Molecular modeling, DFT quantum chemical analysis, and molecular docking on edotecarin, an indolocarbazole anticancer agent

Abstract

Edotecarin is an indolocarbazole class antitumor agent that has significant anticancer effects against various types of cancer, especially lung, breast, and stomach cancer.The conformation analysis of the edotecarin was performed using the PM3 method and six stable conformations were obtained.Afterwards the obtained lowest energy conformation was optimized at the DFT/B3LYP/6-31++G(d,p) level of theory. The vibrational wavenumbers, the highest occupied molecular orbital, the lowest unoccupied molecular orbital and molecular electrostatic potential of the most stable conformer of edotecarin were calculated at the DFT/B3LYP/6-31++G(d,p) level of theory.The molecular docking of the edotecarin molecule against DNA, Topoisomerase I, DNA-Topoisomerase I complex,α₅β₁ and α_IIbβ₃ integrins were performed to reveal its binding modes and binding affinities.

Keywords:

• Anticancer drug
• conformational analysis
• edotecarin
• molecular docking
• molecular modeling

Introduction

Edotecarin, {6-N-(1-hydroxymethyl-2-hydroxy)ethylamino-12,13-dihydro-2,10-dihydroxy-13-(b-D-glucopyranosyl)-5H-indolo[2,3-a]-pyrrolo[3,4-c]-carbazole-5,7(6H)-dione}, was derived from the glucosyl derivative of indolo-carbazole (NB-506) [1]. It is a topoisomerase I (TOPI) inhibitor, induces single-strand DNA cleavage more effectively than NB-506, thus having potential use in solid tumor treatment [1]. In vitro research results indicate that edotecarin is a novel class of TOPI inhibitor with different and superior characteristics compared to other clinically used anticancer drugs [2]. Studies of edotecarin against numerous human xenografts, murine tumors and their lethal toxicity in mice were performed in a study conducted by Arakawa et al. [3]. The antitumor activity of edotecarin was tested in vitro and in vivo to observe the inhibition of tumor growth in models of breast, cervix, pharynx, lung, prostate, colon, stomach and hepatic cancer in a study conducted by Saif et al. Edotecarin was seen to be effective in cells with resistance acquired due to P-glycoprotein [4] and has been shown to demonstrate significant antitumor's activity in human models of xenograft breast cancer and chemically and hormonally affected oncogene-derived breast tumors in a study published in 2007. Moreover, this compound has been shown to improve the effects of docetaxel and capecitabine in xenografts of human breast cancer. Therefore, it has been reported that the edotecarin molecule is an important therapeutic alternative for breast cancer patients as monotherapy or in conjunction with other treatment modalities [5]. The DNA-topoisomerase I complex (DNA-TOPI complex) is stabilized by the edotecarin molecule. This culminated in the failure of cells to unwind DNA and hence preventing them from dividing [3, 6]. A 2010 study by Saif et al. showed that in combination with 5-fluorouracil (5-FU) [7], edotecarin, a novel topoisomerase I inhibitor, has an additional effect on colon cell lines (HCT-116) [7]. Molecules with one or more degrees of freedom of rotation exhibit conformational isomerism leading to stable minimum-energy conformations. Conformation analysis plays an important role in biological activity since most mechanisms of action depend on the compatibility of the active site of the enzyme with the substrate, several methods of structure-activity relationship have been established to describe the three-dimensional structure of drug-like molecules [8].

Literature survey reveals that, although having higly biological activity, no conformational analysis study on edotecarin molecule, and molecular docking simulations of edotecarin against DNA, Topoisomerase I, DNA-Topoisomerase I complex and integrins have been reported so far. In this study, vibration assignments, HOMO-LUMO, frontier molecular orbital (FMO) energies, MEP and molecular docking simulations were performed on the most stable conformer of the edotecarin molecule obtained by applying conformational analysis and then optimization at the DFT/B3LYP/6-31++G(d,p) theory level. Moreover, the drug likeness and ADMET properties of edotecarin were analyzed. These calculations are important to provide insight into molecular and pharmaceutical properties and the interaction mechanisms of edotecarin.

Methods and calculations

The conformational analysis of the edotecarin molecule was performed using Spartan06 program software [9] with the PM3 method [10–13] and six lowest energy conformers within 0-0.76 kcal/mol relative energies were determined. Among them, the lowest energy conformer (I) was then optimized using the Gaussian03 program [14], DFT/B3LYP level of theory [15], and 6-31++G(d,p) basis set. By the scaled quantum mechanical force field approach proposed by Pulay et al. (1983) [16], the harmonic force field of edotecarin was defined using the MOLVIB program [17–18]. The vibration modes' IR intensity, Raman activity and potential energy distribution (PED) were determined; the force fields of the Cartesian coordinate were translated into natural internal coordinates [17, 18]. The calculated Raman activity of edotecarin was converted into Raman intensity by the simulation software Simirra [19]. Lorentzian band shapes with a bandwidth (FWHM) of 10 cm⁻¹ were used for the theoretical calculations. To provide the best fit for the experimental results, the following scale factors for DFT/B3LYP/6-31++G(d,p) level of theory calculations were selected: $$\begin{array}{c}\mathrm{O}-\mathrm{H}\mathrm{ }\text{stretch}\mathrm{ }0.87;\mathrm{ }\mathrm{N}-\mathrm{H}\mathrm{ }\text{stretch}\mathrm{ }0.89;\mathrm{ }\mathrm{C}-\mathrm{H}\mathrm{ }\text{stretch}\mathrm{ }0.91;\mathrm{ }\mathrm{C}=\mathrm{O}\mathrm{ }\text{stretch}\mathrm{ }0.86\\ \mathrm{O}-\mathrm{H},\mathrm{ }\mathrm{N}-\mathrm{H}\mathrm{ }\text{and}\mathrm{ }\mathrm{C}-\mathrm{H}\mathrm{ }\text{deformation}\mathrm{ }0.92;\mathrm{ }\text{all}\mathrm{ }\text{others}\mathrm{ }0.98.\end{array}$$

Results and discussion

Structure

The comparison of the energies of the six lowest energy conformers of edotecarin (Fig. 1) obtained as a result of the conformational analysis is given in Table 1. The differing dihedral angles of these six conformers are given in Table 2 comparatively.

Figure 1. The six conformers with the lowest energy, obtained by conformational analysis of the edotecarin molecule.

Table 1. The energies of the six most stable conformation obtained by conformation analysis.

Conformers	Energy (kcal/mol)	Relative energy (kcal/mol)
(I)	−349.98	0
(II)	−349.50	0.48
(III)	−349.50	0.48
(IV)	−349.46	0.52
(V)	−349.30	0.68
(VI)	−349.22	0.76

Table 2. The dihedral angles differing in the six low energy conformers obtained by conformational analysis.

