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Powerful binders for the D-dimer by conjugation of the GPRP peptide to polypeptides from a designed set: illustrating a general route to new binders for proteins
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
2013 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, no 1, 17-25 p.Article in journal (Refereed) Published
Abstract [en]

The synthetic tetrapeptide GPRP based on the amino-terminal GPR sequence of the fibrin α-chain, binds the D-dimer protein with a dissociation constant KD of 25 μM. The D-dimer protein, a well-known biomarker for thrombosis, contains two cross-linked D fragments from the fibrinogen protein formed upon degradation of the fibrin gel, the core component of blood clots. In order to develop a specific high-affinity binder for the D-dimer protein, GPRP was conjugated via an aliphatic spacer to each member of a set of sixteen polypeptides designed for the development of binder molecules for proteins in general. The binders were individually characterised and ranked using surface plasmon resonance (SPR) analysis. The dissociation constant of the complex formed from the D-dimer and 4-D15L8-GPRP labelled with fluorescein was determined by fluorescense titration and found to be 3 nM, an affinity four orders of magnitude higher than that of free GPRP. According to SPR analysis binding was completely inhibited by free GPRP at mM concentrations and the polypeptide conjugate was therefore shown to bind specifically to the binding site of GPRP. Affinities were further enhanced by dimerisation of the polypeptide conjugates via a bifunctional linker resulting in dissociation constants that were further decreased (affinities increased) by factors of 2-4. The results suggest an efficient route to specific binders for proteins based on short peptides with affinites that need only to be modest, thus shortening the time of binder development dramatically.

Place, publisher, year, edition, pages
2013. Vol. 24, no 1, 17-25 p.
National Category
Organic Chemistry Other Chemistry Topics
Identifiers
URN: urn:nbn:se:uu:diva-183200DOI: 10.1021/bc300186zISI: 000313933700003OAI: oai:DiVA.org:uu-183200DiVA: diva2:562097
Available from: 2012-10-23 Created: 2012-10-23 Last updated: 2017-12-07Bibliographically approved
In thesis
1. High Affinity Synthetic Molecular Binders for Proteins: Design, Synthesis and Evaluation
Open this publication in new window or tab >>High Affinity Synthetic Molecular Binders for Proteins: Design, Synthesis and Evaluation
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the design and synthesis of small molecule derivatives and their polypeptide conjugates as high affinity binders for proteins: the D-dimer protein (D-dimer), a biomarker for diagnosis of thromboembolic diseases; human myeloperoxidase (MPO), a biomarker for cardiovascular diseases; and chitinases, potential targets for asthma therapy. The interactions between the synthetic binder molecules and those proteins were evaluated by surface plasmon resonance (SPR) biosensor analysis and fluorescence spectroscopy. Competition SPR experiments or other methods proved that the small molecule components of the binder molecules were critical for binding and specifically bound to the original binding site of small molecules. The binder molecules consisted of a 42-residue helix-loop-helix polypeptide conjugated to a small molecule via aliphatic spacers of suitable length. The small molecules could be any type of moderately binding structure. In the binder development for the D-dimer, the tetrapeptide GPRP with a dissociation constant Kd of 25 μM was used and the affinity of 4C15L8GPRP obtained was estimated to be approximately 3 nM. In the binder development for MPO, salicylhydroxamic acid (SHA) with Kd of 2 μM was used and the affinity of 4C37L34C11SHA obtained was estimated to be approximately 0.4 nM. In the binder development for chitinases, a theobromine derivative (pentoxifylline) with a Kd of 43±10 μM was used and the affinity of 4C37L34-P obtained was estimated to be considerably higher than that of pentoxifylline. The binder molecules were identified from a 16-membered pool of candidates obtained by conjugating the small molecules to each member of a set of 16 designed polypeptides. The affinities were greatly enhanced by 2-3 orders of magnitude, compared to the small molecule. The polypeptides did not bind to the proteins with measurable affinities. The discovery of these new synthetic binders for protein targets can pave the way to diagnostic tests in vivo or in vitro, independent of antibodies.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 53 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 995
Keyword
polypeptide, conjugates, D-dimer, myeloperoxidase, chitinase
National Category
Organic Chemistry Other Chemistry Topics
Identifiers
urn:nbn:se:uu:diva-183203 (URN)978-91-554-8533-7 (ISBN)
Public defence
2012-12-14, B21, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2012-11-23 Created: 2012-10-23 Last updated: 2013-02-11Bibliographically approved

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Sun, XiaojiaoBaltzer, Lars

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