Thermo‐kinetic analysis space expansion for cyclophilin‐ligand interactions – identification of a new nonpeptide inhibitor using Biacore™ T200

• Published in: FEBS open bio Vol. 7; no. 4; pp. 533 - 549
• Main Authors: Wear, Martin A., Nowicki, Matthew W., Blackburn, Elizabeth A., McNae, Iain W., Walkinshaw, Malcolm D.
• Format: Journal Article
• Language: English
• Published: England John Wiley & Sons, Inc 01.04.2017; John Wiley and Sons Inc; Wiley
• Subjects: Chromatography; Crystallography; cyclophilin‐A; Design; Experiments; inhibitor; Ligands; nonpeptide; Proteins; Sensors; Sodium chloride; Surface plasmon resonance; thermodynamics; United Kingdom > UK; United States > US; thermodynamics; cyclophilin‐A; inhibitor; nonpeptide; surface plasmon resonance
• DOI: 10.1002/2211-5463.12201
• ISSN: 2211-5463

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 in 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 Da, 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 nonpeptide 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‐Da 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 nonpeptide cyclophilin‐A inhibitors. We developed an orientation‐specific stabilisation methodology for generating surface plasmon resonance sensor surfaces of recombinant his‐tagged human cyclophilin‐A. This produced extremely stable and sensitive surfaces that expanded the thermo‐kinetic analysis space, allowed determination of robust thermodynamic parameters for the cyclophilin‐A–cyclosporin‐A interaction and identified a new nonpeptide ligand (2,3‐diaminopyridine) from a fragment library screen. The X‐ray structure of this fragment bound to the active site of cyclophilin‐A was solved to 1.25 Å resolution.