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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5587_Библиотеки_им_академика_М_И_Перельмана.pdf
Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
droxylation. Chemical groups like CN, NO2, CO, etc. of the NSAIs were able to accept a hydrogen bond
from the aromatase active site due to their electronegativities. A hydrophobic spacer group between
the heme coordinating group and the hydrogen bond acceptor moiety was essential for the aromatase
inhibition. However, larger alkyl fragments as spacers disfavored potency due to steric hindrance. For
classes like quinolines, tetralones, flavones, etc, where no Csp3 spacer was available between electron
donating and accepting centers, the activity decreased. However, in case of fluorenes hydrophobicity
might partly compensate the lack of electron accepting groups. The structural requirements for better
activities of these NSAIs are shown in Figure 28.
A new ligand-based strategy combining important pharmacophoric and structural features were
developed according to the postulated aromatase binding mode that may be useful for virtual screening of new potent NSAIs (Neves et al., 2009). Some known NSAIs were used to derive and validate
Figure 28. Structural requirements of NSAIs for better activity
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
pharmacophore model for fast high-throughput in silico screening of anti-aromatase compounds. Common feature pharmacophore model was first developed with fadrozole (4), anastrozole (5), letrozole
(6) and vorozole (7) by using HipHop algorithm. Six possible alignment solutions containing three or
four pharmacophoric points were found. The top two ranked solutions contained four pharmacophore
features (two hydrophobic and two hydrogen bond acceptor groups). The hypotheses revealed that the
imidazole or the triazole nitrogen atom acted as a hydrogen bond acceptor and might coordinate with the
heme iron of aromatase active site. The second hydrogen bond acceptor feature matches either a cyano
group (anastrozole, letrozole, and fadrozole) or the nitrogen atom of a methylbenzotriazole (vorozole).
One of the hydrophobic features superimposed with the phenyl rings of all these molecules, and second
hydrophobic feature depicted the methyl group of anastrozole or the piperidine moiety of fadrozole.
Besides the physicochemical properties of the inhibitor, molecular shape was found to be extremely
important. Most aromatase inhibitors used to build the common features model had a similar shape that
is complementary to the volume of the aromatase active site. Therefore, the shape of letrozole (6) is
converted into a set of inclusion volumes and combined with the pharmacophoric query. Hence, the final
pharmacophore model combined pharmacophoric information (two hydrogen bond acceptors and two
hydrophobic groups) with fragment information (nitrogen containing aromatic heterocycles) and steric
restrictions. All aromatase inhibitors used in the training set matched the final hypothesis. These findings showed that the HBA-shape pharmacophore model was able to identify not only inhibitors from the
training set but also potent NSAIs with different structures. By means of virtual screening and Lipinski
filtering from National Cancer Institute (NCI) database, 29 compounds were selected as hits. Four different types of nitrogen-containing aromatic heterocycles, namely imidazoles, triazoles, pyridines and
pyridazines were retrieved as hits. These nitrogen containing heterocycles are linked to several types
of hydrophobic molecular scaffolds, such as diphenylmethane, arylbenzylsulfane, diphenylimidazole,
pyridinylbenzoimidazole, phenylpyridine, benzyl-1H-indol, acridine, benzo/methylfuran, quinoline, etc.
Biochemical evaluation of these compounds was performed and one compound was found to be more
active than letrozole (6), whereas five other compounds were more active than formestane (1). Docking
study suggested that all these molecules fit well within the binding site cavity of the aromatase enzyme,
and pyridine or imidazole moiety was found to coordinate with the heme group. All molecules have a
hydrogen bond between an acceptor group and Ser478 which was a key residue to the catalysis mechanism of the enzyme. Another hydrogen bond was observed between hydroxyl group and Thr310. The
hydrophobic area was depicted by three phenyl rings docked on an apolar region of the aromatase binding
cavity, defined by hydrophobic residues Val 313 and Leu 477. Additional hydrophobic area had either
phenyl rings or a benzylsulfanyl group fitting a small hydrophobic pocket due to Pro368 and Val370.
The result is represented schematically in Figure 29. This study provided useful information about the
common pharmacophore features of azole NSAIs, binding mode to the active site, steric restrictions
and a drug likeness filter.
Some resveratrol analogs were designed, synthesized and their inhibitory activities against aromatase enzyme were evaluated (Sun et al., 2010). These compounds were subjected to docking, molecular
mechanics energy minimization, computer graphics molecular modeling studies, and these informations
were utilized to design several very potent inhibitors. The aromatase inhibitory activity of these newly
designed compounds was far better than resveratrol. One of these compounds was docked with the aromatase enzyme. The docking result suggested that the amino group of that compound may form hydrogen
bonding with carbonyl group of Ile305 and carboxyl group of Asp309. The ring A might be involved
with the π-π interaction with the indole ring of Trp224 and a CH-π interaction with the methyl group of
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
Figure 29. Schematic representation of the pharmacophore requirements for potent NSAIs
Thr310. The methoxy group of the ring B might form hydrogen bond with the backbone amino group
of Met374 as well as the ring B was involved with methyl group of Val370 through CH-π interaction.
