Combinatorial library
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Detection of peptides lies at the core of bottom-up proteomics analyses. We examined a Bayesian approach to peptide detection, integrating match-based models (fragments, retention time, isotopic distribution, and precursor mass) and peptide prior probability models under a unified probabilistic framework. To assess the relevance of these models and their various combinations, we employed a complete- and a tail-complete search of a low-precursor-mass synthetic peptide library based on oncogenic KRAS peptides. The fragment match was by far the most informative match-based model, while the retention time match was the only remaining such model with an appreciable impact--increasing correct detections by around 8 %. A peptide prior probability model built from a reference proteome greatly improved the detection over a uniform prior, essentially transforming de novo sequencing into a reference-guided search. The knowledge of a correct sequence tag in advance to peptide-spectrum matching had only a moderate impact on peptide detection unless the tag was long and of high certainty. The approach also derived more precise error rates on the analyzed combinatorial peptide library than those estimated using PeptideProphet and Percolator, showing its potential applicability for the detection of homologous peptides. Although the approach requires further computational developments for routine data analysis, it illustrates the value of peptide prior probabilities and presents a Bayesian approach for their incorporation into peptide detection.
Described is a computer-assisted rational design of a DNA-bis-intercalator peptide library. The peptide library of 250 members was prepared and the most powerful binder identified. A value of the binding constant is almost two orders of magnitude higher than that of starting building block-9-aminoacridine. The binder affinity found toward calf thymus DNA is 30-fold of that of human prion peptide 106-126.
- MeSH
- aminakrin chemie MeSH
- design s pomocí počítače MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- DNA chemická syntéza chemie metabolismus MeSH
- financování organizované MeSH
- fluorescenční barviva chemie MeSH
- genová knihovna MeSH
- interkalátory chemie MeSH
- lidé MeSH
- molekulární modely MeSH
- peptidové fragmenty metabolismus MeSH
- priony metabolismus MeSH
- skot MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- techniky kombinatorické chemie MeSH
- thymus chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
Single-chain antibodies (scFv) exhibiting specific binding to Lawsonia intracellularis were isolated from a phagemid library expressing scFvs molecules on the surface of filamentous bacteriophages. For scFv selection whole bacterial cells were used and individual clones were tested in ELISA test. The total of seven unique clones with different fingerprint profiles was isolated. All clones were able to bind specifically in immunofluorescence assay. This is the first report of species specific recombinant antibodies against L. intracellularis.
- MeSH
- financování organizované MeSH
- Lawsonia (bakterie) imunologie MeSH
- lidé MeSH
- peptidová knihovna MeSH
- protilátky bakteriální genetika imunologie izolace a purifikace MeSH
- specificita protilátek MeSH
- variabilní oblast imunoglobulinu genetika imunologie izolace a purifikace MeSH
- Check Tag
- lidé MeSH
We designed a combinatorial library of trifunctional scaffold-derived compounds, which were derivatized with 30 different in-house-made azides. The compounds were proposed to mimic insulin receptor (IR)-binding epitopes in the insulin molecule and bind to and activate this receptor. This work has enabled us to test our synthetic and biological methodology and to prove its robustness and reliability for the solid-phase synthesis and testing of combinatorial libraries of the trifunctional scaffold-derived compounds. Our effort resulted in the discovery of two compounds, which were able to weakly induce the autophosphorylation of IR and weakly bind to this receptor at a 0.1 mM concentration. Despite these modest biological results, which well document the well-known difficulty in modulating protein-protein interactions, this study represents a unique example of targeting the IR with a set of nonpeptide compounds that were specifically designed and synthesized for this purpose. We believe that this work can open new perspectives for the development of next-generation insulin mimetics based on the scaffold structure.
