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At least 415 records · Page 23Linked to original sources
PubMed1998

Tight-binding streptavidin ligands from a cyclic peptide library.

During the screening of a soluble library of cyclo(AXXXXXAE)K-CONH2, a cyclic peptide cyclo(AHPQFPAE)K-CONH2 was identified as a tight-binding ligand (IC50 = 128 nM) and found to bind 1000-fold more tightly than its linear peptide to streptavidin. The results of this study suggest that library screening of conformationally constrained cyclic peptides can be an effective means for the discovery of high affinity ligands.

Amino Acid Sequence↗
PubMed2000

Selection of estrogen receptor beta- and thyroid hormone receptor beta-specific coactivator-mimetic peptides using recombinant peptide libraries.

Steroid and thyroid hormone receptors are members of the superfamily of nuclear receptors (NR) that participate in developmental and homeostatic mechanisms by changes in the transcription of specific genes. These activities are governed by the receptors' cognate ligands and through interaction with the components of the transcriptional machinery. A number of coactivator molecules of the steroid receptor coactivator (SRC)/nuclear receptor coactivator (NCoA) family interact with activation functions within NRs through a conserved region containing helical domains of a core LXXLL sequence and, thereby, participate in transcriptional regulation. Using a mammalian-two-hybrid assay, we show that the thyroid hormone receptor beta (TRbeta) and estrogen receptor beta (ERbeta) have different LXXLL motif preferences for interactions with SRC-1. Using large random and focused (centered on the LXXLL motif) recombinant peptide diversity libraries, we have obtained novel peptide sequences that interact specifically with ERbeta or with TRbeta in a ligand-dependent manner. Random sequence libraries yielded LXXLL-containing peptides, and sequence analysis of selected clones revealed that the preferred residues within and around the LXXLL motif vary significantly between these two receptors. We compared the receptor binding of library-selected peptides to that of peptides derived from natural coactivators. The affinities of selected peptides for the ligand binding domains of ERbeta and TRbeta were similar to the best natural LXXLL motifs tested, but showed a higher degree of receptor selectivity. These selected peptides also display receptor-selective dominant inhibitory activities when introduced into mammalian cells. Finally, by directed mutations in specific residues, we were able to alter the receptor binding preference of these peptides.

Amino Acid Motifs↗
PubMed1996

High-resolution solution structure of two members of a conformationally homogeneous combinatorial peptide library based on the classical zinc-finger motif.

We describe the high-resolution structure by NMR of two peptides that belong to a combinatorial library based on the zinc-finger motif. The library represents, to the best of our knowledge, the first example of a conformationally homogeneous peptide library and was obtained by introducing random residues in five positions of the alpha-helical portion of a 26-residue 'consensus' peptide (CP1) belonging to the Cys(2)-Hys2 zinc-finger family. The result was shown to be a highly homogeneous alpha-helical library (Bianchi et al., 1995). The structures of the parent compound (CPI) and of a representative member (CPlm) that was selected by screening the library with a monoclonal antibody are compared in detail as an example of the very high stability of the zinc-finger scaffold upon sequence variability. The two peptides exhibit an extremely high degree of structural similarity. The use of this type of conformationally constrained combinatorial library might represent a step forward in the design of peptidomimetics, as it considerably accelerates the process of the identification of the spatial relationship among the pharmacophoric groups.

Magnetic Resonance Spectroscopy↗
PubMed2006

A gradient descent algorithm for minimizing amino acid coupling reactions when synthesizing cyclic-peptide libraries.

Combinatorial chemistry has become an invaluable tool in medicinal chemistry for the identification of new drug leads. For example, libraries of predetermined sequences and head-to-tail cyclized peptides are routinely synthesized in our laboratory using the IRORI approach. Such libraries are used as molecular toolkits that enable the development of pharmacophores that define activity and specificity at receptor targets. These libraries can be quite large and difficult to handle, due to physical and chemical constraints imposed by their size. Therefore, smaller sub-libraries are often targeted for synthesis. The number of coupling reactions required can be greatly reduced if the peptides having common amino acids are grouped into the same sub-library (batching). This paper describes a schedule optimizer to minimize the number of coupling reactions by rotating and aligning sequences while simultaneously batching. The gradient descent method thereby reduces the number of coupling reactions required for synthesizing cyclic peptide libraries. We show that the algorithm results in a 75% reduction in the number of coupling reactions for a typical cyclic peptide library.

