Artigo Acesso aberto Revisado por pares

A surface plasmon resonance-based assay for small molecule inhibitors of human cyclophilin A

2005; Elsevier BV; Volume: 345; Issue: 2 Linguagem: Inglês

10.1016/j.ab.2005.06.037

ISSN

1096-0309

Autores

Martin A. Wear, Alan Patterson, Kirk J. Malone, Colin J. Dunsmore, Nicholas J. Turner, Malcolm D. Walkinshaw,

Tópico(s)

Signaling Pathways in Disease

Resumo

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 ∼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 (KdCsA) binding to the immobilized His-CypA was 23 ± 6 nM, with on and off rates of 0.53 ± 0.1 μM−1 s−1 and 1.2 ± 0.1 (×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.

Referência(s)