Development of novel HER2 inhibitors against gastric cancer derived from flavonoid source of Syzygium alternifolium through molecular dynamics and pharmacophore-based screening

Figures & data

Figure 1 (A) Chemical structures of isolated compounds 5-hydroxy-7,4′-dimethoxy-6,8-di-C-methylflavone (1), kaempferol-3-O-β-d-glucopyranoside (2), and kaempferol-3-O-α-l-rhamnopyranoside (3) and (B) cytotoxicities of compounds 1, 2, and 3 in human gastric cancer cells (AGS).

Notes: Cells were exposed to compounds at the concentration range of 5–100 μg/mL, for 48 h at 37°C in an atmosphere of 5% CO₂. Values are represented as mean ± standard deviation of three replicates in each group.

Figure 2 Induction of AGS cell death by compounds 1, 2, and 3 after 48 h of incubation with different concentrations (25, 50, 75, and 100 μg/mL) at 37°C in an atmosphere of 5% CO₂.

Notes: Cells without any treatment were treated as negative control. Cells treated with 0.5% of triton X-100 were treated as positive control morphological changes of AGS cells were observed under light microscope at 100×. Scale bar= 20 μm.

Figure 3 (A) Cell cycle analysis of AGS cells, treated with compounds 1, 2, and 3 at different concentrations (0, 25, 50, and 75 μg/mL, respectively) and incubated for 48 h at 37°C in an atmosphere of 5% CO₂

Notes: Negative control was treated with phosphate-buffered saline. Positive control was treated with 0.5% triton X-100. Harvested cells were stained with PI and subjected to flow cytometric analysis. (B) Effects of compounds 1–3 on cell cycle progression of gastric cancer cells (AGS). The AGS cells were exposed to different concentrations (0, 25, 50, and 75 μg/mL) of compounds 1, 2, and 3. All the values are represented mean ± standard deviation of triplicates of sample.

Table 1 Bonding characterization and binding energies in kcal/mol with various molecular therapeutic targets involved in gastric cancer

