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ABSTRACT: Quantitative Structure-Activity Relationship (QSAR) is based on the hypothesis that changes in molecular
structure reflect changes in the observed response or biological activity. The success of any quantitative structure–activity
relationship model depends on the accuracy of the input data, selection of appropriate descriptors, statistical tools and the
validation of the developed model. Validation is a crucial aspect of QSAR modeling. Validation is the process by which the
reliability and significance of a procedure are established for a specific purpose. Hence in this review we focus on the
importance of validation of QSAR models and different methods of validation.
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512 International Journal of Drug Design and Discovery Volume 2 Issue 3 July – September 2011
The process of QSAR model development can be development at many times. In the QSAR community, the
generally divided into three stages: data preparation, data validation of a model is little more than an assessment of
analysis and model validation (Scheme 1). These steps statistical fit and, occasionally, predictivity using cross-
represent a standard practice of any QSAR modeling and validation techniques. However, it is now being accepted
their implementations are often determined by the that validation is a more important process that includes
researcher’s interests, experience and software availability. assessment of issues such as data quality, applicability of
Acquiring a good quality QSAR model depends on many the model and mechanistic interpretability in addition to
factors, such as the quality of biological data, the choice of statistical assessment20. In this review, we discussed about
descriptors, variable selection, statistical methods and the validation parameters for the QSAR models which are
validation13-18. developed by multiple linear regression (MLR) and partial
Validation is a crucial aspect of any QSAR modeling. It least square regression (PLS).
is the process by which the reliability and relevance of a
procedure are established for a specific purpose19. Formal
validation is one of the most overlooked steps in the model
Importance of validation of QSAR models models35 are (i) cross-validation, (ii) bootstrapping,
(iii) randomization of the response data, and (iv) external
The QSAR models are useful for various purposes validation. Several principles for assessing the validity of
including the prediction of activities of untested chemicals. QSAR models were proposed at an International workshop
The success of drug discovery efforts depends heavily on held in Setubal (Portugal), which were subsequently
the use of structure-activity relationship techniques21. Over modified in 2004 by the OECD Work Programme on
the years of development, many methods, algorithms and QSARs1,3. Against this background, in this review we have
techniques have been discovered and applied in QSAR discussed about the different validation methods of QSAR
studies22. models.
Main challenge in QSAR is to select the group of
descriptors which describe the most critical structural and Validation Methods for QSAR Models
physicochemical features associated with activity. Quality Validation methods are needed to establish the
of biological data and effective descriptor or variable predictiveness of a model on unseen data and to help
selection is an initial essential part of the QSAR modeling
determine the complexity of an equation that the amount
process23. The application of QSAR models is depends on of data justifies. Using the data that created the model (an
statistical significance and predictive ability of these internal method) or using a separate data set (an external
models. The regulatory decisions, justification of usage
method) can help validate the QSAR model (Scheme 2).
and use of a particular QSAR model is depend on the The methods of least squares fit (R2), crossvalidation
model ability to predict unknown chemicals with some
(Q2)36-38 , adjusted R2 (R2adj), chi-squared test (2), root-
known degree of certainity18,24. QSAR model can lead to
mean-squared error (RMSE), bootstrapping and
false prediction of biological activity if the developed
scrambling (Y-Randomization)39,40 are internal methods of
QSAR model is not validated. So validation of QSAR
validating a model. The best method of validating a model
models, after model development, is most important part in
is an external method, such as evaluating the QSAR model
QSAR studies.
on a test set of compounds. These are statistical
QSAR has clearly matured, although it still has a way methodologies used to ensure the models created are sound
to go. The estimation of accuracy of predictions is a critical and unbiased (“good model”). A poor model can do more
problem in QSAR modeling25. Only in this decade, harm than good, thus confirming the model as a “good
validation of QSAR models has received considerable model” is of utmost importance.
attention14-17, 26-34. Four tools of assessing validity of QSAR
compounds) or if there is no test set. For good Randomization test (Scrambling model)
predictability R2 – Q2 value should not exceed 0.3. The The predictive power of the equation is poor when the
equation for CV is observations are not sufficiently independent of each other.
PRESS One way to test for this is by randomization of the
Q2 = 1 – N dependent variables. This procedure ensures that the model
(yi – y m ) 2 is not due to a chance. The set of activity values is
i 1
reassigned randomly to different molecules, and repeating
N
PRESS = (y pred,i – yi ) 2 the entire modeling procedure. This process is repeated
i 1 many times. If the random models activity prediction is
comparable to the original equation, the set of observations
where the yi is the data value(s) not used to construct
is not sufficient to support the model.
the CV model. PRESS is the predictive residual sum of the
squares. The creation of a Scrambled Model39,40 is a unique
method of checking the descriptors used in the model
Beware of Q2 because the bioactivities are randomized ensuring the new
To validate a QSAR model, most of researchers apply the model is created from a bogus data set. The basis for this
LOO or LMO CV procedures. The outcome from this method is to test the validity of the original QSAR model
procedure is a cross-validated correlation coefficient R2 and to ensure that the selected descriptors are appropriate.
