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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
is more potent (IC50 = 1.09 μM) than 7-hydroxyflavanone (IC50 = 3.80 μM), a standardized aromatase
inhibitor. This result was further validated by molecular dynamics simulation analysis and revealed the
high stability and effective binding of screened flavone within the active site of aromatase.
Isoflavone derivatives were found to be effective aromatase inhibitors (Hackett et al., 2005; Su et al.,
2005; Kim, Hackett, & Brueggemeier, 2004). The 2D-QSAR study suggested (Nagar & Saha, 2010c) that
ionization potential, molar refractivity and electron density at atoms C
pivotal to the anti-aromatase activity of these isoflavones. Low electron density at the C
electron density at the O
ing substituent at R
atom might be favorable for aromatase inhibition. Hence, the electron withdraw-
18
and R2 substitutions that imparted more electron density at the O18 increased aroma-
1
tase inhibitory activity. The CoMFA study suggested that the negative charge field at the O
the positive charge field at the C
were important. The substitution pattern at R3 provided more negative
14
electrostatic force field that was favorable for the activity. The substitution pattern of R
favorable steric influence on the O
atom. The unfavorable steric effects are observed at atoms C8 and C9
18
and O18 (Figure 19) might play
14
atom and high
14
atom and
18
imparted positive
2
positions. The CoMSIA study supported the results obtained from the CoMFA study. In addition to the
steric property, the hydrophobicity of the O
atom was also important for the activity. The presence of hydrogen bond acceptors at R
had negative influence on aromatase inhibition. Pharmacophore mapping study suggested that the O
might behave as a hydrogen bond acceptor. Hydrophobic regions around atoms O
atom was important for the activity. Hydrophobicity of the C8
18
and R3 positions
1
atom
11
and C8 are found to be
18
favorable for the activity which was supported by the result of CoMSIA study. The distance between the
hydrogen bond acceptor and the hydrophobic regions were found to be 11.853 Å and 9.278 Å respectively,
whereas the hydrophobic regions are located 5.478 Å away from each other. Structural requirements of
these isoflavones for promising anti-aromatase activity are shown in Figure 19.
Figure 19. Structural and pharmacophoric requirements of isoflavones for anti-aromatase activity
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
Isoflavone compounds were docked into the active site of aromatase enzyme to identify the probable
enzyme interactions for aromatase inhibition (Bonfield et al., 2012). As the enzyme binding pocket is
highly hydrophobic, alkyl chain or aromatic ring might be favorable for enhanced activity. Similarly,
hydrogen bond interactions might play a significant role as the 17-keto oxygen was found to interact
with Met374 backbone amide of aromatase (Ghosh et al., 2009) and the hydroxyl group of Ser478 to
form hydrogen bonding with carbonyl or cyano group containing aromatase inhibitors (Cavalli et al.,
2+
2002; Stefanchi et al., 2011). Again, Fe
in the heme group of aromatase was found to be responsible for
chelating nitrogen heteroatoms of pyridine (Kim et al., 2004), imidazole (Leonetti et al., 2004; Yahiaoui
et al., 2004; Gobbi et al., 2010; Recanatini et al., 2001; Cavalli et al., 2005) or triazole (Leonetti et al.,
2004; Yahiaoui et al., 2004) rings. Docking study of an active compound, 6-methoxyisoflavanone (IC
50
= 0.26 µM) showed that the methoxy group and the Met374 backbone nitrogen lied in a close proximity,
indicating favorable hydrogen bonding interactions. Additionally, a π-π stacking interaction between the
B ring and the heme moiety was observed, which played significant role in aromatase inhibition. But ring
A was not involved in the π-π stacking interaction with the enzyme. Hence, it was inferred that increasing
electron density of ring A provided no beneficial effect on aromatase inhibition though electron donating
methoxy group was observed in the best active compound. Again, it was observed that relatively large
polar substituent at the C
position might be involved in increasing the interaction between the isoflavones
6
and the aromatase pocket. Thus, it was assumed that electron donationg substituent like methoxy and
electron withdrawing substituent like benzo may increase aromatase inhibitory activity. Substitution by
chlorine at C
position increased activity, whereas substitution by t-butyl at this position led to decrease
6
in activity. This phenomenon might be explaind from the electronic and steric properties of the amino
acid residues around C
position of isoflavanones. As this position is spacious and polar due to the
6
presence of Ser478 and Arg115 lying on the pocket, a polar functional group might be favorable at this
position acting through steric and electronic interactions. Additional docking study was also performed
on other fluorinated and bifunctionalized isoflavanones to understand their significance in aromatase
inhibition (Amato et al., 2014). Docking study of the best active bifunctionalized isoflavanone (IC
=
50
0.80 µM) showed that there was a crucial interaction between the pyridyl nitrogen of the compound and
the heme iron atom (distance = 2.5 Å). It was also observed that no hydrogen bonding interaction was
taking place between hydroxyl group side chain of Thr 310 and the pyridyl nitrogen of this compound.
