Pharmacophore Mapping of Selective Binding Affinity of Estrogen

J. Chem. Inf. Model. 2007, 47, 475-487

475

Pharmacophore Mapping of Selective Binding Affinity of Estrogen Modulators through Classical and Space Modeling Approaches: Exploration of Bridged-Cyclic Compounds with Diarylethylene Linkage Subhendu Mukherjee,† Shuchi Nagar,† Sanchita Mullick, Arup Mukherjee, and Achintya Saha* Department of Chemical Technology, University of Calcutta, 92, A.P.C. Road, Kolkata - 700009, India Received September 27, 2006

Research on Selective Estrogen Receptor Modulators (SERMs) has been driven by interest in discovering target selective molecules. In view of such significance, the present work explored the pharmacophores of estrogen receptor (ER) subtypes specific binding affinities of diverse compounds belonging to the category of bridged bicyclic-1,1-diarylethylene derivatives. Implementing classical QSAR and CATALYST based spacemodeling approaches, it has been explored that attachment of aryl ring systems to unsaturated linkages, availability of phenolic hydroxyl group, global hydrophobicity, and stereochemistry of certain functional groups might be important for governing the subtype specific estrogenic behavior of this group of compounds. Supplementing this deduction, critical interfeature distances between hydrogen bond acceptor, hydrophobic, and ring aromatic features along with steric influence are found to primarily influence the ER-subtypes specific binding of this series of compounds. INTRODUCTION

Estrogens have demonstrated remarkable effectiveness in the deterrence and management of pre- and postmenopausal diseases.1,2 The effects of estrogen are mediated by an intranuclear transcription factor called the estrogen receptor (ER),3 of which two structurally similar subtypes (R and β) are presently known. The existence of these two subtypes provide a possible explanation for the tissue selectivity.4 The predominant ER in the female reproductive tract and the mammary glands is ERR, whereas ERβ is primarily present in the vascular endothelial cells, bone, and male prostate tissue. Both the subtypes bind the endogenous estrogen, 17βestradiol (E2), but illustrate different tissue expression and marked response to different agonists and antagonists.5,6 X-ray crystal structures of receptor-E2 complexes have demonstrated that the binding areas of ERR and ERβ are varied by only two amino acids. Leu384/Met421 in ERR are found to be equivalent to Met336/Ile373 in ERβ,7 a classical feature that probably explains the lack of selectivity of E2 in binding to the ER.8 A group of structurally diverse nonsteroidal compounds binding to the ER has been investigated to produce estrogen agonist effects in some tissues, while antagonist effects in others. These classes of compounds are known as selective estrogen receptor modulators (SERMs).9,10 As such, research efforts are being devoted toward developing SERMs that can maintain the benefits of estrogens while avoiding the risks.11 Drug molecules like raloxifene and tamoxifen that have tissue selective ER agonist or antagonist properties are some agents that ensure a safer alternative to estrogen.12,13 There continues to be a serious effort to understand the molecular basis for the tissuesspecific effects of this group of compounds.14 The 1,1-diaryl component is a chemical moiety common to many potential * Corresponding author e-mail: [email protected]. † Both authors contributed equally to this work.

SERMs, such as tamoxifen, toremifene, and idoxifene.15 Our group has also explored this feature to be prime pharmacophore signals for estrogen mediated bioactivities of a different group of compounds, viz., 1-trifluoromethyl-1,2,2triphenylethylenes, bromotriphenylethylenes, and triphenylacrylonitriles through Quantitative Structure-Activity Relationship (QSAR) studies.16-18 QSARs are mathematical methodology, statistically validated, and mostly used to correlate experimental or calculated properties derived from chemical structures with biological activities. They also may be applied to predict the activity values of nonsynthesized compounds structurally related to the training set. Structurally diverse 60 environmental estrogens19 are classified through QSAR studies. Since the different classes of environmental estrogens examined contain no common structural elements, 3D rototranslational descriptors20 are employed to predict activity. Groups of aromatic, phenolic, and aliphatic tetrasubstituted pyrazole,21 raloxifene,22 and xenoestrogens23 are modeled for studying the SAR for binding affinity to the ER, while tetrahydroisoquinolines24 and diphenylnapthyl propylene25 are modeled for ER selective subtypes binding. With the advent of 3D molecular space modeling, a pharmacophore hypothesis can visualize the potential interaction between the ligand and the receptor. A pharmacophore is a set of functional group/fragment types in a spatial arrangement that represents the interaction made in common by a set of small molecular ligands with a protein receptor.26 The pharmacophore concept is based on the kinds of interaction observed in molecular recognition, i.e., hydrogen bonding, charge, and hydrophobic interaction and alternatively can be used as a query in a 3D database search to identify new structural classes of potential lead compounds; and it can serve as a template for generating alignment for 3D QSAR analysis.27 Two types of pharmacophore hypoth-

10.1021/ci600419s CCC: $37.00 © 2007 American Chemical Society Published on Web 03/01/2007

476 J. Chem. Inf. Model., Vol. 47, No. 2, 2007

Figure 1. General structure of bridged-cyclic diaryl compounds.

eses are well established: receptor-based and receptorindependent pharmacophore. Receptor-based pharmacophore mapping of SERMs25,28-30 are commonly employed, but nowadays receptor-independent pharmacophore mapping is growing interestingly for deriving bioactivities of diverse groups of compound, such as aromatase inhibitors,31 serotonin inhibitors,32 and antithormbin33 agents and are now increasingly being handled by automated computational methods, such as CATALYST,34 GASP, and DISCO which are commercially available programs.35-37 Interestingly, there is no such receptor-independent pharmacophore hypothesis identified for SERMs. Consequently, the present work is taken up to study the 1,1-diaryl scaffold as a small ligand38,39 with a view to deduce the active pharmacophore signals based on receptor-independent hypothesis, using the CATALYST program,34 that can eventually aid in apprehending the tissue-specific effects of different compounds containing this unit. Due to the close structural similarity of these groups of compounds with that of many prospective SERMs,15 it might be possible that some kind of pharmacodynamic similarity exists between these groups of compounds. MATERIALS AND METHODS

