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PubMed1999

Combinatorial chemistry, automation and molecular diversity: new trends in the pharmaceutical industry.

Combinatorial chemistry has emerged as a set of novel strategies for the synthesis of large sets of compounds (combinatorial libraries) for biological evaluation. Within a few years combinatorial chemistry has undergone a series of changes in trends, which are closely related to two important factors in libraries: numbers and quality. While the number of compounds in a library may be easily expressed, it is a lot more difficult to indicate the degree of quality of a library. This degree of quality can be split into two aspects: purity and diversity. The changing trends in combinatorial chemistry with respect to the strategies, the technologies, the libraries themselves (numbers and purity aspects) and the molecular diversity are outlined in this paper.

Automation↗

PubMedDate not supplied

Direct-injection NMR (DI-NMR): a flow NMR technique for the analysis of combinatorial chemistry libraries.

A new tool for analyzing compound libraries by NMR has been developed. Aliquots of solution-state samples (between 120 and 350 microL) are directly injected, using a standard liquids handler, into an NMR (LC-NMR) flow probe. Automated NMR software tracks--and suppresses--intense signals arising from the nondeuterated solvents used (if any) and acquires high-sensitivity one-dimensional 1H NMR spectra. An 88-member combinatorial library, dissolved in DMSO and stored in a 96-well microtiter plate, has been analyzed a number of ways using this technique. This nondestructive technique, which we call direct-injection NMR (DI-NMR) and which is embodied in our versatile automated sample changer (VAST) hardware, has proven to be both routine and robust. Our success in automatically acquiring the NMR data for entire plates of library compounds (within 4-8 h) has caused us to develop new ways to display and analyze the resulting NMR data, as will be shown here.

Chemistry, Organic↗

PubMed2005

A convenient method for synthesis of cyclic peptide libraries.

Cyclic peptides have been reported to bind to multiple, unrelated classes of receptor with high affinity. Owing to the robustness of amide bond chemistry, the ability to explore extensive chemical diversity by incorporation of unnatural and natural amino acids, and the ability to explore conformational diversity, through the incorporation of various constraints, arrays of cyclic peptides can be tailored to broadly sample chemical diversity. We describe the combination of a safety catch linker with a directed-sorted procedure for the synthesis of large arrays of diverse cyclic peptides for high-throughput screening.

Amino Acids↗

PubMed2002

Compound library management.

The increasing size of the collections used for drug-discovery purposes has demanded both hardware and software automation of compound management in order to cope with the increasing demands of HTS. Splitting the collection into a number of copies in different formats is a desirable approach to keep a balance between rapid response to the demands and best storage conditions. Flexibility to different assay configurations can be provided with the appropriate selection of liquid handlers, and the informatic management systems should be accessible to keep track of the samples and link them to a variety of information that can help interpret HTS data. In this respect, QC data on the compounds and quality checks of the equipment used are highly desirable. It is also prudent for a large organization with different research sites to have a unified database and compatible plate format and concentration in order to be able to exchange samples and share screening results. The accomplishment of all the previous requirements is the only way to ensure an efficient and effective compound library management.

Computational Biology↗

PubMed1988

Single cell assay with an automated capillary microinjection system.

An automated capillary microinjection system with computer-controlled positioning of the cells and of the capillary, and its applications and advantages, is described. The system is easy in handling and manipulation. About 1500 injections are possible in 1 h, with high reproducibility. In cytoplasmic and nuclear injections more than 90 and 85% of the cells are successfully injected. Using FITC-dextran at a concentration of 0.5% as a fluorescently labeled coinjection marker, 99% of the cells can be retrieved in culture medium even 48 h after injection. The coordinates of the cells are stored in the computer and accuracy in statistical evaluation of experiments is improved in comparison to the manual techniques. Methods for preparation and handling of glass capillaries were developed resulting in reproducible form and significantly reduced clogging rate. The improved characteristics offered by this system are demonstrated in studies leading to the confirmation of existence of an mRNA inhibiting DNA synthesis in cells. Functional screening by cell injections of cDNA libraries and of size-fractionated mRNA molecules can be performed efficiently with the automated microinjection system.

Autoradiography↗

PubMed1991

Identification of clinical isolates of gram-negative nonfermentative bacteria by an automated cellular fatty acid identification system.