S no	Compound	Dock score (S)	Binding affinity (pKi)	Binding energy (kcal/mol) (MM-GB/VI)	Bonding interaction	Bond length (Å)	Bond type
Epidermal growth factor receptor (EGFR) (PDB id: 4HJO)
1	Compound 1	−11.44	8.558	−11.33	Met769 N121730181217301812173018O	3.05	H-acc
2	Compound 2	−16.02	10.93	−27.79	Thr766 OG1217301812173018-O	2.56	H-don
					Thr830 OG1217301812173018-O	2.57	H-acc
					Asp831 OD1217301812173018--H	1.77	H-acc
					Asp831 OD1217301812173018--H	1.51	H-acc
					Lys721 NZ1217301812173018--O	2.60	H-acc
3	Compound 3	−18.41	10.84	−29.49	Met769 O121730181217301812173018H	1.74	H-don
					Arg817 O121730181217301812173018-H	1.71	H-don
					Thr830 OG1217301812173018--O	2.69	H-acc
					Thr830 OG1217301812173018---O	3.07	H-acc
					Lys721 NZ1217301812173018--O	2.83	H-acc
					Lys721 NZ1217301812173018--O	2.57	H-acc
Human epidermal growth factor receptor (HER2) (PDB id: 3PP0)
4	Compound 1	−15.2	5.493	−19.2	Met96 O121730181217301812173018-H	1.67	H-don
					Met96 N121730181217301812173018-O	2.75	H-don
5	Compound 2	−28.2	10.29	−33.28	Ser23 O121730181217301812173018-H	1.55	H-don
					Thr93 OG1217301812173018---O	2.83	H-don
					Lys31 NZ1217301812173018---O	2.66	H-acc
					Thr93 OG121730181217301812173018O	2.85	H-acc
					Arg144 NE1217301812173018--O	2.49	H-acc
					Arg144 NH1217301812173018--O	2.63	H-acc
6	Compound 3	−22.8	6.93	−30.10	Ser23 O121730181217301812173018-H	1.62	H-don
					Asp158 OD1217301812173018H	1.90	H-don
					Arg144 cation12173018aren
Vascular endothelial growth factor receptor 2 (VEGFR2) (PDB id: 4AG8)
7	Compound 1	−12.18	9.24	−10.53	Lys868 NZ1217301812173018--O	3.09	H-acc
8	Compound 2	−14.42	11.29	−12.73	Ile1025 O121730181217301812173018H	1.67	H-don
					Lys868 NZ1217301812173018--O	2.29	H-don
9	Compound 3	−11.56	7.26	−25.78	Glu885 OE1217301812173018-H	1.31	H-don
					Asp1046 OD12173018---H	1.30	H-don
Cyclin A (PDB id: 4FX3)
10	Compound 1	−11.53	8.028	−20.82	Glu28 OE1217301812173018---H	1.61	H-don
					Asn23 ND1217301812173018--O	2.66	H-acc
11	Compound 2	−17.09	11.50	−25.91	Glu28 OE1217301812173018---H	1.66	H-don
					Val29 OE1217301812173018--H	2.03	H-don
					Ser245 O1217301812173018---H	2.06	H-don
					Lys65 NZ1217301812173018--O	2.78	H-acc
					Ser247 N121730181217301812173018O	2.72	H-acc
12	Compound 3	−15.48	11.13	−26.48	Glu28 OE1217301812173018---H	1.53	H-don
					Leu243 O1217301812173018--H	1.71	H-don
					Ser245 O1217301812173018---H	1.78	H-don
					Lys65N Z1217301812173018--O	2.68	H-acc
					Lys196 NZ1217301812173018O	2.86	H-acc
Caspase 3 (PDB id: 2XYP)
13	Compound 1	−8.17	6.25	−20.50	Arg207 NE1217301812173018--O	2.97	H-acc
					Arg207 cation12173018--aren
14	Compound 2	−15.58	11.39	−27.09	Thr62 OG1217301812173018--O	2.73	H-don
					Ser120 O1217301812173018---H	1.93	H-don
					Thr204 OH1217301812173018-O	2.73	H-don
					Arg64 NE1217301812173018--O	3.01	H-acc
					Gln161 NE1217301812173018-O	3.22	H-acc
15	Compound 3	−16.23	10.22	−22.36	Thr62 OG1217301812173018--O	2.26	H-don
					Ser120 O1217301812173018---H	1.91	H-don
					His121 ND1217301812173018---O	2.92	H-don
					Arg64 NE1217301812173018--O	3.02	H-acc
					Gln161 NE1217301812173018-O	3.30	H-acc
Caspase 6 (PDB id: 2WDQ)
16	Compound 1	−12.44	6.74	−20.56	Arg164 NH1217301812173018--O	2.77	H-acc
					Arg164 NE1217301812173018--O	3.16	H-acc
17	Compound 2	−15.25	10.47	−27.54	Gln137 OE1217301812173018---H	1.81	H-don
					Cys163 O121730181217301812173018H	2.43	H-don
					Glu214 OE1217301812173018-H	1.68	H-don
					Cys163 O1217301812173018---H	2.78	H-acc
					Arg164 NH1217301812173018-O	2.37	H-acc
18	Compound 3	−16.50	8.65	−29.41	Ala129 O121730181217301812173018H	1.72	H-don
					Cys163 N121730181217301812173018O	2.69	H-acc
					Arg164 NH1217301812173018-O	2.65	H-acc
					Arg164 NE1217301812173018-O	2.93	H-acc
					Thr199 N121730181217301812173018O	2.81	H-acc
Caspase 8 (PDB id: 1QTN)
19	Compound 1	−8.15	5.52	−19.02	Thr503 O121730181217301812173018-H	1.67	H-don
20	Compound 2	−18.38	10.48	−33.15	Gly318 O121730181217301812173018-H	1.59	H-don
					Asp319 OD1217301812173018--H	1.57	H-don
					Tyr324 OH1217301812173018--O	2.82	H-don
					Lys253 NZ1217301812173018---O	2.76	H-acc
					Ser567 N121730181217301812173018--O	2.67	H-acc
21	Compound 3	−15.59	11.59	−24.44	Lys253 NZ1217301812173018---O	1.79	H-don
					Gly318 O121730181217301812173018-H	1.55	H-don
					Tyr324 OH1217301812173018--O	2.74	H-don
					Thr503 OG1217301812173018-O	2.72	H-acc

Figure 4 (A) Structural alignment of compounds 1–3 depicted with pharmacophore features; (B) three-dimensional pharmacophore features generated from complex of human growth factor receptor-2-compound 2; and (C) the retrieved best 10 hits and their RMSDs with compound 2.

Abbreviation: RMSD, root mean square deviation.