This docking result is in agreement with the crystallographic data (Ghosh et al., 2009). These docking
results were used for designing new analogs. The amino group of the ring A was replaced with hydrogen
bond donating groups like hydroxy and aminomethyl groups that might have similar interactions with
Asp309 and Ile305. A hydrogen bond acceptor located near the ring B may form hydrogen bond with
the carbonyl group of residue Leu477. As many of these aromatase inhibitors possess imidazole and
triazole groups that could coordinate to the iron atom, the azole function was incorporated into stelbene
framework. Most of the resveratrol analogs with an amino group on the para position of the ring A
exhibited significant aromatase inhibition. Substitution of para amino group of the ring A with nitro,
halogen, hydroxy, nitrile, acetyl and aminomethyl groups resulted inactive compounds. Changing of
the para amino position also reduced the activity. Reduction of the CH=CH double bond of resveratrol
analog influenced the favorable activity to some extent. The para amino group of the ring A had greater
importance than the methoxy group of the ring B. Incorporating imidazole group played important role
by coordinating with heme iron. Structural requirements of these resveratrol analogs for higher antiaromatase activity are shown in Figure 30.
The QSAR studies for exploring structural requirements of the dichlorophenyl compounds (Nagy,
Tokarski, & Hopfinger, 1994) for better active NSAIs are represented schematically in Figure 31. The
distance (D) of the nitrogen atom in the variable heterocycle from the core carbon atom was the most
important factor for higher activity. To maximize inhibitor potency, the optimum distance between the
nitrogen and carbon, was about 3.6 Å for these analogs. The field calculations coupled with the QSAR
studies suggested that this nitrogen (located from 3.6 Å distance from the core carbon) acted as a hydrogen
bond acceptor. Two possible three-dimensional pharmacophores were proposed for effective aromatase
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
Figure 30. Structural requirements of resveratrol analogs for anti-aromatase activity
inhibitors. All these QSAR models developed showed that the distance (D) between the methane core
carbon and the heterocyclic nitrogen was the crucial factor for aromatase inhibition. The steric bulk of
the substituents about the chiral carbon center markedly limited conformational flexibility so that the
global minimum energy conformation was energetically the only plausible conformation which could
be considered an ‘active’ conformation. The distance (D) and the integrated potential field difference
(∆F) of specific region in space correlated with aromatase inhibitory activity. It suggested that hydrogen
bonding or strong electrostatic interaction force between the nitrogen in the inhibitor heterocycle and
the receptor was important for the higher activity. Conformational and field analyses data also indirectly
supported that dipole properties of the heterocycle did not correlate with the aromatase inhibitory activity. Pharmacophore mapping study suggested that hydrogen bond acceptor regions were located at the
heteroaromatic ring and in the hydroxyl group. No intermolecular hydrogen bonding was likely to occur
as the site was within the receptor pocket.
A ligand-based approach was used to generate pharmacophore models derived from highly active
and selective P450 19 inhibitors (Schuster et al., 2006). In the common feature pharmacophore model
generation, hydrophobic aromatic (HAR) and ring aromatic (RA) features were considered as most
frequent for active aromatase inhibitors. Hiphop pharmacophore model was generated with two ring
aromatic and two HBA+F+Cl features. A Hyporefine pharmacophore model was also derived with
these compounds with in vitro activities ranging from 40 nM to >250 μM. The HypoRefine hypothesis
was generated with one RA, one hydrophobic, two HBA+F+Cl features, and one excluded volume. The
Hyporefine model was able to predict 90% of all tested compounds. Using the Hiphop model as search
queries and employing a sophisticated filtering procedure, the drug likeness of the test compounds were
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
Figure 31. Structural requirements of the dichlorophenyl compounds for NSAIs
increased and two compounds were discovered. The models were used as database search queries to
identify active compounds from the NCI database. The pharmacophoric requirements for better active
compounds are shown in Figure 32.
