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

Abstract

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.

Spongionella Secondary Metabolites Regulate Store Operated Calcium Entry Modulating Mitochondrial Functioning in SH-SY5Y Neuroblastoma Cells

“…Cyp D and in consequence inhibits the formation of mPTP opening [22,37]. Therefore, since Spongionella compounds shown the same effect than CsA, Cyp D was checked as cellular target for them.…”

Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallography Enhances Defenses against Influenza A Virus in Vivo

“…Use of Ni-NTA biosensors has not found widespread use for the study of His-tagged proteins in part because of the relative instability of the nickel complex (40). For example, investigators found that it was necessary to covalently couple monomeric His-tagged cyclosporine A to the biosensor, circumventing the potential advantages of the tag (41). It is likely that the unusually stable binding of trimeric NCRD proteins to the Ni-NTA biosensor results from cooperative interactions of the three hexahistidine tags of each trimer with the Ni 2ϩ -activated NTA surface.…”

Developing Antagonists for the Met-HGF/SF Protein–Protein Interaction Using a Fragment-Based Approach

“…SPR competition experiments were performed as essentially described previously (36). Met 928 was immobilized to a CM5 chip using the amine coupling method.…”