Interaction of cepharanthine with immobilized heat shock protein 90α (Hsp90α) and screening of Hsp90α inhibitors

Abstract

Heat shock protein 90α (Hsp90α) immobilized on aminopropyl silica gels was prepared via the N- or C-terminal, which was termed Hsp90α-NT or Hsp90α-CT, respectively. Binding interactions of biscoclaurine alkaloids (cepharanthine (CEP), berbamine (BBM), isotetrandrine (ITD), and cycleanine (CCN)) with Hsp90α were examined using the Hsp90α-NT or -CT columns by frontal and zonal chromatography studies. The dissociation constants of CEP, BBM, ITD, and CCN to Hsp90α-NT were estimated to be 5.3, 18.6, 46.3, and 159 μM, respectively, by frontal chromatography techniques. Similar results were obtained with the Hsp90α-CT column. These data suggest that these biscoclaurine alkaloids interact with the middle domain of Hsp90α. This was confirmed by demonstrating that CEP competed with endothelial nitric oxide synthase at the middle domain of Hsp90α, where it was shown to have a dissociation constant of 15 nM. Furthermore, the Hsp90α-NT column was applied for preliminary screening of natural Hsp90α inhibitors by zonal chromatography studies.

Fig. 1

Structures of CEP, BBM, ITD, and CCN.

Fig. 2

Determination of the dissociation constants of CEP to (a) Hsp90α-NT and (b) Hsp90α-CT columns by frontal chromatography techniques. LC conditions: column, Hsp90α-NT or Hsp90α-CT (20 × 5.0 mm i.d.); mobile phase, 10 mM Tris–HCl buffer (pH 7.4) containing various concentrations of CEP; flow rate, 0.2 ml/min; column temperature, 25 °c; detection, 282 nm; injection volume, 5 ml.

Fig. 3

Relationship for competitive displacement of CEP by eNOS. LC conditions: column, Hsp90α-NT (20 × 5.0 mm i.d.); mobile phase, 10 mM Tris–HCl buffer (pH 7.4) containing various concentrations of eNOS; flow rate, 0.2 ml/min; column temperature, 25 °c; detection, 210 nm; amount loaded, 500 ng.

Fig. 4

Plots of the retention times of natural products on Hsp90α-NT and control-NT columns. LC conditions: column, Hsp90α-NT or control-NT (20 × 5.0 mm i.d.); mobile phase, 10 mM Tris–HCl buffer (pH 7.4); flow rate, 0.2 ml/min; column temperature, 25 °c; detection, 210 nm; amount loaded, 500 ng. Symbols: 1, CEP; 2, BBM; 3, ITD; 4, CCN; 5, quercetin; 6, curcumin; 7, wogonin; 8, glycyrrhizin; 9, baicalin; 10, baicalein; 11, ginsenoside.