Virtual Screening and Optimization of Novel mTOR Inhibitors for Radiosensitization of Hepatocellular Carcinoma

Figures & data

Figure 1 (A) Pharmacophore model based on the reported mTOR inhibitors and the lead compound B170422 screened out by the pharmacophore model; (B) schematic of the binding mode of B170422 with mTOR.

Figure 2 Hits obtained by virtual screening.

Figure 3 Superposition of three-dimensional protein crystal structures of mTOR and PI3Kα. Red: mTOR (4JSV); green: PI3Kα (6OAC). The ATP binding pocket of mTOR and PI3Kα is represented by solid tube; the other parts are represented by ribbon.

Figure 4 Representative mTOR inhibitors and PI3K inhibitors.

Table 1 Structures, Enzyme Inhibitory Activities, and Selectivities of 1a–1d and 2a–2j

	R1	R2	R3	mTOR IC50 (nM)^a	PI3Kα IC50 (nM)^a	Selectivity
1a				167	1578	9
1b				50	793	16
1c				16	516	32
1d				1562	>10,000	>6
2a				7.1	895	126
2b				124	437	4
2c				139	1055	8
2d				23	228	10
2e				72	254	4
2f				28	193	7
2g				313	844	3
2h				359	3203	9
2i				92	2681	29
2j				184	952	5

Note:^aAll values represent the mean of two independent assays.

Table 2 Structures, Enzyme Inhibitory Activities, and Selectivities of 3a–3h

	R1	R2	R3	mTOR IC50 (nM)^a	PI3Kα IC50 (nM)^a	Selectivity
3a				260	3562	14
3b				2211	>10,000	>5
3c				246	703	3
3d				932	7365	8
3e				479	2766	6
3f				529	433	0.8
3g				1774	>10,000	>6
3h				326	738	2

Notes: ^aAll values represent the mean of two independent assays.

Figure 5 (A) Schematic of the binding mode of 2a with mTOR; (B) image of the mTOR surface around 2a.

Scheme 1 The synthetic route of 2a. Reagents and conditions: (A) NaOH, ethyl alcohol, rt; (B) triethylamine, methylbenzene, 100°C; (C) ethyl alcohol, 80°C.

Table 3 Comparison of the Inhibitory Activity of 2a on mTOR Enzyme and MHCC97-H Cells at Different Concentrations (Considering the Experimental Error, the Number of Decimal Places Has Been Optimized)

Concentration of 2a (μM)	mTOR Enzyme Inhibition Rate (%)	Cell Inhibition Rate (%)
10	98.2 ± 7.2	52.5 ± 3.8
3	96.4 ± 4.1	37.7 ± 6.5
1	94.3 ± 6.2	29.4 ± 5.3
0.3	87.9 ± 5.3	17.7 ± 1.7
0.1	74.8 ± 2.7	8.1 ± 2.3
0.03	67.2 ± 4.5	5.3 ± 1.5
0.01	54.7 ± 3.6	3.2 ± 0.7
0.003	41.1 ± 1.7	2.3 ± 0.3

Figure 6 Compound 2a enhances the sensitivity of MHCC97-H cells to IR. MHCC97-H cells pretreated with 2a were treated with the indicated dose (0, 2, 4, or 8 Gy) of 60Co-γ IR and were examined by colony formation experiments. (A) Experimental results are shown as images of colonies; (B) experimental results are shown as inhibition rates (mean ± SD). *p<0.05 versus solvent control or 2a.

Figure 7 Compound 2a enhances DNA DSBs induced by 60Co-γ IR in MHCC97-H cells. (A) 2a enhances the γ-H2aX foci induced by IR (4 Gy) in nuclear of MHCC97-H cells; (B) the total area of granules of γ-H2aX foci (mean ± SD). *p<0.05.