A novel multi-target inhibitor harboring selectivity of inhibiting EGFR T790M sparing wild-type EGFR

Abstract

Non-Small Cell Lung Cancer (NSCLC) is driven by a variety of deregulated kinases and the development of multi-target inhibitor for multiple signaling pathways or multiple steps is required. Here, we reported that ZWM026, an indolocarbazoles analogue, derived from mangrove in coastal marine wetland, exhibited selectivity and reversibility against T790M mutant over wild-type EGFR in naturally occurring NSCLC cells and constructed NIH-3T3 cells. It simultaneously inhibited activities of HER2, HER3, HER4 and RET but was different from current multi-target kinase inhibitors. There was no activity in protein kinase C (PKC) family which is generally recognized as molecule target of indolocarbazoles. ZWM026 had more potent activities against gefitinib sensitizing, non-sensitizing and rare EGFR mutant NSCLC cells and constructed NIH-3T3 cells. ZWM026 induced apoptosis and exerted a synergistic effect by combining with cisplatin in NCI-H1975 cells. In summary, we identified a novel reversible multi-target inhibitor which could serve as a promising lead compound of drug development for NSCLC.

Keywords: EGFR T790M; NSCLC; ZWM026; multi-target inhibitor.

Figure 1

Structures of ZWM026 and PKC412.

Figure 2

ZWM026 selectively inhibits EGFR-T790M and spares with EGFR-WT in cells. (A) ZWM026 inhibits EGFR phosphorylation and downstream signaling pathways in NCI-H1975 cells (left) and A549 cells (right). Cells were treated with increasing concentrations of ZWM026 for 30 minutes and lysed as described Methods. The resulting extracts were probed with the indicated antibodies. (B) ZWM026 inhibits EGF-induced EGFR phosphorylation and downstream signaling pathways in NCI-H1975 cells (left) and A549 cells (right). Cell lines were cultured in serum-starved medium for 10 hours followed by indicated compounds treatment. Cell lines were stimulated by EGF (250 ng/ml) for 15 minutes and then were analyzed by western blotting as above. (C, D) Efficacy comparisons of ZWM026, gefitinib, vandetanib and PKC412 against phosphor-EGFR in NCI-H1975 cells and A549 cells. (E, F) Efficacy comparisons of ZWM026, gefitinib, vandetanib and PKC412 against phosphor-EGFR in constructed NIH-3T3 stably expressed EGFR-L858R/T790M and EGFR-WT. (G, H) Efficacy comparisons of ZWM026, gefitinib, vandetanib and PKC412 against HCC827 cell lines harboring the sensitive mutant EGFR. Blots are quantified in Figure 2 (D), (F) and (H), respectively. (I) Molecule docking of ZWM026 binding to EGFR-T790M. EGFR x-ray structure was retrieved from the Protein Data Bank (PDB code 3IKA) and the molecular modeling was conducted using Sybyl × 1.10. Compound ZWM026 was docked into the active site of the T790M mutant EGFR. The rings of ZWM026 formed hydrophobic interaction with the LEU718, VAL726, ALA743, MET790 GLU791, MET793 and LEU844. (J) Reversibility assessments for ZWM026 and PKC412. Cells were treated with ZWM026 and PKC412 at 1 μM for 16 hours. Then removed the culture medium containing compounds, and EGFR expression was determined by Western Blotting after washing the culture at 0, 1, 3, 6, 24 hours.

Figure 3

Activities of ZWM026 against other kinases in H1975 cells. (A-H) Efficacy comparisons of ZWM026, gefitinib, vandetanib and PKC412 against phosphorylation HER2 (A), HER3 (B), HER4 (E) and RET (F) in NCI-H1975 cell lines. Cells were treated with increasing concentrations of indicated compounds for 30 minutes and the cell extracts were probed with the indicated antibodies. Blots are quantified in Figure 3 (C), (D), (G) and (H), respectively. (I) Efficacy comparisons of ZWM026 and PKC412 on phosphorylation of HER2 in constructed NIH-3T3 stably expressed HER2-WT. The experiment methods are same as above.

Figure 4

ZWM026 potently and selectively inhibits growths of several different NSCLC cells. ZWM026 inhibits growths in various tumor cell lines, including NSCLC cells with EGFR-mutant and EGFR-WT, as well as some normal cell lines. Cell viability was analyzed using MTT assay after 72 hours of treatment. The data are expressed as mean ± SD from three or more independent experiments.

Figure 5

ZWM026 induces different death way of cells and exerts synergistic effect combined with cisplatin. (A, B) Activities of ZWM026 against cleaved-PARP, LC3B and phosphor-PKCα/βII in H1975 and A549 cell lines. Cells were treated with ZWM026 and gefitinib at indicated concentration for 48 hours. The resulting extracts were probed with the indicated antibodies. (C) Efficacy comparisons of ZWM026 and PKC412 against phosphor-EGFR and phosphor-PKCα/βII in constructed NIH-3T3 stably expressed EGFR-L858R/T790M and EGFR-WT. Cells were treated with the compounds for 30 minutes and lysed as described methods. (D) ZWM026 in combination with cisplatin induces the synergistic inhibition of proliferation. We examined the effects of ZWM026 and cisplatin, either alone or in combination, on growth inhibition of H1975 cells by the MTT assay (The CI value was calculated according to the CompuSyn software). The CI > 1 is antagonistic, CI = 1 is additive and CI < 1 is synergistic. *p < 0.05, combination vs. ZWM026, **p < 0.01, combination vs. cisplatin.