Second interaction (distance = 3.82 Å) was observed between the amino acid Arg115 and the ring A of
the isoflavanone. The docking observations for promoting aromatase inhibitory activity of isoflavone
are represented in Figure 20.
Some azoles and tetralone derivatives were subjected to CoMFA analysis (Recanatini & Cavalli,
1998). The CoMFA model suggested that the phenyl ring along with the meta and para position of azole
derivatives is suitable for steric substitution. This is in agreement with previous results (Recanatini,
1996). The steric zone around the azole derivatives was found to be important for the higher aromatase
inhibitory activity than tetralone derivatives, whereas steric interactions around whole molecules were
found to be unfavorable for the activity. The positive electrostatic regions around the carbonyl oxygen
atom of the tetralone derivatives as well as around the phenyl ring of the less active azoles were found
to be unfavorable. This study also supported the previous results (Recanatini, 1996). Favorable negative
electrostatic regions were observed mostly in π-electron containing the negative azole ring functions
which favors aromatase inhibitory activity. Structural requirements of these azoles and tetralone derivatives for hifher activity are shown in Figure 21.
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427
Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
Figure 20. Docking interactions of the isoflavones for obtaining potent aromatase inhibitory activity
Figure 21. Structural requirements of azoles and tetralones for promising active NSAIs
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
The first X-ray crystal structure of aromatase bound to androstenedione was reported in 2009 (Ghosh
et al., 2009). In the absence of X-ray crystal structure, homology modeling is frequently performed to
obtain three-dimensional structure of the desired protein. In 2000, Cavalli et al. reported construction of
the active site of the cytochrome P450 human aromatase by homology modeling technique using cytochrome p450-CAM (PDB: 1PHD) as the template structure (Cavalli et al., 2000). A number of residues
were found to be invariant or highly conserved in all the P450s. These include Gly60, Arg112, Gly249,
Thr252, Glu287, Arg290, Arg299, Phe350, Gly353, His355, Cys357, Gly359, Ala363 and Leu375.
