The three-dimensional quantitative structure-activity relationship (3D-QSAR) model of sulfonamide analogs binding a monoclonal antibody (MabSMR) produced against sulfamerazine, was carried out by comparative molecular field analysis (CoMFA). The affinities of MabSMR, expressed as Log10IC50, for 17 sulfonamide analogs were determined by competitive fluorescence polarization immunoassay (FPIA). Removal of two outliers from the initial set of 17 sulfonamide analogs improved the predictability of the models. The 3D-QSAR model of 15 sulfonamides resulted in q2cv values of 0.600, and r2 values of 0.995, respectively. This novel study combining FPIA with CoMFA demonstrates that multidisciplinary research can be used as a useful tool to investigate antigen-antibody interactions and provide information required for design of novel haptens, which may result in new antibodies with properties already optimized by an antibody-based immunoassay

Congming Wu

Haiyang Jiang

Jianzhong Shen

Linli Cheng

Ross C. Beier

Xingyuan Cao

Xinling Yang

Zhanhui  Wang

Zhenpeng  Kai

English

Nature Precedings

Manuscript for Nature Precedings
Three Dimensional Quantitative Structure-Activity Relationships of 
Sulfonamides Binding Monoclonal Antibody by Comparative Molecular Field 
Analysis 
Zhanhui Wang1, Xingyuan Cao1, Zhenpeng Kai2, Ross C. Beier3, Congming Wu1, Haiyang Jiang1, 
Linli Cheng1, Jianzhong Shen1*, Xinling Yang2
1College  of  Veterinary  Medicine,  China  Agricultural  University,  Beijing  100094,  People’s 
Republic of China,  and National Reference Laboratories for Veterinary Drug Residue, District 
Haidian, Beijing 100094, People’s Republic of China. 
2College of Science, China Agricultural University, Beijing, 100094, People’s Republic of China.
3Southern Plains Agricultural Research Center, Agricultural Research Service, U.S. Department of 
Agriculture, 2881 F & B Road, College Station, Texas 77845-4988,
*Corresponding author: 
Jianzhong Shen
China Agricultural University, Beijing 100094
Phone: 8610-6273-2803
Fax: 8610-6273-1032
E-mail: wangzhanhui@cau.edu.cn
Abstract
The  three-dimensional  quantitative  structure-activity  relationship  (3D-QSAR)  model  of 
sulfonamide analogs binding a monoclonal  antibody (MabSMR) produced against sulfamerazine, 
was carried  out  by comparative molecular  field  analysis  (CoMFA).  The affinities  of  MabSMR, 
expressed as Log10IC50, for 17 sulfonamide analogs were determined by competitive fluorescence 
polarization immunoassay (FPIA). Removal of two outliers from the initial set of 17 sulfonamide 
analogs  improved the  predictability  of  the  models.  The  3D-QSAR model  of  15 sulfonamides 
resulted in q2cv values of 0.600, and r2 values of 0.995, respectively. This novel study combining 
FPIA with  CoMFA demonstrates that multidisciplinary research can be used as a useful tool to 
investigate  antigen-antibody interactions  and provide  information required  for  design  of  novel 
haptens, which may result in new antibodies with properties already optimized by an antibody-
based immunoassay.
INTRODUCTION
Sulfonamides are widely used to control a number of diseases in the animal industry and 
aquaculture, as well as used for animal growth-promotion.1 The presence of sulfonamide residues 
in foods of animal origin or the environment constitutes a potential hazard for humans due to the 
increasing incidence of microbial resistance and the risk of allergic reactions. The availability of 
reproducible, sensitive and rapid methods for screening sulfonamides in foodstuffs is essential. 
The antibody-based analytical methods, called immunoassays, have proven to be useful as simple, 
fast and sensitive tools for detecting and quantifying sulfonamides in a variety of matrices.2,3 The 
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antibody is key reagent in any format of immunoassay. However, the affinity and specificity of the 
generated antibodies often have un-uniform properties.  The  conventional  process  for  antibody 
production, when carried out without careful theoretical considerations, primarily focuses on new 