Table 2 Lipinski’s parameter, drug score, drug likeness, and adverse effects of isolated compounds 1–3 and the best hits computed with MOLINSPERATION and OSIRIS server

Compound name	CLogP	Solubility	Weight (kDa)	HBD	HBA	b_rotN	Drug likeness	Drug score	TPSA	Mutagenic	Tumorigenic	Irritant	Reproductive effect
Compound 1	3.57	−4.17	326.0	2	5	3	−3.22	0.23	64.99	No	Mild	No	No
Compound 2	0.0	−2.49	448.0	5	9	4	−2.68	0.42	186.3	No	No	No	No
Compound 3	0.93	−2.99	432.0	6	8	3	−2.8	0.77	166.1	No	No	No	No
Best leads
ZINC67903192	−0.91	−2.69	594.0	9	15	6	1.58	0.54	245.2	No	No	No	No
ZINC59763389	−0.31	−2.82	564.0	8	13	5	−2.89	0.33	225.0	No	No	No	No

Abbreviations: CLogP, log octanal/water partition coefficient; weight, molecular weight; HBD, H-bond donor atoms; HBA, H-bond acceptor atoms; b_rotN, number of rotatable bonds; TPSA, topological polar surface area.

Table 3 Bonding characterization of similar pharmacophore hits for compound 2 from ZINC database against HER2

S no	Ligand name	Structure	RMSD (Å)	Bonding interaction	Bond length	Bond type	Dock score (S)	Binding energy (kcal/mol) (MM-GB/VI)	Binding affinity (pKi)
1	ZINC67903192		0.538	Met801 O1217301812173018H	2.20	H-don	−32.2	−40.13	11.21
				Asp863 OD12173018--H	1.26	H-don
				Lys736 NZ12173018---O	2.59	H-acc
				Leu726 CD12173018--O	3.01	H-acc
2	ZINC59763389		0.792	Ser728 O1217301812173018-H	3.79	H-don	−28.4	−34.24	10.33
				Met801 O1217301812173018H	2.19	H-don
				Tyr803 O1217301812173018H	1.80	H-don
				Asp863 OD12173018-H	2.02	H-don
				Met801 N12173018---O	2.92	H-acc
				Lys736 N212173018--O	2.50	H-acc
3	ZINC85816423		0.601	Ser728 O1217301812173018-H	1.66	H-don	−27.2	−26.23	9.62
				Asp808 OD12173018-H	1.75	H-don
				Arg849 NE12173018---O	2.78	H-acc
				Arg849 NH12173018--O	3.09	H-acc

Abbreviation: RMSD, root mean square deviation.

Figure 5 Backbone root mean square deviation values (A), root mean square fluctuations of backbone atoms (C), radius of gyration of backbone atoms (D), solvent accessible surface area (E) of HER2a, HER2b, HER2c, HER2d, and HER2e, and backbone RMSD and SASA values (B and F) for compounds 1, 2, 3, and ZINC67903192 during 20 ns molecular dynamic simulations.

Notes: Black, HER2a; red, HER2b; green, HER2c; blue, HER2d; yellow, HER2e.
Abbreviations: HER, human growth factor receptor; RMSD, root mean square deviation.

Figure 6 (A) Cα RMSF values of HER2, HER2-compound 1, HER2-compound 2, HER2-compound 3, and HER2-ZINC67903192 were plotted against residue numbers; (B) the profile of Cα atomic fluctuations of key H-bonding residues in the catalytic site of HER2.

Notes: Black, HER2a; red, HER2b; green, HER2c; blue, HER2d; yellow, HER2e.
Abbreviations: HER, human growth factor receptor; RMSF, root mean square fluctuation.

Figure 7 Secondary structural changes in HER2a, HER2b, HER2c, HER2d, and HER2e (A, B, C, D, and E, respectively) during 20 ns of molecular dynamic simulations, reported through Database of Secondary Structure of Proteins software.

Abbreviation: HER, human growth factor receptor.

Figure 8 Molecular interactions of compounds 1, 2, 3 and structural hit ZINC67903192 with tyrosine kinase domain of human growth factor receptor-2 (A, B, C, and D, respectively).

Figure 9 (A) The snapshots of docking pose of compound 2 and total H-bond intensity at various time scale intervals in 20 ns complex molecular dynamic simulations with human growth factor receptor-2; (B) hydrogen bond diagram between compound 2 and active site residues, namely, Leu726, Gly726, Ser728, Gly729, and Met801 of human growth factor receptor-2.

Note: Black squares indicate the presence of H-bond and white squares correspond to the absence of H-bond throughout the time scale of 20 ns molecular dynamic simulations.

Figure 10 (A) The snapshots of docking pose of ZINC67903192 and total H-bond intensity at various time scale intervals in 20 ns complex molecular dynamic simulations with human growth factor receptor-2; (B) hydrogen bond diagram between ZINC67903192 and active site residues, namely, Leu726, Gly727, Ser728, Met801, Thr862, and Asp863 of human growth factor receptor-2.

Note: Black squares indicate the presence of H-bond and white squares correspond to the absence of H-bond throughout the time scale of 20 ns molecular dynamic simulations.