Two structurally different non-steroidal aromatase inhibitors, i.e., S-fadrozole and a pyridyltetralone
derivative were docked. Hydrophobic interactions were observed between the tetrahydropyridine moiety
of S-fadrozole and the side chains of Ile305, Ala306 and Thr310. The phenyl ring of S-fadrozole may
contribute to the binding of the enzyme by a number of possible hydrophobic contacts with the side
chains of Val313, Thr310, Val369 and Val370. The -CN group appears to be H-bonded to the hydroxyl
group of Ser478, and thus confirming the important role of hydrogen bond acceptor functions present in
the corresponding position of fadrozole like inhibitors (Furet et al., 1993). This result was supported by
the other model (Koymans, Moereels, & Bossche, 1995), where it was observed that cyano-substituted
phenyl ring of S-fadrozole binds in a region surrounded by Asp309, Ser478 and His480. In case of the
pyridyltetralone derivative, interaction was observed with pyridine nitrogen and the heme iron. A largely
hydrophobic pocket made up by the side chains of Ile305, Val369, Val370 and Leu477 hosts the tetralone
moiety of this inhibitor. The remarkable difference between the S-fadrazole and the pyridyltetralone is that
the Ser478 side chain protrudes inside the cavity without any H-bond interaction. A CoMFA model was
further developed on a related series of non-steroidal inhibitors. In case of S-fadrazole, the p-cyanophenyl
ring was found to be surrounded by a favorable steric region, whereas in case of the pyridyltetralone
derivatives the unfavorable steric regions at the tetralone moiety are important for aromatase inhibitory
activity. Steric unfavorable region at the tetrahydropyridine moiety of S-fadrazole was also important
for higher aromatase inhibition. The structural requirements of these analogs for better active NSAIs are
depicted schematically in Figure 22.
Some aromatase inhibitors were synthesized and assayed followed by the 3D-QSAR study (Castellano et al., 2008). As per the work, analysis of the GOLPE PLS coefficients plots revealed that three
areas (A, B, and C in Figure 23) surrounding the ligand-based aligned molecules were important for
the activity. In all these molecules of the training set, a region was always occupied by the negatively
charged substituents, thus highlighting the importance of this area in modulating the inhibitory potency.
In fact, the area, i.e., ring A might be related to significant electrostatic and hydrogen bond interactions. On the other hand, the poor importance of large polyhedrons in the C region might suggest that
this region was not significant for the activity. Nevertheless, most of the training set molecules were
highly superimposed in this area leading to low standard deviation on the grid points surrounding it. As
a matter of fact, the PLS algorithm considered the regions corresponding to the missing polyhedrons as
poorly correlating with the training set activities. Again, the azole function of the B position might be
responsible for coordinating with the heme iron of aromatase, which has a positive impact on aromatase
inhibition. The structural requirements for better aromatase inhibitory activity are shown in Figure 23.
Diarylalkyl-imidazole and -triazole derivatives were found to be promising aromatase inhibitors
(Karjalainen et al., 2000). The QSAR model (Nagar & Saha, 2010b) revealed the importance of van der
Waals atomic and molecular surface areas, presence of hydrogen atoms, importance of size and potential
energy for the anti-aromatase activity of these compounds. The CoMFA study showed that regions near
the imidazole or the triazole ring, and the ring A as well as substitution in the diarylalkyl chain imparted
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
Figure 22. Structural requirements of S-fadrozole and pyridyltetralone derivative for NSAIs
Figure 23. Important structural features required for better NSAIs
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
steric influence which favors the activity. The presence of the positive charges around the imidazole or the
triazole ring and the negative charges around the two phenyl rings favored the activity. Hydrophobicity
and electrophilic substitutions at the C
atom were found to be favorable for the activity. The CoMSIA
4
study suggested that the presence of acceptor region near the ring C had negative impact, whereas the
presence of the same near the ring B at C
the ring C and the long aryl chain attached to the ring A, area near atoms C
position was favorable for the activity. The steric fields around
15
and C17 of the ring B had
16
positive effects. Other regions of ring B and further substitution on that long chain imparted negative
steric influence towards anti-aromatase activity. Favorable hydrophobic region is observed around the
ring A, whereas the hydrophobic area around the C
of the positive charge at atoms C
favored the activity. The presence of donor groups in the vicinity of the ring A was found to be fa-
C
15
, C7 and C8 of the ring C and negative charge substitution at the atom
14
atom was unfavorable for activity. The presence
18
vorable but donor groups at substituted long alkyl chain attached to ring A disfavored the activity. The
results were adjudged through receptor-dependent modeling studies (Karkola & Wahala, 2009; Saberi
et al., 2006), where it was observed that molecular hydrophobicity and hydrogen bond interactions
played pivotal role in aromatase inhibition. The pharmacophore mapping of these molecules showed
the importance of one hydrogen bond acceptor (HBA), one ring aromatic (RA) and two hydrophobic
aromatic (HA) features. In hyporefine hypothesis, one excluded volume (EV) was observed to be another
steric feature important for anti-aromatase activity. In the hyporefine model, the distance between the
acceptor feature and the ring aromatic feature was 13.030 Å, whereas both of the hydrophobic aromatic
features were located 12.417 Å and 6.689 Å away from the acceptor feature, respectively. Both of the
hydrophobic aromatic features were located 7.176 Å and 8.958 Å away from the ring aromatic feature
respectively, whereas the hydrophobic aromatic features were separated by a distance of 7.486 Å. The
study revealed that high electronegative hydrogen bond acceptor group at the C
atom, aromatic rings
15
like imidazole or triaole with hydrophobic character and steric interaction in the ring B are crucial for
the activity. Structural requirements for potent aromatase inhibitors of diarylalkyl-imidazole and -triazole
derivatives are shown in Figure 24.
