A surface plasmon resonance-based assay for small molecule inhibitors of human cyclophilin A.
A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr approximately 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K(dCsA)) binding to the immobilized His-CypA was 23+/-6 nM, with on and off rates of 0.53+/-0.1 microM(-1) s(-1) and 1.2+/-0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.
Wear
Martin A
MA
Institute of Structural and Molecular Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR, UK.
Patterson
Alan
A
Malone
Kirk
K
Dunsmore
Colin
C
Turner
Nicholas J
NJ
Walkinshaw
Malcolm D
MD
D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods*; D021984:Cyclophilin A / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016572:Cyclosporine / Q000032:analysis / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000737:chemistry; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D005453:Fluorescence; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008970:Molecular Weight; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012996:Solutions; D055672:Static Electricity; D020349:Surface Plasmon Resonance; D013997:Time Factors; D017186:Titrimetry
D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D008024:Ligands; D011994:Recombinant Proteins; D012996:Solutions; D006639:Histidine; D016572:Cyclosporine; D021984:Cyclophilin A; D009571:Nitrilotriacetic Acid