Virtual Screening Guided Design, Synthesis and Bioactivity Study of Benzisoselenazolones (BISAs) on Inhibition of c-Met and Its Downstream Signalling Pathways

Abstract

c-Met is a transmembrane receptor tyrosine kinase and an important therapeutic target for anticancer drugs. In this study, we designed a small library containing 300 BISAs molecules that consisted of carbohydrates, amino acids, isothiourea, tetramethylthiourea, guanidine and heterocyclic groups and screened c-Met targeting compounds using docking and MM/GBSA. Guided by virtual screening, we synthesised a series of novel compounds and their activity on inhibition of the autophosphorylation of c-Met and its downstream signalling pathway proteins were evaluated. We found a panel of benzisoselenazolones (BISAs) obtained by introducing isothiourea, tetramethylthiourea and heterocyclic groups into the C-ring of Ebselen, including 7a, 7b, 8a, 8b and 12c (with IC₅₀ values of less than 20 μM in MET gene amplified lung cancer cell line EBC-1), exhibited more potent antitumour activity than Ebselen by cell growth assay combined with in vitro biochemical assays. In addition, we also tested the antitumour activity of three cancer cell lines without MET gene amplification/activation, including DLD1, MDA-MB-231 and A549. The neuroblastoma SK-N-SH cells with HGF overexpression which activates MET signalling are sensitive to MET inhibitors. The results reveal that our compounds may be nonspecific multitarget kinase inhibitors, just like type-II small molecule inhibitors. Western blot analysis showed that these inhibitors inhibited autophosphorylation of c-MET, and its downstream signalling pathways, such as PI3K/AKT and MARK/ERK. Results suggest that bensoisoselenones can be used as a scaffold for the design of c-Met inhibiting drug leads, and this study opens up new possibilities for future antitumour drug design.

Keywords: benzoisoselenone; c-Met inhibition; docking; molecular dynamics simulation; virtual screening.

Figure 1

Structure of the known c-Met inhibitors and Ebselen.

Scheme 1

Reagents and conditions: (i) NH₂(CH₂)_nBr, Et₃N, THF, rt, 5 h, 50–70%; (ii) thiourea, THF, 70 °C, overnight, 40–60%; (iii) tetramethylthiourea, THF, 70 °C, overnight, 55%; (iv) N¹-(6-bromohexyl)-N¹-methylbenzene-1,4-diamine, Et₃N, THF, rt, 5 h, 35–37%; (v) thiourea, THF, 70 °C, overnight, 45–53%; (vi) tetramethylthiourea, THF, 70 °C, overnight, 45–55%; (vii) 9a~b, Et₃N, THF, rt, 5 h, 50–70%; (viii) HCl, DCM, 3 h, 90–95%; (ix) R₁NH₂, Et₃N, THF, rt, 5 h, 50–65%; (x) 1-(3-bromopropyl) piperidine, K₂CO₃, THF, rt, overnight, 40%.

Figure 2

Effects of the tested compounds on cell viability of EBC-1 cancer cell line. (A) Percentage of viable cells (EBC-1) after 72 h exposure to the compounds at a concentration of 25 μM compared to the compound-free control received an equal volume of DMSO (100% viability). Each value was calculated from two independent experiments. The competitive inhibitory activity was expressed as an inhibition rate. (B) Growth inhibition effect of compounds 3d, 7a, 7b, 8a, 8b, 12c and 12d on EBC-1 cells. EBC-1 cells were seeded into 96-well cell culture plates and allowed to grow overnight. Thereafter, cells were treated with vehicle control (DMSO) or the compounds at the indicated concentration for 72 h. After treatment, 10 μL resazurin were added to the culture medium. After incubation at 37 °C for 5 h, fluorescence intensity was measured at a 530 nm excitation wavelength and a 590 nm emission wavelength using a Synergy HT photometer.

Figure 3

Effects of compounds 3d, 7a, 8a, 8b and 12c on cell viability of four cancer cell lines. Percentage of viable cells (SK-N-SH, DLD1, MDA-MB-231 and A549) after 72 h exposure to the compounds at a concentration of 25 μM. Each value was calculated from two independent experiments. The competitive inhibitory activity was expressed as an inhibition rate.

Figure 4

Effects of Ebselen and compounds on p-c-Met, p-AKT and p-ERK in EBC-1 cancer cells. (A) Western blot analysis in the EBC-1 cells treated with concentrations near the IC₅₀ values of compounds for 4 h. Ebselen was unable to inhibit the phosphorylation of c-Met at 25 μM. (B) Representative Western blot analysis in the EBC-1 cells treated with increasing concentrations of compounds 3d, 7a, 7b, 8b and 12c, as indicated. All of the tested compounds could inhibit the phosphorylation of Met, AKT and ERK in a dose-dependent manner.

Figure 5

The binding mode of compounds in the ATP binding pocket of c-Met protein in two-dimensional panel. The green full line indicates the π–π stacking interaction, the purple arrow indicates the hydrogen bond and the fuchsia line indicates the ionic bond. The grey shade highlights the solvent exposure region of the small compounds. (A) The binding mode of compound 7a. (B) The binding mode of compound 7b. (C) The binding mode of compound 8a. (D) The binding mode of compound 8b. (E) The binding mode of compound 12c.

Figure 6

Analysis of the stability of compound/c-Met over 50 ns (from molecular dynamics simulations). (A) The binding mode of compound 7a (yellow) in the binding site of c-Met (cyan and green) and its binding pocket. (B) The hydrogen bond interactions of 7a–MET1160 (yellow), 7a–ASP1204 (red and green), 7a-ASN1209 (black), 7a-ASP1222 (blue) and 7a–PHE1223 (brown). (C) The binding mode of compound 8a (yellow) in the binding site of c-Met (cyan and green) and its binding pocket. (D) The hydrogen bond interactions of 8a–MET1211 (black) and the ionic bond of 8a–GLU1127 (red). (E) The binding mode of compound 12c (yellow) in the binding site of c-Met (cyan and green) and its binding pocket. (F) The hydrogen bond interactions of 12c–ASP1222 (black) and 12c–PHE1223 (red and green).