Algorithms↗
PubMed2003

Isolation of lung tumor specific peptides from a random peptide library: generation of diagnostic and cell-targeting reagents.

Discovery of ligands specific to receptor(s) on a surface of a cancer cell could impact clinical issues including functional diagnosis and cell-specific drug delivery. Using a phage display approach, we have isolated 20-mer peptide ligands that bind to 3 different human lung tumor cell lines, NCI-H1299, NCI-H2009, and A549. The panning protocol is unbiased with no selection pressure towards binding a particular cellular receptor. The isolated phage bind to their target cells 24-300 times better than a control phage. Furthermore, the isolated peptides display remarkable cell-specificities and are able to discriminate between normal and cancerous cells as well as different lung tumor cells. The cell-specificities are not coincident with tumor classes indicating that the peptides are able to recognize cell-surface features that are not represented within the classification of tumor type. The isolated peptides are functional outside of the context of the phage and multimerization of the peptide increases its affinity for its given cell type, thus expanding their utility in clinical situations.

Biomarkers, Tumor↗
PubMed2004

Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer.

OBJECTIVE: Prostate cancer cells secrete the unique protease human glandular kallikrein 2 (hK2) that represents a target for proteolytic activation of cytotoxic prodrugs. The objective of this study was to identify hK2-selective peptide substrates that could be coupled to a cytotoxic analogue of thapsigargin, a potent inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase pump that induces cell proliferation-independent apoptosis through dysregulation of intracellular calcium levels. METHODS: To identify peptide sequence requirements for hK2, a combination of membrane-bound peptides (SPOT analysis) and combinatorial chemistry using fluorescence-quenched peptide substrates was used. Peptide substrates were then coupled to 8-O-(12[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin (L12ADT), a potent analogue of thapsigargin, to produce a prodrug that was then characterized for hK2 hydrolysis, plasma stability, and in vitro cytotoxicity. RESULTS: Both techniques indicated that a peptide with two arginines NH2-terminal of the scissile bond produced the highest rates of hydrolysis. A lead peptide substrate with the sequence Gly-Lys-Ala-Phe-Arg-Arg (GKAFRR) was hydrolyzed by hK2 with a Km of 26.5 micromol/L, kcat of 1.09 s(-1), and a kcat/Km ratio of 41,132 s(-1) mol/L(-1). The GKAFRR-L12ADT prodrug was rapidly hydrolyzed by hK2 and was stable in plasma, whereas the GKAFRR-L peptide substrate was unstable in human plasma. The hK2-activated thapsigargin prodrug was not activated by cathepsin B, cathepsin D, and urokinase but was an excellent substrate for plasmin. The GKAFRR-L12ADT was selectively cytotoxic in vitro to cancer cells in the presence of enzymatically active hK2. CONCLUSION: The hK2-activated thapsigargin prodrug represents potential novel targeted therapy for prostate cancer.

Amino Acid Sequence↗
PubMed1997

Mimotopes of polyreactive anti-DNA antibodies identified using phage-display peptide libraries.

Three monoclonal IgG2a anti-DNA polyreactive autoantibodies, derived from lupus-prone mice (NZB x NZW)F1, were studied by surface plasmon resonance (BIAcore) analysis using three different synthetic double-stranded (ds) oligonucleotides of 25, 30, and 25 base pairs (bp). These monoclonal antibodies (mAb) exhibited dissociation rate constants (k(off)), ranging from 0.0001 (mAb F14.6 and F4.1) to 0.01/s (mAb J20.8) and k(on) ranging from 2 x 10(5) to 2 x 10(6) /M/s. The screening of a constrained random peptide library displayed on M13 bacteriophages on these mAb allowed the determination of the specific consensus motifs (mimotopes) for mAb F14.6 and J20.8, but not for mAb F4.1. No cross-reaction was observed between F14.6- and J20.8-specific peptides (and vice versa). Binding of all phages selected on F14.6 was inhibited with 700 ng/ml soluble DNA. The binding of one group of peptides selected on J20.8 was inhibited by 400 ng/ml soluble DNA, of a second group by 2500 ng/ml, while binding of a third group could not be inhibited. The determined consensus sequences do not match with known sequences. Peptides specific for F14.6 share negative charges and aromatic rings that may mimic a DNA backbone, while peptides selected with J20.8 do not bear any negative charge, implying a different kind of molecular recognition, for example hydrogen or salt bonds. The peptides selected on J20.8 also bind serum antibodies from human patients with systemic lupus erythematosus. In addition, BALB/c mice immunized with some of the selected phages exhibit high serum titers of IgG3 anti-dsDNA antibodies, further supporting the hypothesis that peptide epitopes may mimic an oligonucleotide structure.

