J. Chil. Chem. Soc., 56, Nº 4 (2011)
Figure 1: Structures of isoflavans derivatives 1, 2 and 3, quercetin 4 and
3,4-dihydroxybenzoic acid 5 used in the in vitro assays with 15-sLOX and the
docking studies.
Figura2: (A) Coordination of 1c to the non-heme iron atom inside the
binding site through its catecholic hydroxyl groups ; (B) Insertion of ring B of
inhibitors 1a,1b, and 1d superimposed into the hydrophobic pocket formed by
Enzyme rates of formation of HPETE from arachidonic acid at 25oC in
the presence of increasing concentrations of the isoflavans inhibitor yielded
sigmoidal curves that allowed the determination of the corresponding IC50
values with errors smaller than 5%. In order to validate our protocol and the
obtained IC50 values, we first performed a control with quercetin 4 under the
same conditions of our kinetics experiments. We obtained for this compond
an IC50 value of 53 µM, very similar to the reported value of 50 µM found in
literature [17].The table 1 lists the IC50 values obtained for the seven isoflavans
derivatives, for the sake of comparison, the corresponding values for quercetin
4 and for the 3,4-dihydroxybenzoic acid 5 are also given. Studies suggest that
quercetin is presumably degraded to 3,4-dihydroxybenzoic acid [18], so that
the latter was used in our docking studies as a compound reference for the
investigation of the enzyme-inhibitor interactions, since a crystal structure of 5
bound to the 15-sLOX enzyme is also available (pdb code: 1N8Q) [18].
Ile572, Ile770 and Val769
.
In summary, the results showed an increased inhibitor activity of many
of the isoflavans, when compared with quercetin 4 and 3, 4-dihydroxybenzoic
acid 5, the docking studies revealed that the most active isoflavans were bound
to the active site of the enzyme by coordination of their catecholic hydroxyl
groups to the non-heme iron atom, reproducing the same behavior found in the
crystalline structure of 5 bound to the enzyme [18]. The resulting inhibitory
activity of these isoflavans would then be a compromise betweent these two
factors, chelation of the central Fe+3 cation and accomodation of the substituted
ring B within a hydrophobic pocket formed by Leu565, Leu773, Val769 ,Ile572 and
Ile770 fragments.
ACKNOWLEDGMENTS
Table 1: IC50 values of 15-sLOX inhibitors 1-5.
Financial support from DICYT-USACH project # 021041MC is gratefully
acknowledged
Isoflavans
IC50(µM)
28 ± 0.2
26 ± 0.1
18 ± 0.1
60 ± 2.0
65 ± 1.5
24 ± 0.1
90 ± 1.5
53 ± 2.0
74*
1a
1b
1c
1d
2
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Docking results showed that the most potent isoflavans (1a, 1b, 1c and
3a) chelated the central iron atom through their catecholic hydroxyl groups,
similarly to what was observed for 3,4-dihydroxybenzoic acid 5. For these
inhibitors, the average distance between the catecholic oxygens and the iron
atom was 5 Å, in good agreement with the distance obtained for compound 5 in
the active site of the enzyme [18]. The fact that suppression of this catecholic
pattern, as in compound 2, led to an increase of the IC value, suggests that this
is an important structural feature for the inhibitory ac5t0ivity of these isoflavans.
Figure 2 shows isoflavans 1a-d bound to the same active site. All
inhibitors are coordinated to the non-heme iron cation through the catecholic
hydroxyl groups, a structural requirement that is emphasized in figure 2a. In
addition, isoflavans 1a-d, through aromatic rings C and B, may generate further
interactions with hydrophobic fragments in the active site, such as Leu565, Ile572
,
Phe576, Val769, Ile770 and Leu773. Hydrophobic interactions with Leu565 and Ile572
have been suggested to account for the activities of eugenol derivatives, as
potent 15-sLOX inhibitors [20]. The insertion of the aromatic B ring of these
inhibitors into a hydrophobic pocket formed by Ile572, Ile770 and Val769 (Figure
2b) may provide additional interactions between the isoflavans and the active
site, thus strengthening its binding to the enzyme.
These two structural features may cooperate positively to the inhibitory
activity of the isoflavans, as is the case with compounds 1a, 1b, 1c and 3a.
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may be the result of opposing trends. Accomodation of the substituted ring B
of these compounds into the hydrophobic pocket may require conformational
adjustments that lead ultimately to a weaker interaction between the catecholic
oxygens and the non-heme iron of the enzyme. The result is an IC50 value that
approaches the value of the reference inhibitor 5
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