High Affinity Synthetic Molecular Binders for Proteins: Design, Synthesis and Evaluation
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 995
polypeptide, conjugates, D-dimer, myeloperoxidase, chitinase
Organic Chemistry Other Chemistry Topics
IdentifiersURN: urn:nbn:se:uu:diva-183203ISBN: 978-91-554-8533-7OAI: oai:DiVA.org:uu-183203DiVA: diva2:562113
2012-12-14, B21, BMC, Husargatan 3, Uppsala, 13:15 (English)
Nilsson, Ulf, Professor
Baltzer, Lars, ProfessorNorberg, Thomas, Professor
List of papers