hapten  design  and  extensive  screening  protocols,  is  limited.  There  is  considerable  interest  in 
understanding the structural basis of antibody-analyte complex interactions. A method that can 
provide useful  information about the topological  properties  of a hapten can be very useful  in 
producing an antibody with the desired affinity and specificity. 
In  this  paper,  the  3D-QSAR  techniques  based  on  CoMFA,  were  used  to  describe  the 
quantitative  binding  affinities  of  sulfonamides  towards  MabSMR.  The  methods  are  useful  in 
determining  the  most  important  features  of  antigen-antibody  binding,  and  therefore,  provide 
insights into the wide-range of variations in affinity values among sulfonamide structural analogs. 
This work may develop knowledge of interactions that govern drug/antibody binding, and may 
help in the design of novel, enhanced antibodies by recombinant techniques.4
EXPERIMENTAL SECTION
Fluorescence  Polarization  Immunoassay.
 The  tracer  used  in  binding  affinity 
determinations,  fluorescein  isothiocyanate  (FITC) labelled  sulfamethazine  (SMZ-FITC), was 
synthesized and purified by thin layer chromatography (TLC).5 The binding affinity (IC50) values 
of MabSMR with 17 sulfonamides were determined by a previously developed FPIA.5
CoMFA  analysis.  Minimum  energy  conformations  of  all  17 sulfonamide  analogs  were 
calculated using the Minimize module of Sybyl 7.0 following the same process previously used for 
fluoroquinolone modeling.6 For CoMFA calculations7, the alignment molecules were placed in a 
3D-cubic lattice with a 2 Å grid in x, y and z directions. The default sp3-hybridized carbon atom 
with +1 charge was selected as the probe atom for the calculation of the steric (Lennard-Jones 6–
12 potential) and electrostatic fields (Coulombic potential) around the aligned molecules with a 
distance-dependent dielectric constant at all lattice points. Values of steric and electrostatic energy 
were  truncated  to  30  kcal  mol–1.  PLS  methodology  was  used  for  all  3D-QSAR  analysis  to 
determine the significance of the models. Quality of the final CoMFA models was measured by 
two statistical parameters: q2cv and r2. 
RESULTS AND DISCUSSION
Determination of IC50 Values Using FPIA.
The  affinity  of  MabSMR for  all  sulfonamide  analogs  was  expressed  as  IC50 values.5 The 
structures, IC50,  and Log10IC50 values of all sulfonamides are summarized in Table 1. The only 
difference among the sulfonamide analogs lies  in  the  diverse  array of  R-groups  linked to  the 
nitrogen at position 7 (Figure 1b, Table 1). Simple inspection of IC50  values reveals that the R-
group is of primary importance for MabSMR binding to the sulfonamide analogs (Table 1). Since 
MabSMR was  produced  to  the  sulfamerazine  hapten,  the  sulfonamides  with  close  structural 
similarity, such as sulfamethazine, demonstrated lower IC50 values than that of other analogs. Even 
the addition of a methyl group at position 3 on the pyrimidine ring, as seen in sulfamethazine, 
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reduced  the  affinity  compared  to  sulfamerazine  by  about  3.5-fold  (Figure  1b,  Table  1).  The 
favorable MabSMR binding requirement for a methyl group at position 5 on the pyrimidine ring was 
illustrated by the IC50 value of sulfadiazine (137 ng mL–1), which does not have a methyl group at 
position 5 and has a reduced affinity by 7.2-fold compared to the hapten, sulfamerazine. The size 
influence of the groups at positions 3 and 5 on binding affinity can be observed by comparing the 
IC50 value of sulfamethazine to that of sulfadimethoxine (Table 1). It is interesting that the methoxy 
substituted analog,  sulfameter, binds MabSMR with  a 40-fold lower affinity than sulfamerazine. 
However, the effect of the methoxy oxygen atom at position 4 of the pyrimidine ring on MabSMR 
binding is not understood. But the importance of the pyrimidine ring can be shown based on the 