In the present work, 14 compounds belonging to the categories of bridged bicyclic-1,1-diarylethylenes39 (Figure 1a) and 20 compounds belonging to oxabicyclic-1,2-diarylethylenes38 (Figure 1b) have been considered (Table 1) for modeling aspects through classical approach. The relative binding affinities (RBA) of these compounds to the ER subtypes (R and β) have been considered as the biological activity and implemented as logarithmic functions (pRBA) for modeling purpose. The primary objective of the work undertaken had been to generate relationships between the structure and corresponding activity through multiple linear regression (MLR) approach and deduce a pharmacophore map through receptor-independent space modeling technique. In the foremost section of the study, 3D structure of the molecules are energy minimized in MOPAC using the AM1 method to locate their global minima conformer, and subsequent calculation of different molecular properties, such as physiochemical, electronic, topological, and spatial and structural features are estimated for classical modeling. The partial charge is calculated using the Extended Huckel approach,40 and E-state indices41 of all the atoms are generated using a JAVA-based program,42 while other descriptors are generated using Chem3D Pro 5.040 and Tsar 3.3.43 MLR is performed using standard and forward stepwise regression methods.44 QSAR model origination is accom-

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plished by correlation analysis, and statistical parameters of the regression equation considered are as follows: r or R (correlation coefficient), EV (explained variance), F (variance ratio), df (degree of freedom), s (standard error of estimate), and AVRES (average of absolute values of calculated residuals). Leave-one-out (LOO) cross-validation45 is performed that generated PRESS (predictive residual sum of squares), SDEP (standard deviation of error of predictions), Presav (average of absolute value of predicted residuals), and Q2 (cross-validated variance). On the contrary, groups of compounds (n ) 34) containing these bridged-cyclic diaryl scaffolds have been taken up for receptor-independent pharmacophore space modeling studies with regards to binding affinities at the ERR and ERβ. The chemical features optimized for exploring the spatial pharmacophore maps of these groups of compound are hydrogen bond (HB) acceptor (a) and donor (d), hydrophobic (p), and ring aromatic (r). The pharmacophore models (hypothesis), generated by CATALYST,34 consist of an array of features necessary for bioactivity of the ligands arranged in 3D space that can explain the variance in activity of the molecules w.r.t. geometric localization of the chemical features present in them. To be retrieved as a hit, a candidate ligand must possess appropriate functional groups which can simultaneously reside within the respective tolerance spheres of the pharmacophoric features. Each feature is associated with a weight (a measure of its proposed importance to the pharmacophore as a whole), and the better the overall superimposition of functional groups of the molecule to the appropriate features of the pharmacophore, the higher the score of the fit.46 The different control parameters employed for hypothesis generation (called a HypoGen process) are spacing, uncertainty, and weight variation. Spacing is a parameter representing the minimum interfeatures distance that may be allowed in the resulting hypotheses. In the generated hypothesis, each feature signifies some degree of magnitude of the compound’s activity. The level to which this magnitude is explored by the hypothesis generator is controlled by the weight variation parameter. This is varied in some cases from 1 to 2. In other cases, the default value of 2 is generally considered. The uncertainty parameter reflects the error of prediction and denotes the standard deviation of a prediction error factor called the error cost. A default value of 3 is considered as the uncertainty parameter in the present work. While generating hypothesis, a total cost function is minimized comprising of three terms, viz., weight cost, error cost, and configuration cost. Weight cost is a value that increases as the weight variation in the model deviates from an ideal value of 2. The deviation between the estimated activities of the training set and their experimentally determined values is the error cost. A fixed cost (ideal hypothesis cost) depends on the complexity of the hypothesis space being optimized and is denoted as the configuration cost. The configuration cost is equal to the entropy of hypothesis space. The CATALYST program also calculates the cost of a null hypothesis that assumes no relationship in the data, and the experimental activities are normally distributed about their mean. Accordingly, the greater the difference between the total and the null costs, it is more likely that the hypothesis does not reflect a chance correlation. The minimum difference between the total and null

PHARMACOPHORE MAPPING

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J. Chem. Inf. Model., Vol. 47, No. 2, 2007 477

Table 1. Structural Features of Bridged-Cyclic Scaffolds

compd no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14

R1 OH F H OH H OH OH OH OH OH OH OH OH OH

bridged cyclesa

a

substituents and ring systems R2 H H H OH OH H H H H H H H H H

BC k k k k k l m n o p q k r r

a

C1-C15b U U U U U U U U U U U S U S

compd no.

substituents V

W

X

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

OH OH OH OH OH OH OH OH OH OH OH H H H OH H H

OH OH OH OH OH OH OH H H OMe OMe H H H OH H H

H H H H H H H OH OH H H H H H H H H

SO3Ph SO3Ph (R) SO2Ph SO2CH2CH3 CO2Et

Y

31

OH

OH

H

32

H

H

H

33

H

H

H

Z

y-z

H H H

S S S S S S S S S S S S S S S S S

SO3-Ph-OH

H

S

CO2Me

Ph

U

CO2Me

Ph-OH

U

sss sts SO3Ph (mix.) SO2Ph SO3Ph (mix.) SO2Ph SO3Ph SO2Ph sus SO3-Ph-OMe SO3-Ph-OH SO3-Ph-OH (R)

H H H H CO2Et H H H H H H

Bridged cycles. b See C15 in structures k-r. S: saturated; U: unsaturated; Y-substituent not mentioned denotes β configuration.

costs is taken as 60 bits for a hypothesis optimization. The generated hypothesis is further validated to nullify overprediction of bioactivities for inactive compounds through a process known as Hyporefine. In this process, the steric interactions of compounds are considered in the hypothesis generation, and if steric properties are crucial for bioactivities, then these are portrayed in the validated (refined) hypothesis. The quality of hypothesis generated is adjudged through a cross-validation technique using CatScramble.34 The validation procedure is based on Fischer’s randomization test,47 where the biological activity data are randomized within a fixed chemical data set and the HypoGen process is initiated. By logic, the hypothesis generated prior to scrambling should be better to attest for a good pharmacophore model. In the present work, the number of conformer generation is limited to a maximum of 250 using the ‘best conformer generation’ method with 20 kcal/mol of energy cutoff. The spacing is varied from 60 to 250 pm. The biological activities are expressed as RBA to the ER subtypes. The HypoGen algorithm is forced to find pharmacophore models that contain at least one and at most two of all the features.

RESULTS

Classical Modeling. A structure-activity relationship has been drawn, investigating physiochemical (partition coefficient, hydrophobicity, steric, and moments), electronic (atomic and partial charge functions, orbital energies), and electrotopological (E-state indices) features of molecular architecture for characterization of unique pharmacophore features. In all the regressional models, the 95% confidence intervals are shown in parentheses, and the F-values are significant at the 99% confidence level. The regression constants for all relations are significant at 95% (except superscripted with #). (a) Bridged-Cyclic Diarylethylenes. While modeling RBA of bridged-cyclic diarylethylene derivatives (Figure 1a), the best single variate model for binding to the R-subtype could be developed with the charge function of atom C1 (Ch1) that explained 37.097% variance in activity, and the statistical quality of the relation is estimated to be

r ) 0.648; r2 ) 0.419; s ) 0.463; n ) 14


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Table 2. Statistical Qualities of Best QSAR Relations for Binding Affinity of Bridged-Cyclic Scaffolds correlation statistics eq no.

n

R

R2

1 2 3 4 5 6 7

14 14 14 20 20 20 20

0.928 0.944 0.919 0.942 0.946 0.984 0.911

0.862 0.891 0.845 0.886 0.896 0.968 0.830

prediction statistics

EV (%)

F

df

s

AVRES

PRESS

SDEP

Presav

Q2

80.052 84.315 77.602 86.520 86.770 96.016 79.769

14.042 18.470 12.260 41.660 32.140 121.490 25.971

4.9 4.9 4.9 3.16 4.15 4.16 3.16

0.261 0.241 0.132 0.360 0.180 0.182 0.357

0.184 0.156 0.085 0.254 0.120 0.129 0.260

1.595 1.270 0.460 3.047 0.930 0.934 2.889

0.340 0.301 0.181 0.390 0.215 0.216 0.380

0.303 0.253 0.143 0.315 0.154 0.168 0.0067

0.640 0.740 0.545 0.830 0.800 0.800 0.759

Table 3. Intercorrelation Matrix of Independent Variables Used in the Models compound

variables

S1

S6

S11

S15

S19

PCh8

PCh20

I12-OH

bridged-bicyclic diarylethylenes

S1 S6 S11 S15 S19 PCh8 PCh20 I12-OH

1.00

1.00 1.00

0.50 0.01 1.00

0.53 0.54 0.17 1.00

0.10 0.11 0.14 0.06 1.00

0.29 0.22 0.42 0.41 0.12 1.00

0.02 0.04 0.12 0.27 0.20 0.28 1.00

0.05 0.04 0.23 0.27 0.23 0.16 0.16 1.00

compound

variables

Ch5

Ch15

Ch16

Ch17

ELUMO

PMIy

CSO3Ph

bridged oxabicyclic diarylethylenes

Ch5 Ch15 Ch16 Ch17 ELUMO PMIy CSO3Ph

1.00

0.12 1.00

0.39 0.89 1.00

0.90 0.31 0.50 1.00

0.17 0.27 0.20 0.02 1.00

0.17 0.29 0.35 0.26 0.44 1.00

0.14 0.03 0.13 0.17 0.56 0.10 1.00

and in the case of a bivariate relationship, the best significant relationship has been explored with Ch1 and an indicator, Np (no. of free terminal atoms, excluding hydrogens in the molecule), that explained 63.252% variance in activity. The quality of this relationship has been estimated to be

R ) 0.830; R2 ) 0.689; s ) 0.354; n ) 14 But, the best relationship for ER binding affinity to R-subtype has been deduced to be

[pRBA]R ) 5.219((1.917)PCh20 - 0.784((0.119)S15 0.454((0.091)S19 + 0.739((0.196)I12-OH + 3.900((0.381) (1) where S15, S19, and PCh20 are the E-state indices of atoms C15 and C19, and the Extended Huckel atomic partial charge on C20, respectively. I12-OH indicates the presence of a hydroxyl group at the C12 atom. For binding affinities to the β-subtype, the best univariate relation has again been deduced with Ch1 that explained 48.103% variance in activity, and the statistical quality of the relationship is estimated to be

r ) 0.722; r ) 0.521; s ) 0.439; n ) 14 2

and similarly the best bivariate significant relationship has been explored with Ch1 and Np that explained 77.199% variance in activity. The quality of this relationship has been estimated as

R ) 0.898; R2 ) 0.807; s ) 0.291; n ) 14

The best relationship for binding affinity to ERβ has been found to be

[pRBA]β ) 7.088((1.735)PCh20 - 0.797((0.107)S1 0.535((0.086)S19 + 0.822((0.177)I12-OH + 3.190((0.272) (2) where S1 is the E-state index of atom C1. In terms of selectivity (ERβ/ERR) toward binding affinity to the receptor subtypes of the compounds investigated, the best significant single variate relation has been explored with a charge function at atom C8 (Ch8) that explained 29.471% variance in activity, and the statistical quality of the relationship is estimated to be

r ) 0.591; r2 ) 0.350; s ) 0.234; n ) 14 and in the case of a bivariate relationship, the best significant relationship has been explored with Ch8 and the E-state index of atom C4 that explained 50.436% variance in activity. The quality of this relationship has been estimated as

R ) 0.762; R2 ) 0.581; s ) 0.196; n ) 14 But, the overall best relationship for selective binding affinity to ER (β/R) has been found to be

[pRBA]β/R ) 1.628((0.314)S6 + 0.051((0.020)S11 28.670((5.528)PCh8 + 3.232((0.899)PCh20 22.819((4.744) (3)


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Table 4. Calculated and Predicted Activities of Bridged-Cyclic Diaryl Compounds from the QSAR Models selective ER subtype binding affinity ERR compd no. 1 2 3 4 5 6 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

obsg

ERβ

calcg h

0.290 1.398h 1.509h 0.967h 1.337h 0.614h 1.013h 2.046h 1.244h 2.000h 1.824h 1.168h 2.495h 3.968i 3.613i 2.806i 2.000i 1.322i 1.301i 1.114i 2.954i 1.838i 2.991i 1.832i 1.832i 0.903i 1.146i 2.505i 1.342i 1.398i 3.342i 0.845i 1.079i

a

0.679 1.476a 1.305a 0.833a 1.463a 0.921a 0.824a 2.126a 1.201a 1.914a 2.134a 0.998a 2.279a 3.299b 3.358b 2.361b 1.822b 1.752b 1.307b 1.269b 3.070b 1.951b 2.906b 2.304b 1.918b 0.879b 1.210b 2.632b 1.798b 1.791b 3.474b 0.460b 0.572b

predg a

0.798 1.515a 1.201a 0.805a 1.526a 1.025a 0.779a 2.397a 1.188a 1.651a 2.404a 0.961a 2.019a 3.164b 3.276b 2.293b 1.760b 1.826b 1.309b 1.315b 3.111b 1.958b 2.891b 2.432b 1.938b 0.872b 1.236b 2.653b 1.897b 1.883b 3.512b 0.349b 0.444b

obsg

selectivity, ERβ/ERR

calcg h


c

0.442 1.344c 1.108c 0.673c 1.339c 0.815c 0.566c 1.850c 0.905c 1.974c 1.969c 0.494c 1.929c 2.010d 1.222d 0.772d 0.721d 0.714d 0.506d 0.609d 1.160d 0.569d 1.190d 0.822d 1.306d 0.215d 0.024d 0.880d 0.410d 0.481d 0.992d 0.489d 0.598d

predg c

0.421 1.374c 0.929c 0.663c 1.484c 0.923c 0.482c 1.733c 0.969c 1.624c 2.050c 0.499c 1.808c 1.837d 1.258d 0.773d 0.695d 0.733d 0.530d 0.655d 1.139d 0.569d 1.209d 0.824d 1.501d 0.225d 0.071d 0.857d 0.406d 0.491d 0.957d 0.505d 0.626d

obsg

calcg h


e

3.025 2.965e 3.052e 2.731e 2.769e 2.873e 2.897e 2.877e 2.549e 3.025e 2.969e 2.133e 2.617e 3.281 f 3.474 f 2.365 f 1.840 f 2.003 f 1.650 f 1.681 f 3.201 f 2.033 f 3.003 f 2.044 f 2.111 f 1.020 f 1.641 f 2.806 f 2.020 f 1.917 f 3.320 f 1.359 f 1.494 f

predg 2.964e 2.983e 3.090e 2.682e 2.806e 2.868e 2.898e 2.964e 2.577e 2.933e 2.955e 1.864e 2.647e 3.391 f 3.506 f 2.265 f 1.781 f 2.037 f 1.596 f 1.675 f 3.054 f 2.069 f 2.933 f 2.117 f 2.139 f 1.102 f 1.555 f 2.841 f 2.041 f 1.914 f 3.361 f 1.311 f 1.444 f

a As per eq 1. b As per eq 4. c As per eq 2. d As per eq 6. e As per eq 3. f As per eq 7. g obs ) observed values; calc ) calculated values; pred ) predicted values. h Reference 19. i Reference 20.

where S6, S11, and PCh8 indicate E-state indices of atoms C6 and C11 and the Extended Huckel atomic partial charge on C8, respectively. (b) Bridged-Oxabicyclic Diarylethylenes. While modeling RBA of bridged-oxabicyclic diarylethylene derivatives, the best univariate model for binding affinity to the R-subtype of ER could be developed with a charge function of atom C16 (Ch16) that explained 53.441% variance in activity, and the statistical quality of the relation is estimated to be

r ) 0.748; r ) 0.559; s ) 0.660; n ) 20 2

and in the case of a bivariate relationship, the best significant relationship has been explored with the charge functions of atoms C6 (Ch6) and C16 (Ch16) that explained 71.830% variance in activity. The quality of this relationship has been estimated to be

R ) 0.865; R2 ) 0.748; s ) 0.514; n ) 20 The best relationship deduced for ERR binding affinity could explain 86.520% activity variance with good predictive property, and the relation is

[pRBA]R ) - 6.253((0.950)Ch5 1.053((0.392)Ch16 - 3.240((0.711)ELUMO 3.045((0.604) (4)

where Ch5 and ELUMO indicate the atomic charge function of C5 and the lowest unoccupied molecular orbital energy, respectively. In the case of β-receptor subtype binding, the best single variate model could be developed with an indicator, CSO3Ph (configuration of -SO3Ph substitution at Y) that explained 46.855% variance in activity, and statistical quality of the relation is estimated to be

r ) 0.705; r2 ) 0.496; s ) 0.360; n ) 20 and in the case of a bivariate relationship, the best significant relationship has been explored with CSO3Ph and charge function of atom C3 (Ch3) that explained 71.442% variance in activity, and the quality of this relationship has been estimated to be

R ) 0.863; R2 ) 0.744; s ) 0.264; n ) 20 But, for binding affinities to the ERβ, the best relation developed is

[pRBA]β ) 0.835((0.226)Ch17 - 2.309((0.457)Ch5 + 1 × 10-4((4 × 10-5)PMIy + 1.083((0.130)CSO3Ph 0.193((0.164)# (5) where Ch17 and PMIy indicate the atomic charge function of


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Figure 2. Observed, calculated, and predicted values as per eq 1.






C17 and the principal moment of inertia along the y-Cartesian coordinate axis, respectively. In this model (eq 5), the intercept is found statistically insignificant, and deletion of the same did not affect the relation but could explain as much as 96% activity variance, and the relationship is

For selectivity toward the binding affinity for ER (β/R), the best univariate model could be developed with ELUMO that explained 33.247% variance in activity, and the statistical quality of the relationship is estimated to be

[pRBA]β ) 0.249((0.062)Ch17 - 0.551((0.113)Ch5 + 0.363((0.108)PMIy + 0.419((0.050)CSO3Ph (6)

r ) 0.606; r2 ) 0.368; s ) 0.648; n ) 20 and in the case of a bivariate relationship, the best significant relation has been explored with the atomic charge function


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Table 5. Hypotheses Parameters Observed in Successive Runs for ERR Binding Affinity of Bridged-Cyclic Diaryl Analogs costd

run parameters run no.

spacing (pm)

input featuresa

hypothesis no.

pharmacophore features in generated hypothesisa

configuration

total

∆

R

rmsde

1

250

a, d, p, r

2

180

a, d, p, r

3

100

a, d, p, r

4

10

a, d, p, r

5

60

a, d, p, r

6

60

a, p, r

7

60

d, p, r

8b

60

a, d, p, r

9c

60

a, d, p, r

10b,c

60

a, d, p, r

1 3 1 2 1 3 1 2 3 1 2 3 1 2 1 2 1 3 1 2 1 3

a, p, 2 × r d, p, 2 × r a, p, 2 × r d, p, 2 × r a, p, 2 × r d, p, 2 × r a, p, 2 × r d, p, 2 × r d, p, 2 × r a, p, 2 × r a, p, 2 × r d, p, 2 × r a, p, 2 × r a, p, 2 × r d, p, 2 × r d, p, 2 × r a, p, 2 × r d, p, 2 × r a, p, 2 × r d, p, 2 × r a, p, 2 × r d, p, 2 × r

10.532 10.532 11.381 11.381 11.577 11.577 12.104 12.104 12.104 11.577 11.577 11.577 9.947 9.947 10.140 10.140 23.187 23.187 23.187 23.187 34.796 34.796

112.963 115.778 110.847 115.62 110.407 116.564 112.879 117.03 117.201 110.407 112.963 116.564 111.314 114.769 113.643 114.413 122.661 127.862 124.71 130.368 135.197 140.647

71.617 68.802 73.733 68.96 74.173 68.016 71.701 67.550 67.379 74.173 71.617 68.016 73.266 69.811 70.937 70.167 61.919 56.718 59.87 54.212 49.383 43.933

0.916 0.901 0.932 0.906 0.936 0.902 0.927 0.903 0.901 0.936 0.923 0.902 0.923 0.903 0.909 0.906 0.932 0.904 0.934 0.903 0.938 0.909

1.137 1.232 0.994 1.199 0.994 1.224 1.059 1.221 1.228 0.994 1.089 1.224 1.093 1.216 1.175 1.201 1.028 1.213 1.014 1.218 0.980 1.179

a a: HB acceptor; d: HB donor; p: hydrophobic factor; r: ring aromatic. b Weight variation changed to 1 from default value. c Variable tolerance changed to 1 from default value. d Null cost ) 184.580; ∆ ) null cost - total cost. e rmsd: rms deviation; n ) 25.

Table 6. Hyporefine Parameters Observed for Hypothesis 1 of Run 5 in ERR Binding Affinity of Bridged-Cyclic Diaryl Analogs costb

run parameters

c

run no.

spacing (pm)

input featuresa

hypothesis no.


configuration

total

∆

R

rmsdc

11

60

a, d, p, r

1 2 3

a, 2 × p, r, e d, 2 × p, r, e a, p, 2 × r, e

12.173 12.173 12.173

110.532 112.568 115.743

74.048 72.012 68.837

0.938 0.928 0.909

0.976 1.052 1.179

a a: HB acceptor; d: HB donor; p: hydrophobic factor; r: ring aromatic; e: excluded volume. b Null cost ) 184.580; ∆ ) null cost - total cost. rmsd: rms deviation; n ) 25.

at C5 (Ch5) along with ELUMO that explained 75.299% variance in activity. The quality of this relationship has been estimated to be

Table 7. Results of CatScramble Cross-Validationa of Run 5 (Hypothesis 1) for ERR Binding Affinity of Bridged-Cyclic Diaryl Analogs b

R ) 0.883; R2 ) 0.719; s ) 0.393; n ) 20

cost trial/ spreadsheet no. configuration fixed

But, the best relation for binding selectivity toward ER (β/R) has been explored to be

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

[pRBA]β/R ) 0.961((0.441)Ch17 5.734((0.875)Ch5 - 3.711((0.534)ELUMO 2.202((0.512) (7) where Ch17 indicates atomic charge function of C17. The statistical parameters of the best relations (eqs 1-7) are described in Table 2, and independent variables used in various equations are not intercorrelated (r < 0.5) (Table 3). The calculated and predicted activities from the equations have been delineated in Table 4 and Figures 2-7. Pharmacophore Space Modeling. The results of this study have been depicted in Tables 5-13. The hypothesis 1 of run number 5 (Table 5) has been adjudged to be the best pharmacophore hypothesis for binding affinity to ERR, while that of run number 13 for hypothesis 1 (Table 8) has been adjudged to be the best hypothesis for binding affinity to

10.288 17.711 11.808 17.362 10.739 17.774 16.230 14.520 18.885 19.253 11.777 17.306 17.318 10.357 14.154 16.943 18.939 17.471 11.851

96.389 103.872 97.968 103.522 96.900 103.935 102.39 100.68 108.046 105.414 97.937 103.467 103.479 96.518 100.314 103.104 105.10 103.631 98.012

total

∆

R

rmsdc

166.88 161.925 173.574 179.31 166.719 154.60 181.634 157.302 171.799 168.913 136.728 175.142 162.557 184.504 156.374 175.285 164.103 150.684 169.781

17.700 22.655 11.006 5.270 17.861 29.980 2.946 27.278 12.781 15.667 47.852 9.438 22.023 0.076 28.206 9.295 20.477 33.896 14.799

0.547 0.650 0.498 0.496 0.553 0.704 0.461 0.661 0.589 0.608 0.785 0.566 0.642 0.353 0.665 0.531 0.643 0.730 0.538

2.373 2.154 2.457 2.462 2.363 2.012 2.515 2.127 2.291 2.251 1.755 2.340 2.173 2.653 2.116 2.402 2.173 1.937 2.395

95% confidence level. b Null cost ) 184.580; ∆ ) null cost total cost. c rmsd: rms deviation. a


MUKHERJEE

ET AL.

Table 8. Hypotheses Parameters Observed in Successive Runs for ERβ Binding Affinity of Bridged-Cyclic Diaryl Analogs costd

run parameters run no.

spacing (pm)

input featuresa

hypothesis no.


configuration

total

∆

R

rmsde

12

250

a, d, p, r

13

180

a, d, p, r

14

100

a, d, p, r

15

10

a, d, p, r

16

180

a, p, r

17

180

d, p, r

18b

180

a, d, p, r

19c

180

a, d, p, r

20b,c

180

a, d, p, r

1 3 1 2 1 3 1 3 1 2 1 2 1 2 1 3 1 3

a, p, 2 × r d, p, 2 × r a, p, 2 × r d, p, 2 × r a, p, 2 × r d, p, 2 × r a, p, 2 × r d, p, 2 × r a, p, 2 × r a, p, 2 × r d, p, 2 × r d, p, 2 × r a, p, 2 × r d, p, 2 × r a, p, 2 × r d, p, 2 × r a, p, 2 × r d, p, 2 × r

10.445 10.445 11.208 11.208 11.407 11.407 11.981 11.981 9.599 9.599 9.603 9.603 22.818 22.818 22.818 22.818 34.428 34.428

104.781 106.049 103.911 105.691 104.243 107.002 106.411 106.919 103.311 104.003 104.355 105.636 115.396 118.578 116.526 120.88 131.953 132.863

110.659 109.391 111.529 109.749 111.197 108.438 109.029 108.521 112.129 111.437 111.085 109.804 100.044 96.862 98.914 94.560 83.487 82.577

0.969 0.964 0.976 0.969 0.975 0.964 0.969 0.967 0.971 0.969 0.967 0.962 0.977 0.963 0.977 0.961 0.963 0.960

0.791 0.857 0.707 0.796 0.707 0.862 0.798 0.817 0.767 0.800 0.817 0.875 0.682 0.862 0.680 0.901 0.863 0.901

a a: HB acceptor; d: HB donor; p: hydrophobic factor; r: ring aromatic. b Weight variation changed to 1 from default value. c Variable tolerance changed to 1 from default value. d Null cost ) 215.439; ∆ ) null cost - total cost. e rmsd: rms deviation; n ) 25.

Table 9. Hyporefine Parameters Recorded for Hypothesis 1 of Run 13 in ERβ Binding Affinity of Bridged-Cyclic Diaryl Analogs run parameters spacing (pm) input featuresa

run no. 21

c

180

hypothesis no.


configuration

1 2 3

a, p, 2 × r, e d, 2 × p, r, e a, 2 × p, r, e

11.944 11.944 11.944

a, d, p, r

costb total 103.713 104.821 105.062

∆

R

rmsdc

111.727 110.619 110.378

0.979 0.975 0.974

0.652 0.713 0.727

a a: HB acceptor; d: HB donor; p: hydrophobic; r: ring aromatic; e: excluded volume. b Null cost ) 215.439; ∆ ) null cost - total cost. rmsd: rms deviation; n ) 25.

Table 10. Results of CatScramble Cross-Validationa of Run 13 (Hypothesis 1) for ERβ Binding Affinity of Bridged-Cyclic Diaryl Analogs trial/ spreadsheet no. configuration 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

11.365 15.203 9.614 11.244 17.535 13.655 13.703 10.021 14.879 13.726 9.791 12.222 15.472 15.460 11.521 17.312 13.909 10.581 14.556

costb fixed

total

∆

R

rmsdc

97.525 101.363 95.775 97.404 103.894 99.815 99.863 96.181 101.04 99.886 95.951 98.382 101.633 101.62 97.682 103.472 100.069 96.742 100.716

168.231 180.876 224.344 181.43 153.201 181.062 167.615 169.606 199.368 171.929 214.495 193.937 181.432 166.674 202.696 182.156 161.377 192.597 199.741

47.208 34.563 -8.905 34.009 62.238 34.377 47.824 45.833 16.071 43.510 0.944 21.502 34.007 48.765 12.743 33.283 54.062 22.842 15.698

0.687 0.628 0.150 0.605 0.80 0.617 0.701 0.673 0.526 0.682 0.312 0.523 0.628 0.711 0.447 0.634 0.733 0.520 0.497

2.356 2.521 3.204 2.579 1.945 2.549 2.311 2.397 2.758 2.372 3.079 2.762 2.520 2.277 2.898 2.507 2.205 2.768 2.812

a 95% confidence level. b Null cost ) 215.439; ∆ ) null cost total cost. c rmsd: rms deviation.

ERβ. This has been characterized in terms of the highest cost difference, the lowest root-mean-square divergence (rmsd), and the best correlation coefficient. The generated best hypotheses are validated to nullify overprediction of bioactivities for inactive compounds through Hyporefine. In this

process, the steric interactions of compounds are considered in the hypothesis generation and are portrayed in the validated (refined) hypothesis (run nos. 11 and 21). The mapped pharmacophore features for binding affinity to the receptor subtypes (R and β) are described in Table 11 and Figures 8 and 9, and estimated fit scores are delineated in Figures 10 and 11, respectively. The quality of hypothesis generated is adjudged through a cross-validation technique using Fischer’s randomization test,47 where the biological activity data are randomized within a fixed chemical data set and the HypoGen process is initiated to explore possibilities of other hypotheses of predictive values (Tables 7 and 10). The predicted hypothesis for R and β ER binding are further tested with some estrogenic and nonestrogenic compounds as reference compounds, and the fit score of the compounds are listed in Table 13. The binding affinity of bridged-cyclic diaryl scaffolds toward the ER subtypes (R and β) demonstrated the importance of HB acceptor (a), hydrophobic (p), and ring aromatic (r) features along with the steric influence (e) of the compounds. In the hyporefined models, the distance between a and p constraints are observed to be 8.496 Å and 7.830 Å, while the separation of a and r constraints are found to be 7.080 Å and 7.699 Å for R- and β-subtypes binding, respectively (Table 11, Figures 8 and 9). DISCUSSION

Reasonably well predictable models for binding affinity to ER-subtypes of diaryl compounds have been obtained with


OF

ESTROGEN MODULATORS


Table 11. Features Obtained from Refined Pharmacophore Hypothesesa Hyporefine for ERR p1 p2 a r1 e

interfeature distance (Å)b p2 a r1

p1 0.000

5.587 0.000

8.496 2.964 0.000

6.349 5.149 7.080 0.000

e

Hyporefine for ERβ

10.299 6.617 5.707 8.614 0.000

p1 a r1 r2 e

p1 0.000

interfeature distance (Å)b a r1 r2 7.830 0.000

5.832 2.820 0.000

5.978 7.699 4.975 0.000

e 6.195 8.016 5.476 4.060 0.000

a Run nos. 11 and 21. b p1 ) hydrophobic1; p2 ) hydrophobic2; a ) hydrogen bond acceptor; r1 ) ring aromatic 1; r2 ) ring aromatic 2; e ) excluded volume.

Table 12. Observed and Estimated Activities of Training Set for Bridged-Cyclic Diaryl Analogs ER binding affinity (RBA) R-subtypea

a

compd no. 1 2 3 4 5 6 7 8 9 10 11 12 15 16 17 18 19 20 21 22 23 24 25 29 32

β-subtypea

obs

est run 5 (hypo 1)

est Hyporefine (hypo 1)

1.95 25.00 32.26 9.26 21.74 4.12 21.28 10.31 111.11 17.54 100.00 66.67 107.53 243.90 1.56 × 103 1.00 × 104 4.76 × 104 5.00 × 104 7.69 × 104 1.11 × 103 1.45 × 104 1.02 × 103 1.47 × 104 3.13 × 103 454.545

14.0 12.0 15.0 9.3 14.0 17.0 26.0 11.0 50.0 18.0 270.0 9.1 1.1 × 103 230.0 2.2 × 103 7.9 × 103 1.4 × 104 1.2 × 105 1.4 × 104 990.0 3.2 × 103 4.1 × 103 4.7 × 103 4.9 × 103 960.0

4.2 4.2 4.9 2.7 3.8 2.4 5.8 4.3 1.6 4.7 9.8 6.1 15.0 3.5 6.9 1.9 × 103 4.6 2.1 × 103 150.0 1.7 2.6 3.5 14 5.8 4.7

obs

est run 13 (hypo 1)

est Hyporefine (hypo 1)

3.32 19.23 28.57 5.35 11.2 3.16 4.74 8.00 76.92 5.29 125.00 76.92 588.24 6.67 × 103 1.70 × 104 1.30 × 104 2.78 × 104 4.00 × 104 5.88 × 104 4.76 × 103 2.70 × 104 9.09 × 103 1.51 × 104 1.00 × 104 6.25 × 103

8.3 9.3 9.6 9.4 11.0 9.3 10.0 4.3 38.0 17.0 110.0 19.0 2.5 × 103 4.5 × 103 7.2 × 103 2.1 × 104 1.8 × 104 4.1 × 104 4.2 × 104 7.8 × 103 7.7 × 103 1.1 × 104 1.4 × 104 1.0 × 104 1.6 × 104

1.6 1.5 1.4 1.1 1.6 0.9 1.0 1.0 1.0 1.2 2.0 3.3 1.6 × 104 210.0 250.0 2.5 × 104 280.0 2.8 × 104 1.9 × 104 4.0 40.0 830.0 1.2 × 104 490.0 340.0

obs: observed activity; est: estimated activity.

cross-validated variance (CVV) exceeding 50%. The models generated for bridged-cyclic diaryl derivatives account for more than 75% variance in observed activity with low estimation errors. However, the best relations (eqs 1 and 2) revealed the importance of atoms C19, C20 and the presence of a hydroxyl group23 at C12 of bridged-cyclic diaryl-5hydroxy analogs for binding affinities to both the ER subtypes. Furthermore, the ethylenic fragment (atom C15 in eq 1 and atom C1 in eq 2) signifies the impact for binding to both the ERR and ERβ. The negative coefficient of S19 indicates that increase in the E-state value of atom C19 or an increment in the e- density in the vicinity of this atom will result in enhanced binding to both subtypes. A negative coefficient of the binary indicator also suggests that the presence of a hydroxyl group at atom C12 (in ring B) is further favorable for binding affinity to both the ER subtypes. Again, the positive coefficient of the partial charge at atom C20 signifies that amplification of the partial charge or more electronegativity at C20 will result in decreased binding to the receptor surface, which is selectively more in ERβ than in ERR subtype. Negative coefficients of S15 (eq 1) and S1

(eq 2) explain that an increase in e- densities around atoms C15 and C1 (ethylenic fragment) will result in enhanced binding to both ER subtypes. Thus, judging from eqs 1 and 2, it can be inferred that the presence of the hydroxyl group at C12, electronic distributions in the vicinity of atoms C19 and C20 of bridged-cyclic diaryl-C5-hydroxy analogues, and atoms C15 and C1 (ethylenic fragment) are essential requirements for binding at the ER-ligand binding regions, irrespective of the subtypes. The significant contributions of atoms C15, C19, and C20 in either case, as obtained during the course of the present investigation, showed the importance of the cyclic ring system for ER binding of these groups of compounds. The importance of atoms C1 and C15 can be conceptualized as the influence of unsaturation in the C1C15 linkage. The significance of an ethylenic fragment in nonsteroidal estrogens has been explored in a series of diverse compounds containing the diarylhydroxy unit.16-18,39 In terms of binding selectivity to ER (β/R), the best model (eq 3) generated with 78% binding selectivity indicated that increase in E-state values of atoms O6 and C11 will decrease selectivity of ERβ over ERR. Consequently, substituents that


MUKHERJEE

ET AL.

Table 13. Fit Scores of Training, Test, and Reference Compounds fit score ERR binding

seta

compd no./name

Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Tr Ts Ts Ts Ts Ts Ts Ts Ts Ts ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref

1 2 3 4 5 6 7 8 9 10 11 12 15 16 17 18 19 20 21 22 23 24 25 29 32 13 14 26 27 28 30 31 33 34 raloxifene tamoxifene estradiol cyclofenil tetrahydroisoquinoline clidinium phenytoin norgestrel acetaminophine ephedrine morphine propranolol testosterone stanalone letrozole a

ERβ binding

Hyporefine Hyporefine run 5 (hypo 1, run 13 (hypo 1, (hypo 1) run 11) (hypo 1) run 21) 13.410 13.390 13.410 13.420 13.403 13.186 13.187 13.487 13.656 13.650 12.414 13.426 13.564 13.210 13.426 10.440 13.570 10.050 12.025 13.272 12.770 13.275 13.103 13.302 13.158 13.297 13.374 10.559 10.391 10.023 10.666 10.685 10.556 10.736 13.071 13.308 9.684 13.371 12.768 7.136 8.421 7.139 6.694 6.522 6.655 7.138 6.859 7.137 10.117

12.956 12.955 12.892 13.154 13.000 13.204 12.813 12.946 13.378 12.906 12.588 12.793 12.396 13.031 12.740 10.293 12.917 10.236 11.409 13.360 13.170 13.033 12.438 12.819 12.911 12.839 13.093 10.418 10.032 10.093 10.334 11.017 10.255 11.374 13.059 13.359 10.056 13.059 13.326 7.015 7.779 8.619 6.917 6.521 8.035 7.897 6.992 8.523 9.586

13.436 13.421 13.430 13.444 13.420 13.192 13.241 13.302 13.407 12.992 12.516 13.360 12.689 12.461 12.555 10.253 13.454 10.097 12.273 12.595 12.112 12.196 12.107 12.945 13.082 13.347 13.435 10.449 10.195 9.235 10.507 12.787 9.850 13.048 12.922 13.256 9.707 13.400 12.402 7.004 7.814 7.019 6.685 6.797 6.396 7.003 6.623 6.981 10.166

13.427 13.467 13.499 13.603 13.432 13.689 13.645 13.652 13.632 13.564 13.334 13.121 10.443 11.314 11.237 10.247 11.193 10.194 10.366 13.039 12.034 10.273 10.566 10.953 11.107 13.509 13.397 10.187 10.185 9.674 10.357 10.186 10.274 10.503 12.710 13.048 8.333 13.605 12.691 7.014 7.962 7.019 6.928 6.773 7.988 7.016 6.478 7.018 9.960

Tr ) training set; Ts ) test set; ref ) reference compound.

tend to decrease the e- density around these atoms should result in increased selectivity. The importance of the 5-phenolic hydroxyl group for selective estrogenic activity is demonstrated earlier.48 The negative coefficient of PCh8 and the positive coefficient of PCh20 correspondingly signify that increase and decrease in electronegativity around these atoms should result in an enhanced selectivity profile. From the observation, atom C20 appeared to be a fragment pivotal for both ER binding as well as ERβ selectivity. The best relations explored for subtypes binding affinity of bridged-oxabicyclic diarylethylenes in eqs 4 and 6 have brought into the picture the importance of charge functions of atoms C5, C16 and C17, overall electron affinity along with

Figure 8. Pharmacophore features (hypothesis 1 of run nos. 5 and 11) for RBA to ERR. Mapped features are HB acceptor (a), hydrophobic (p), ring aromatic (r), and excluded volume (e); green: raloxifene, yellow: compn no. 1; blue: E2.

the orientation, and conformational rigidity of the oxabicyclic core for binding affinities to the ER subtypes. These relations revealed that increased negative charge on atom C5 (influenced by the V-substituent) shall increase the binding affinity to both the receptor subtypes, since Ch5 has negative contribution. Further the negative contribution of Ch16 in eq 4 and the positive contribution of Ch17 in eq 6 depicted that a decrease and an increase of negative charges at atoms C16 (influenced by the Y-substituent) and C17 (influenced by the Z-substituent) will cause lowering of the receptor binding affinity to ERR and ERβ, respectively. A negative contribution of ELUMO in eq 4 revealed that enhanced electron affinity of the molecules is detrimental for binding to ERR, whereas conformational rigidity enhanced the binding affinity of the ligands to ERβ as is evident from the coefficient of PMIy in eq 6. The positive contribution of PMIy indicated that increase in the value of this parameter can increase the binding affinity to ERβ. PMIy values of compounds 15 and 16 are 5563.300 and 4881.240 g/mol Å2, and as such the former is more potent in binding to ERβ. Eq 6 also indicated the significance of configuration of the -SO3Ph (phenyl sulfonate) group attached to atom C16 in affecting the receptor binding affinity and demonstrated that β-configuration is favorable for binding affinity to ERβ. In terms of binding selectivity toward ER (β/R), the best model (eq 7) can portray that increased negative charge on atom C5 (influenced by V-substituent), decreased negative charge on atom C17 (influenced by Z-substituent), and overall decrease in electron affinity of the molecule will favor the selectivity. Thus electron affinity and the orientation and conformational rigidity of oxabicyclic diarylethylenes along with Y- and Z-substituents differentiate the binding affinity to ERsubtypes. The quality of best hypothesis generated in either case for pharmacophore space modeling studies on RBA are significant with regards to cost differences, correlation coefficients, and rmsd recorded. The best hypothesis (hypo 1, run no. 5) and hyporefine (hypo 1 of run no. 11) on the same for ERR binding demonstrated around 94% correlation with the


OF

ESTROGEN MODULATORS


Figure 9. Pharmacophore features (hypothesis 1 of run nos. 13 and 21) for RBA to ERβ. Mapped features are HB acceptor (a), hydrophobic (p), ring aromatic (r), and excluded volume (e); green: raloxifene, yellow: compd no. 6; blue: E2.

Figure 10. Estimated versus fit score from hyporefine of ERR binding.

Figure 11. Estimated versus fit score from hyporefine of ERβ binding.

binding affinity of compounds, while that for ERβ (hypo 1 of run nos. 13 and 21) is to the extent of 98%. It has been deduced that HB acceptor, hydrophobic, ring aromatic, and steric features might function as prime biophores for ER binding affinity to both subtypes. However, the receptor subtype specific binding of compounds might arise due to geometrical distances between these features within the compounds. From Table 11 and Figures 8 and 9, the interbiophore distances between similar features can be perceived to be widely varying, which clearly differentiate the binding affinity to both ER subtypes. HB donor is not figured out as a significant contributor toward the ER-binding affinity of bridged-cyclic diaryl analogs. From Tables 7 and 10 for CatScrambling based on the cross-validation of both

ERR and ERβ binding affinity respectively, it is observed that none of the spreadsheets generated better parameters in comparison to the original hypotheses. The cross-validation results clearly indicated the superiority of the hypotheses selected and also provide strong confidence on the initial pharmacophore hypo 1 of both the cases. The results also demonstrated superior predictive ability of the models for binding affinity to ER subtypes, while tested with reference compounds (Table 13). The standard SERMs are highly fitted (fit score >12), with both the models for the binding affinity to the ER subtypes, while non-SERMs, including E2,9 showing low fit score (

Pharmacophore Mapping of Selective Binding Affinity of Estrogen

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