An automated cellular fatty acid (CFA) bacterial identification system, Microbial Identification System (MIS; Microbial ID, Newark, Del.), was compared with a conventional system for the identification of 573 strains of gram-negative nonfermentative bacteria. MIS identifications were based exclusively on the CFA composition following 22 to 26 h of growth at 28 degrees C on Trypticase soy agar. MIS identifications were listed with a confidence measurement (similarity index [SI]) on a scale of 0 to 1.0. A value of greater than or equal to 0.5 was considered a good match. The MIS correctly listed as the first choice 478 of 532 (90%) strains contained in the data base. However, only 314 (59%) had SI values of greater than or equal to 0.5. Of the 54 strains in which there was not agreement, 37 belonged to the genera Acinetobacter, Moraxella, or Alcaligenes or were Pseudomonas pickettii. Reproducibility studies suggest that SI variation is most likely a function of a difference in culture age at the time of analysis, which is due to the relatively low temperature and time of incubation. Other discrepancies were attributable to insufficiently characterized library entries or an inability to differentiate chemotaxonomically closely related species. The MIS, as the first automated CFA identification system, is an accurate, efficient, and relatively rapid method for the identification of gram-negative nonfermentative bacteria. The development of a CFA library with the media and incubation conditions routinely used for the isolation of clinical pathogens could further decrease the identification time and provide an increase in accuracy.

Bacteriological Techniques↗

PubMed2005

Rescue and in situ selection and evaluation (RISE): a method for high-throughput panning of phage display libraries.

Phage display has proven to be an invaluable instrument in the search for proteins and peptides with optimized or novel functions. The amplification and selection of phage libraries typically involve several operations and handling large bacterial cultures during each round. Purification of the assembled phage particles after rescue adds to the labor and time demand. The authors therefore devised a method, termed rescue and in situ selection and evaluation (RISE), which combines all steps from rescue to binding in a single microwell. To test this concept, wells were precoated with different antibodies, which allowed newly formed phage particles to be captured directly in situ during overnight rescue. Following 6 washing steps, the retained phages could be easily detected in an enzyme-linked immunosorbent assay (ELISA), thus eliminating the need for purification or concentration of the viral particles. As a consequence, RISE enables a rapid characterization of phage-displayed proteins. In addition, this method allowed for the selective enrichment of phages displaying a hemagglutinin (HA) epitope tag, spiked in a 10(4)-fold excess of wild-type background. Because the combination of phage rescue, selection, or evaluation in a single microwell is amenable to automation, RISE may boost the high-throughput screening of smaller sized phage display libraries.

Bacteriophages↗

PubMed2004

Technological advances in high-throughput screening.

High-throughput screening (HTS) is the process of testing a large number of diverse chemical structures against disease targets to identify 'hits'. Compared to traditional drug screening methods, HTS is characterized by its simplicity, rapidness, low cost, and high efficiency, taking the ligand-target interactions as the principle, as well as leading to a higher information harvest. As a multidisciplinary field, HTS involves an automated operation-platform, highly sensitive testing system, specific screening model (in vitro), an abundant components library, and a data acquisition and processing system. Various technologies, especially the novel technologies such as fluorescence, nuclear-magnetic resonance, affinity chromatography, surface plasmon resonance, and DNA microarray, are now available, and the screening of more than 100,000 samples per day is already possible. Fluorescence-based assays include the scintillation proximity assay, time-resolved energy transfer, fluorescence anisotropy, fluorescence correlation spectroscopy, and fluorescence fluctuation spectroscopy. Fluorescence-based techniques are likely to be among the most important detection approaches used for HTS due to their high sensitivity and amenability to automation, giving the industry-wide drive to simplify, miniaturize, and speed up assays. The application of NMR technology to HTS is another recent trend in drug research. One advantage afforded by NMR technology is that it can provide direct information on the affinity of the screening compounds and the binding location of protein. The structure-activity relationship acquired from NMR analysis can sharpen the library design, which will be very important in furnishing HTS with well-defined drug candidates. Affinity chromatography used for library screening will provide the information on the fundamental processes of drug action, such as absorption, distribution, excretion, and receptor activation; also the eluting curve can give directly the possibility of candidate drug. SPR can measure the quantity of a complex formed between two molecules in real-time without the need for fluorescent or radioisotopic labels. SPR is capable of characterizing unmodified biopharmaceuticals, studying the interaction of drug candidates with macromolecular targets, and identifying binding partners during ligand fishing experiments. DNA microarrays can be used in HTS be used to further investigate the expression of biological targets associated with human disease, which then opens new and exciting opportunities for drug discovery. Without doubt, the addition of new technologies will further increase the application of HTS in drug screening and its related fields.

Animals↗

PubMed2004

Harnessing the potential of chemical defenses from antimicrobial activities.

