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A synthetic polypeptide conjugate from a 42-residue polypeptide and salicylhydroxamic acid binds human myeloperoxidase with high affinity
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry. (Lars Baltzer)
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.
2012 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 18, no 12, 731-739 p.Article in journal (Refereed) Published
Abstract [en]

Myeloperoxidase (MPO) is a 150 kD tetrameric heme protein consisting of two heavy chains and two light chains, which ispresent in neutrophils, white blood cells, at concentrations between 2% and 5% and plays an important role in the innateimmune system. The MPO concentration in serum or plasma has been shown to be linked to the risk for cardiovasculardiseases, and MPO is considered to be a high potential diagnostic biomarker. To develop a molecule that binds MPO,salicylhydroxamic acid (SHA), a substrate analog inhibitor of MPO with a KD=2uM, was conjugated to a designed set of42-residue polypeptide scaffolds via 9- and 11-carbon atom aliphatic spacers to form 20 different protein binder candidates,and their interactions with MPO were evaluated by surface plasmon resonance analysis. The polypeptide conjugate4C37L34C11SHA was found to bind to MPO with an affinity that could be estimated to have a dissociation constant of around400 pM, nearly four orders of magnitude higher than that of SHA. Inhibition of binding to MPO by free SHA was observed incompetition experiments demonstrating that the binding of the polypeptide conjugate is dominated by the interactions ofSHA with the heme cavity. Although still in the future, the discovery of these new synthetic binders for MPO suggests aroute to clinical diagnostic tests in vivo or in vitro, independent of antibodies.

Place, publisher, year, edition, pages
2012. Vol. 18, no 12, 731-739 p.
National Category
Organic Chemistry Other Chemistry Topics
Identifiers
URN: urn:nbn:se:uu:diva-183195DOI: 10.1002/psc.2459ISI: 000311053700003OAI: oai:DiVA.org:uu-183195DiVA: diva2:562096
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, XiaojiaoYang, JieNorberg, ThomasBaltzer, Lars

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