A diverse set of NSAIs were subjected to the docking and 3D-QSAR studies (Roy & Roy, 2010b).
The docking study suggested that the ligands bind with both polar, i.e. Arg115, Arg375, Asp309,
Asp371, Ser478, Thr310, Asp371, Glu302 and non polar, i.e. Ala306, Ala307, Ile133, Ile305, Leu477,
Met374, Phe134, Phe221, Trp224, Val369, Val370, Val373 amino acids. This study was in an agreement with the crystallographic data that showed androstenedione-bound human placental aromatase
formed a hydrogen bond with the amide of Met374 (Ghosh et al., 2009). The docking results indicated
the important interactions observed in the active site cavity of aromatase are Met374, Arg115, Ile133,
Ala306, Thr310, Asp309, Val370 and Ser478. All these compounds are found to form hydrogen bond
with the amide backbone of Met374. Moreover, the nitro group containing compounds are found to form
hydrogen bond with Arg115, whereas the cyano group containing compounds formed hydrogen bond
with the hydroxyl group of Thr310. Apart from forming hydrogen bonds, van der Waals interactions
were observed with different amino acids like Ala306, Thr310, Trp224, Val370, Ile133, Phe134, Leu372
and Val373. The azole moiety was found to be responsible for coordinating with the heme iron. Steric
bumps of these compounds with different amino acids, like Asp309, Thr310, Met374, Arg115, Ser478
and Val370 might be responsible for poor activity of these compounds. The QSAR study suggested that
the optimum number of hydrogen bond acceptor should be less than or equal to 2 favored aromatase
binding. This result supported the docking results also. The QSAR study also suggested the importance
of different shape, structural, topological and electrotopological state index that were crucial for aroma-
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
Figure 24. Structural and pharmacophoric requirement of dialkyl azoles for promising anti-aromatase
activity
tase inhibition. Optimum hydrophobicity (aLogP < 4.7) of the ligand might play vital role for aromatase
inhibition. The validated QSAR study also suggested that the cyano group containing compounds might
form hydrogen bond with Met374 which is supported by the previously published result (Castellano et
al., 2008). Structural requirements for better anti-aromatase activity are shown in Figure 25.