affinity of MabSMR for sulfamerazine (IC50 = 19 ng mL–1), which is 2–4 orders of magnitude higher 
than the affinity for other sulfonamide analogs where the pyrimidine ring was substituted with a 
different heterocyclic ring. MabSMR has a higher binding affinity for sulfamerazine, sulfamethazine 
and sulfadiazine, all which contain a  pyrimidine ring at position 7, and the inhibition curves of 
these three sulfonamides including sulfadimethoxine are shown in Figure 2.
It is well known that antigen-antibody formation is mainly dependent on molecular shape, 
defined by the geometry and low-energy interactions.8 In an effort to determine which structural 
and electronic effects were primarily important for MabSMR-sulfonamide binding, studies using 
advanced molecular modeling techniques were undertaken. The contribution of R-group structure 
to MabSMR-sulfonamide complex formation was analyzed by using CoMFA. 
CoMFA Analysis. 
The CoMFA model was used to correlate variability in MabSMR binding affinities to variations in 
sulfonamide analog molecular structure. An improved CoMFA model (M4) was obtained with 15 
sulfonamide  analogs  but  without  sulfanilamide  and  sulfaphenazole.  The  Model  exhibited  a 
satisfactory predictive ability with a cross-validate q2cv value of 0.600, non-cross-validated r2 value 
of  0.995 and standard error  of  the  estimate  of  0.071. The contributions of  the  steric  and the 
electrostatic  fields  to  binding  affinity  were  55.8% and  44.2%,  respectively,  by PLS  analysis, 
indicating a strong relationship between the sulfonamide analogs structure and binding affinity. In 
the case of sulfonamide-MabSMR complexes, steric interactions dominated the contribution toward 
the observed binding variations. There is a very good agreement between the experimental and 
predicted values.  Table 1 lists  experimental  binding affinities,  predicted binding affinities  and 
residual values (defined as the difference between experimental and predicted binding affinity) by 
CoMFA. 
Figures 3 and 4 feature steric and electrostatic contour plots, respectively, of CoMFA that 
show where the changes in steric and electrostatic fields are associated with MabSMR binding to 
sulfonamide analogs. Greater values were correlated with more bulk near the green contours and 
with less bulk near the yellow contours (Figure 3); whereas, more positive charge was correlated 
with the blue contours, and more negative charge with the red contours (Figure 4). Some of the 
most noticeable features in Figure 3 are the presence of large green areas that indicate improved 
binding affinity with increased steric tolerance in the region of sulfamerazine near the NH2 group 
at position 17 and the NH group at position 7 (see Figure 1). However, even more attention was 
focused on the R-group (see Figure 1a), since all sulfonamide analogs had differences in the R-
group. The green steric contours for CoMFA shown in Figure 3 localized near position 3 and 4 of 
the pyrimidine ring indicate that bulky substituents in this region of the R-group will enhance 
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binding  affinity.  However,  the  introduction  of  the  methoxyl  group  in  the  pyrimidine  ring  at 
position 4 decreased the binding affinity as seen with sulfameter. This may be due to the effects of 
other fields like electrostatic, hydrophobic or hydrogen-bonding that may play an important role in 
the interactions of the sulfonamide analogs and MabSMR. Yellow-colored and green-colored regions 
near position 5 (the pyrimidine ring methyl), in respect to the proportion of the two colors, show 
that small groups at position 5 of the R-group increases binding affinity.
The electrostatic field of the CoMFA model is shown in Figure 4 with sulfamerazine as a 
reference. The large blue area above the molecule from position 13 to position 18 represents where 
a positive Gasteiger-Hückel charge is favored (see Figure 1b). Red-colored regions near positions 3 
and 5 show that suitable electronegative groups in these areas of the R-group are favored to bind 