Dihedral (^o)	(I)	(II)	(III)	(IV)	(V)	(VI)
O1-C16-N12-C23	−135.8	−136.0	−136.1	−136.5	−137.3	−137.1
C34-N14-N15-C40	118.3	118.1	118.3	−112.9	110.4	−115.0
N14-N15-C40-C44	−139.7	−140.2	−139.7	−111.1	−100.7	66.3
C22-C20-O4-H57	66.0	66.2	66.3	65.9	68.8	68.8
C17-C19-O3-H56	75.8	75.9	75.4	75.8	62.7	62.7
C16-C17-O2-H55	−172.8	−174.6	−173.2	−172.7	−73.6	−74.0
C20-C22-C30-O5	−163.1	−163.7	−163.4	−163.4	−163.2	−163.1
C22-C30-O5-H63	−65.2	−66.1	−64.7	−65.0	−65.1	−65.4
C33-C36-O8-H65	−179.3	−0.7	−179.4	−179.6	179.3	179.5
C39-C42-O9-H70	179.9	179.9	0.4	−179.9	−179.9	179.8
N15-C40-C44-O11	73.3	73.4	73.6	−67.0	−65.8	63.2
C40-C44-O11-H72	−57.3	−57.1	−57.4	76.5	75.8	−80.1
N15-C40-C43-O10	−77.1	−77.0	−77.1	60.4	60.3	−54.5
C40-C43-O10-H71	63.1	63.9	63.1	46.7	45.6	70.2

Following the conformation analysis, the lowest energy conformer (I) was optimized using the DFT/B3LYP level of theory and the 6-31++G(d,p) base set and the most stable molecular geometry was reached. The optimized geometry of edotecarin molecule obtained by DFT/B3LYP/6-31++G(d,p) level of theory was given in Fig. 2. In Table 3 the optimized geometry parameters of edotecarin molecule were given and in Table 4 some of the optimized parameters were compared with available related data.

Figure 2. The most stable geometric structure of the edotecarin molecule obtained with the DFT/B3LYP/6-31++G(d,p) level of theory.

Table 3. Edotecarin's optimized geometry parameters * were calculated using 6-31++G(d,p) basis set at DFT/B3LYP level of theory.

Atoms		Atoms		Atoms		Atoms		Atoms		Atoms		Atoms
R(1,16)	1.435	R(11,44)	1.415	R(19,47)	1.097	R(31,39)	1.394	A(16,1,22)	113.1	A(40,15,61)	113.3	A(17,19,47)	108.6
R(1,22)	1.444	R(11,72)	0.974	R(20,22)	1.534	R(33,36)	1.396	A(17,2,55)	108.4	A(1,16,12)	109.6	A(20,19,47)	108.3
R(1,52)	2.058	R(12,16)	1.438	R(20,48)	1.105	R(33,53)	1.083	A(19,3,56)	109.2	A(1,16,17)	109.1	A(4,20,19)	111.4
R(2,17)	1.416	R(12,18)	1.404	R(21,25)	1.445	R(35,37)	1.390	A(20,4,57)	108.2	A(1,16,45)	109.0	A(4,20,22)	108.3
R(2,55)	0.968	R(12,23)	1.406	R(21,26)	1.403	R(35,54)	1.084	A(30,5,63)	108.3	A(12,16,17)	114.3	A(4,20,48)	110.1
R(3,19)	1.433	R(13,24)	1.385	R(22,30)	1.524	R(36,37)	1.408	A(36,8,65)	110.0	A(12,16,45)	106.7	A(19,20,22)	109.4
R(3,56)	0.970	R(13,31)	1.391	R(22,49)	1.100	R(37,58)	1.087	A(42,9,70)	110.0	A(17,16,45)	108.0	A(19,20,48)	109.1
R(4,20)	1.418	R(13,52)	1.013	R(23,25)	1.417	R(38,41)	1.391	A(43,10,71)	109.8	A(2,17,16)	107.3	A(22,20,48)	108.5
R(4,57)	0.969	R(14,15)	1.384	R(23,33)	1.394	R(38,59)	1.084	A(44,11,72)	108.9	A(2,17,19)	111.4	A(18,21,25)	106.7
R(5,30)	1.417	R(14,32)	1.405	R(24,27)	1.432	R(39,42)	1.396	A(16,12,18)	127.0	A(2,17,46)	110.7	A(18,21,26)	118.2
R(5,63)	0.968	R(14,34)	1.412	R(25,35)	1.403	R(39,60)	1.085	A(16,12,23)	122.6	A(16,17,19)	109.3	A(25,21,26)	135.1
R(6,32)	1.227	R(15,40)	1.489	R(26,28)	1.406	R(40,43)	1.534	A(18,12,23)	107.9	A(16,17,46)	108.8	A(1,22,20)	108.9
R(6,71)	1.934	R(15,61)	1.016	R(26,32)	1.477	R(40,44)	1.536	A(24,13,31)	109.1	A(19,17,46)	109.3	A(1,22,30)	106.7
R(7,34)	1.219	R(16,17)	1.544	R(27,28)	1.399	R(40,62)	1.095	A(24,13,52)	121.4	A(12,18,21)	109.1	A(1,22,49)	109.1
R(8,36)	1.369	R(16,45)	1.094	R(27,29)	1.448	R(41,42)	1.410	A(31,13,52)	125.8	A(12,18,24)	130.6	A(20,22,30)	114.0
R(8,65)	0.966	R(17,19)	1.534	R(28,34)	1.481	R(41,64)	1.087	A(15,14,32)	125.4	A(21,18,24)	120.2	A(20,22,49)	109.4
R(9,42)	1.369	R(17,46)	1.101	R(29,31)	1.420	R(43,66)	1.095	A(15,14,34)	122.0	A(3,19,17)	111.3	A(30,22,49)	108.7
R(9,70)	0.966	R(18,21)	1.427	R(29,38)	1.403	R(43,67)	1.105	A(32,14,34)	111.9	A(3,19,20)	111.6	A(12,23,25)	109.3
R(10,43)	1.416	R(18,24)	1.399	R(30,50)	1.092	R(44,68)	1.095	A(14,15,40)	117.6	A(3,19,47)	104.2	A(12,23,33)	128.7
R(10,71)	0.975	R(19,20)	1.531	R(30,51)	1.102	R(44,69)	1.103	A(14,15,61)	109.5	A(17,19,20)	112.4	A(25,23,33)	122.0

* Bond lengths (R) in Å and angle (A) in degree (^o).

Table 4. Comparison between the optimized parameters of the edotecarin molecule and the experimental [20, 21] parameters of the carbazole molecule and also the theoretical [22] parameters of the geometric 4-hydroxy carbazole molecule.

Atoms	Edotecarin	carbazole [20, 21]	4–hydroxy carbazole [22]
R(9,42) ; R(8,36)	1.369; 1.369		1.368
R(9,70) ; R(8,65)	0.966; 0.966		0.962
R(26,28)	1.406	1.395	1.391
R(27,28)	1.399	1.400	1.404
R(21,26)	1.403	1.398	1.400
R(18,21)	1.427	1.390	1.391
R(18,24)	1.399	1.395	1.394
R(13,24); R(12,18)	1.385; 1.404	1.414	1.386
R(24,27)	1.432	1.404	1.419
R(13,31); R(12,23)	1.391; 1.406	1.414	1.384
R(31,39); R(23,33)	1.394; 1.394	1.395	1.394
R(29,31); R(23,25)	1.420; 1.417	1.404	1.421
R(39,42); R(33,36)	1.396; 1.396	1.390	1.391
R(39,60); R(33,53)	1.085; 1.083	0.897	1.084
R(41,42); R(36,37)	1.410; 1.408	1.398	1.402
R(38,41); R(35,37)	1.391; 1.390	1.395	1.391
R(41,64); R(37,58)	1.087; 1.087	1.070	1.084
R(29,38); R(25,35)	1.403; 1.403	1.400	1.401
R(38,59); R(35,54)	1.084; 1.084	0.893	1.085
R(27,29); R(21,25)	1.448; 1.445	1.467	1.452

Molecular electrostatic potential

Dipole moment, electronegativity, and partial charges are associated by molecular electrostatic potential (MEP). It gives a simple way to understand a molecule's relative polarity. It is very beneficial in recognizing the electrophilic and nucleophilic reaction sites for the identification of interactions with hydrogen bonding. Negative electrostatic potential refers to the attracting of the proton by the molecular concentrated electron density (from lone pairs, pi-bonds, etc). Positive electrostatic potential corresponds to repulsion of the positive charge (proton) in regions where low electron density exists. The big differences in red and blue means the more the molecule is polar. MEP were calculated for the investigated molecule (red as negative, blue as positive) at the optimized geometry obtained using DFT method at B3LYP/6-31++G(d,p) level of theory. Fig. 3 shows the molecular electrostatic potential surface of edotecarin. The red and blue colors regions represents the minimum electrostatic potential and the maximum of electrostatic potential, respectively.

