Chalcones isolated from Angelica keiskei inhibit cysteine proteases of SARS-CoV

Figures & data

Figure 1. Chemical structures of isolated alkylated chalcones from A. keiskei.

Table 1. Inhibitory effects of isolated alkylated chalcones (1–9) and coumarins (10–13) on cell-free trans-cleavage and cell-based cis-cleavage activities of SARS-CoV 3CL^pro.

	Cell-free cleavage		Cell-based cleavage
Compounds	IC50 (µM)^a*	Inhibition type (Ki, µM)	IC50 (µM)^a*	CC50 (µM)†	SI‡
1	39.4 ± 5.2	Competitive (57.8 ± 15.1)	11.9 ± 2.8	15.3 ± 1.6	1.3
2	81.4 ± 8.5	Competitive (85.9 ± 11.6)	50.8 ± 3.0	23.0 ± 2.4	0.4
3	38.4 ± 3.9	Competitive (89.9 ± 24.5)	5.8 ± 0.6	20.4 ± 3.8	3.5
4	34.1 ± 4.8	Competitive (29.2 ± 11.7)	32.6 ± 2.2	28.8 ± 6.3	0.6
5	26.6 ± 5.2	Competitive (34.8 ± 7.9)	9.3 ± 1.2	62.0 ± 1.3	6.7
6	11.4 ± 1.4	Competitive (16.1 ± 2.5)	7.1 ± 0.8	65.6 ± 1.7	9.2
7	22.2 ± 6.5	Competitive (43.8 ± 8.2)	8.6 ± 2.6	36.3 ± 0.6	4.2
8	129.8 ± 10.3	Competitive (267.3 ± 50.1)	NT¶	NT	NT
9	44.1 ± 1.3	Competitive (76.1 ± 12.0)	9.8 ± 2.3	62.6 ± 6.3	6.4
10	45% at 200 µM	NT	NT	NT	NT
11	40% at 200 µM	NT	NT	NT	NT
12	10% at 200 µM	NT	NT	NT	NT
13	40% at 200 µM	NT	NT	NT	NT

*,†All compounds were examined in a set of triplicates experiment; IC₅₀ (50% inhibitory concentration) values of compounds represent the concentration that caused 50% enzyme activity loss; CC₅₀ (50% cytotoxic concentration) was the concentration giving half the OD 570–630 nm of mock cells in MTT assay.

‡SI means selective index (CC₅₀/IC₅₀).

¶Not tested.

Figure 2. (A) Effects of compounds 1–9 on the activity of SARS-CoV PL^pro for proteolysis of substrate. (B) Relationship of the hydrolytic activity of SARS-CoV PL^pro as function of enzyme concentrations at different concentration of compound 1.

Table 2. Inhibitory effects of isolated alkylated chalcones (1–9) and coumarins (10–13) on SARS-CoV PL^pro.

	PL^pro, IC50 (µM)*
	LRLRGG-AMC
Compounds	IC50 (μM)	Inhibition type (Ki, µM)	Deubiquitination activity	DeISGylation activity
1	13.0 ± 0.9	Mixed (7.9 ± 1.4)	16.0 ± 4.5	10.9 ± 1.8
2	26.0 ± 1.5	Noncompetitive (15.2 ± 0.7)	15.4 ± 9.8	9.2 ± 0.1
3	11.7 ± 3.2	Noncompetitive (10.7 ± 0.7)	10.2 ± 2.8	6.1 ± 1.4
4	5.6 ± 0.5	Noncompetitive (5.4 ± 1.7)	4.9 ± 0.5	1.1 ± 0.6
5	19.3 ± 1.8	Noncompetitive (10.7 ± 1.4)	13.1 ± 6.6	8.2 ± 1.3
6	1.2 ± 0.4	Noncompetitive (1.2 ± 0.06)	2.6 ± 0.7	5.2 ± 0.4
7	11.7 ± 0.3	Noncompetitive (7.3 ± 0.3)	17.4 ± 0.6	7.9 ± 1.5
8	46.4 ± 7.8	Noncompetitive (32.5 ± 2.4)	44.1 ± 1.2	10.2 ± 1.1
9	21.1 ± 5.6	Noncompetitive (12.9 ± 0.5)	30.2 ± 4.5	11.3 ± 1.9
10	40% at 200 µM	NT†	18.0 ± 3.2	5.6 ± 1.3
11	30% at 200 µM	NT	73.3 ± 13.2	18.8 ± 3.8
12	30% at 200 µM	NT	53.8 ± 8.1	33.1 ± 6.4
13	80% at 200 µM	NT	13.0 ± 1.9	12.1 ± 2.6

*All compounds were examined in a set of experiments repeated three times; IC₅₀ values of compounds represent the concentration that caused 50% enzyme activity loss.

†Not tested.

Figure 3. Graphical determination of the type of inhibition for compound 6. (A) Lineweaver–Burk plot for inhibition of compound 6 on SARS-CoV 3CL^pro. (B) Dixon plot for inhibition of compound 6 on SARS-CoV 3CL^pro. (C) Lineweaver–Burk plot for inhibition of compound 6 on SARS-CoV PL^pro. (D) Dixon plot for inhibition of compound 6 on SARS-CoV PL^pro.

Figure 4. In silico molecular docking analysis of compound 6 binding to SARS-CoV 3CL^pro (A) and PL^pro (B).

Table 3. Inhibitory effects of isolated alkylated chalcones (1–9) and coumarins (10–13) on other proteases.

	Cysteine protease, IC50 (µM)*	Serine protease, IC50 (µM)*
Compounds	Papain	Trypsin	Chymotrypsin
1	35.2 ± 3.9	20% at 200 µM	111.9 ± 4.8
2	41.2 ± 3.2	134.4 ± 10.7	102.8 ± 4.2
3	58.9 ± 4.1	20% at 200 µM	N.A
4	103.6 ± 15.8	20% at 200 µM	N.A
5	32.1 ± 1.0	10% at 200 µM	N.A
6	4.8 ± 0.4	143.3 ± 5.7	273.4 ± 16.8
7	43.5 ± 13.6	133.4 ± 13.1	N.A
8	47.3 ± 11.2	51.6 ± 8.4	76.5 ± 3.8
9	20% at 200 µM	20% at 200 µM	N.A
10	N.A	N.A	N.A
11	N.A	N.A	N.A
12	N.A	N.A	N.A
13	N.A	N.A	N.A

*All compounds were examined in a set of experiments repeated three times; IC₅₀ values of compounds represent the concentration that caused 50% enzyme activity loss.

N.A., No activity.