Ligand based (LB) and structure based (SB) models were generated on a diverse set of NSAIs
(Muftuoglu & Mustata, 2010) and both of these models were merged to get a new model that produced
a combined pharmacophoric requirements to differentiate active and inactive NSAIs. The LB pharmacophore model consisted two hydrogen bond acceptors (A) and two hydrophobic aromatic (HYARO)
features, whereas the SB pharmacophore included one acceptor (A) and two hydrophobic aromatic
(HYARO) features including eleven excluded volumes (EV). In case of LB pharmacophore model, the
hydrogen bond acceptor features were separated by a distance of 2.11 Å, whereas the HYARO features
were separated by a distance of 4.46 Å. The distance between one acceptor and the HYARO features
were 1.14 Å and 5.40 Å respectively, whereas the distances between second acceptor with the HYARO
features were 3.24 Å and 7.20 Å, respectively. In case of SB pharmacophore model, the HYARO features
were located from the acceptor feature at the distance of 3.16 Å and 6.99 Å respectively, whereas the
HYARO features were separated by a distance of 4.46 Å. Amalgamated pharmacophore model consisted
of two acceptors (A), two hydrophobic aromatic (HYARO) features and eleven excluded volumes (EV).
In this model, the hydrogen bond acceptors were separated by a distance of 2.06 Å, whereas the HYARO
features were separated by a distance of 4.46 Å. The distances between one acceptor and the HYARO
features were 1.14 Å and 5.36 Å respectively, whereas the distances between second acceptor with the
HYARO features were 3.19 Å and 7.10 Å, respectively. All these three models successfully identified
training and test sets AIs. The LB model could predict 90% molecules, whereas the SB model and the
merged model could predict 100% test set AIs. The merged pharmacophore model was able to predict
these inhibitors and used to determine the active and the inactive compounds. This approach could be
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
Figure 32. Important pharmacophoric features required for better NSAIs
very useful for identifying lead discovery with improved inhibitory activity through 3D database search.
Docking study analyzed the predictions for interactions between aromatase enzyme and two of the most
potent inhibitors, i.e., verozole (7) and 3-[imidazol-1-yl-(4-nitrophenyl)-methyl]-chromen-4-one (42).
The results showed that dock score and interaction energy values for the two nonsteroidal AIs were
comparable to the preferred substrate androstenedione. Interaction of heteroatom with heme coordination was found to be implicated in each NSAIs. The docking study might be used to virtually evaluate
the potency of new NSAIs. The pharmacophore requirements for potent NSAIs are shown in Figure 33.
Benzofuran derivatives were also found to be effective and potential aromatase inhibitors (Vinh et al.
1999; Saberi et al., 2006; Vinh et al., 2000; Saberi, Shah, & Simons, 2005). The QSAR study (Nagar et
al., 2009) revealed that the electron donating group at the C
groups at R′′′, hydrogen bond accepting character of the O
atom (Figure 35) or the electron withdrawing
1
atom and the CN group substitution at the C14
7
atom as well as the decreasing steric effect might influence anti-aromatase activity. The pharmacophore
mapping study of these molecules was well supported by the QSAR data. The best pharmacophore model
showed the importance of three hydrogen bond acceptors, one hydrophobic feature and one excluded
volume. The hydrogen bond acceptor features were separated from a distance of 5.348 Å, 10.367 Å and
14.025 Å respectively, whereas the hydrophobic feature was located 9.496 Å, 6.867 Å and 6.427 Å away
from the hydrogen bond acceptor features, respectively. The distances between the excluded volume and
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
Figure 33. The ligand-based and structure-based pharmacophore requirements for potent NSAIs
Figure 34. Structural requirements of benzofurans for potent aromatase inhibitiory activity
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
the three hydrogen bond acceptor features and one hydrophobic feature were 5.330 Å, 4.328 Å, 9.850 Å
and 7.896 Å, respectively. The pharmacophore mapping study suggested that hydrogen bond acceptor
region at atoms C
, O7 and C14 was important for the activity, which was supposed to form the hydrogen
3
bonding interaction with the receptor. A hydrophobic character was necessary for the aromatase inhibitory activity. The structural requirements of these benzofurans for promising anti-aromatase activity are
shown in Figure 34.
Benzcyclo derivatives were found to be effective aromatase inhibitors (Ulmschneider et al., 2005).
The 2D-QSAR study (Nagar & Saha, 2010a) suggested that the nucleophilic substitution at the C
(Figure 35), the electronegative substitution at the C
atom, the positive atomic charge at the C5 atom,
4
atom
2
the decreasing electronic charge at the atom number 13 and the increasing hydrophobicity of these
molecules were important for anti-aromatase activity. The presence of the nitrogen atom at the C ring
3+
might have close interaction with Fe
of the heme to block the active site (Saberi, Shah, & Simons,
2005). Polar groups were found to be effective due to hydrophobic interactions in the active site cavity with the nonpolar amino acids like Ala306, Val370, Leu372, Val373, Met374, Leu377 and Leu477
(Karkola & Wahala, 2009; Roy & Roy, 2010b). The CoMFA study suggested that presence of the electropositive groups at C
due to the pattern of rings B and C as well as unsaturation at the C
, C5 and C13 positions favored aromatase inhibitory activity. Resonance effects
3
atom may impart positive charge
5
field at these positions. It was supported by the docking studies where it was observed that the electropositive substituent at the C
atom might have binding interactions with residues, like Leu372, Met374
3
and Leu377 of aromatase enzyme (Ghosh et al., 2009). Similarly, the presence of the electronegative
group at the C
atom favored activity. The electronegative substituent at the C4 atom was lying close to
4
Leu477 residue of aromatase enzyme and had some negative electrostatic interaction with it. Again, the
Figure 35. Structural requirements of benzocyclo derivatives for potent anti-aromatase activity
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
presence of the nitrogen atom at the heterocyclic C ring might have interaction with Fe3+ of the heme
as well as the residue Ala306 in the active site of aromatase enzyme (Saberi, Shah, & Simons, 2005).