Figure 3. Molecular electrostatic potential (MEP) of Edotecarin obtained by DFT/B3LYP/6- 31++G(d,p).

The color code of the edotecarin map changes in a range of −2.108 V (dark red) to 2.108 V (dark blue). Fig. 3 shown that, the regions with positive potential value are predominantly positioned as possible nucleophilic attack sites above the hydrogen atoms belonging to the OH group. Also, as possible sites for electrophilic attack, the negative potential regions are located around oxygen atoms.

Vibrational spectral analysis

Edotecarin molecule (C₂₉H₂₈N₄O₁₁) has 72 atoms thus, 210 vibrational modes with C1 symmetry all modes are IR and Raman active. The wavenumbers of the vibrational modes of the title molecule, based on calculated potential energy distributions, IR and Raman intensities, are identified in Table 5. The simulated Raman and IR spectra of Edotecarin are shown in Fig. 4.

Figure 4. Calculated IR (a) and Raman (b) spectra of Edotecarin.

Table 5. Calculated wavenumbers (cm^-1), calculated IR, Raman intensities (I) and the potential energy distribution of the vibrational modes of the Edotecarin.

		DFT/B3LYP 6-31++G(d,p)			PED % Edotecarin 6-31++G(d,p)
Assignment		νcal^S	I(IR)	I(Ra)	PED
1	νOH	3571	104	10	νOH(99)
2	νOH	3570	147	10	νOH(99)
3	νOH	3552	32	2	νOH(100)
4	νOH	3545	70	2	νOH(99)
5	νOH	3536	47	2	νOH(99)
6	νOH	3520	25	1	νOH(99)
7	νOH	3448	403	6	νOH(98)
8	νOH	3428	409	6	νOH(94)
9	νNH	3391	268	3	νNH(97)
10	νNH	3339	7	2	νNH(100)
11	νCH	3086	11	2	νCH(98)
12	νCH	3082	9	3	νCH(98)
13	νCH	3074	4	3	νCH(99)
14	νCH	3063	5	3	νCH(99)
15	νCH	3030	19	9	νCH(98)
16	νCH	3029	20	9	νCH(98)
17	νCH	2991	9	3	νCH(97)
18	νCH	2951	74	8	vCH(97)
19	νCH	2945	19	4	νCH(93)
20	νCH	2938	8	3	νCH(97)
21	νCH	2935	16	2	vCH(99)
22	νCH	2929	9	2	vCH(98)
23	νCH	2889	36	1	νCH(95)
24	νCH	2870	13	2	νCH(97)
25	νCH	2853	38	3	νCH(93)
26	νCH	2841	111	6	νCH(92)
27	νCH	2817	34	3	νCH(97)
28	νCH	2816	46	4	νCH(94)
29	νCO	1689	312	28	νCO(75)
30	νCC	1654	5	100	νCC(53)+νCN(6)
31	νCO+ νCC	1651	959	91	νCO(29)+νCC(24)
32	νCC	1645	68	60	νCC(46)+νCO(8)
33	νCO	1633	54	28	νCO(42)+νCC(18)
34	νCC	1604	56	8	νCC(61)
35	νCC	1602	89	8	νCC(59)
36	νCC	1587	138	6	νCC(48)+νCO(6)
37	νCC	1521	9	12	νCC(21)+νCO(7)+δCCH(6)
38	νCC	1511	37	10	νCC(29)+νCO(8)+δCCH(7)
39	νCC	1491	155	5	νCC(36)
40	δNNH	1484	6	4	δNNH(13)+δCNH(10)+δHCH(10)+νCC(5)
41	νCC	1474	2	8	νCC(13)+δCCH(10)
42	δHCH	1468	3	5	δHCH(28)+δOCH(6)+ΓNCCH(12)+ΓHCCH(6)+δOCH(5)
43	δHCH	1465	10	4	δHCH(33)+δOCH(13)+ΓOCCH(12)+ΓCCCH(6)
44	δNNH	1461	31	3	δNNH(16)+δHCH(15)+δCNH(12)
45	νCC	1457	191	2	νCC(23)+νNC(11)+δCCH(5)
46	νCN+δNCH	1434	59	4	νCN(8)+δNCH(8)+δCNH(6)+ΓCOCH(5)
47	νCC	1419	101	13	νCC(18)+νCN(5)
48	νNN	1410	82	13	νNN(29)+νCN(15)
49	νCC	1405	101	9	νCC(16)+δCOH(9)+νCN(5)
50	νCN	1401	115	7	νCN(12)+νNN(5)
51	δCOH	1396	58	6	δCOH(9)+νCC(8)+δOCH(7)+ΓHCCH(6)
52	δCOH	1395	16	6	δCOH(18)+νNN(9)+δOCH(5)
53	δCOH	1394	14	6	δCOH(19)+δOCH(5)
54	νCC	1384	24	4	νCC(6)+δCCH(5)
55	δCCH	1379	11	4	δCOH(17)+νCC(14)+δCCH(18)+δOCH(10)
56	νCC	1376	37	4	νCC(19)
57	νCC	1372	54	5	νCC(22)
58	νCC	1361	66	9	νCC(6)
59	νCC	1354	207	16	νCC(23)
60	δCOH	1352	10	18	δCOH(11)+δCCH(5)
61	δCOH	1351	59	19	δCOH(8)+δCCH(7)+δOCH(5)
62	νCN	1339	187	46	δCOH(10)+νCN(12)
63	δCOH	1338	11	45	δCOH(43)+δOCH(12)+δCCH(8)
64	νCC	1331	46	33	νCC(9)+νCN(5)+δCOH(5)
65	δCOH	1321	8	16	δCOH(14)+νCC(7)
66	δCOH	1318	14	14	δCOH(36)+δOCH(15)+δCCH(8)
67	δCCH	1316	17	14	δCOH(10)+δCCH(14)
68	δCCH	1307	42	20	νCO(20)+δCCH(21)+νCN(7)
69	νCO	1301	42	17	νCO(26)+νCN(6)+νCC(6)
70	δCOH	1280	4	3	δCOH(14)+δCCH(12)+δOCH(8)+ΓCNCH(13)
71	δCOH	1274	5	3	δCOH(21)+δCCH(16)+ΓCOCH(9)+δOCH(6)
72	νCN	1258	229	18	νCN(18)+νCC(6)
73	δCOH	1241	38	6	δCOH(17)+νCC(8)+δCCH(6)+νCN(6)
74	δCOH	1238	83	6	δCOH(43)
75	δCOH	1231	50	8	δCOH(40)
76	δCCH	1230	17	9	δCCH(20)+δCOH(15)+ΓNNCH(6)
77	δCCH	1225	30	11	δCCH(18)+νCO(9)+νCN(5)
78	δCOH	1217	23	8	δCOH(20)+δCCH(7)+νCO(7)
79	δCOH	1213	74	9	δCOH(10)
80	δCOH	1202	57	7	δCOH(9)+δCCH(8)+νCC(7)
81	δCOH	1198	23	6	δCOH(20)+δCCH(6)
82	δCOH	1182	10	6	δCOH(31)+δCCH(10)+δOCH(6)+νCO(5)+νCC(5)
83	δCOH	1181	9	7	δCOH(14)+νCO(9)+δCCH(7)
84	δCOH	1172	1	4	δCOH(29)
85	δCOH	1169	7	4	δCOH(30)
86	δCOH	1148	170	12	δCOH(57)+νCC(8)
87	νNN	1139	128	15	νNN(10)+δCOH(7)+νCC(6)+νCN(5)
88	δCOH	1136	29	12	δCOH(24)+δCCH(18)+νCO(8)
89	νCO	1130	2	9	νCO(34)+νCC(17)+δCOH(7)
90	δCCH	1128	31	9	δCCH(17)+νCC(8)+νCN(8)
