Synthetic Combinatorial Libraries Research Articles

Studies on the synthesis and applications of synthetic oligonucleotide combinatorial libraries

Studies on the synthesis and applications of synthetic oligonucleotide combinatorial libraries

Rapid identification of compounds with enhanced antimicrobial activity by using conformationally defined combinatorial libraries.

We have combined the strength of our synthetic combinatorial library approach for the rapid identification of highly active compounds with prior knowledge of the relationship between the antimicrobial activities of individual peptides with specific induced conformations in order to identify new peptides with enhanced activity relative to a starting known antimicrobial sequence. In the current study, conformationally defined combinatorial libraries were generated based on an 18-mer antimicrobial peptide known to be induced into an alpha-helical conformation in a lipidic environment. Not only were novel sequences readily identified with 10-fold increases in activity, but detailed information about the structure-activity relationships of the peptides studied was also obtained during the deconvolution process. By using circular dichroism spectroscopy it was found that the individual 18-mer peptides could be induced into alpha-helical conformations on interaction with the cell lipid layer and/or sialic acids, which could result in bacterial cell lysis due to perturbation of the lipid packing of the cell wall.

Tea bag synthesis of positional scanning synthetic combinatorial libraries and their use for mapping antigenic determinants.

AbstractPositional scanning synthetic combinatorial libraries (PS-SCLs) are useful in identifying the amino acid sequences of antigenic determinants recognized by monoclonal antibodies (MAbs). These libraries are typically composed, in total, of tens of millions of nonsupport-bound peptides, and can be readily screened using enzyme-linked immunosorbent assay (ELISA) to determine specific sequences that bind to a target antibody. From our studies using peptide libraries, we have found that MAbs exhibit a broad range of specificities, ranging from those recognizing only conservative substitutions at one or two positions in the antigenic determinant to antibodies that recognize sequences completely unrelated to the parent antigen while having comparable affinities (1–10). The information derived from the screening of PS-SCLs is similar to that of “fingerprint” profiles using individual substitution analogs at each position of the antigenic determinant (11). For example, the more specific a position is, the fewer amino acids in the peptide mixtures that will be found to be effective. On the other hand, if there are many active peptide mixtures for a given defined position, then that position can be considered redundant or replaceable. In other instances, there may be several amino acids that are conservative replacements of each other that have equal activities. Additionally, since the diversities of PS-SCLs consist of millions of sequences, the extent of the multiple binding specificities of a given antibody can be readily addressed in a systematic manner. Finally, it should be noted that a single PS-SCL can be used to identify antigenic determinants and high-affinity sequences for many different antibodies.KeywordsAntigenic DeterminantPeptide MixturePeptide LibraryCompetitive ELISAIndividual PeptideThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Identification of a hexapeptide inhibitor of the human immunodeficiency virus integrase protein by using a combinatorial chemical library.

Integration of human immunodeficiency virus (HIV) DNA into the human genome requires the virus-encoded integrase (IN) protein, and therefore the IN protein is a suitable target for antiviral strategies. To find a potent HIV IN inhibitor, we screened a "synthetic peptide combinatorial library." We identified a hexapeptide with the sequence HCKFWW that inhibits IN-mediated 3'-processing and integration with an IC50 of 2 microM. The peptide is active on IN proteins from other retroviruses such as HIV-2, feline immunodeficiency virus, and Moloney murine leukemia virus, supporting the notion that a conserved region of IN is targeted. The hexapeptide was also tested in the disintegration reaction. This phosphoryl-transfer reaction can be carried out by the catalytic core of IN alone, and the peptide HCKFWW was found to inhibit this reaction, suggesting that the hexapeptide acts at or near the catalytic site of IN. Identification of an IN hexapeptide inhibitor provides proof of concept for the approach, and, moreover, this peptide may be useful for structure-function analysis of IN.

