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Design, Synthesis and Characterization of Small Molecule Inhibitors and Small Molecule: Peptide Conjugates as Protein Actors
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes different aspects of protein interactions. Initially the function of peptides and their conjugates with small molecule inhibitors on the surface of Human Carbonic Anhydrase isoenzyme II (HCAII) is evaluated.

The affinities for HCAII of the flexible, synthetic helix-loop-helix motif conjugated with a series of spacered inhibitors were measured by fluorescence spectroscopy and found in the best cases to be in the low nM range. Dissociation constants show considerable dependence on linker length and vary from 3000 nM for the shortest spacer to 40 nM for the longest with a minimum of 5 nM for a spacer with an intermediate length. A rationale for binding differences based on cooperativity is presented and supported by affinities as determined by fluorescence spectroscopy. Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC) spectroscopic experiments with 15N-labeled HCAII were used for the determination of the site of interaction.

The influence of peptide charge and hydrophobicity was evaluated by surface plasmon resonance experiments. Hydrophobic sidechain branching and, more pronounced, peptide charge was demonstrated to modulate peptide – HCAII binding interactions in a cooperative manner, with affinities spanning almost two orders of magnitude.

Detailed synthesis of small molecule inhibitors in a general lead discovery library as well as a targeted library for inhibition of α-thrombin is described. For the lead discovery library 160 members emanate from two N4-aryl-piperazine-2-carboxylic acid scaffolds derivatized in two dimensions employing a combinatorial approach on solid support.

The targeted library was based on peptidomimetics of the D-Phe-Pro-Arg showing the scaffolds cyclopropane-1R,2R-dicarboxylic acid and (4-amino-3-oxo-morpholin-2-yl)- acetic acid as proline isosters. Employing 4-aminomethyl-benzamidine as arginine mimic and different hydrophobic amines and electrophiles as D-phenylalanine mimics resulted in 34 compounds showing IC50 values for α-thrombin ranging more than three orders of magnitude with the best inhibitor showing an IC50 of 130 nM. Interestingly, the best inhibitors showed reversed stereochemistry in comparison with a previously reported series employing a 3-oxo-morpholin-2-yl-acetic acid scaffold.

Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi , 2005.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 960
Keyword [en]
Synthetic receptor, Peptide inhibitor conjugate, Peptide protein surface interactions, General Lead Discovery Library, Combinatorial Chemistry, Solid Phase Chemistry, Targeted Library, Thrombin inhibitor, Fluorescence, Surface Plasmon Resonance
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:liu:diva-3943ISBN: 91-85457-00-0 (print)OAI: oai:DiVA.org:liu-3943DiVA: diva2:20483
Public defence
2005-09-16, Planck, Fysikhuset, Campus Valla, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2005-09-21 Created: 2005-09-21 Last updated: 2009-03-06
List of papers
1. The binding of human Carbonic Anhydrase II by functionalized folded polypeptide receptors
Open this publication in new window or tab >>The binding of human Carbonic Anhydrase II by functionalized folded polypeptide receptors
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2005 (English)In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 12, no 11, 1245-1252 p.Article in journal (Refereed) Published
Abstract [en]

Several receptors for human carbonic anhydrase II (HCAII) have been prepared by covalently attaching benzenesulfonamide carboxylates via aliphatic aminocarboxylic acid spacers of variable length to the side chain of a lysine residue in a designed 42 residue helix-loop-helix motif. The sulfonamide group binds to the active site zinc ion of human carbonic anhydrase II located in a 15 Å deep cleft. The dissociation constants of the receptor-HCAII complexes were found to be in the range from low micromolar to better than 20 nM, with the lowest affinities found for spacers with less than five methylene groups and the highest affinity found for the spacer with seven methylene groups. The results suggest that the binding is a cooperative event in which both the sulfonamide residue and the helix-loop-helix motif contribute to the overall affinity.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13359 (URN)10.1016/j.chembiol.2005.08.018 (DOI)
Available from: 2005-09-21 Created: 2005-09-21 Last updated: 2017-12-13
2. Electrostatic and hydrophobic contributions to protein: peptide surface interactions
Open this publication in new window or tab >>Electrostatic and hydrophobic contributions to protein: peptide surface interactions
Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-13360 (URN)
Available from: 2005-09-21 Created: 2005-09-21 Last updated: 2010-01-13
3. Solid-phase synthesis of libraries generated from a 4-phenyl-2-carboxy-piperazine Scaffold
Open this publication in new window or tab >>Solid-phase synthesis of libraries generated from a 4-phenyl-2-carboxy-piperazine Scaffold
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2001 (English)In: Journal of Combinatorial Chemistry, Vol. 3, no 6, 546-553 p.Article in journal (Refereed) Published
Abstract [en]

Strategies for finding novel structures of therapeutical interest are discussed. The rationale for the selection of the two scaffolds N4-(m-aminophenyl)-piperazine-2-carboxylic acid E and N4-(o-aminophenyl)-piperazine-2-carboxylic F is described. The synthesis of the appropriate precursors to scaffold E and F and their use in solid-phase chemistry are described. A 160-member library was produced combining these novel piperazine scaffolds with eight sulfonyl chlorides/acid chlorides and 10 amines. The compound library prepared was analyzed using LC-MS, showing the expected base peak in all wells at an average purity of 82%.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13467 (URN)10.1021/cc010013o (DOI)
Available from: 2005-11-24 Created: 2005-11-24
4. Synthesis and SAR of Thrombin Inhibitors Incorporating a Novel 4-Amino-Morpholinone Scaffold: Analysis of X-ray Crystal Structure of Enzyme Inhibitor Complex
Open this publication in new window or tab >>Synthesis and SAR of Thrombin Inhibitors Incorporating a Novel 4-Amino-Morpholinone Scaffold: Analysis of X-ray Crystal Structure of Enzyme Inhibitor Complex
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2001 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, Vol. 46, no 19, 3985-4001 p.Article in journal (Refereed) Published
Abstract [en]

A 4-amino-2-carboxymethyl-3-morpholinone structural motif derived from malic acid has been used to mimic d-Phe-Pro in the thrombin inhibiting tripeptide d-Phe-Pro-Arg. The arginine in d-Phe-Pro-Arg was replaced by the more rigid P1 truncated p-amidinobenzylamine (Pab). These new thrombin inhibitors were used to probe the inhibitor binding site of α-thrombin. The best candidate in this series of thrombin inhibitors exhibits an in vitro IC50 of 0.130 μM. Interestingly, the stereochemistry of the 4-amino-2-carboxymethyl-3-morpholinone motif is reversed for the most active compounds compared to that of a previously reported 2-carboxymethyl-3-morpholinone series. The X-ray crystal structure of the lead inhibitor cocrystallized with α-thrombin is discussed.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13362 (URN)10.1021/jm0307990 (DOI)
Available from: 2005-09-21 Created: 2005-09-21 Last updated: 2009-05-28

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