91	νCO	1122	53	10	νCO(65)+νCC(10)
92	δCCH	1121	399	10	δCOH(8)+δCCH(19)
93	νCN	1116	8	8	νCO(8)+νCN(21)
94	νCO	1103	53	5	νCO(52)+νCC(20)
95	νCO	1102	71	5	νCO(42)+νCN(26)
96	νCC	1091	27	3	νCC(32)+νCO(23)+δCOH(5)
97	νCO	1090	26	3	νCO(29)+νCC(14)+δCOH(12)+νCN(12)
98	νCO	1084	153	2	νCC(26)+νCO(33)+δCOH(9)
99	νCO	1075	35	3	νCO(65)
100	νCO	1069	44	3	νCO(30)+νCC(16)+δCOH(12)+νCN(11)
101	νCC	1063	79	4	νCC(15)+νCN(6)
102	νCO	1053	137	2	νCO(46)+νCC(15)+δCOH(8)
103	νCO	1044	51	2	νCC(24)+νCO(30)+δCOH(11)
104	νCO	1036	94	2	νCO(12)+νCN(9)
105	νCO	1034	101	2	δCOH(13)+νCO(21)+νCC(9)
106	νCO	1031	112	2	νCO(55)+ δCOH(7)
107	νCO	1023	41	1	νCO(55)+νCC(23)
108	νCC	998	50	1	δCOH(15)+νCC(34)
109	νCC	983	33	2	νCC(19)+νCO(6)+δCCC(5)
110	ΓCCCH	968	2	2	ΓHCCH(39)+ΓCCCH(43)
111	ΓHCCH	968	1	2	ΓHCCH(53)+ΓCCCH(30)
112	νCO	961	85	4	νCO(33)+δCOH(9)+νCC(7)
113	νCC	959	46	4	νCC(30)+νCO(9)+δCCC(8)+δCCH(12)
114	νCC	911	20	1	νCC(5)
115	νCC	891	2	7	νCC(23)+δCCH(16)+νCN(7)
116	νCC	886	19	9	νCC(10)+νCN(7)+δCCH(6)
117	νCO	880	43	4	νCO(7)+νCN(6)+νCC(5)
118	δOCN	858	5	1	δOCN(11)+νCO(5)+δOCC(5)
119	νCN	844	29	3	νCN(9)+δCNN(5)
120	ΓOCCH	832	31	1	ΓOCCH(32)+ΓNCCH(27)+ΓCCCH(24)
121	ΓOCCH	830	24	1	ΓOCCH(31)+ΓNCCH(26)+ΓCCCH(22)
122	ΓCCCH	807	26	1	ΓOCCH(25)+ΓCCCH(42)+ΓHCCH(11)
123	ΓCCCH	805	18	2	ΓCCCH(75)+ΓHCCH(6)
124	νCO	792	10	10	νCO(8)+νCC(6)
125	νCO	787	21	9	νCO(6)+νCC(5)+νCN(5)
126	νCC	765	9	1	νCC(12)
127	ΓCCCO	753	0	1	ΓCCCO(15)+ΓCCCC(7)
128	ΓCCCO	748	9	1	ΓCCCO(11)+ΓCCCC(6)
129	δCCC	747	16	1	δCCC(7)+νCO(5)
130	ΓCCCO	743	3	1	ΓCCCO(9)
131	ΓCCCO	729	20	1	ΓCCCO(16)+ΓNNCO(12)+ΓCNNC(5)
132	ΓNCCN	725	2	1	ΓNCCN(9)+ΓCNCC(6)
133	δCCC	677	1	4	δCCC(3)
134	νCN	671	19	7	νCN(4)
135	δCCC	662	16	5	δCCC(5)
136	ΓCCNH	657	18	7	νCN(10)+δCNH(7)+ΓCCNH(12)+δNNH(5)
137	νCC	636	40	5	νCC(4)
138	ΓCCCH	631	5	4	ΓCCCH(8)+ΓCCCC(7)
139	ΓCCCH	629	20	3	ΓCCCH(14)+ΓCCCC(8)
140	ΓHOCH	615	13	8	ΓHOCH(5)+ΓHOCC(5)+δOCC(5)
141	ΓHOCH	610	154	11	ΓHOCH(37)+ΓHOCC(19)
142	ΓHOCH	599	47	4	ΓHOCH(41)+ΓHOCC(19)
143	ΓHOCH	592	1	3	ΓHOCH(5)
144	ΓHOCH	588	205	3	ΓHOCH(56)+ΓHOCC(27)
145	δCNN	582	8	3	δCNN(6)
146	ΓHNCC	577	79	4	ΓHNCC(57)
147	νCO	563	1	2	νCO(4)
148	ΓHNCC	551	45	1	ΓHNCC(6)
149	δCCC	545	16	2	δCCC(5)
150	δCCN	542	0	2	δCCN(3)
151	δCCO	526	6	1	δCCO(6)
152	δCCC	503	23	0	δCCC(5)
153	ΓCCNH	493	24	1	CCNH(8)+δCCO(7)+δCNN(6)+δOCO(6)+ΓCNNH(5)+ΓHOCH(5)
154	ΓCCOH	474	72	1	ΓCCOH(38)+ΓHOCH(13)
155	ΓCCOH	457	19	1	ΓCCOH(15)
156	ΓCCOH	452	59	1	ΓCCOH(33)+ΓHOCH(9)
157	ΓCCOH	447	39	1	ΓCCOH(12)
158	δCCO	444	7	1	δCCO(19)
159	νCC	430	0	2	νCC(5)
160	ΓCCCC	422	0	2	ΓCCCC(3)
161	δCCO	412	4	2	δCCO(13)
162	ΓHOCH	405	16	3	ΓHOCH(30)+ΓCCOH(11)
163	νCC	403	7	3	νCC(11)
164	ΓHOCH	396	116	2	ΓHOCH(58)+ΓCCOH(22)
165	ΓCCOH	383	50	2	ΓCCOH(48)+ΓHOCH(18)
166	ΓCCOH	379	26	3	ΓCCOH(14)+ΓHOCH(6)
167	δCNN	374	17	2	δCNN(18)+δCCN(8)+ΓCNNH(5)
168	ΓCCOH	351	15	2	ΓCCOH(5)
169	ΓCCOH	339	106	3	ΓCCOH(96)
170	δOCC	337	30	4	δOCC(7)+δOCN(5)+δOCO(5)+νNN(5)
171	ΓCCOH	333	102	4	ΓCCOH(94)
172	νCC	330	2	5	νCC(5)
173	ΓCCOH	323	22	3	ΓCCOH(52)+ΓHOCH(11)
174	ΓCCOH	321	3	3	ΓCCOH(18)+ΓHOCH(7)
175	ΓCCOH	312	5	2	ΓCCOH(22)+ΓHOCH(7)
176	ΓCCOH	307	6	2	ΓCCOH(18)+ΓHOCH(7)
177	ΓCCNH	295	12	2	ΓCCNH(18)+ΓHNCH(8)+δCNN(8)+ΓCNNH(6)
178	ΓHOCH	294	11	2	ΓHOCH(11)
179	νCC	277	5	5	νCC(6)
180	ΓNCCC	261	1	4	ΓNCCC(2)
181	ΓCCCO	259	1	5	ΓCCCO(5)
182	ΓCCOH	254	3	8	ΓCCOH(15)
183	ΓCCOH	249	4	6	ΓCCOH(20)+ΓHOCH(6)
184	ΓCCOH	236	18	2	ΓCCOH(27)+ΓHOCH(6)+δCCO(5)
185	ΓHOCH	230	3	2	ΓHOCH(10)
186	νO..H	223	1	2	νO..H(15)
187	νO..H	196	3	2	νO..H(21)+ δCCC(7)
188	νO..H	188	2	2	νO..H(14)
189	νO..H	175	1	2	νO..H(13)
190	ΓHOCH	160	5	3	ΓHOCH(4)
191	νO..H	156	1	3	νO..H(10)
192	ΓNCCO	153	4	3	ΓNCCO(6)+ΓNCCH(5)
193	νO..H	144	3	3	νO..H(8)
194	νO..H	132	3	5	νO..H(9)+ΓCNNC(7)+ΓNCCO(5)
195	ΓCCCO	121	7	5	ΓCCCO(6)+ΓNCCO(6)+ΓCCCH(6)
196	ΓOCCC	116	0	5	ΓOCCC(4)
197	ΓNNCC	103	1	10	ΓNNCC(3)
198	ΓCCOH	101	2	11	ΓCCOH(4)
199	νO..H	101	2	11	νO..H(7)
200	ΓCCCH	94	2	9	ΓCCCO(8)+ΓOCCO(8)+ΓCCCH(13)+ΓOCCH(12)
201	ΓCNNC	84	1	13	ΓCNNC(8)+ΓCNNH(7)
202	ΓCNNH	68	0	23	ΓCNNH(8)+ΓCNNC(6)
203	ΓCCCC	67	1	23	ΓCCCC(4)
204	ΓNNCC	46	0	66	νO..H(8)+ΓNNCC(13)+ΓHOCH(5)+ΓCCOH(5)
205	ΓCNNH	42	0	84	ΓCNNH(7)+ΓCNCO(6)
206	νO..H	38	0	82	νO..H(9)+ΓCOCN(8)+ΓNCCC(6)
207	ΓCNCO	31	1	148	νO..H(10)+ΓCNCO(16)+ΓCNCC(13)+ΓCNCH(5)
208	νO..H	29	1	150	νO..H(14)
209	ΓHOCH	19	1	116	ΓNNCC(19)+ΓHOCH(21)+ΓCCOH(10)+ΓCCNH(6)
210	ΓCNCC	14	1	182	ΓCNCO(12)+ΓCNCC(29)+ΓCNCH(19)