The CoMSIA study supported similar pattern of the electrostatic interaction of the CoMFA model. In
addition to electrostatic interactions at atoms C
substituent at the C
atom was important as that might have some binding interactions with residues,
2
and C4, CoMSIA study suggested that electronegative
3
like Arg115, Ile133 and Val373 at the active site of aromatase enzyme. The electronegative substituent
at the C
atom might bind with the receptor through H-bonding with Val373 and bump interaction with
2
Arg115. Hydrophobicity was also playing a vital role to the aromatse inhibition. The polar groups at
atom might have hydrophobic interaction with the non-polar amino acid residues, like Met374 or
the C
3
Leu372 (Karkola & Wahala, 2009). The C
non-polar amino acid, Leu477. As the electronegative substitution at C
it might be inferred that the electronegative substituent at C
hydrophobic interaction (Sun et al., 2010). The steric influence at atoms C
atom was also essential for the hydrophobic interactions with
4
atom was also observed, hence,
4
atom was more preferable rather than the
4
, C2, C12 and C13 was found to
1
be favorable. The six member aromatic B ring is found to be more favorable than the five member ring
system due to mesomeric effect. The unsaturation between atoms C
and C7 decreased steric hindrance
10
and is favorable for the activity (Roy & Roy, 2010b). Pharmacophore mapping suggested that the four
hydrophobic features (HY1, HY2, HY3 and HY4) were important for enzyme inhibition. HY1 feature
was located 4.269 Å, 7.005 Å and 6.780 Å away from HY2, HY3 and HY4 features respectively, whereas
the HY2 feature was located 3.718Å and 5.595Å away from HY3 and HY4 features, respectively. The
distance between HY3 feature and HY4 feature was 3.717Å. The aromatic rings and aliphatic side chain
in the ring A behaved as promising hydrophobic regions. The hydrophobic substitutions at the C
atom increased inhibitory activity by increasing the binding interaction with amino acid residues
the C
3
atom or
2
in the active site cavity. These observations are well supported by the other modeling studies (Neves
et al., 2009; Saberi et al., 2006). The structural requirements of these benzocyclo derivatives for better
anti-aromatase activity are shown in Figure 35.
Low molecular weight compounds from wild mushrooms were docked in aromatase enzyme and the
potential SAR studies for the compounds with the best estimated inhibition constants were discussed
for each compound family (Froufe, Abreu, & Ferreira, 2011). The compound 4-O-caffeoylquinic was
found to be the top-ranked potential inhibitors for aromatase enzyme. Each family of low molecular
weight compounds from wild mushrooms, like phenolic acid derivatives (benzoic and cinnamic acid
derivatives and flavonoids), vitamins (tocopherols and ascorbic acid), carotenoids, sugars and fatty acids
were docked against aromatase. It was found that benzoic acid derivatives were appeared to have no
significant inhibitory activity against aromatase. Apart from 4-O-caffeylquinic and 5-O-caffeylquinic,
cinnamic acid derivatives were not found to have significant activity. In case of flavonoids, quercetin
was found to be the best compound to inhibit aromatase. The top-ranked compound 4-O-caffeoylquinic
occupied the space of the aromatic rings of androstenedione and quinic acid might have polar contacts
with heme group of aromatase through the carboxylic acid moiety. The compound may mimic androstenedione by forming hydrogen bond with Arg115 and peptide bond between Val373 and Met374. This
might be possible due to one oxygen atom of 4-O-caffeoylquinic which was positioned on the same
space occupied by the oxygen atom of one of carbonyl group of androstenedione. In case of vitamins
and sugars, β-tocopherol and linoleic acid, respectively were found to be active against aromatase as far
as the estimated K
(µM) value was concerned. Important structural features required for higher anti-
i
aromatase activity are shown in Figure 36.
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
Figure 36. Important structural features of natural compounds from mushroom for anti-aromatase activity
Figure 37. Schematic representation for designing potential and selective non-steroidal aromatase inhibitors
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