Peptide, peptidomimetic, and organic synthetic combinatorial libraries

Medicinal Research ReviewsVolume 15, Issue 6 p. 481-496 Article Peptide, peptidomimetic, and organic synthetic combinatorial libraries Jutia Eichler, Jutia Eichler Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorJon R. Appel, Jon R. Appel Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorSylvie E. Blondelle, Sylvie E. Blondelle Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorColette T. Dooley, Colette T. Dooley Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorBarbara Döurner, Barbara Döurner Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorJohn M. Ostresh, John M. Ostresh Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorEnrique Péarez-Payá, Enrique Péarez-Payá Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorClemencia Pinilla, Clemencia Pinilla Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorRichard A. Houghten, Corresponding Author Richard A. Houghten Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121 Phone: 619–455-3803; fax: 619–455–3804Search for more papers by this author Jutia Eichler, Jutia Eichler Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorJon R. Appel, Jon R. Appel Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorSylvie E. Blondelle, Sylvie E. Blondelle Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorColette T. Dooley, Colette T. Dooley Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorBarbara Döurner, Barbara Döurner Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorJohn M. Ostresh, John M. Ostresh Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorEnrique Péarez-Payá, Enrique Péarez-Payá Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorClemencia Pinilla, Clemencia Pinilla Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Search for more papers by this authorRichard A. Houghten, Corresponding Author Richard A. Houghten Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121 Phone: 619–455-3803; fax: 619–455–3804Search for more papers by this author First published: November 1995 https://doi.org/10.1002/med.2610150603Citations: 31AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume15, Issue6November 1995Pages 481-496 RelatedInformation

Blocking of CD4 cell receptors for the human immunodeficiency virus type 1 (HIV-1) by chemically modified bovine milk proteins: potential for AIDS prophylaxis.

The chemical transformation of synthetic combinatorial libraries to increase the diversity of compounds of medicinal interest was reported recently. Chemical modification of natural products represents a complementary approach to accomplish this aim. Modification of lysines by aromatic acid anhydrides, preferentially by 3-hydroxyphthalic and trimellitic anhydrides and trimellitic anhydride chloride, converted commonly available proteins (human and bovine serum albumin and casein) into potent inhibitors of (i) binding between the HIV-1 gp 120 envelope glycoprotein and the CD4 cell receptor, probably owing to their binding to CD4, and (ii) infection by HIV-1. Modified bovine milk proteins are also potent HIV-1 inhibitors and may have potential for anti-HIV-1 prophylaxis.

Mapping the detailed specificity of a calcium-dependent monoclonal antibody through the use of soluble positional scanning combinatorial libraries: Identification of potent calcium-independent antigens

The detailed specificity of monoclonal antibody M1, which has been reported to bind in a calcium-dependent manner to the 'FLAG' sequence DYKDDDDK-NH2, was examined using soluble hexa- and decapeptide positional scanning synthetic combinatorial libraries (PS-SCLs) made up of 52 x 10(6) and 4 x 10(12) different sequences, respectively. To study the influence of calcium on the specificity of this antigen-antibody interaction, each PS-SCL was screened in the presence and absence of calcium using a competitive ELISA. Overall, peptide mixtures had greater inhibitory activity against mAb M1 binding to FLAG in the absence of calcium. A total of 16 individual hexapeptides were identified, all of which contained the motif-DYK_K_(-), and were recognized by mAb M1 in the absence of calcium with 50- to 100-fold higher affinity than the FLAG octapeptide (IC50 = 273 nM). On average, the same set of peptides bound 10-fold less effectively in the presence of calcium. Upon screening the decapeptide PS-SCL in the absence of calcium, lysine was also more active in the fifth position than the original aspartic acid. Based on the screening results, 24 individual decapeptides were prepared and were found to have activities 10- to 100-fold higher than the FLAG octapeptide in the absence of calcium. The specificity of lysine at the fifth position in the antigen-antibody interaction was further examined by synthesizing and assaying substitution analogs at this position for the octapeptide and hexapeptide forms of the FLAG sequence, as well as for two hexapeptides identified from the PS-SCL. Truncation analog analysis was also carried out on the FLAG octapeptide to determine optimal antigen length for antibody binding. Overall, lysine at the fifth position could be substituted with ornithine with no significant loss in activity, and peptide length was not a critical factor for antibody binding in the absence of calcium. Also, the octapeptide having lysine at the fifth position in place of the aspartic acid had the same activity in the presence or absence of calcium. This study demonstrates the ease and effectiveness of PS-SCLs over individual peptide analogs for the examination of the degree of cross-reactivity for a given monoclonal antibody as well as for the identification of novel, high-affinity peptides.

Library of libraries: approach to synthetic combinatorial library design and screening of "pharmacophore" motifs.

