Thermo-kinetic analysis space expansion for cyclophilin-ligand interactions - identification of a new nonpeptide inhibitor using Biacore™ T200
| Autor: | Malcolm D. Walkinshaw, Elizabeth A. Blackburn, Matthew W. Nowicki, Martin A. Wear, Iain W. McNae |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Inhibitor nonpeptide Protein Data Bank (RCSB PDB) Isomerase General Biochemistry Genetics and Molecular Biology 03 medical and health sciences thermodynamics Surface plasmon resonance Cyclophilin Research Articles cyclophilin‐A 030102 biochemistry & molecular biology biology Chemistry Biochemistry Genetics and Molecular Biology(all) Active site Ligand (biochemistry) Combinatorial chemistry Cyclophilin-A Nonpeptide inhibitor Crystallography 030104 developmental biology Covalent bond biology.protein Thermodynamics surface plasmon resonance Protein ligand Research Article |
| Zdroj: | FEBS Open Bio Wear, M, Nowicki, M, Blackburn, E, McNae, I & Walkinshaw, M 2017, ' Thermo-kinetic analysis space expansion for cyclophilin-ligand interactions; identification of a new non-peptide inhibitor using Biacore™ T200 ', FEBS Open Bio . https://doi.org/10.1002/2211-5463.12201 |
| ISSN: | 2211-5463 |
| Popis: | We have established a refined methodology for generating surface plasmon resonance sensor surfaces of recombinant his-tagged human cyclophilin-A. Our orientation-specific stabilisation approach captures his-tagged protein under “physiological conditions” (150 mM NaCl, pH 7.5) and covalently stabilises it on Ni2+-nitrilotriacetic acid surfaces, very briefly activated for primary amine-coupling reactions, producing very stable and active surfaces (≥ 95 % specific activity) of cyclophilin-A. Variation of protein concentration with the same contact time allows straightforward generation of variable density surfaces, with essentially no loss of activity, making the protocol easily adaptable for studying numerous interactions; from very small fragments, ~ 100 Daltons, to large protein ligands. This new method results in an increased stability and activity of the immobilised protein and allowed us to expand the thermo-kinetic analysis space, and determine accurate and robust thermodynamic parameters for the cyclophilin-A:cyclosporin-A interaction. Furthermore, the increased sensitivity of the surface allowed identification new non-peptide inhibitor of cyclophilin-A, from a screen of a fragment library. This fragment, 2,3-diaminopyridine, bound specifically with a mean affinity of 248 ± 60 µM. The X-ray structure of this 109 dalton fragment bound in the active site of cyclophilin-A was solved to a resolution of 1.25 Å (PDB ID: 5LUD), providing new insight into the molecular details for a potential new series of non-peptide cyclophilin-A inhibitors. |
| Databáze: | OpenAIRE |
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