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

Figures & data

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).

Table 1. Docking score and residues involved in binding interactions of compound 1 with PARP1, PARP2, and tubulin.

Compound	Docking score (kcal/mol)	Residues involved in hydrogen bonds (bond distance)	Residues involved in hydrophobic & other interactions
PARP1 (PDB ID: 5DS3)
1	−8.7	–	His862, Ala880, Tyr896, Ala898, Lys903, Tyr907
PARP2 (PDB ID: 4TVJ)
1	−9.5	–	Ala446, Tyr462, Ala464, Lys469, Tyr473
Tubulin (PDB ID: 1SA0)
1	−8.7	–	Gln11, Ala12, Ala99, Tyr224

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.

Table 2. In-vitro growth inhibition (IC₅₀ in µM) effects of the compounds (1–21).

Compound code	IC50 values (Ishikawa cell lines, µM ± SD^a)
1	3 ± 1.09
2	1.5 ± 0.67
3	2.1 ± 0.89
4	>10
5	>10
6	>10
7	6.3 ± 1.08
8	3.2 ± 1.04
9	3 ± 0.785
10	>10
11	>10
12	>10
13	2.03 ± 1.10
14	1.0 ± 0.40
15	1.5 ± 1.08
16	>10
17	>10
18	>10
19	>10
20	>10
21	>10
X	>10
Olaparib	3.91 ± 1.06
Combretastatin	4.21 ± 0.78

^aSD: standard deviation, all experiments were independently performed at least three times

Figure 8. IC₅₀ values (PARP inhibition) of compound 14, Olaparib and Veliparib.

Table 3. PARP1 and PARP2 inhibitory activity of compound 14.

Compound	IC50 (nM)
	PARP1	PARP2
14	74.6	109
Olaparib	0.1	0.11
Veliparib (ABT-888)	1.44	5.58

Table 4. Tubulin inhibitory activity of compound 14 (Treatment concentrations – 0.1, 1, 5, 10 μM).

Compound	IC50 (µM, Tubulin polymerisation)^a
14	1.4 ± 0.3
CA-4	1.3 ± 0.7

^aIC₅₀ values were shown as the mean ± SD of three independent experiments

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.

Table 5. Docking score and residues involved in binding interactions of compound 14 with PARP1, PARP2 and tubulin.

Compound	Docking score (kcal/mol)	Residues involved in hydrogen bonds (bond distance)	Residues involved in hydrophobic & other interactions
PARP1 (PDB ID: 5DS3)
14	−9.4	Gly863 (3.07 Å)	His862, Ala880, Tyr889, Tyr896, Ala898, Tyr907
PARP2 (PDB ID: 4TVJ)
14	−9.9	Arg444 (3.19 Å & 3.27 Å)	Glu335, Asp339, His428, Arg444, Tyr462, Tyr473
Tubulin (PDB ID: 1SA0)
14	−8.0	Thr145 (3.10 Å), Tyr224 (2.90 Å)	Ala12, Asp98, Glu183

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.

Table 6. ADME parameters of compound 14 predicted using the SwissADME online tool.

Property	Predicted value
Molecular formula	C19H11FO3
Molecular weight (g/mol)	306.29
Number of heavy atoms	23
Number of H-bond acceptors	4
Number of H-bond donors	0
Number of rotatable bonds	2
Topological polar surface area (Ų)	43.35
Molar refractivity	85.58
Partition coefficient (Log P)	4.14
Log S (Estimated solubility)	−5.02
Solubility class	Moderately soluble
Bioavailability score	0.55
PGP substrate	No
Log Kp (skin permeation)	−5.08 cm/s
Drug-likeness (Lipinski’s rule of five)	Yes
Number of violations	0
Pan-assay interference structures (PAINS) alerts	0