Construction of synthetic combinatorial libraries is described that allows for the generation of a library of motifs rather than a library of compounds. Peptide libraries based on this strategy were synthesized and screened with model targets streptavidin and anti-beta-endorphin antibody. The screens resulted in observation of expected motifs providing evidence of the effectiveness of the suggested approach.

Synthetic combinatorial libraries: Views on techniques and their application

This paper reviews synthetic combinatorial libraries. Particular emphasis is placed on one-bead-one-compound libraries, although issues relating to all synthetic techniques are touched upon. The discussion is focused on questions relating to synthetic diversity, drug discovery assays and structure determination techniques.

Kinetic characterization of a peptide inhibitor of trypsin isolated from a synthetic peptide combinatorial library

Recently Eichler and Houghten 1 have described the use of a combinatorial peptide library to optimize small trypsin inhibitors. Our kinetic analysis of the most inhibitory peptide, YYGAKIYRPDKM, reveals that it is an inefficiently cleaved substrate for trypsin due to a low k cat. possesses a relatively low K M, and may exhibit nonproductive binding.

The antimicrobial activity of hexapeptides derived from synthetic combinatorial libraries

A series of peptides identified through the use of synthetic hexapeptide combinatorial libraries (represented by the formula Ac-RRWWCO-NH2) were examined for their antimicrobial activity against five different micro-organisms. Their toxicity was also evaluated in an in vitro haemolytic assay. The peptides showed activity against the five micro-organisms, although higher activities were found against Gram-positive bacteria. Both growth inhibition and cell viability assays were carried out to demonstrate the bactericidal activities of these peptides against two of the micro-organisms tested. The dimeric cystine forms of these peptides were shown to have biological activities identical to the monomeric forms.

Deconvolution of combinatorial libraries for drug discovery: a model system.

Iterative synthesis and screening strategies have recently been used to identify unique active molecules from complex synthetic combinatorial libraries. These techniques have many advantages over traditional screening methods, including the potential to screen large numbers of compounds to identify an active molecule while avoiding analytical separations and structural determination of unknown compounds. It is not clear, however, whether these techniques identify the most active molecular species in the mixtures and, if so, how often. Two key factors which may affect success of the selection process are the presence of many active compounds in the library with a range of activities and the chosen order of unrandomization. The importance of these factors has not been previously studied. Moreover, the impact of experimental errors in determination of subset activities or in randomization during library synthesis is not known. We describe here a model system based on oligonucleotide hybridization that addresses these questions using computer simulations. The results suggested that, within achievable experimental and library synthesis error, iterative deconvolution methods generally find either the best molecule or one with activity very close to the best. The presence of many active compounds in a library influenced the profile of subset activities, but did not preclude selection of a molecule with near optimal activity.

Application of a dual color detection scheme in the screening of a random combinatorial peptide library

Selectide Technology is a random synthetic combinatorial library method in which millions of random compounds are screened in parallel for their ability to bind to a tagged macromolecular target. The library consists of millions of beads and each individual bead expresses a unique chemical compound such as a peptide. In the standard enzyme-linked colorimetric detection scheme, the positive bead which turns color is isolated for microsequencing. In this paper, a dual color detection scheme using two sequential orthogonal probes is described. This dual color system enables one to rapidly differentiate false positive beads from true positive beads, resulting in a much more efficient use of the microsequencer.

A review of the utility of soluble peptide combinatorial libraries

This paper reviews the preparation and use of soluble synthetic combinatorial libraries (SCLs) made up of millions of peptide and nonpeptide sequences for the identification of highly active individual compounds. First presented in 1991, SCLs have been prepared in a number of different lengths and formats, and are composed entirely of L-, D-, and unnatural amino acids. Also, existing peptide libraries have been chemically transformed to yield large diversities of nonpeptidic compounds. This review encompasses the published work from this laboratory using SCLs for the identification of antigenic sequences recognized by monoclonal antibodies, novel peptide agonists and antagonists to opioid receptors, new trypsin inhibitors, novel antibacterials, and compounds that inhibit melittin's hemolytic activity. SCLs offer a fundamental, practical advance in the study of interactions between peptide and nonpeptide sequences and their biochemical or pharmacological targets.