Amino Acid Sequence↗
PubMed2005

Isolation of a mycoplasma-specific binding peptide from an unbiased phage-displayed peptide library.

An important goal in medicine is the development of methods for cell-specific targeting of therapeutic molecules to pathogens or pathogen-infected cells. However, little progress has been made in cell-specific targeting of bacterially infected cells. Using a phage display approach, we have isolated a 20-mer peptide that binds to Mycoplasma arginini infected pancreatic beta-cells in tissue culture. This peptide binds to M. arginini infected beta-cells 200 times better than a control phage and is specific for the infected cells. Furthermore, transferring the M. arginini contamination to another cell line renders the newly infected cell line susceptible to peptide binding. Immunolocalization experiments suggest that the peptide is binding to M. arginini adhered to the cell surface. The free synthetic peptide retains its binding in the absence of the phage vehicle and tetramerization of the peptide increases its affinity for the infected cells. Efforts have been made to use this peptide to eliminate Mycoplasma from infected cell lines using ferromagnetic beads coated with the selected peptide. A ten-fold reduction of infection was accomplished with one fractionation via this approach. Our results suggest that this peptide, isolated from an unbiased selection, may be of utility for the detection and reduction of Mycoplasma infection in cultured cells. Furthermore, a general implication of our findings is that phage display methods may be useful for identifying peptides that target a broad array of other biological pathogens in a specific fashion.

Bacteriophages↗
PubMed2002

Characterization and sequence confirmation of unnatural amino acid containing peptide libraries using electrospray ionization mass spectrometry.

The content of 24 12-membered heptapeptide libraries was investigated using capillary liquid chromatography coupled with an electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometer. Adjustment of the chromatographic parameters led to the separation of most of the components. Extraction of the [M + 2H]2+ ions allowed us to demonstrate the presence of all expected species in the library and to evaluate their relative abundance in the mixture. Rapid sequence confirmation was achieved by subtraction of product ion spectra, a way to eliminate common ions and to simplify the spectra for interpretation. This technique can also easily be applied to other libraries consisting of components with a common core.

Amino Acid Sequence↗
PubMed2003

Epitope selection from an uncensored peptide library displayed on avian leukosis virus.

Phage display libraries have provided an extraordinarily versatile technology to facilitate the isolation of peptides, growth factors, single chain antibodies, and enzymes with desired binding specificities or enzymatic activities. The overall diversity of peptides in phage display libraries can be significantly limited by Escherichia coli protein folding and processing machinery, which result in sequence censorship. To achieve an optimal diversity of displayed eukaryotic peptides, the library should be produced in the endoplasmic reticulum of eukaryotic cells using a eukaryotic display platform. In the accompanying article, we presented experiments that demonstrate that polypeptides of various sizes could be efficiently displayed on the envelope glycoproteins of a eukaryotic virus, avian leukosis virus (ALV), and the displayed polypeptides could efficiently attach to cognate receptors without interfering with viral attachment and entry into susceptible cells. In this study, methods were developed to construct a model library of randomized eight amino acid peptides using the ALV eukaryotic display platform and screen the library for specific epitopes using immobilized antibodies. A virus library with approximately 2 x 10(6) different members was generated from a plasmid library of approximately 5 x 10(6) diversity. The sequences of the randomized 24 nucleotide/eight amino acid regions of representatives of the plasmid and virus libraries were analyzed. No significant sequence censorship was observed in producing the virus display library from the plasmid library. Different populations of peptide epitopes were selected from the virus library when different monoclonal antibodies were used as the target. The results of these two studies clearly demonstrate the potential of ALV as a eukaryotic platform for the display and selection of eukaryotic polypeptides libraries.

Amino Acid Sequence↗