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PubMed1994

Peptide libraries: determination of relative reaction rates of protected amino acids in competitive couplings.

In the solid phase preparation of synthetic peptide libraries, equimolarity of the resultant peptides in the mixture simplifies the identification of active compounds. Two primary methods for the preparation of combinatorial peptide mixtures are currently used. In the first method, the starting resin is divided into equal aliquots, individual amino acids are coupled to each aliquot, and the resin is then recombined. This process is repeated for each position. However, due to the physical process, each resin bead contains only one peptide sequence. Statistically, for mixtures of longer sequences, an ever-increasing amount of resin is necessary to ensure complete representation of each peptide in the library. Thus, each peptide will be represented in the library if a sufficient number of resin beads are used. In addition, the concentration of each peptide in the library depends on both the number of mixture positions in the library and the amount of resin used. In the second method, mixtures of amino acids are coupled simultaneously at each addition step. The proportion of each amino acid in the reaction mixture is varied inversely to its reaction rate such that, ideally, an equimolar mixture of each peptide is synthesized. An advantage of this method over the previous method is that each peptide is ensured to be represented in the library, although not necessarily in equimolar amounts. It is known that not only do the coupling rates of each amino acid vary, but the coupling rates of individual amino acids also change when coupled to different amino acid resins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed2000

Two-dimensional screening of the Wageningen chicken BAC library.

We have constructed a Bacterial Artificial Chromosome (BAC) library that provides 5.5-fold redundant coverage of the chicken genome. The library was made by cloning partial HindIII-digested high-molecular-weight (HMW) DNA of a female White Leghorn chicken into the HindIII site of the vector pECBAC1. Several modifications of standard protocols were necessary to clone efficiently large partial HindIII DNA fragments. The library consists of 49,920 clones arranged in 130 384-well plates. An average insert size of 134 kb was estimated from the analysis of 152 randomly selected BAC clones. The average number of NotI restriction sites per clone was 0.77. After individual growth, DNA was isolated of the pooled clones of each 384-well plate, and subsequently DNA of each plate was isolated from the individual row and column pools. Screening of the Wageningen chicken BAC library was performed by two-dimensional PCR with 125 microsatellite markers. For 124 markers at least one BAC clone was obtained. FISH experiments of 108 BAC clones revealed chimerism in less than 1%. The number of different BAC clones per marker present in the BAC library was examined for 35 markers which resulted in a total of 167 different BAC clones. Per marker the number of BAC clones varied from 1 to 11, with an average of 4.77. The chicken BAC library constitutes an invaluable tool for positional cloning and for comparative mapping studies.

Animals↗

PubMed2006

Construction of human ScFv phage display library against ovarian tumor.

In order to construct a single chain fragment variable (ScFv) phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes (VH) and light chain variable region genes (VL) were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E. coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 x 10(9) cfu/microg was obtained. After amplification with helper phage, the titer of antibody library reached 5 x 10(12) cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

Cell Surface Display Techniques↗

PubMed1985

Screening of lambda library for differentially expressed genes using in vitro transcripts.

We present an improved approach to the screening of eucaryotic libraries for differential gene expression. Previous techniques have generated probe via the harvesting of cellular poly(A)+ RNA and synthesizing labeled cDNA probe using reverse transcriptase. In our approach we prepare labeled RNA probe via in vitro transcription. Unlike cDNA preparation, in vitro transcription (i) directly reflects the ongoing rate of gene expression, and (ii) allows one to assess expression of genes whose transcripts are not polyadenylated. To make this approach practical for the screening of a large library, we modified and optimized existing in vitro transcription techniques, enhancing manyfold the [alpha-32P]UTP incorporation into mRNA, while almost completely suppressing rRNA incorporation. In addition, we developed a simple procedure for making precise replicate dot blots of very large quantities of lambda-phage library DNA. By combining our techniques of in vitro transcription and replicate blotting, we are able to approach detection of a twofold difference in gene expression over a greater than 1000-fold range in overall expression. Our single-clone amplification and dot-blotting technique resulted in nearly the same number of lambda-phage DNA copies per dot for all members of the library. This feature allows us to assign genes to different expression classes, as well as to detect any alterations in expression. We demonstrate our approach by screening the drosophila genomic DNA library with in vitro transcripts from drosophila tissue culture cells. Screening of the entire drosophila genomic library at the 99% probability level is readily achieved.

