be obtained for free and has advantages over other methods.

Cataract↗

PubMedDate not supplied

Mass spectrometry in combinatorial chemistry.

In the fast expanding field of combinatorial chemistry, profiling libraries has always been a matter of concern--as illustrated by the buoyant literature over the past seven years. Spectroscopic methods, including especially mass spectrometry and to a lesser extent IR and NMR, have been applied at different levels of combinatorial library synthesis: in the rehearsal phase to optimize the chemistry prior to library generation, to confirm library composition, and to characterize after screening each structure that exhibits positive response. Most of the efforts have been concentrated on library composition assessment. The difficulties of such analyses have evolved from the infancy of the combinatorial concept, where large mixtures were prepared, to the recent parallel syntheses of collections of discrete compounds. Whereas the complexity of the analyses has diminished, an increased degree of automation was simultaneously required to achieve efficient library component identification and quantification. In this respect, mass spectrometry has been found to be the method of choice, providing rapid, sensitive, and informative analyses, especially when coupled to chromatographic separation. Fully automated workstations able to cope with several hundreds of compounds per day have been designed. After a brief introduction to describe the combinatorial approach, library characterization will be discussed in detail, considering first the solution-based methodologies and secondly the support-bound material analyses.

Chemistry, Organic↗

PubMed2004

Automated high throughput ADME assays for metabolic stability and cytochrome P450 inhibition profiling of combinatorial libraries.

Early determinations of pharmaceutical properties can serve as predictors of a compound's likely development success. Our laboratory has implemented high throughput in vitro absorption, distribution, metabolism and excretion (ADME) assays which address absorption, metabolism, and physico-chemical properties in an effort to identify potential development liabilities early, thereby minimizing discovery to market attrition. In response to the throughput demands of parallel synthesis, we have incorporated a SAGIAN core robotics system for the determination of both metabolic stability in human liver microsomes (HLMs) and cytochrome P450 (CYP450) inhibition. This automated solution has led to an increase in capacity, throughput and reliability for both in vitro assays. The SAGIAN core robotics system integrates devices such as liquid handlers, plate hotels and incubators through the use of an ORCA robotic arm. The HLM stability assay utilizes a Multimek 96-channel pipettor for liquid handling. The incubation plates are transferred off-line for final semi-quantitative analysis using high throughput parallel LC/MS. The CYP inhibition method combines both liquid handlers and an integrated fluorescence plate reader to perform single concentration percent inhibition assays for 88 compounds. Cytochrome P450 inhibition is measured for both CYP3A4 and CYP2D6 isozymes. This system represents a fully integrated approach to high throughput ADME evaluation in support of drug discovery. The core system concept creates a plug-and-play approach, which combines a series of modular stations to build a robotic platform, which is flexible, upgradable, and easily reconfigured when assays change or are newly developed. The application of these strategies as a means of assessing metabolic stability and CYP inhibition of synthetic libraries is discussed.

Combinatorial Chemistry Techniques↗