Modelling inhibition of avian aromatase by azole pesticides

^†Presented at the 8th International Symposium on Computational Methods in Toxicology and Pharmacology Integrating Internet Resources, CMTPI-2015, 21–25 June 2015, Chios, Greece.

Figures & data

Figure 1. Sequence alignment of C. japonica, G. gallus, T. guttata, O. mykiss and human aromatases.

Figure 2. (a) The alignment of homology-modelled proteins of C. japonica, G. gallus, T. guttata and O. mykiss with human protein. (b) Comparison of the important residues at the catalytic site of the aromatase enzyme in the studied species. In the three bird species, V373 in humans is replaced by I373, while in O. mykiss it is replaced by T373. L372 in human is replaced by F372 in O. mykiss.

Figure 3. Structures of 22 azoles and related compounds considered in this study.

Figure 4. (a) Docking of fadrozole (S), (b) imazalil (S) and (c) vorozole (S) at the active site of human aromatase.

Figure 5. Docking of fadrozole (S), imazalil (S) and vorozole (S) at the C. japonica aromatase (a–c), at the G. gallus aromatase (d–f), at the T. guttata aromatase (g–i), and at the O. mykiss aromatase (j–l).

Table 1. Interactions of fadrozole, imazalil and vorozole with the active site residues of human, bird, and fish aromatases.

Species	Compound	Site
Human		R115	I133	F134	F221	W224	T310	I313	V370	L372	V373	M374	L477	H480	HEM600
	Fadrozole	HB	–	HY^a	–	–	–	–	HY	HY	–	HB^b	HY	–	Ar^c/M^d
	Imazalil	–	HY	HY	HY	HY	–	–	HY	HY	HY	HY	HY	–	M
	Vorozole	–	HY	HY	HY	HY	–	–	HY	–	–	HB	HY	–	M/Ar
C. japonica		R115	I133	F134	F221	W224	T310	I313	V370	L372	I373	M374	L477	H480	HEM600
	Fadrozole	HB	HY	–	–	–	–	–	HY	HY	–	HB	HY	–	M/Ar
	Imazalil	–	HY	HY	HY	HY	–	–	HY	HY	HY	HY	HY	–	M
	Vorozole	–	HY	HY	HY	HY	–	–	HY	–	–	HB	–	–	M/Ar
G. gallus		R115	I133	F134	F221	W224	T310	I313	V370	L372	I373	M374	L477	H480	HEM600
	Fadrozole	–	–	HY	–	–	–	–	HY	–	–	HB	–	–	M/Ar
	Imazalil	–	HY	HY	HY	HY	–	–	–	–	HY	HY	HY	–	M
	Vorozole	–	HY	HY	HY	HY	HY	–	HY	–	–	–	–	–	M/Ar
T. guttata		R115	I133	F134	F221	W224	T310	I313	V370	L372	I373	M374	L477	H480	HEM600
	Fadrozole	–	–	–	–	–	–	–	HY	–	–	HB	HY	–	M/Ar
	Imazalil	–	HY	HY	HY	HY	–	–	HY	HY	HY	HY	HY	–	M
	Vorozole	–	HY	–	HY	HY	–	–	HY	–	–	–	HY	HY	M/Ar
O. mykiss		R115	I133	F134	F221	W224	T310	I313	V370	F372	T373	M374	L477	H480	HEM600
	Fadrozole	–	–	HY	–	–	–	–	HY	–	–	HB	HY	–	M/Ar
	Imazalil	–	HY	HY	HY	HY	–	HY	HY	–	-	HY	HY	–	M
	Vorozole	–	HY	–	HY	HY	–	HY	HY	–	–	–	HY	–	M

^a hydrophobic interaction;

^b hydrogen bond interaction;

^c aromatic;

^d metal.

Table 2. Docking scores (Kcal/mol) vs. inhibition of human recombinant CYP19 (aromatase) for 18 azoles.

Compound^a	Docking scores (closed)	Docking scores (open)	IC50^b [μM]	pIC50
Flusilazole	−6.12	−4.82	0.055	1.26
Imazalil	−5.37	–	0.072	1.14
Myclobutanil	−5.75	–	0.47	0.33
Penconazole	−6.03	–	0.85	0.07
Epoxiconazole	−5.19	−3.83	1.44	−0.16
Propiconazole	−4.74	–	3.2	−0.51
Tebuconazole	−5.12	–	5.8	−0.76
Cyproconazole	−4.38	–	8.5	−0.93
Triadimenol	−4.32	–	12.6	−1.10
Triadimefon	−4.62	−4.5	17.5	−1.24
Hexaconazole	−4.67	−3.95	35	−1.54
Bifonazole	−7.17	−5.84	0.019	1.72
Miconazole	−6.67	–	0.064	1.19
Fadrozole	−7.78	−5.22	0.0076	2.12
Letrozole	−7.29	−5.17	0.015	1.82
Prochloraz	−6.23^p	−4.81	0.047	1.33
Clotrimazole	−5.8^p	−4.61	0.11	0.96
Ketoconazole	−5.23^p	−4.35	5.6	−0.75

^a See Figure 3 for the structures;

^b Concentration of azole required to reduce product fluorescence by a factor of two [25];

p = predicted docking score.

Table 3. Docking scores (Kcal/mol) of azoles in the aromatases of the different studied species.