Animals↗

PubMed1994

Two-stage selection of sequences from a random phage display library delineates both core residues and permitted structural range within an epitope.

Libraries of random peptides can be screened to identify species which interact with antibodies or receptors. Similarly, maps of native molecular interactions can frequently be deduced by screening a limited set of peptide fragments derived from sequences within a native antigen or ligand. However, the existence of cross-reactive sequences that mimic original epitopes and the limited replaceability of amino acid residues suggest that the sequence space accessible by a receptor can be much broader. Definition of this space is of particular importance where structural information is required for peptidomimetic or drug design. We have used a two-stage selection scheme to expand the sequence space accessible by a phage display library and to define peptide epitopes of the anti-FLAG octapeptide monoclonal M2 antibody. Affinity selection of a primary library of 2 x 10(6) random decapeptides identified a non-contiguous core of three residues in the binding motif Tyr-Lys-Xaa-Xaa-Asp. A second stage library with 2 x 10(7) individual clones bearing the core motif but with the remaining flanking and internal residues re-randomized permitted access to a broader sequence space represented in a library equivalent to several orders of magnitude larger. Data here demonstrate that extended access to binding sequence space permitted by multi-stage screening of phage display libraries can reveal not only essential residues required for ligand binding, but also the ligand structural range permitted within the receptor binding pocket.

Amino Acid Sequence↗

PubMed1995

The use of dedicated peptide libraries permits the discovery of high affinity binding peptides.

The motif for peptide binding to monoclonal antibody mAb A16, which is known to be directed against glycoprotein D of Herpes simplex virus type 1, was determined using two dedicated peptide libraries. As a starting point for this study we used an A-16 binding lead sequence, which had previously been obtained from a phage display peptide library (Schellekens et al., 1994). Binding studies with different length variants of this peptide identified a 12mer as a suitable lead compound for our library study. Two incomplete dedicated resin-bound synthetic peptide libraries were generated. Both consisted of 2 x 10(6) 12mers, in which positions were alternately fixed (amino acids identical to the lead sequence) and random. The libraries were screened with mAb A16 and beads with binding peptides were sequenced using Edman degradation. This resulted in a unique peptide binding motif, essentially comprising a 7mer core sequence. Comparison of the sequence of the natural epitope with the binding motif revealed that its sequence was identical to the motif except for one position. Substitution of a methionine in the natural epitope by a tyrosine or a phenylalanine at that position, as dictated by the motif, resulted in a peptide with an affinity for binding to mAb A16 about 50 times higher than that of the natural epitope. Thus, if a lead sequence is available, the use of incomplete, dedicated synthetic peptide libraries provides a fast and powerful tool for the detection of high affinity peptides.

Amino Acid Sequence↗

PubMed1995

A case study in ontology library construction.

The goal of our work is to facilitate the development of medical knowledge-based systems by providing a library of reusable ontologies. The availability of such a library reduces the amount of knowledge acquisition required to create knowledge bases of new applications, and makes it easier to connect a knowledge-based system to existing data bases. This article presents a case study in constructing such a library. The emphasis is on studying the principles that underly the internal structure of the library as well as on the process of constructing and using the library. We envision that, in the future, application ontologies can be constructed by the selection and refinement of generic ontologies and domain ontologies from such a library.

Artificial Intelligence↗

PubMed1996

'Mutational SURF': a strategy for improving lead compounds identified from combinatorial libraries.