The seven OH stretching modes (νOH) of the edotecarin were computed in the 3571-3428 cm⁻¹ region (with PED contribution 94-100%). This mode for the 4-hydroxy carbazole molecule was observed at 3393 cm⁻¹ in the FTIR spectrum and calculated at 3854 cm⁻¹ at the DFT/B3LYP/6-311++G(d,p) level of theory [22]. Morever in the Gly-Tyr molecule, the OH stretching modes were observed at 3566 and 3446 cm⁻¹ in the FTIR spectrum and at 3577 and 3448 cm⁻¹ in the Raman spectrum [23].

The molecule has two N-H groups. These N-H stretching vibrations were calculated at 3391 and 3339 cm⁻¹ as a pure mode (with PED contribution 100-97%). The N-H stretching mode for the 4- hydroxy carbazole molecule was observed at 3210 cm⁻¹ in the FTIR spectrum and was calculated with the DFT/B3LYP/6-311++G(d,p) level of theory at 3670 cm⁻¹ [22]. This mode was computed at 3542 cm⁻¹ for the 2,7-dimethoxy-9H-carbazole-3-carbaldehyde molecule using the B3LYP/LanL2Z level of theory [24], for Gly-Tyr dipeptide the N-H stretching vibrations were observed at 3321 and 3210 cm⁻¹ in the Raman and at 3326 cm⁻¹ in the FTIR spectra [23]. Our results are in Accord with the previous findings.

The CH stretching vibrations of edotecarin were computed in the range of 3086-2816 cm⁻¹ with 92-99% PED contributions. The CH stretching vibrations of 4-hydroxy carbazole molecule were observed at 3053 and 3061 cm⁻¹ in FTIR and Raman spectra, respectively and was calculated in the range of 3199-3149 cm⁻¹ using the DFT/B3LYP/6-311++G(d,p) level of theory [22]. In addition, the CH stretching vibrations of 2,7-dimethoxy-9H-carbazole-3-carbaldehyde molecule were found in the range of 3110-2905 cm⁻¹ in calculations using the B3LYP/LanL2Z theory level [24]. For 9-p-tolyl-9H-carbazole-3-carbaldehyde molecule, the CH stretching modes were observed in the 3056-2764 cm-1 range in the FTIR spectrum[25], and for the CLTOAB molecule, the CH stretching modes were observed in the 2994-2850 cm⁻¹ and 2993-2846 cm⁻¹ ranges, in the ATR-IR and Raman spectra respectively [26].

The vibrational wavenumbers of the C = O stretching modes of the edotecarin were computed at 1689 and 1633 cm⁻¹ with 75% and 42% PED contributions, respectively. The same vibrations were calculated at 1619 and 1603 cm⁻¹ for the 2,7-dimethoxy-9H-carbazole-3-carbaldehyde molecule using the B3LYP/LanL2Z level of theory [24]. The C = O stretching vibration of the 9-p-tolyl-9H-carbazole-3-carbaldehyde molecule was observed at 1686 and 1692 cm⁻¹ in the FTIR and Raman spectra, respectively [25], and the C = O stretching modes of the Gly-Tyr dipeptide were observed in the 1698-1635 cm⁻¹ and 1692-1630 cm⁻¹ ranges, in the FT-IR and Raman spectra, respectively.

The computed 1468 and 1465 cm⁻¹ wavenumbers were assigned to HCH in-plane angle bending vibrations of edotecarin, in accord to PED values. For the Val-Met dipeptide [27] the HCH in plane bending vibrations were experimentally observed at 1451 and 1437 cm⁻¹ and computed at 1455 and 1438 cm⁻¹and for CLTOAB, were calculated in the range of 1487-1432 cm⁻¹ and observed in the 1484-1416 cm⁻¹.

