Identification of the first-in-class dual inhibitors of human DNA topoisomerase IIα and indoleamine-2,3-dioxygenase 1 (IDO 1) with strong anticancer properties

Figures & data

Figure 1. Human topoisomerase Iiα inhibitors based on thiosemicarbazide core^6–8.

Table 1. Chemical structures of compounds characterised by the most potent affinity towards human DNA topoisomerase II.

Compound No.	Chemical structure	Free energy of binding [kcal/mol]
1		−11.0
2		−11.8
3		−10.8
4		−10.7
5		−10.3
6		−10.1
7		−10.1
8		−10.4
9		−10.2
10		−10.3
11		−10.0
12		−10.2
Native ligand		−14.4

Table 2. Inhibition of human DNA topoisomerase Iiα by compounds 1-3 and etoposide.

	IC50 [µM] ± SD
1	71.04 ± 4.81
2	43.11 ± 2.27
3	76.80 ± 4.85
Etoposide	123.36 ± 5.42

IC₅₀ values represent median inhibitory effects of compounds 1-3 and etoposide against relaxation activity of human DNA topoisomerase IIα. Compounds 4-10 had no inhibitory activity (or had only marginal activity) in this assay.

Table 3. Antiproliferative activity of compounds 1-3 and etoposide examined in BrdU assay.

	IC50 [µg/mL] ± SD
	Etoposide	1	2	3
MDA-MB-231	>100	26.29 ± 0.99	>100	>100
MCF-7	54.94 ± 1.82	1.19 ± 0.04	33.16 ± 1.05	32.54 ± 1.98
SCC-25	21.21 ± 0.74	1.10 ± 0.02	1.06 ± 0.04	4.89 ± 0.22
A549	2.60 ± 0.11	0.23 ± 0.01	0.83 ± 0.03	0.25 ± 0.01
AGS	45.99 ± 2.61	0.51 ± 0.02	62.34 ± 3.04	43.92 ± 1.94
T98G	29.51 ± 0.93	0.72 ± 0.02	6.19 ± 1.83	6.28 ± 0.37
LS180	>100	4.68 ± 0.17	10.39 ± 0.77	10.56 ± 0.24
FaDu	12.82 ± 0.27	1.04 ± 0.04	7.11 ± 0.34	9.04 ± 0.39
A375	9.18 ± 0.15	0.96 ± 0.03	0.85 ± 0.04	0.56 ± 0.03
HT-29	19.93 ± 0.25	0.44 ± 0.03	9.47 ± 0.51	13.14 ± 0.81
Human skin fibroblasts	55.69 ± 3.01	22.38 ± 0.94	24.17 ± 1.40	32.11 ± 1.75

BrdU incorporation was tested after 24 and 48 h incubation of the cells with the investigated compounds. However, due to strong inhibition of BrdU incorporation by compounds 1-3, the results obtained after 48 h of incubation (IC₅₀ values tend to zero for most of the cancer cell lines) were omitted for clarity.

Table 4. Cytotoxicity of the investigated compounds 1-10 and etoposide against a panel of cancer cell lines measured by using MTT assay after 24 and 48 h incubation.

	IC50 [µg/mL]*
		Etoposide	1	2	3	4	5	6	7	8	9	10
MDA-MB-231	24h	>100	9.21	7.64	10.47	>100	>100	>100	>100	>100	>100	83.42
	48h	40.31	8.04	7.07	6.02	32.48	56.31	16.84	69.37	17.23	>100	68.12
MCF-7	24h	>100	9.82	9.18	7.67	>100	>100	>100	>100	>100	>100	64.11
	48h	6.15	8.45	8.85	5.48	19.42	28.13	15.93	24.84	12.74	16.37	20.64
SCC-25	24h	59.07	7.06	4.73	4.76	>100	>100	25.98	42.41	>100	32.19	52.41
	48 h	23.72	5.22	3.12	3.87	40.86	17.32	9.78	24.32	29.47	34.48	23.86
A549	24h	>100	3.87	5.84	5.37	>100	>100	20.77	>100	>100	>100	92.63
	48h	15.59	2.27	5.78	5.13	>100	>100	23.45	>100	>100	>100	32.84
AGS	24h	50.79	5.92	5.32	5.54	>100	>100	23.46	>100	>100	>100	29.46
	48 h	29.77	2.69	2.96	4.31	63.87	63.92	29.77	18.76	>100	19.66	21.06
T98G	24h	>100	6.98	5.53	3.35	55.47	>100	8.41	11.45	97.22	>100	47.23
	48h	17.06	2.27	4.85	2.94	7.26	30.08	9.78	10.66	11.82	17.39	30.63
LS180	24h	>100	4.57	2.87	3.12	>100	>100	>100	>100	>100	>100	72.43
	48 h	11.71	2.54	2.24	2.70	55.62	19.84	10.41	24.68	41.15	19.82	12.55
FaDu	24h	12.54	7.26	2.75	2.49	>100	>100	12.89	33.52	>100	28.16	20.12
	48 h	10.73	2.01	2.54	2.18	39.25	31.69	14.77	22.47	12.81	19.53	29.74
A375	24h	10.21	4.57	8.96	1.80	>100	89.31	>100	>100	>100	>100	47.92
	48 h	6.48	1.82	2.47	0.56	14.81	38.45	7.98	13.88	9.45	8.14	30.15
HT-29	24h	94.49	8.96	8.96	7.37	25.01	45.23	22.78	47.38	>100	>100	32.94
	48 h	11.30	5.33	5.33	3.77	>100	12.30	6.78	7.18	14.77	5.23	32.16
HEK-293	24h	37.85	17.60	15.59	27.54	>100	78.14	>100	67.15	>100	67.03	63.71
	48h	29.12	9.61	10.03	9.19	>100	24.59	60.13	32.70	23.42	23.03	19.50

*SD values were omitted for clarity.

Figure 2. The exemplary, representative poses of the three ligands interacting with binding cavity of topoisomerase II. All depicted poses, characteristic of a given ligand, exhibit similar level of binding energies (see discussion in the text). (A) compound 1; (B) compound 2; (C) compound 3.

Figure 3. The exemplary, energetically-favourable poses of the three lead compounds identified during docking study: (A) compound 1; (B) compound 2; (C) compound 3.The ligand molecules are shown as thick sticks whereas all the closest amino-acid residues (within the distance of 0.4 nm) are represented by thin sticks. The description of the interaction types is given in the text.

Table 5. Inhibitory effect of compounds 1-3 against indoleamine-2,3-dioxygenase 1 (IDO 1).

	IDO 1 inhibition (%) ± SD
1	91.37 ± 1.82
2	41.37 ± 2.47
3	21.43 ± 1.05
Epacadostat	99.58 ± 0.43

The compounds were tested in a fixed dose of 50 µg/ml. Since compounds 1-3 were devoid of inhibitory effect towards IDO 2 and TDO (also examined during the assay), therefore these results are not included in the table.

Figure 4. Validation of the docking procedure. The comparison of the position of the ligand present in the crystal structure of protein deposited in PDB:3qx3 (colored by atom type) and the position predicted during docking simulations (colored in orange). Hydrogen atoms are omitted.