Synthesis and testing of mixtures of compounds in a combinatorial library offers the potential of much greater throughput than the 'one compound, one well' approach. When mixtures of compounds are screened, however, pooling and deconvolution strategies must be employed to identify the most active compound in the library. The possibility exists that the most active compound will not be identified. We have developed a theoretical model of library deconvolution using the well characterized properties of nucleic acid hybridization to calculate activities of individual molecules in libraries of more than 250,000 compounds. Calculations using this model have been employed to evaluate strategies for pooling and deconvolution. In the presence of errors in synthesis and testing, iterative deconvolution or position scanning sometimes identified a compound with sub-optimal activity. We describe a procedure called 'mutational SURF' in which 'mutants' of the selected compound are individually synthesized and tested. Simulations of mutational SURF using our model libraries suggest that mutational SURF provides an efficient method for improving the activity of lead compounds identified from combinatorial libraries.

Base Sequence↗

PubMed2006

On the risk of false positive identification using multiple ion monitoring in qualitative mass spectrometry: large-scale intercomparisons with a comprehensive mass spectral library.

Analysts involved in qualitative mass spectrometry have long debated the minimum data requirements for demonstrating that signals from an unknown sample are identical to those from a known compound. Often this process is carried out by comparing a few selected ions acquired by multiple ion monitoring (MIM), with due allowance for expected variability in response. In a few past experiments with electron-ionization mass spectrometry (EI-MS), the number of ions selected and the allowable variability in relative abundance were tested by comparing one spectrum against a library of mass spectra, where library spectra served to represent potential false positive signals in an analysis. We extended these experiments by carrying out large-scale intercomparisons between thousands of spectra and a library of one hundred thousand EI mass spectra. The results were analyzed to gain insights into the identification confidence associated with various numbers of selected ions. A new parameter was investigated for the first time, to take into account that a library spectrum with a different base peak than the search spectrum may still cause a false positive identification. The influence of peak correlation among the specific ions in all the library mass spectra was also studied. Our computations showed that (1) false positive identifications can result from similar compounds, or low-abundance peaks in unrelated compounds if the method calls for detection at very low levels; (2) a MIM method's identification confidence improves in a roughly continuous manner as more ions are monitored, about one order of magnitude for each additional ion selected; (3) full scan spectra still represent the best alternative, if instrument sensitivity is adequate. The use of large scale intercomparisons with a comprehensive library is the only way to provide direct evidence in support of these conclusions, which otherwise depend on the judgment and experience of individual analysts. There are implications for residue chemists who would rely on standardized confirmation criteria to assess the validity of a given confirmatory method. For example, standardized confirmation criteria should not be used in the absence of interference testing and rational selection of diagnostic ions.

Journal Article↗

PubMed2003

Designing repeat proteins: modular leucine-rich repeat protein libraries based on the mammalian ribonuclease inhibitor family.

We present a novel approach to design repeat proteins of the leucine-rich repeat (LRR) family for the generation of libraries of intracellular binding molecules. From an analysis of naturally occurring LRR proteins, we derived the concept to assemble repeat proteins with randomized surface positions from libraries of consensus repeat modules. As a guiding principle, we used the mammalian ribonuclease inhibitor (RI) family, which comprises cytosolic LRR proteins known for their extraordinary affinities to many RNases. By aligning the amino acid sequences of the internal repeats of human, pig, rat, and mouse RI, we derived a first consensus sequence for the characteristic alternating 28 and 29 amino acid residue A-type and B-type repeats. Structural considerations were used to replace all conserved cysteine residues, to define less conserved positions, and to decide where to introduce randomized amino acid residues. The so devised consensus RI repeat library was generated at the DNA level and assembled by stepwise ligation to give libraries of 2-12 repeats. Terminal capping repeats, known to shield the continuous hydrophobic core of the LRR domain from the surrounding solvent, were adapted from human RI. In this way, designed LRR protein libraries of 4-14 LRRs (equivalent to 130-415 amino acid residues) were obtained. The biophysical analysis of randomly chosen library members showed high levels of soluble expression in the Escherichia coli cytosol, monomeric behavior as characterized by gel-filtration, and alpha-helical CD spectra, confirming the success of our design approach.

Amino Acid Sequence↗

PubMed1999

Physico-chemical and biological analysis of true combinatorial libraries.