Compound	Human	C. japonica	G. gallus	T. guttata	O. mykiss
Flusilazole	−6.12	−5.08	−4.93	−6.24	−6.04
Imazalil	−5.37	−4.67	−4.42	−4.595	−4.78
Myclobutanil	−5.75	−4.52	−4.13	−4.5	−5.21
Penconazole	−6.03	−5.36	−5.6	−5.76	−5
Epoxiconazole	−5.19	−4.77	−4.38	−4.77	−5.48
Propiconazole	−4.74	−4.75	−4.11	−4.83	−4.51
Tebuconazole	−5.12	−5.51	−5.4	−5.12	−5.59
Cyproconazole	−4.38	−4.58	−4.37	−4.25	−4.63
Triadimenol	−4.32	−4.82	−4.65	−5.39	−4.39
Triadimefon	−4.62	−4.96	−5.12	−5.75	−5.81
Hexaconazole	−4.67	−3.5	−4.2	−4.44	−4.15
Bifonazole	−7.17	−7.3	−6.35	−7.45	−6.46
Miconazole	−6.67	−6.3	−6.31	−5.795	−6.5
Fadrozole	−7.78	−6.35	−6.43	−7.04	−7.7
Letrozole	−7.29	−7.04	−7.3	−7.27	−7.43
Prochloraz	−6.23	−5.7	−5.6	−6.0	−6.0
Clotrimazole	−5.8	−5.4	−5.3	−5.6	−5.7
Ketoconazole	−5.23	−5.0	−4.8	−5.2	−5.2

Figure 6. Correlation between the docking scores of 18 azoles on the human aromatase and the corresponding human recombinant CYP19 inhibitory activity values (Table 2).

Table 4. Docking scores (Kcal/mol) and inhibition of aromatase activities in brain and ovarian microsomes of O. mykiss [28].

Number	Compound^*	Docking score (close)	Brain IC50 [μM]	pIC50 brain	Ovarian IC50 [μM]	pIC50 ovary
1	Imazalil	−4.78	0.43	0.37	0.32	0.49
2	Propiconazole	−4.51	0.9	0.05	0.9	0.05
3	Triadimenol	−4.39	11	−1.04	26	−1.41
4	Fenbuconazole	−4.5	1.3	−0.11	0.21	0.68
5	Fenarimol	−4.12	6	−0.78	18	−1.26
6	Prochloraz	−6.0^**	1.3	−0.11	1	0
7	Clotrimazole	−5.7^**	0.011	1.96	0.016	1.8
8	Difenoconazole	−5.54	70	−1.85	29	−1.46
9	Androstatrienedione	−8.93	0.015	1.82	0.00025	3.60
10	4-OH androstenedione	−8.58	0.009	2.05	0.00015	3.82

^* Structures displayed in Figure 3

^** predicted docking score (see text).

Figure 7. Differences in the residuals obtained for the 10 brain pIC₅₀ values in O. mykiss (Table 4).

Figure 8. Structures of 24 additional azoles and related compounds considered in this study.

Table 5. Docking scores (Kcal/mol) of 24 azoles on aromatases of the different studied species.

Compound	Human	C. japonica	G. gallus	T. guttata	O. mykiss
Anastrazole	−6.57	−6.29	−5.9	−6.41	−6.23
Azaconazole	−6.08	−5.47	−5.72	−5.6	−5.01
BitertanolSS	−5.81	−5.73	−5.34	−5.8	−4.94
Bromuconazole	−5.99	−5.82	−5.45	−5.78	−5.49
Climbazole	−5.1	−5.68	−5.91	−5.5	−5.48
Diclobutrazole	−5.99	−5.69	−5.7	−6.13	−5.2
Diniconazole	−5.47	−5.23	−6.16	−6.36	−5.84
Etaconazole	−5.98	−4.87	−5.35	−5.66	−5.56
Fluquinconazole	−4.95	−4.46	−4.84	−4.72	−4.58
Flutriafol	−6.04	−6.86	−6.01	−5.19	−6.32
Imibenconazole	−5.95	−6.12	−5.87	−5.97	−5.75
Metconazole	−5.6	−4.6	−5.94	−6.13	−5.41
Nuarimol	−6.07	−6.09	−6.29	−6.17	−5.96
Oxpoconazole	−3.12	−3.02	−2.93	−3.23	−3.22
Prothioconazole	−5.77	−4.95	−6.23	−4.98	−5.9
Quinconazole	−5.44	−6.13	−5.28	−4.92	−5.88
Simeconazole	−6.1	−5.8	−5.37	−6.05	−5.63
Tetraconazole	−5.34	−5.18	−4.79	−5.57	−4.96
Triflumizole	−4.98	−4.04	−5.5	−4.59	−4.03
Triticonazole	−5.74	−5.74	−5.84	−5.95	−5.84
Uniconazole	−6.1	−5.43	−5.97	−5.89	−6.5
Furconazole	−5.5	−5.49	−5.21	−5.73	−5.38
Ipconazole	−3.61	−3.72	−3.89	−4	−4.03
Pyrifenox	−5.47	−5.12	−5.78	−5.62	−5.21

Figure 9. Correlation matrix of the score values on the aromatases of the different studied species for the 24 compounds in Figure 8.

Figure 10. Graphical display of the score values obtained for the 46 studied molecules (x-axis) on the aromatases of human, C. japonica, G. gallus, T. guttata and O. mykiss.

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