Flavone-based dual PARP-Tubulin inhibitor manifesting efficacy against endometrial cancer

Abstract

Structural tailoring of the flavone framework (position 7) via organopalladium-catalyzed C-C bond formation was attempted in this study. The impact of substituents with varied electronic effects (phenyl ring, position 2 of the benzopyran scaffold) on the antitumor properties was also assessed. Resultantly, the efforts yielded a furyl arm bearing benzopyran possessing a 4-fluoro phenyl ring (position 2) (14) that manifested a magnificent antitumor profile against the Ishikawa cell lines mediated through dual inhibition of PARP and tubulin [(IC₅₀ (PARP1) = 74 nM, IC₅₀ (PARP2) = 109 nM) and tubulin (IC₅₀ = 1.4 µM)]. Further investigations confirmed the ability of 14 to induce apoptosis as well as autophagy and cause cell cycle arrest at the G2/M phase. Overall, the outcome of the study culminated in a tractable dual PARP-tubulin inhibitor endowed with an impressive activity profile against endometrial cancer.

Keywords: Flavone; PARP; benzopyran; endometrial cancer; inhibitor; tubulin.

Graphical abstract

Figure 1.

Reported flavonoid-based PARP inhibitors.

Figure 2.

Validation of the docking protocol. The docking protocol was validated via redocking the co-crystallized ligands. The re-docked ligands (yellow) produced a pose similar to those of the co-crystallized ligands (red) (A- PARP1; B- PARP2 and C- Tubulin).

Figure 3.

Interaction analysis of 1 with PARP1. (A) Overlay of 1 (yellow) with co-crystallized ligand (red). (B) Orientation of 1 in the active site. (C) 3D docked pose of 1. (D) 2D docked pose of 1 showing hydrophobic interactions with PARP1.

Figure 4.

Interaction analysis of 1 with PARP2. (A) Overlay of 1 (yellow) with co-crystallized ligand (pink). (B) Orientation of 1 in the active site of PARP2 protein. (C) 3D docked pose of 1. (D) 2D docked pose of 1 showing hydrophobic interactions.

Figure 5.

Interaction analysis of 1 with tubulin. (A) Overlay of 1 (yellow) with co-crystallized ligand (pink). (B) Orientation of 1 in the active site of tubulin. (C) 3D docked pose of 1. (D) 2D docked pose of 1 showing hydrophobic interactions with tubulin.

Figure 6.

Design strategy.

Figure 7.

IC₅₀ value of compound 14 (Treatment concentrations- 2,4,6 and 8 µM).

Scheme 1.

Reagents and conditions: (A) NaOH, Ethanol, rt, 24h; (B) I₂, DMSO, reflux, 24 Wh; (C) aryl/heteroaryl boronic acids, Pd(PPh₃), Dioxane: Water (9:3), reflux, 100 °C, 2h.

Figure 8.

IC₅₀ values (PARP inhibition) of compound 14, Olaparib and Veliparib.

Figure 9.

Effect of compound 14 on the cell wall and nuclear morphology of Ishikawa cells.

Figure 10.

Morphology of untreated Ishikawa cells (control) and those treated with 0.1, 0.5, and 1 µM of compound 14.

Figure 11.

(A) Cleavage of LC-3 (autophagy marker) (B) Protein expression was measured by western blotting after 24 h treatment. The intensity of the band indicates down regulation of proteins in the cells. Data represented as the mean of three independent experiments. Statistical significance is represented as follows: ns - non-significant, * p < 0.05, *** p < 0.001.

Figure 12.

Results of Rhodamine staining assay.

Figure 13.

Results of DCFDA staining.

Figure 14.

Effect of compound 14 on cell cycle proteins regulating G2/M progression at indicated concentrations. Statistical significance is represented as follows: ns - non-significant, *p < 0.05, ** p < 0.01.

Figure 15.

(A) Expression levels of proteins (B) Protein expression was measured by western blotting after 24 h treatment. The intensity of the band indicates the down-regulation of proteins in the cells. Data represented as the mean of three independent experiments. Statistical significance is represented as follows: ns - non-significant, *p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 16.

Interaction analysis of 14 with PARP1. (A) Overlay of 14 (yellow) with co-crystallized ligand (red). (B) Orientation of 14 in the active site of PARP1 protein. (C) 3D docked pose of 14 showing hydrogen bond interaction. (D) 2D docked pose of 14 showing hydrogen bond and hydrophobic interactions in the active site of PARP1 protein.

Figure 17.

Interaction analysis of 14 with PARP2. (A) Overlay of 14 (yellow) with co-crystallized ligand (pink). (B) Orientation of 14 in the active site PARP2 protein. (C) 3D docked pose of 14 showing hydrogen bond interaction. (D) 2D docked pose of 14 showing hydrogen bond and hydrophobic interactions in the active site of PARP2 protein.

Figure 18.

Interaction analysis of 14 with tubulin. (A) Overlay of 14 (yellow) with co-crystallized ligand (pink). (B) Orientation of 14 in the active site of tubulin protein. (C) 3D docked pose of 14 showing hydrogen bond interactions. (D) 2D docked pose of 14 showing hydrogen bond and hydrophobic interactions in the active site of tubulin protein.

Figure 19.

Docked pose showing binding interactions of olaparib with PARP1.

Figure 20.

Docked pose showing binding interactions of olaparib with PARP2.

Figure 21.

Docked pose showing binding interactions of combretastatin A4 with tubulin.