A diverse set of molecules comprising NSAIs and SAIs were subjected to different univariate (statistical and histogram analysis) and multivariate (decision tree and principal component analysis) approaches
by using quantum chemical and molecular descriptors to understand the origin of anti-aromatase properties of these molecules (Nantasenamat et al., 2013a). Fragment based substructure analysis provided
important information about the fragment important to be an active as well as inactive for aromatase
inhibition. It is observed that five of the top ten active fragments contain azole ring function, amongst
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
Figure 25. Structural requirements for anti-aromatase activity of non steroidal compounds
them the top ranked fragment contains 1, 3 imidazole ring system as per molecular score. Apart from
that, 1, 2, 4-triazole ring system was found to be an active fragment. On the other hand, 4-[(4-cyanophenyl) methyl] benzonitrile, a substructure of third-generation NSAI, letrozole (6), was found to be
an important active fragment. Interestingly, it was observed that all the top ten inactive fragment were
composed of 5-hydroxy-2-phenyl-4H,10H-pyrano[2,3-f ]chromen-4-one, fusion of flavones and 4Hpyran ring. Comparing the mean activity and greater percentage of molecular fragments occurring in
the active set, it was assumed that the NSAIs are more effective that SAIs. This chemical space analysis
might be beneficial for future designing of potential active NSAIs. The structures of the top ten active
and inactive fragments according to the molecular score as per the study are shown in Figure 26.
Large scale QSAR study was performed on the same structurally diverse aromatase inhibitors (SAIs
and NSAIs), described in last paragraph, based on Monte Carlo approach (Worachartcheewan et al.,
2014). Such type of QSAR models might be very useful as far as the structural point of view, as this
approach directly dealt with the relation between the biological activity and the SMILES notation encoding molecular structure. In this approach, the data was divided into four types of random split sets,
i.e., sub-training, calibration, test, and validation sets. QSAR models were judged by parameters, like R
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2
Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
Figure 26. Active and inactive fragments with their molecular score important for aromatase inhibition
and Q2 which correspond to the goodness-of-fit and the goodness-of-prediction parameters, respectively.
Fischer (F) ratio also provided further estimate of the predictive ability of the model. The statistical
results obtained from different splits and data sub-sets indicated that the QSAR model was reliable and
significant. The SMILES molecular fragments might also be useful for prediction and interpretation of
the origins of aromatase inhibitory activities. The top ranking fragments for imparting anti-aromatase
activity include the presence of the cyclic ring in the molecular structure, absence of halogens, presence
of double bond and oxygen atoms together with double bonds those are not connected in the structure.
Similarly, the top ranking fragments for reducing anti-aromatase activity were branching in the molecule,
presence of nitrogen and double bonds those are not connected in the structure and presence of oxygen
atoms connected through double bonds. This Monte Carlo type of QSAR technique might also be beneficial for providing the mechanistic information of the molecular fragments for influencing aromatase
inhibitory activity.
A QSAR study on 1-substituted 1, 2, 3- triazole analogs of letrozole was done (Nantasenamat et al.,
2013b) using multiple linear regression (MLR), artificial neural network (ANN) and support vector
machine (SVM). Geometry optimizations of these compounds were done by AM1 method followed by
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Ligand- and Structure-Based Drug Design of NSAIs in Breast Cancer
DFT method. Quantum chemical and molecular descriptors were selected for MLR model development.
After removal of the outliers, the best model was subjected to ANN and SVM techniques for parameter
optimization. This model might be useful to predict new aromatase inhibitors of this class. Among all
the multivariate analyses performed, SVM was found to be more superior to MLR and ANN. The SVM
provided the highest accuracy, whereas the MLR model could be easily interpretable. Three descriptors,
i.e., number of rings, hydrophobicity and HOMO-LUMO energy gap were found to be important for
aromatase inhibition. Decreasing values of these three parameters may be beneficial for the aromatase
inhibition. Structural requirements for higher aromatase inhibitory activity of this class of compounds
are shown in Figure 27.
A mechanism-based SAR categorization framework highlighting the important chemical and structural
features responsible for aromatase inhibition was developed (Petkov et al., 2009). The steroidal scaffold
is found to be the most prominent when the structure of the target chemical is similar to androstenedione
and testosterone. The NSAIs should possess heteroatoms like N, O and S usually in a diazole or triazole
ring so that these can coordinate the heme iron of aromatase enzyme and interfere with the steroid hy-
Figure 27. Important structural features of 1-substituted 1, 2, 3- triazole analogs for anti-aromatase activity
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