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Polypeptide conjugates that bind chitinases
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.
Department of forest mycology and pathology, swedish university of agricultural sciences, sweden.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Polypeptide conjugates formed from members of a designed set of polypeptides and the chitinase inhibitors pentoxifylline, C4B3 and C5B1 were evaluated for affinity and selectivity towards chitinases. The polypeptide conjugate 4C37L34-P was found to recognize and bind chitinases from the fungal species Aspergillus nidulans and Neurospora crassa, as well as from the fungus Trichoderma viride and the bacterial strain Streptomyces griseus. The small organic molecule pentoxifylline and the polypeptide 4C37L34 both contributed to binding of the chitinases and the affinity of the polypeptide conjugate was significantly enhanced in comparison with that of pentoxifylline with an affinity that was on the order of 2-3 orders of magnitude higher than that of pentoxyfylline. In view of the large numbers of chitinases present in nature the recognition and detection of groups of chitinases using one binder molecule for many enzymes may be an advantageous approach.

National Category
Organic Chemistry Other Chemistry Topics Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-183201OAI: oai:DiVA.org:uu-183201DiVA: diva2:562099
Available from: 2012-10-23 Created: 2012-10-23 Last updated: 2012-11-23
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)
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Supervisors
Available from: 2012-11-23 Created: 2012-10-23 Last updated: 2013-02-11Bibliographically approved

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