Combinatorial libraries offer new sources of compounds for the research of pharmacological agents such as receptor ligands, enzyme inhibitors or substrates and antibody-binding epitopes. The present review stresses the main roles played by both physico-chemical analysis, particularly when complex mixture of compounds are synthesized as libraries, and biological analysis from which active compounds are identified. After a brief discussion of semantic problems related to the designation of the product mixtures, the physico-chemical analysis of mixtures is reviewed with special emphasis on mass spectrometric techniques. These methods are able both to give a representative view of a library composition and to identify single critical compounds in large libraries. Then the biological screening of such combinatorial libraries is critically discussed with respect to the power and limitations of the methods used for the identification of the active components. Special attention is given to the complex process of library deconvolution. It is pointed out that while combinatorial techniques have evolved towards sophisticated high-tech methods, simple and robust biochemical tests should be used to deconvolute. From a large panel of published examples, a set of trends are identified which should help investigators to choose the most appropriate assay for the discovery of new entities.

Chemistry, Pharmaceutical↗

PubMed2002

Cocaine detoxification by combinatorially substituted beta-cyclodextrin libraries.

Per-6-substituted- and A,C,E(F)-tri-6-substituted-6-deoxy-beta-cyclodextrin (beta-CD) libraries were generated using solution-phase combinatorial chemistry techniques starting from the corresponding iodo precursors and different combinations of individual amine nucleophiles. Using a high throughput electrospray mass spectrometry (ESMS) screen to monitor the hydrolysis of cocaine, certain libraries showed the ability to specifically hydrolyze the methyl ester of cocaine, with the most active per-6-substituted beta-CD library I producing complete hydrolysis in 24h. The cocaine hydrolytic activity in this series showed structure-activity relationships which appeared to involve specific interaction between the amine side chains and the cocaine molecule. Comparison of the composition of the most active per-6-substiuted beta-CD libraries and A,C,E(F)-tri-6-substituted-6-deoxy-beta-CD libraries (I and XV) showed three common side chains (3, 4, and 5), suggesting that active side chains in the tri-substituted beta-CD library might be predicted from evaluation of the more easily prepared per-6-substituted-6-deoxy-beta-CD series.

Cocaine↗

PubMedDate not supplied

Designing targeted libraries with genetic algorithms.

In combinatorial synthesis, molecules are assembled by linking chemically similar fragments. Because the number of available chemical fragments often greatly exceeds the number that can be used in one synthetic experiment, one needs a rational method for choosing a subset of desirable fragments. If a combinatorial library is to be targeted against a particular biological activity, virtual screening methods can be used to predict which molecules in a virtual library are most likely to be active. When the number of possible molecules in a virtual library is very large, genetic algorithms (GAs) or simulated annealing can be used to quickly find high-scoring molecules by sampling a small subset of the total combinatorial space. We previously demonstrated how a GA can be used to select a subset of fragments for a combinatorial library, and we used topology-based methods of scoring. Here we extend that earlier work in three ways. (1) We demonstrate use of the GA with 3D scoring methods developed in our laboratory. (2) We show that the approach of assembling libraries from fragments in high-scoring molecules is a reasonable one. (3) We compare results from a library-based GA to those from a molecule-based GA.

Algorithms↗

PubMed2006

Emerging chemical and biological approaches for the preparation of discovery libraries.

The term of combinatorial chemistry has come to embrace all types of small-molecule library strategies. In both academic and industrial settings, the development of many combinatorial techniques has been driven by a desire to generate diverse libraries of molecules. Recently, chemical approaches have been reported that range from alternative library design concepts to new synthetic procedures that allow for selective product formation in extremely short reaction times. Concurrently, biological techniques have made great strides by adapting naturally occurring strategies for library preparation and screening. With proper understanding of library design, combinatorial endeavors and molecular libraries will continue to impact on the development of the next generation of tools for modern medicine.

Combinatorial Chemistry Techniques↗

PubMed2000

Evaluation of electrospray transport CID for the generation of searchable libraries.

