Structure activity relationships and the binding mode of quinolinone-pyrimidine hybrids as reversal agents of multidrug resistance mediated by P-gp

Abstract

P-gp-associated multidrug resistance is a major impediment to the success of chemotherapy. With the aim of finding non-toxic and effective P-gp inhibitors, we investigated a panel of quinolin-2-one-pyrimidine hybrids. Among the active compounds, two of them significantly increased intracellular doxorubicin and rhodamine 123 accumulation by inhibiting the efflux mediated by P-gp and restored doxorubicin toxicity at nanomolar range. Structure-activity relationships showed that the number of methoxy groups, an optimal length of the molecule in its extended conformation, and at least one flexible methylene group bridging the quinolinone to the moiety bearing the pyrimidine favored the inhibitory potency of P-gp. The best compounds showed a similar binding pattern and interactions to those of doxorubicin and tariquidar, as revealed by MD and hybrid QM/MM simulations performed with the recent experimental structure of P-gp co-crystallized with paclitaxel. Analysis of the molecular interactions stabilizing the different molecular complexes determined by MD and QTAIM showed that binding to key residues from TMH 4-7 and 12 is required for inhibition.

Figure 1

Chemical structures and labelling of the tested compounds.

Figure 2

Inhibition on P-gp doxorubicin (Dox) outward transport by different concentrations of compounds 5c (A) and 7b (B), determined by accumulation assay in Lucena 1 and (C) at 20 μM of both compounds in Lucena 1 and K562 cells. Doxorubicin-associated intracellular fluorescence significantly increased in Lucena 1 by treatments with selected compounds but not in K562. Significant differences relative to the respective negative control were determined by using a paired one-tailed Student’s t test (***p < 0.001, **p < 0.01, *p < 0.05).

Figure 3

Inhibition on P-gp rhodamine 123 (Rho123) outward transport by different concentrations of compounds 5c (A) and 7b (B) determined by accumulation assay in Lucena 1 and (C) at 20 μM of both compounds in Lucena 1 and K562 cells. Rhodamine 123 associated intracellular fluorescence significantly increased in Lucena 1 by treatments with selected compounds but not in K562. Significant differences relative to the respective negative control were determined by using a paired one-tailed Student’s t test (**p < 0.01, *p < 0.05).

Figure 4

Dose–response curves for restoring sensitivity in Lucena 1 treated with doxorubicin (Dox) as an antineoplastic drug in the absence or presence of (A) compound 5c and (B) compound 7b. In Lucena 1 cells, doxorubicin toxicity was significantly increased when compounds 5c and 7b were co-administered from 0.04 and 0.08 µM, respectively (p < 0.05). The same assay was performed in K562 to discard additional effects other than P-gp. Values are expressed as means ± SE of at least three independent experiments.

Figure 5

(A) Distant view of the P-gp surrounded by a molecular surface (probe 1.4 Å) colored according to its hydrophobicity (orange → most hydrophobic). The green cube indicates the docking region; the dotted yellow rectangle is zoomed in (B). (B) Superimposition of doxorubicin (yellow) and the co-crystallized chemotherapeutic drug, Taxol (magenta).

Figure 6

(A) Superimposition of the docked structures of the active compounds 4b, 5b–c, 6a–b, 7a–c, 10 and 11, together with the chemotherapeutic substrate doxorubicin and tariquidar. (B) The same for poor or inactive compounds 2, 2b–c, 3a–c, 5, 8, 8a, 9 and 9a.

Figure 7

(A,B) Two views of the P-gp rotated about 80 degrees around the vertical axis; they are zoomed in (C,D. (C) Superimposition of the most populated clusters from a representative trajectory for compound 7b (red tones), doxorubicin (green tones) and compound 8 (blue tones). (D) The same for compounds 7b and 8 and for Taxol (gray tones).

Figure 8

Decomposition of the MD free energy of binding in terms of per residue contribution. Residues showing the most negative peaks correspond to stronger stabilizations: (A) doxorubicin; (B) tariquidar; (C) compound 5c, (D) compound 5.

Figure 9

Sum of charge density values (atomic units) at the critical intermolecular binding points between P-gp and ligands with different levels of activity 3, 5a, 5c, 7b and 8. Total interactions obtained for the different complexes are shown as a function of the different transmembrane (TMH) domains of the receptor.

Figure 10

Structures of doxorubicin, tariquidar and compounds 5c, 7b and 5a partitioned into three portions: moiety 1 (blue), linker (green) and moiety 2 (light blue).

Figure 11

Sum of the charge density values (atomic units) at the critical intermolecular bond points between P-gp and doxorubicin, tariquidar, 5a, 5c and 7b. The total of interactions established is shown according to the contributions of the three portions of each molecule.