Farmer's peptidomimetic approach: A target for synthetic combinatorial libraries

Two combinatorial libraries of 1296 compounds each were synthesized from two sets of carboxylic acid building blocks and two diamino acid scaffolds. The library was designed to produce low-molecular-weight compounds in a soluble form, to be assayed as potential ligands for peptidergic receptors.

An All D-Amino Acid Opioid Peptide with Central Analgesic Activity from a Combinatorial Library

A synthetic combinatorial library containing 52,128,400 D-amino acid hexapeptides was used to identify a ligand for the mu opioid receptor. The peptide, Ac-rfwink-NH2, bears no resemblance to any known opioid peptide. Simulations using molecular dynamics, however, showed that three amino acid moieties have the same spatial orientation as the corresponding pharmacophoric groups of the opioid peptide PLO17. Ac-rfwink-NH2 was shown to be a potent agonist at the mu receptor and induced long-lasting analgesia in mice. Analgesia produced by intraperitoneally administered Ac-rfwink-NH2 was blocked by intracerebroventricular administration of naloxone, demonstrating that this peptide may cross the blood-brain barrier.

Identifying substrate motifs of protein kinases by a random library approach.

Protein phosphorylation is an important posttranslational modification process that plays a crucial role in signal transduction. There are many protein kinases involved in cell signaling. However, substrate motifs of many protein kinases in signal transduction are not well-known. Traditional methodologies for identifying these motifs are often difficult and inefficient. In the present study, we developed a novel approach for discovering linear substrate motifs of protein kinases. This method is based on the screening of random synthetic combinatorial peptide libraries on beads where each bead expresses only one peptide entity [Lam et al. (1991) Nature 354, 82-84]. We chose cyclic AMP-dependent protein kinase (cAPK) as a model system in the present study since it is a well-studied enzyme. Random pentapeptide and heptapeptide libraries were screened with the addition of [gamma-32P]ATP and cAPK. 32P-Labeled peptide-beads were then isolated for microsequencing. The identified peptide motif for cAPK was RRXS and is identical to that reported in the literature. Kinetic studies of the best three peptides indicate that they are efficient substrates for cAPK discovered from random synthetic combinatorial peptide libraries. Our results also suggest that this method is potentially useful for identifying substrate motifs of various protein kinases with high sensitivity and specificity. In addition, this method can also be used as a general method for identifying linear substrate motifs for various posttranslational modifications.

Electrospray Mass Spectrometry and Multiple Sequence Analysis of Synthetic Peptide Libraries

The determination of the composition and purity of synthetic combinatorial libraries of free peptides requires analytical techniques that are especially suited for the challenging task of mixture analysis. Qualitative and semi-quantitive information on the composition of complex mixtures can be obtained by electrospray mass spectrometry with a maximum of reliability, accuracy, and fastness. Especially in combination with tandem mass spectrometry, contaminations with by-products (e.g., peptides with uncleaved side-chain protecting groups) can be easily revealed. Multiple sequence analysis based on Edman degradation allows a quantitative determination of the composition of libraries as well as the determination of coupling efficiencies during the synthesis of a library.

Rapid identification of novel opioid peptides from an N-acetylated synthetic combinatorial library

Rapid identification of novel opioid peptides from an N-acetylated synthetic combinatorial library

Identification of antimicrobial peptides by using combinatorial libraries made up of unnatural amino acids

The use of water-soluble synthetic peptide combinatorial libraries permits the systematic examination of tens to hundreds of millions of peptides in existing microdilution assays. In the present study, we prepared and determined the antistaphylococcal activities of two new synthetic peptide combinatorial libraries (one N-acetylated, the other not) composed of tetrapeptides having one position defined and the remaining three positions made up of mixtures of L-, D-, and unnatural amino acids (a total of 58 different amino acids). These libraries, when used in conjunction with an iterative selection process, allowed for the development of a series of individually defined tetrapeptides with high levels of activity against Staphylococcus aureus. The activities of the final individual peptides against two additional strains of gram-positive bacteria (methicillin-resistant S. aureus and Streptococcus sanguis), a gram-negative bacterium (Escherichia coli), as well as the yeast Candida albicans were also determined. Cell viability assays showed that the identified peptides are bacteriostatic against both S. aureus and E. coli.