This paper reports the preliminary investigation of performance-based standard conditions that have been developed for electrospray ionization mass spectrometry. Using performance-based standard criteria, reproducible spectra can be obtained by CID in the electrospray-transport region and searched using a database created using the same performance criteria. To generate library-searchable mass spectra, the instrument was tuned to standard conditions that correspond to low, mid, and high fragmentation energies. The instrument was tuned using ion ratios relative to a given peak in a tune compound for each energy level. The library was evaluated using a set of 22 benzodiazepines. The CID libraries were found to be reproducible, both on the same instrument and on different instruments of the same type. Also, the libraries were found to be independent of flow rate and solvent system. The library was expanded to include 16 sulfonylurea herbicides and tested using spiked water samples. Performance of the library was tested over a concentration range of 2 orders of magnitude using sulfonylurea standards.

Benzodiazepines↗

PubMedDate not supplied

Combinatorial library of peptide isosters based on Diels-Alder reactions: identification of novel inhibitors against a recombinant cysteine protease from Leishmania mexicana.

A combinatorial split-and-mix library of peptide isosters based on a Diels-Alder reaction was synthesized as a "one-bead-two-compounds" library and encoded by ladder synthesis for facile analysis by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. In the "one-bead-two-compounds" library approach, each bead contains a library member as a putative protease inhibitor along with a fluorescence-quenched substrate for the protease. When the library was screened with CPB2.8 DeltaCTE, a recombinant cysteine protease from L. mexicana, several beads containing compounds with inhibitory activity could be selected from the library and analyzed by MALDI-TOF MS for structure elucidation. Two types of inhibitors were revealed. One novel class of inhibitors had the bicyclic Diels-Alder product isosteric element incorporated internally in a peptide, while the other type was an N-terminal alpha,beta-unsaturated ketone Michael acceptor used as starting material for the Diels-Alder reaction. Selected hit sequences and constructed consensus sequences based on the observed frequencies of amino acids in different subsites were resynthesized and assayed in solution for inhibitor activity and were shown to have IC(50) values in the high nanomolar to low micromolar range.

Animals↗

PubMedDate not supplied

Synthesis and screening of libraries of synthetic tripodal receptor molecules with three different amino acid or peptide arms: identification of iron binders.

A novel selectively deprotectable triazacyclophane scaffold was used for the design and split-mix synthesis of two libraries of solid-phase bound tripodal synthetic receptors possessing three different amino acid or peptidic arms. In the synthesis of the first library, the two outer arms consisted of amino acid Ala, Arg, Asp, Gln, Gly, Lys, Phe, Ser, Tyr, or Val and the middle arm consisted of amino acid Asn, Glu, His, Leu, or Pro. The second library contained amino acid and/or (di)peptide arms. The arms were different in all library members. The first outer arm consisted of amino acid(s) Ala, Arg, Gln, Phe, or Ser, the second outer arm consisted of amino acid(s) Asp, Gly, Lys, Tyr, or Val, and the middle arm consisted of amino acid(s) Asn, Glu, His, Leu, or Pro, leading to a 27 000 member library of synthetic tripodal receptor molecules. In on-bead screening experiments, a remarkable selectivity of some library members for Fe(3+) was observed and decoding of their structures by Edman degradation revealed consensus sequences with structural resemblance to non-heme iron proteins.

Amino Acids↗

PubMedDate not supplied

Design and synthesis of a diverse morpholine template library.

Efficient and general procedures have been developed for the solution-phase preparation of substituted morpholine derivatives, and a library has been produced around generic structure 1. This library was designed with proprietary modeling software for use as a general screening library. The 30 R1 reagents were phenols, and the 275 R2 reagents were taken from five different reagent classes, giving a variety of product classes in the final library of 8250 potential products. All of the library members were generated from a common intermediate, mesylate (5), which was synthesized efficiently, in bulk, in three steps from N-benzylethanolamine (2). High-throughput chemistry using robotics was carried out to produce the 7907 library members, which were individually characterized by reversed-phase LC/MS analysis.

Aldehydes↗