Resistance to the drugs used in the treatment of many infectious diseases is increasing, while microbial infections are being found to be responsible for more life-threatening diseases than previously thought. Despite a large investment in the invention and application of high-throughput screening techniques involving miniaturization and automation, and a diverse array of strategies for designing and constructing various chemical libraries, relatively few new drugs have resulted. Natural products, however, have been a major source of drugs for centuries. Since some of them are produced by organisms as a result of selection in favour of improved defense against competing deleterious microorganisms, in principle they would be less likely to incur resistance. Furthermore, the production of those defensive secondary metabolites is inducible because their original function is a response to environmental challenges. Moreover, symbioses, co-habitation associations between two or more different species of organisms, are universal in nature, and the production of secondary metabolites by symbiotic microbes may be an important adaptation allowing microbes to affect their hosts. Therefore, co-culture strategies, using combinations of plant cell-pathogenic microbes, plant cell-endophytes (or symbionts), and symbiont-pathogenic microbes, based on the principles of chemical defense and the known mechanisms of organism interactions, may be an efficient general approach in the search for new anti-microbial drugs.

Animals↗

PubMed2003

Parallel high-throughput accurate mass measurement using a nine-channel multiplexed electrospray liquid chromatography ultraviolet time-of-flight mass spectrometry system.

A nine-channel multiplexed electrospray (MUX) liquid chromatography ultraviolet time-of-flight mass spectrometry (LC/UV/TOFMS) system has been used to simultaneously measure accurate masses of eluting components from eight parallel gradient LC columns. Accuracies better than 5 and 10 ppm were achieved for 50 and 80% of samples, respectively, from a single batch analysis of ten plates (960 samples) of a Fmoc-Asp(OtBu)-OH and reserpine mixture. Combinatorial library compounds were analyzed using this parallel high-throughput system in both positive and negative modes to rigorously verify expected products and identify side products. A mass accuracy of 10 ppm root mean square (RMS) is routinely obtained for combinatorial library samples from this high-throughput accurate mass LC/MS system followed by automated data processing. This mass accuracy is critical in revealing combinatorial synthesis problems that would be missed by unit mass measurement.

Alanine↗

PubMed1991

Amino terminal sequence of the mitochondrial protein mtDBP-C: similarity with nonhistone chromosomal proteins HMG 1 and 2.

We have previously reported the characterization of a DNA-binding protein isolated from Xenopus laevis mitochondria (mtDBP-C). The amino terminal sequence of this protein (26 residues) has been determined by automated Edman degradation and used to search for sequence similarity with the NBRF library. A segment of 17 amino acids displays 47.1% of identity with proteins HMG-1 and 2 of various vertebrate species.

Amino Acid Sequence↗

PubMedDate not supplied

Design and characterization of libraries of molecular fragments for use in NMR screening against protein targets.

We have designed four generations of a low molecular weight fragment library for use in NMR-based screening against protein targets. The library initially contained 723 fragments which were selected manually from the Available Chemicals Directory. A series of in silico filters and property calculations were developed to automate the selection process, allowing a larger database of 1.79 M available compounds to be searched for a further 357 compounds that were added to the library. A kinase binding pharmacophore was then derived to select 174 kinase-focused fragments. Finally, an additional 61 fragments were selected to increase the number of different pharmacophores represented within the library. All of the fragments added to the library passed quality checks to ensure they were suitable for the screening protocol, with appropriate solubility, purity, chemical stability, and unambiguous NMR spectrum. The successive generations of libraries have been characterized through analysis of structural properties (molecular weight, lipophilicity, polar surface area, number of rotatable bonds, and hydrogen-bonding potential) and by analyzing their pharmacophoric complexity. These calculations have been used to compare the fragment libraries with a drug-like reference set of compounds and a set of molecules that bind to protein active sites. In addition, an analysis of the overall results of screening the library against the ATP binding site of two protein targets (HSP90 and CDK2) reveals different patterns of fragment binding, demonstrating that the approach can find selective compounds that discriminate between related binding sites.

Algorithms↗

PubMedDate not supplied

Screening for basic drugs in 2-mL urine samples by dual-plate overpressured layer chromatography and comparison with gas chromatography-mass spectrometry.

A dual-plate overpressured layer chromatography (OPLC) method was evaluated for broad-scale screening of basic drugs in 2-mL autopsy urine samples. Extraction was carried out by mixed-mode solid-phase extraction, and identification was based on automated comparison of corrected Rf values (hRfc) and in situ UV spectra with library values by dedicated software. The day-to-day precision of hRfc values was good in both OPLC1 and OPLC2 systems with median relative standard deviations of 2.4% and 3.4%, respectively. Both Rf and hRfc values were independent of the amount of analyte (0.5-10 microg) applied to the plate. Detection limits were determined for 47 drug substances in 2-mL urine samples, and they varied between 0.05 and 3.5 mg/L with a median of 1.0 mg/L. The performance of OPLC was evaluated by analyzing 30 autopsy urine samples by both OPLC and gas chromatography-mass spectrometry (GC-MS). The majority of findings by OPLC were in agreement with GC-MS. Some substances with low concentrations were not detected by OPLC, whereas GC-MS failed to detect a few polar substances. The OPLC method thus provides an alternative for current planar and column liquid chromatographic drug screening methods with the possibility of lowering detection limits by using a larger sample size.