The calculated N-N stretching vibrations of edotecarin were obtained as mixed vibrations at 1410 and 1139 cm⁻¹, with 29% and 10% PED contributions, respectively. For the benzyl (imino (1H-pyrazol-1-yl) methyl) carbamate molecule, by using B3LYP/6-31G(d,p) level of theory, the N-N stretching modes were computed at 1269 (46% PED) and 1203 cm⁻¹ (77% PED) [28]. In the study on the 3-amino-1,2,4-triazole molecule, the N-N stretching vibrations were estimated at 1247; 1146; 1066; 1023 and 952 cm⁻¹ with 10, 15, 12, 32 and 10% PED contributions, respectively, using with B3LYP/6-311++G(d,p) level of theory and the corresponding bands were observed at 1266; 1153; 1047; 1026 and 926 cm⁻¹, respectively in the FTIR spectrum [29].

Frontier molecular orbital analysis

The frontier molecular orbitals, namely, the highest energy occupied orbital (HOMO) and the lowest energy empty orbital (LUMO), are the molecular orbitals that participate in the chemical reactions or interactions with other species. Their energy difference (gap) gives information about the reactivity of the molecule and allow us to comprehend the wavenumbers they emit/reflect [30].

By Time-Dependent Density Functional Theory (TD-DFT) using B3LYP/6-31++G(d,p) basis set, the energies of the frontier molecular orbitals of edotecarin molecule was determined. The energy gap between the ground energy level and the first excited state of edotecarin was found to be 2.769 eV, which indicated the presence of the charge transfer interaction in the molecule and reflected the biological activity of the molecule.

The percentage of contributions of atomic orbitals to HOMO and LUMO (frontier orbitals) was estimated using the program GaussSum (Version, 3.0) [31]. Table 6 gives the measured transition energies, oscillator strengths and main contributions. Fig. 5 displays the schematic diagrams of the frontier HOMO and LUMO orbitals of edotecarin. The HOMO-LUMO energy difference is calculated to be 4.8 and 4.21 eV by DFT/B3LYP/6-31G(d) calculations in a report on carbazole and benzocarbazole, respectively [32].

Figure 5. The atomic orbital HOMO-LUMO composition of the frontier molecular orbital for Edotecarin.

Table 6. Selected calculated positions of the pure electronic transitions, oscillator strengths (f) and major contributions.

Excited State	Energy (eV)	Wavelength (nm)	Osc. Strength (f)	major contributions (%)
1	2.769	447.76	0.0906	HOMO->LUMO (96%) H-1->L + 1 (3%)
2	2.7933	443.86	0.0373	H-1->LUMO (93%) H-2->LUMO (2%), HOMO->L + 1 (2%)
3	3.3793	366.89	0.0095	H-2->LUMO (83%) H-5->LUMO (4%), H-3->LUMO (8%)

In silico molecular docking studies of edotecarin as anticancer agent

Edotecarin (J-107088) is an DNA topoisomerase-1 inhibitor, and is known to interact with DNA and additionally stabilize the DNA-TOPI complex [33, 34]. Edotecarin is a derivative of a compound called NB-506, an indolocarbazole antitumor agent, but edotecarin is a strong and selective TOPI inhibitor that is more effective at inducing single-stranded DNA cleavage than NB-506 [4, 35, 36].

In order to investigate the interactions of edotecarin molecule with the target DNA, TOPI and TOPI-DNA complex, molecular docking simulations were performed. Moreover, the molecular docking simulations of edotecarin with the target proteins α_IIbβ₃ and α₅β₁ integrins were also performed to reveal its anticancer properties

The docking study of the edotecarin molecule was carried out using AutoDockVina [37] and the crystal structures of DNA (PDB ID: 1BNA) and Topoisomerase I/DNA Complex (PDB ID: 1A36) were obtained with reference to the protein database [38–40]. Before the docking analysis, DNA was modified for the docking analysis by extracting water molecules and adding polar hydrogens, and the DNA charges of Kollman were determined. Partial charges of the edotecarin molecule were calculated using the Geistenger method and the active region of DNA is defined as in size 40 Å × 40 Å × 40 Å.

The optimized structure of the edotecarin molecule, calculated in gas phase, was found to bind to the nucleic acids DC9, DG10, DC11, DG12, DG16 and DA17 by hydrogen bonds and Pi-Anion interaction (See Fig. 6). The result shows that the binding affinity (ΔG) of the interaction of edotecarin with DNA is −8.1 kcal/mol. The identified active site of DNA was in accordance with the literature [41–43]. The interactions between the edotecarin molecule and nucleic acids are described below:

Figure 6. Molecular docked models of edotecarin with DNA (a),The interactions between the edotecarin and DNA are labeled using colored dashed lines(b) (-8.1 kcal/mol).

Unfavorable acceptor-acceptor interaction with a length of 2.71 Å between DC9 and the oxygen atom; Hydrogen bond interaction with a length of 2.44 Å between DC10 and oxygen atom; Carbon hydrogen bond interaction with a length of 3.67 Å between DC11 and oxygen atom; Pi-Anion interaction between DG12 and edotecarin molecule at lengths of 4.67, 4.31, 4.90 Å; Hydrogen bond interactions between DG16 and oxygen atom with lengths of 2.45, 2.51 and 2.19 Å; Hydrogen bond interaction between DA17 and hydrogen atom with lengths of 2.77 and 2.38 Å.

In a study performed by Das et al., the DNA docking of various piperidines that have antitumor activity were investigated by molecular dynamics method [44]. The active site of DNA determined in DNA-piperidine complexes, is compatible with the active site of DNA where edotecarin interacts, found in this study. Hydrophobic interaction and hydrogen bonding have been determined to contribute to stabilizations of DNA-piperidine complexes [44].

Before the docking analysis, the same preparation was made for Topoisomerase I, i.e. DNA and water molecules were extracted from the Topoisomerase I/DNA Complex, polar hydrogens were added and Topoisomerase I 's Kollman charges were calculated, and Topoisomerase I was prepared for docking analysis. And also the partial charges of the Edotecarin molecule were calculated using the Geistenger method. Using CAVER [45], the active site of Topoisomerase I can be determined at max. Probe. The grid size is then specified as 40 Å × 40Å × 40Å for docking calculation. Optimized structure of the edotecarin molecule, determined with DFT/B3LYP/6-31++G(d,p) level of theory in the gas phase, binds to the amino acids of TOPI (ARG364, GLN421, ASN491, LYS493, THR498, ALA499, THR501, LYS532 ve ASP533) with −8.9 kcal/mol binding affinity(ΔG) (Fig. 7).

Figure 7. (a) and (b) Different representations of Edotecarin docking with TOPI. Dotted lines indicate interactions (-8.9 kcal/mol).

Interactions between the edotecarin molecule and Topoisomerase I are described below: Unfavorable acceptor-acceptor interaction with a length of 2.8 Å between ARG364 and the oxygen atom; Carbon hydrogen bond interaction with a length of 3.71 Å between GLN421 and edotecarin molecule; Unfavorable donor-donor interaction with a length of 1 Å between ASN491 and the Hyrogen atom; Between LYS493 and edotecarin molecule: Pi-Cation; Pi-Donor hydrogen bond interaction with a length 2.85 Å, Pi-Cation interaction with a 3.58 Å length, Pi-Cation interaction with a 4.02 Å length; Pi-Sigma interaction with a length of 3.99 Å between THR498 and edotecarin molecule; Unfavorable donor-donor interaction with a length of 1.41 Å between ALA499 and the edotecarin molecule and Pi-Alkyl interaction with a length of 5.09 Å; Hydrogen bond interaction with a length of 1.86 Å between THR501 and oxygen atom; Between LYS532 and edotecarin molecule: Pi-Cation interaction with a length 3.78 Å, Pi-Alkyl interaction with a 5.16 Å length, Pi-Alkyl interaction with a 4.37 Å length and Pi-Alkyl interaction with a 3.71 Å length; Pi-Anion interaction with a length of 4.82 Å between ASP533 and edotecarin molecule.

