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Thermo-kinetic analysis space expansion for cyclophilin-ligand interactions - identification of a new non-peptide inhibitor using Biacore ™ T200

DOI: 10.1002/2211-5463.12201 DOI Help

Authors: Martin A. Wear (University of Edinburgh) , Matthew Nowicki (University of Edinburgh) , Elizabeth A. Blackburn (University of Edinburgh) , Iain W. Mcnae (University of Edinburgh) , Malcolm D. Walkinshaw (University of Edinburgh)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Febs Open Bio

State: Published (Approved)
Published: February 2017
Diamond Proposal Number(s): 7613

Open Access Open Access

Abstract: 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 to determine accurate and robust thermodynamic parameters for the cyclophilin-A:cyclosporin-A interaction. Furthermore, the increased sensitivity of the surface allowed identification of a 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: 5LUD), providing new insight into the molecular details for a potential new series of non-peptide cyclophilin-A inhibitors.

Journal Keywords: Cyclophilin-A; surface plasmon resonance; thermodynamics; non-peptide; inhibitor

Subject Areas: Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography