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High Affinity Synthetic Molecular Binders for Proteins: Design, Synthesis and Evaluation
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
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 [en]
polypeptide, conjugates, D-dimer, myeloperoxidase, chitinase
National Category
Organic Chemistry Other Chemistry Topics
Identifiers
URN: urn:nbn:se:uu:diva-183203ISBN: 978-91-554-8533-7 (print)OAI: oai:DiVA.org:uu-183203DiVA: diva2:562113
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
List of papers
1. 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
Open this publication in new window or tab >>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
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.

National Category
Organic Chemistry Other Chemistry Topics
Identifiers
urn:nbn:se:uu:diva-183200 (URN)10.1021/bc300186z (DOI)000313933700003 ()
Available from: 2012-10-23 Created: 2012-10-23 Last updated: 2017-12-07Bibliographically approved
2. A synthetic polypeptide conjugate from a 42-residue polypeptide and salicylhydroxamic acid binds human myeloperoxidase with high affinity
Open this publication in new window or tab >>A synthetic polypeptide conjugate from a 42-residue polypeptide and salicylhydroxamic acid binds human myeloperoxidase with high affinity
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.

National Category
Organic Chemistry Other Chemistry Topics
Identifiers
urn:nbn:se:uu:diva-183195 (URN)10.1002/psc.2459 (DOI)000311053700003 ()
Available from: 2012-10-23 Created: 2012-10-23 Last updated: 2017-12-07Bibliographically approved
3. Polypeptide conjugates that bind chitinases
Open this publication in new window or tab >>Polypeptide conjugates that bind chitinases
Show others...
(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:nbn:se:uu:diva-183201 (URN)
Available from: 2012-10-23 Created: 2012-10-23 Last updated: 2012-11-23

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