In a recent study (Dario et al.), the interaction of the vanadyl complex, an antitumor agent, with TOPI was investigated using molecular docking simulations and showed interactions of the vanadyl complex at different sites of TOPI [46]. It was reported that the vanadyl complex interacted with the amino acids LEU485, ASN539, LEU629, ASN631, GLN713 and ILE714 in the external region of TOPI and interacted with LYS493, GLU495, GLY496, THR498, ALA499, ASP500, GLU561, ASP562 ve ASP563 residues in the lips region of TOPI. The complex interacted with GLU492, GLN421 and GLU494, in the center channel between the lips, where the DNA is bound for the activity of the protein. The GLN421 and ALA499 amino acids of TOPI where vanady complex interacted, in the center channel between lips and in lips regions, respectively, are the amino acids that edotecarin interacts. Thus docking simulations indicates that active site, where edotecarin docked, is the central channel between the lips close to lips region.

Before the docking analysis, the same process was made DNA-TOPI complex, i.e. water molecules were extracted, polar hydrogens were added, and DNA-TOPI complex 's Kollman charges were calculated, and DNA-TOPI complex was prepared for docking analysis. The active site of the DNA-TOPI complex was also determined according to the max probe value using the CAVER program [45]. Then, grid size was defined as 60 Å × 60 Å × 60 Å for docking calculation.

Optimized edotecarin molecule structure, determined by DFT/B3LYP/6-31++G(d,p) gas-phase theory, binds to DNA-TOPI amino acid complexes (ASN711, ILE714, LEU721, ILE743 ve ASN745) and also binds to DNA residues (DG12, DA13, DA14, DT109 ve DT110) (Fig. 8). It is quite consistent with literature studies [47–50]. The binding affinity of this interaction (ΔG) is −10.7 kcal/mol.

Figure 8. (a) Structural model of DNA-TOPI complexes with ligand edotecarine binding site (green sphere), (b)The interactions between the edotecarin and DNA-TOPI complexes are labeled using colored dashed lines (-10.7 kcal/mol).

The results revealed that edotecarin is bound to the DNA with weak non-covalent interactions. More specifically it is bonded to DNA with conventional hydrogen bonding, carbon-hydrogen bond, pi-alkyl, pi-sigma and unfavorable donor-donor interactions. The details of the interactions are given below:

Unfavorable donor-donor interaction, 1.97 Å in length, between ASN711 and the edotecarin molecule; hydrogen bond interaction with ILE714 (2.49 Å); pi-alkyl interaction (5.46 Å) with LEU721; conventional hydrogen bond (2.42 Å) and carbon hydrogen bond (3.49 Å) interactions with ILE743; hydrogen bond interaction (2.33 Å) with ASN745; hydrogen bond interaction (2.39) Å with DG12; hydrogen bond interactions (2.59 Å and 3.02 Å) with DA13; hydrogen bond interaction (2.24 Å) with DA14; hydrogen bond (2.58 Å) and pi-sigma (3.77 Å) interactions with DT109; hydrogen bond interaction (2.75 Å) with DT110.

In order to examine the anti-proliferation effect of the edotecarin molecule for the anticancer function, molecular docking studies in target proteins α₅β₁ and α_IIbβ₃ integrins were performed.

After the α₅β₁ integrin (PDB ID: 4WK0) was prepared for molecular docking, the active side was determined at max probe by CAVER [45]. The 3 D docked view of edotecarin in α₅β₁ integrin and the estimated interactions are shown in Fig. 9. The predicted binding affinity for α₅β₁ integrin is −9.1 kcal/mol (K_d = 0.212 µM). The interactions can be summarized as follows:

Figure 9. The 3 D docked view of the optimized structure of edotecarin in α₅β₁ integrin. The ligand interaction diagrams of receptor ligand complexes are Show (binding affinity -9.1 kcal/mol).

Unfavorable donor-donor interaction (1.48 Å) with ASP293; hydrogen bond (1.81 and 1.98 Å) and unfavorable acceptor-acceptor (2.7 Å) interactions with ASP300; pi-anion interactions (4.62, 4.01 and 3.85 Å) with GLU246; pi-alkyl interactions (5.39, 4.43 and 4.13 Å) with LYS174; hydrogen bond interaction (2.98 Å) with GLU171; pi-cation interaction (4.99 Å) with ARG423; hydrogen bond interaction (2.59 Å) with ARG420 and pi-alkyl interaction (5.33 Å) with PRO357.

In our previous study on the interaction of cationic pentapeptide, Glu-Gln-Arg-Pro-Arg, with α₅β₁ integrin [51], it was found the the ligand interacted with PHE21,SER22,VAL23,ARG106, PHE168, GLU171, SER234, VAL235, LYS269, TYR287, LEU355, SER417 and ARG420, through salt bridge, attractive charge, hydrogen bond, carbon hydrogen bond, unfavorable positive-positive, unfavorable donor-donor interactions. The active site, where cationic pentapeptide docked [51] was found to be highly compatible with the active site found by our calculations.

After the α_IIbβ₃ integrin (PDB ID: 3ZDX) was prepared for molecular docking, the active side was determined at max probe by CAVER [45]. The 3 D docked view of edotecarin in α_IIBβ₃ integrin and the estimated interactions are shown in Fig. 10. The predicted binding affinity for α_IIBβ₃ integrin is −7.9 kcal/mol (K_d = 1.6 µM). The interactions can be summarized as follows:

Figure 10. The 3 D docked view of the optimized structure of edotecarin in α_IIbβ₃ integrin. The ligand interaction diagrams of receptor ligand complexes are Show (binding affinity -7.9 kcal/mol).

Unfavorable donor-donor interaction (1.89 Å) with LYS27; hydrogen bond interaction (2.2 Å) with ASP24; unfavorable acceptor-acceptor (3.0 Å) and carbon-hydrogen bond interactions (3.47 Å) with SER99; pi-alkyl interactions (4.36, 4.47, 4.55 ve 4.94 Å) with ALA424; pi-alkyl interaction (5.26 Å) with PRO362 and unfavorable donor- donor interaction (1.67 Å) with THR296.

In the previous study on the docking simulations of cationic pentapeptide, Glu-Gln-Arg-Pro-Arg, with α_IIbβ₃ integrin [51], it was found the the ligand interacted with LEU23, ASP24, PHE25, ALA95, SER96, SER99, PHE171, SER173, TYR237, SER238, ARG261, HIS291, VAL293, ILE360, SER420, LEU421, ARG422 residues of integrin, through salt bridge, attractive charge, hydrogen bond, carbon hydrogen bond, unfavorable positive-positive, unfavorable donor-donor interactions. The active site of integrin, where cationic pentapeptide docked [51] was found to be highly compatible with the active site found by our calculations.

The strong binding affinities of endotecarin molecule to the integrins α₅β₁ and α_IIbβ₃, with K_d values of 0.21 µM and 1.6 µM, respectively, shows its the anti-cancer activity.

Analysis of toxicological and physicochemical properties

The program can calculate lipophilicity (expressed as clogP), solubility in water (expressed as logS), molecular weight(MW), drug-likeness indices and drug scores. Moreover, the toxicological properties of the compounds may be shown also. CLogP is a central parameter in drug discovery and environmental toxicity research and this value must not be greater than 5.0 [52, 53]. As increasing molecular weight decreases absorption, maintaining less molecular weight is an essential part of the drug development process. The solubility (logS) properties of a substance in aqueous solution influence the absorption and distribution characteristics. The solubility of a compound is predicted using the logS-based OSIRIS method. The preferred value is more than −4 [52, 53].