- MeSH
- azidy chemická syntéza chemie MeSH
- inzulin analogy a deriváty chemie metabolismus MeSH
- knihovny malých molekul chemická syntéza chemie metabolismus farmakologie MeSH
- měď analýza MeSH
- molekulární struktura MeSH
- receptor inzulinu chemie metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- techniky kombinatorické chemie * MeSH
- techniky syntézy na pevné fázi MeSH
- vazba proteinů MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
An efficient and high-yielding solid phase synthesis of a small library of imidazolidin-2-ones and imidazol-2-ones was carried out employing a high chemo- and regioselective gold-catalyzed cycloisomerization as a key step. Polymer-supported amino acids derivatized with several alkyne functionalities combined with tosyl- and phenylureas have been subjected to gold-catalysis exhibiting exclusively C-N bond formation. The present work proves the potential of solid phase synthesis and homogeneous gold catalysis as an efficient and powerful synthetic tool for the generation of drug-like heterocycles.
The development of canine immunotolerant monoclonal antibodies can accelerate the invention of new medicines for both canine and human diseases. We develop a methodology to clone the naive, somatically mutated variable domain repertoire from canine B cell mRNA using 5'RACE PCR. A set of degenerate primers were then designed and used to clone variable domain genes into archival "holding" plasmid libraries. These archived variable domain genes were then combinatorially ligated to produce a scFv M13 phage library. Next-generation long-read and short-read DNA sequencing methodologies were developed to annotate features of the cloned library including CDR diversity and IGHV/IGKV/IGLV subfamily distribution. A synthetic immunoglobulin G was developed from this scFv library to the canine immune checkpoint receptor PD-1. This synthetic platform can be used to clone and annotate archived antibody variable domain genes for use in perpetuity in order to develop improved preclinical models for the treatment of complex human diseases.
- MeSH
- antigeny CD279 imunologie MeSH
- jednořetězcové protilátky * imunologie genetika MeSH
- lidé MeSH
- monoklonální protilátky imunologie genetika MeSH
- nádory imunologie terapie MeSH
- peptidová knihovna MeSH
- psi MeSH
- rekombinantní proteiny imunologie genetika MeSH
- translační biomedicínský výzkum MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Galectin-3 (Gal-3), a member of the β-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galβ1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N',N″-diacetyllactosamine (GalNAcβ1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.
- MeSH
- aminocukry chemická syntéza chemie metabolismus MeSH
- galektin 3 antagonisté a inhibitory metabolismus MeSH
- glykoproteiny chemická syntéza chemie metabolismus farmakologie MeSH
- glykosylace MeSH
- lidé MeSH
- ligandy MeSH
- oligosacharidy chemická syntéza chemie metabolismus MeSH
- sérový albumin hovězí chemická syntéza chemie metabolismus farmakologie MeSH
- skot MeSH
- techniky kombinatorické chemie MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
xxiv, 516 s. : il.
- Klíčová slova
- Farmakologie, Léčiva,
- MeSH
- farmaceutická chemie MeSH
- farmakologie MeSH
- racionální návrh léčiv MeSH
A piperazine amide linker for cyclative cleavage from solid support and its use in the traceless solid-phase synthesis of dihydroquinoxalinones are described. Piperazine was attached to Wang resin via a carbamate linkage and acylated with Fmoc-amino acids. Following Fmoc group removal, resin-bound amines were reacted with 1-fluoro-2-nitrobenzenes. The nitro group of the resulting 2-nitroanilines was reduced, and acyclic precursors, in contrast to traditional ester-type linkage, remained attached to the resin. Target dihydroquinoxalinones were obtained either by acid- or microwave-mediated cyclative cleavage. The synthesis provided crude compounds of high purity and enabled the preparation of stable immobilized linear intermediates. The linker is suitable for combinatorial synthesis of compound libraries.
- MeSH
- acylace MeSH
- amidy chemie MeSH
- aminokyseliny chemie MeSH
- chinoxaliny chemická syntéza chemie MeSH
- fluoreny chemie MeSH
- piperaziny chemie MeSH
- syntetické pryskyřice chemie MeSH
- techniky kombinatorické chemie metody MeSH
- techniky syntézy na pevné fázi metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