Chromatography, Liquid↗

PubMed2005

Identification of nitric oxide donors by biomimetic HTS application.

Metalloporphyrin catalyzed biomimetic oxidation was used for the identification of nitric oxide (NO) donors with diverse chemical structure. Methodology was validated by testing known NO donors. Efficient automation of the test allowed us to investigate a subset of our corporate library. Several hits identified in this campaign were validated in both the chemical and also microsomal model that revealed all hits to be active in the biological system, as well. One of the hits showed comparable activity to V-PYRRO/NO, the prototypic liver selective NO donor.

Animals↗

PubMed1998

Structural and comparative analysis of the mouse gene for pigment epithelium-derived factor (PEDF).

PURPOSE: Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a member of the serine protease inhibitor supergene family. PEDF promotes both neuronal phenotype in cultured Y79 cells and neuronal survival in cultured cerebellar granulocytes. The purpose of this study was to clone the mouse PEDF gene and to determine its structure and levels of expression in different tissues. METHODS: The mouse PEDF cDNA was cloned from a mouse liver cDNA library using human PEDF cDNA as a probe. The mouse PEDF gene was cloned from a mouse ES genomic P1 library. DNA sequencing was performed using a PE-Applied Biosystems model 373 automated fluorescent sequencer. RESULTS: The mouse PEDF cDNA is 1461 bp in length and contains an open reading frame of 417 amino acids. The mouse PEDF gene spans approximately 13 kb and, like the human, it is fragmented into 8 exons. The splice sites follow the AG/GT consensus rule. Southern blot analysis indicates that the mouse genome contains only one gene for PEDF. Northern blot analysis shows the presence of the PEDF transcript in a broad range of adult mouse tissues with liver showing the highest level of expression. CONCLUSIONS: The mouse and human PEDF promoters share overall 27% similarity but are nearly identical between mouse +86 to -166 and human +102 to -96. The present study will allow us to move from in vitro experiments to in vivo studies through the development of a "knock-out" mouse model.

Animals↗

PubMedDate not supplied

Evaluation of human-readable annotation in biomolecular sequence databases with biological rule libraries.

MOTIVATION: Computer-based selection of entries from sequence databases with respect to a related functional description, e.g. with respect to a common cellular localization or contributing to the same phenotypic function, is a difficult task. Automatic semantic analysis of annotations is not only hampered by incomplete functional assignments. A major problem is that annotations are written in a rich, non-formalized language and are meant for reading by a human expert. This person can extract from the text considerably more information than is immediately apparent due to his extended biological background knowledge and logical reasoning. APPROACH: A technique of automated annotation evaluation based on a combination of lexical analysis and the usage of biological rule libraries has been developed. The proposed algorithm generates new functional descriptors from the annotation of a given entry using the semantic units of the annotation as prepositions for implications executed in accordance with the rule library. RESULTS: The prototype of a software system, the Meta_A(nnotator) program, is described and the results of its application to sequence attribute assignment and sequence selection problems, such as cellular localization and sequence domain annotation of SWISS-PROT entries, are presented. The current software version assigns useful subcellular localization qualifiers to approximately 88% of all SWISS-PROT entries. As shown by demonstrative examples, the combination of sequence and annotation analysis is a powerful approach for the detection of mutual annotation/sequence inconsistencies. AVAILABILITY: Results for the cellular localization assignment can be viewed at the URL http://www.bork. embl-heidelberg.de/CELL_LOC/CELL_LOC.html.

Algorithms↗

PubMed2002

Spreadsheets for automated data collection, analysis, and report generation for diagnostic medical physics: publicly available on the world wide web.

A library of spreadsheets has been developed to facilitate the practice of diagnostic physics quality assurance. Each spreadsheet follows a standard template and uses the highest ranking controlling authority (within the United States) for pass/fail criteria and testing procedures. Sheets are now available for CT (computed tomography), MR (magnetic resonance), US (ultrasound), screen-film mammography, stereotactic breast, radiographic, fluoroscopic, computed radiography, film digitizer, and display quality control. Use is made of spreadsheet "workbooks" so that each testing event is a single sheet in the workbook. Thus, results over the lifetime of the device are gathered in a single file, and historical control charts are gathered on the first sheet. The spreadsheets are available at http://radweb.mcis.washington.edu/~sglanger, and are released under the Gnu (a recursive acronym. Gnu's Not Unix) public license. It is expected that others will add improvements, and they are expected and requested to submit them back to the author to be shared with the diagnostic physicist community at large.

Diagnostic Imaging↗

PubMed2005

Accurate multiplex polony sequencing of an evolved bacterial genome.

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.

Acrylic Resins↗