TPSA is a molecular polar surface region and the transport properties of a drug are defined by it. This property is described by polar atoms and is predicted using the various classical 3 D PSA methods and the sum of tabulated surface contributions of polar fragments [52, 53]. Drug-likeness is described on the basis of the molecular characteristics of the drugs advertised. The drug-likeness is the sum of the molecule score values of the fragments present. The positive drug-likeness value suggests that the molecule being tested mainly contains fragments that are commonly found in the products being marketed [52, 53].

Using the OSIRIS Property Explorer (2010) [53] software, which offers an overall risk prediction drug score, the approximate toxicity risk (Mutagenic, Tumorigenic, Irritant, Reproductive effect) values of Edotecarin and certain essential physicochemical properties (cLogP,Solubility,MW,TPSA,Druglikeness,Drug-score) have been calculated and given in Table 7.

Table 7. Osiris's estimation of title compounds toxicity hazards and physicochemical properties.

Compound	Toxicity risks				Physicochemical properties
	Mutagenic	Tumorigenic	Irritant	Reproductive Effect	cLogP	Solubility	MW	TPSA	Druglikeness	Drug-score
Edotecarin	no indication	no indication	no indication	no indication	−5.14	−1.64	626	235.7	−0.71	0.39

Absorption analysis of target molecule

The blood-brain barrier (BBB) is a barrier that separates circulating blood from the central nervous system (CNS) [54], predicting BBB permeable compounds is critical to identifying toxic substances that can harm the brain and to design drug molecules that can reach its target in the CNS [55]. Human intestinal absorption (HIA) estimation is an important goal for the production of oral drugs. The need for fast and effective models for predicting HIA and other biopharmaceutical properties has greatly increased in vitro parallel synthesis and screening of candidate compounds in drug development for pharmacological activity [56–58]. Extensive studies have revealed that Caco-2 cell culture is widely used in vitro cell culture models for absorption screening assay of new drug and is a valuable index [59–61]. P-glycoprotein (Pgp) is an essential protein in the cell membrane that removes from the cell several foreign substances [62]. As such, the pharmacokinetic effects of drugs are a significant criterion of this. Chemotherapy resistance in cancer cells is specifically regulated by overexpression of P-glycoprotein (Pgp) [63]. It is therefore of great importance to recognize substances that can either be transported out of the cell by Pgp (substrates) or hinder the function of Pgp (inhibitors) [55]. Cytochrome P450 (CYP450) enzymes are important to the processing of cholesterol, steroids, etc. They are also used for the detoxification of foreign chemicals and for drug metabolism [55, 64, 65]. Mutagens are chemicals that induce cancer-leading abnormal genetic mutations. A typical way of determining the mutagenicity of a chemical is the Ames test [66]. This procedure has become the benchmark for chemical and drug safety evaluation and has been used to test thousands of molecules [55]. In Ref [67] probability values for Blood-Brain Barrier and Human Intestinal Absorption of Doxorubicin molecule with anticancer activity, were calculated as 0.9951, 0.8092, respectively. In this study, probability values were calculated as 0.6453 and 0.7758, respectively.

The silico module admetSAR [68] was used to define various pharmacokinetic criteria, such as absorption, delivery, metabolism, excretion and toxicity, were examined for the scrutinized edotecarin molecule. Results from its ADMET analysis are presented in Table 8.

Table 8. Prediction of ADMET profiles of the edotecarin ADMET predicted profile – classification.

Model	Result	Probability
Absorption
Blood-Brain Barrier	BBB-	0.6453
Human Intestinal Absorption	HIA+	0.7758
Caco-2 Permeability	Caco2-	0.8020
P-glycoprotein Substrate	Substrate	0.5778
P-glycoprotein Inhibitor	Non-inhibitor	0.7463
	Non-inhibitor	0.8176
Renal Organic Cation Transporter	Non-inhibitor	0.8845
Distribution
Subcellular localization	Mitochondria	0.5868
Metabolism
CYP450 2C9 Substrate	Non-substrate	0.8730
CYP450 2D6 Substrate	Non-substrate	0.8282
CYP450 3A4 Substrate	Substrate	0.5508
CYP450 1A2 Inhibitor	Non-inhibitor	0.9370
CYP450 2C9 Inhibitor	Non-inhibitor	0.8943
CYP450 2D6 Inhibitor	Non-inhibitor	0.9178
CYP450 2C19 Inhibitor	Non-inhibitor	0.9167
CYP450 3A4 Inhibitor	Non-inhibitor	0.9095
CYP Inhibitory Promiscuity	Low CYP Inhibitory Promiscuity	0.9638
Excretion
Toxicity
Human Ether-a-go-go-Related Gene Inhibition	Weak inhibitor	0.8688
	Inhibitor	0.5613
AMES Toxicity	AMES toxic	0.5160
Carcinogens	Non-carcinogens	0.8519
Fish Toxicity	High FHMT	0.9753
Tetrahymena Pyriformis Toxicity	High TPT	0.9179
Honey Bee Toxicity	Low HBT	0.7860
Biodegradation	Not ready biodegradable	0.9887
Acute Oral Toxicity	III	0.5730
Carcinogenicity (Three-class)	Non-required	0.6273
ADMET Predicted Profile --- Regression
Model	Value	Unit
Absorption
Aqueous solubility	-2.2387	LogS
Caco-2 Permeability	-0.3147	LogPapp, cm/s
Toxicity
Rat Acute Toxicity	2.2228	LD50, mol/kg
Fish Toxicity	1.5975	pLC50, mg/L
Tetrahymena Pyriformis Toxicity	0.3045	pIGC50, ug/L

Conclusion

Conformational analysis of the edotecarin molecule was performed using a semi-experimental method PM3 and then obtained the lowest energy conformer (I) was optimized with the DFT/B3LYP/6-31++G(d,p) level of theory. Since protein-ligand interactions play a vital role in drug design, in this study, the docking simulations were performed for evaluation of the biological activity of the title molecule, using its most stable structure. The docking with DNA, TOP-I and DNA-TOPI complex was investigated in order to examine the inhibitory activity of the edotecarin molecule. The binding affinities of edotecarin to DNA, TOP-I and DNA-TOPI complex were obtainded −8.1, −8.9 and −10.7 kcal/mol, respectively, that corresponded to K_d = 1.24 µM, K_d= 0.3 µM and K_d= 0.015 µM, respectively. The predicted results support the exhibited antineoplastic activity of the edotecarin molecule. Moreover the predicted strong binding affinities of endotecarin molecule to the integrins α₅β₁ and α_IIbβ₃, with K_d values of 0.21 µM and 1.6 µM, respectively, are relevant to its the anti-cancer activity. The molecular docking predictions show that, the ligand edodecarin has good anti-tumor properties. The drug likeness and ADMET properties of edotecarin showed that molecule has good pharmacokinetic profiles.

This research indicates that the edotecarin can be further researched and evaluated for treatment and control techniques and for anticancer effects against different forms of cancer, in particular lung, breast, and stomach cancer.

Conflict of interests

The authors declare that they have no conflict of interests.

Additional information

Funding

This work was supported by the Scientific Research Projects Coordination Unit of Istanbul University [ÖNAP-2423].