Fig 1.
Screening for compounds with the ability to overcome Pgp-mediated MDR.
A. Expression of Pgp in parental and multidrug-resistance cell line pairs. Both pairs of cell lines were analyzed through western blotting for the expression of Pgp (MDR1). B. Flow chart for primary/secondary screening and identification of hits.
Fig 2.
Secondary screening with 118 compounds in both pairs of parental and resistant cell lines.
Growth inhibition of A2780/A2780-Pac-Res (A.) and KB-3-1 and KB-V1 (B.) cell line pairs by 15 hits confirmed in the secondary screening at 1μM concentration. C. Growth inhibition of both the cell line pairs by paclitaxel.
Table 1.
Resistant Index (RI) of selected inhibitors in parental and resistant cell line pairs.
Fig 3.
Calcein AM Efflux assays in Pgp-overexpressing multidrug-resistant cell lines.
Calcein retention in KB-3-1/KB-V1 and A2780/A2780-Pac-Res parental and resistant cell line pairs. Calcein retention in the presence of DMSO, verapamil and CHIR-124 was determined through FACS analysis (A) or immunofluorescence (B) as described in the materials and methods.
Fig 4.
Effect of tyrphostin-9, brefeldin A and elesclomol on Pgp inhibition using calcein AM efflux assay.
Calcein retention in parental and resistant cells was determined through FACS analysis following treatment with the indicated concentrations of tyrphostin-9, brefeldin A (A) and elesclomol (B).
Fig 5.
The docking of CHIR-124 and PIK-75 with Pgp.
(A) Binding of CHIR-124 in the NBD1 of Pgp (PDB code: 4Q9H) (B) 2D presentation of the interactions of CHIR-124 with the amino acid residues of NBD1 (C) Binding of PIK-75 in the NBD1 of Pgp (D) 2D presentation of the interactions of PIK-75 with the amino acid residues of NBD1. (E) Inhibition of ATPase activity of Pgp by CHIR-124 and PIK-75. ATPase activity was measured using Pgp-Glo assay (Promega) as described in materials and methods. Samples were treated with 100 μM Na3VO4, 200 μM Verapamil, 2.5 μM CHIR-124 and PIK-75 or left untreated. Fold change in verapamil-treated and compound treated Pgp activity was calculated according to the manufacturer’s instructions.