MabSMR. This interpretation is born out by the greater binding affinity of sulfadimethoxine, having 
methoxy  groups  at  positions  3  and  5,  compared  with  sulfameter,  sulfamethoxypyridazine,  or 
sulfachloropyridazine, which are substituted with an electronegative atom at position 4.
CONCLUSIONS
This paper provides a QSAR of CoMFA, studies on 17 sulfonamide analogs binding MabSMR 
produced to sulfamerazine. The developed CoMFA models had excellent agreement with 15 of the 
17 sulfonamides studied. Results from this multidisciplinary research can also provide insights into 
key  structural  elements  required  to  design  new  haptens  for  development  of  more  desirable 
antibodies.
ACKNOWLEDGMENTS
The Authors thank Jordan R. Beier for technical assistance. This work is supported by the 
grants  from State Key Program of National  Natural  Science of China (No.30830082),  Beijing 
Excellent Doctoral Dissertation Fund (YB20081001902), and state project 863 (2008AA10Z423-
4).
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NH2 S
O
O
NH R
a
N S
O
O
N
N
N
CH3
H
H H
b 3
4
1
2
56
7
8
9
10
11
1213
14
15 16
17
18
Figure 1 Schematic a) backbone of sulfonamides and b) pharmacophore of sulfamerazine.
0.01 0.1 1 10 100 1000 10000 100000
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100
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140
160
180
200
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(m
P
)
Drug standard Concentration (ng mL-1)
 Sulfamerazine
 Sulfamethazine
 Sulfadiazine
 Sulfadimethoxine
Figure 2 Representative examples of competition ELISA determination of the binding specificities 
of  Mab.  The  calibration  curves  show  the  ability  of  sulfamerazine,  sulfamethazine, 
sulfadiazine and sulfadimethoxine to inhibit SMZ-FITC binding to Mab. Each point is 
the average of three determinations. 
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Figure 3 CoMFA contour plots of steric field contributions of sulfonamides binding to Mab. Green 
contours  indicate  regions  where  bulky  groups  increase  antibody  affinity,  and  yellow 
contours indicate regions where bulky groups decrease antibody affinity.
Figure 4 CoMFA contour plots of electrostatic field contributions of sulfonamides binding to Mab. 
Blue contours indicate regions where positive charged groups increase antibody affinity, 
and red contours indicate regions where negatively charged groups increase antibody 
affinity.
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Table 1. Experimental and Predicted Sulfonamides Binding Affinity to MabSMR
drugsa R IC50 Log10IC50(exp.) CoMFA
Log10IC50 (pre.) residual
SMR
N
N
CH3
19 1.28 1.24 0.04
SMZ
N
N
CH3
CH3
65.7 1.82 1.87 -0.05
SDZ
N
N 137 2.14 2.10 0.04
SDM
N
N
OCH3
OCH3
528 2.72 2.77 -0.05
SME N
N
OCH3
760 2.88 2.87 0.01
STZ
N
S 1000 3.00 3.03 -0.03
SMP
N N
OCH3 1056 3.02 3.01 0.01
SMO
N
O CH3
CH3
1118 3.05 3.01 0.04
SPY
N
1189 3.07 3.10 -0.03
SQX N
N
1538 3.20 3.20 0.00
SCP
N N
Cl 3800 3.58 3.55 0.03
SMT
N N
S CH3 6333 3.80 3.83 -0.03
SMX
N
O CH3
9048 3.96 3.94 0.02
SMM N
N
OCH3
12667 4.10 4.09 0.01
SFX NO
H3C CH3
19000 4.28 4.27 0.01
SPA NN >190000 - - -
SAM H >190000 - - -
a
 The abbreviations  are  as  follows:  Sulfamerazine  (SMR),  sulfamethazine  (SMZ),  sulfadiazine  (SDZ), 
sulfadimethoxine  (SDM),  sulfameter  (SME),  sulfathiazole  (STZ),  sulfamethoxypyridazine  (SMP),  sulfamoxole 
(SMO),  sulfapyridine  (SPY),  sulfaquinoxaline  (SQX),  sulfachloropyridazine  (SCP),  sulfamethizole  (SMT), 
sulfamethoxazole  (SMX),  sulfamonomethoxine  (SMM),  sulfisoxazole  (SFX),  sulfaphenazole  (SPA),  and 
sulfanilamide (SAM).
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Three Dimensional Quantitative Structure-Activity Relationships of Sulfonamides Binding Monoclonal Antibody by Comparative Molecular Field Analysis

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Three Dimensional Quantitative Structure-Activity Relationships of Sulfonamides Binding Monoclonal Antibody by Comparative Molecular Field Analysis

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