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A highly selective polypeptide binder for human Acetylcholine esterase
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Swedish Defence Research Agency, Division of CBRN Defence and Security.
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(English)Manuscript (Other academic)
Abstract [en]

A highly selective high-affinity polypeptide conjugate binder for human Acetylcholine Esterase (hAChE) has been obtained by coupling a derivative of acridine, a known medium-affinity inhibitor of hAChE, to each member of a 16-membered set of 42-residue polypeptide scaffolds. The best candidate, 4-C10L17-Ac, was identified to have a KD of 10 nM or less in an assay where each polypeptide conjugate was titrated with hAChE in 50 mM phosphate buffer at pH 7.0 and 298K. It was found in a sandwich ELISA to have high selectivity for hAChE in cerebrospinal fluid. Targeting the active site of hAChE by a polypeptide conjugate binder presents a special problem as it is buried deep inside the protein in a cavity that is approximately 20 Å deep. In order to permit simultaneous and cooperative binding of the acridine and the polypeptide to the active site and the AChE surface a fourteen atom spacer was needed. The 9-aminoacridine group was linked to the side chain of a lysine residue in each polypeptide via a spacer prepared from two aminohexanoic acid fragments. The results reinforce the impression that polypeptide conjugates are excellent alternatives to currently used protein binder technologies in diagnostic and therapeutic applications and that the conjugation of enzyme inhibitors to polypeptide scaffolds is a strategy of general applicability in the design of high-affinity binders for enzymes.

URN: urn:nbn:se:uu:diva-101405OAI: oai:DiVA.org:uu-101405DiVA: diva2:212938
Available from: 2009-04-25 Created: 2009-04-25 Last updated: 2011-12-07
In thesis
1. Polypeptide Conjugates as High-affinity Binders for Proteins
Open this publication in new window or tab >>Polypeptide Conjugates as High-affinity Binders for Proteins
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A novel concept for protein recognition has been developed. The recognition unit is a hybrid molecule obtained by conjugation of a small organic molecule to a synthetic polypeptide selected from a 16-membered set of 42 amino acid residue sequences. The sequences are unordered and have no prior relation to the target proteins. The concept is based on the hypothesis that a small set of sequences capable of hydrophobic interactions, hydrogen bonding and electrostatic interactions can yield a binder for any selected protein, provided that the small molecule shows medium affinity or better and is reasonably selective.

The concept has been illustrated by the design, synthesis and evaluation of binders for three different proteins, the C-reactive protein, CRP, human Carbonic anhydrase II, HCAII, and Acetylcholine esterase, AChE. Highly efficient binders for CRP have been developed by conjugation of a derivative of the natural ligand, phosphocholine, to the side chain of one of the amino acids in each polypeptide. The binders in the set show a wide range of affinities for CRP and the tightest binder, 4-C10L17-PC6, binds almost irreversibly. Selected binders have been evaluated in human serum, where they capture CRP with high selectivity.High-affinity binders have been developed for HCAII, and the selectivity evaluated by extraction of the protein from blood. The binder 4-C37L34-B, a polypeptide conjugated to a spacered benzenesulphonamide residue, was able to extract Carbonic anhydrases specifically and to discriminate between the two isoforms of human Carbonic anhydrase. The conjugation of an acridine derivative to a polypeptide via a 14 atom spacer has been shown to yield a binder with high affinity and selectivity for AChE. The selectivity was demonstrated by extraction of AChE from Cerebrospinal fluid.

This thesis focuses on the development of a fast and reliable procedure for the construction, selection and evaluation of protein binders, with the ambition to develop a technology that is applicable to the development of binders for all proteins.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 47 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 650
Protein recognition, polypeptide conjugates, peptide, molecular interactions, C-reactive protein, Acetylcholine esterase, Human Carboinc anhydrase
National Category
Chemical Sciences
Research subject
Organic Chemistry
urn:nbn:se:uu:diva-101406 (URN)978-91-554-7542-0 (ISBN)
Public defence
2009-06-12, B:21, BMC, Husargatan 3, Uppsala, 10:15 (English)
Available from: 2009-05-20 Created: 2009-04-25 Last updated: 2009-05-20Bibliographically approved

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Österlund, Lars
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