These new models (Scram-models) are created using the
(Q2). Frequently, Q2 is used as a criterion of both
same descriptors as the original model, yet the bioactivities
robustness and predictive ability of the model. Many
are changed. After each Scram-model is created, validation
authors consider high Q2 (for instance, Q2 > 0.5) as an is performed using the methods mentioned earlier. To
indicator or even as the ultimate proof of the high ensure that the Scram-models are truly random, the process
predictive power of, the QSAR model. They do not test the of changing the bioactivities can be repeated and as each
models for their ability to predict the activity of new Scram-model is created its R2 and Q2 values. Each
compounds of an external test set (i.e., compounds that time the R2 and Q2 values of the Scram-models are
have not been used in the QSAR model development). substantially lower further enforces that the true QSAR
There are several examples of recent publications, in which model is sound. The basis of using this method is to
the authors claim that their models have high predictive validate the original QSAR model because the Scram-
ability without validating them by use of an external test models are created using the original descriptors and bogus
set42-46. Some authors validate their models by the use of bioactivities. The model would be in question if there was
a strong correlation (R2 > 0.50)50 between the randomized
only one or two compounds that were not used in QSAR
bioactivities and the predicted bioactivities, specifically
model development47,48 and still claim that their models are
that the model is not responsive to the bioactivities.
highly predictive. However, it has been found that if a test
set with known values of biological activities is available External validation
for prediction, no correlation may exist between the
Several authors have suggested that the only way to
predicted and observed activities for the test set49,14.
estimate the true predictive power of a QSAR model is to
Bootstrapping compare the predicted and observed activities of an
Bootstrapping is another method of internal validation (sufficiently large) external test set of compounds that
where samples are selected randomly from the data set. In were not used in the model development49,50, 51-53. The
the simplest form of bootstrapping, instead of repeatedly problem in external validation is how can we select the
analyzing subsets of the data, sub samples of the data are training and test set? Roy et al. clearly discussed that how
repeatedly analyzed. Each sub sample is a random sample we can solve this problem in one of their article22.
with replacement from the full sample. In a typical To estimate the predictive power of a QSAR model,
bootstrap validation, K groups of size n are generated by a Golbraikh and Tropsha recommended use of the following
repeated random selection of n objects from the original statistical characteristics of the test set14: (i) correlation
data set. Some of these objects can be included in the same coefficient R between the predicted and observed
random sample several times, whereas other objects may activities; (ii) coefficients of determination (R2) [54]
never be selected. The model obtained from n randomly (predicted vs. observed activities r02, and observed vs.
selected objects is used to predict the target properties for predicted activities r0'); (iii) slopes k and k' of the
the excluded samples. A high average Q2 in the bootstrap regression lines through the origin. They consider a QSAR
validation is a demonstration of the model robustness.
516 International Journal of Drug Design and Discovery Volume 2 Issue 3 July – September 2011
model is predictive, if the following conditions are values with intercept value set to zero. A value of r2m is
satisfied14: greater than 0.5 may be taken as an indicator of good
R2 pred > 0.6, external predictability.
Unlike external validation parameters (R2pred etc.), the
r2 – r02 / r2 < 0.1, r2 – r0 ,2 / r2 < 0.1 and
r2m (overall) statistic is not based only on limited number
0.85 < k < 1.15 or 0.85 < k’ < 1.15. of test set compounds. It includes prediction for both test
set and training set (using LOO predictions) compounds.
The predictive ability of the selected model was also
Thus, this statistic is based on prediction of comparably
confirmed by external R2 pred. A value of R2 pred is
large number of compounds. The r2m (overall) statistic may
greater than 0.6 may be taken as an indicator of good
be advantageous when the test set size is considerably
external predictability.
small and regression based external validation parameter
test
(yexp – y pred ) 2 may be less reliable and highly dependent on individual
R 2Pr ed 1– i 1 test set observations. The r2m (overall) statistic may be
test
(yexp – y tr )2 used for selection of the best predictive models from
i 1
among comparable models are obtained, where some
Where y tr is the average value for the dependent models show comparatively better internal validation
parameters and some other models show comparatively
variable for the training set.
superior external validation parameters.
The lack of the correlation between Q2 and R2 was
Other validation parameter named as R2p to check the
noted in, Kubinyi et al.49, Novellino et al.51, Norinder52,
acceptability of the selected model has been reported by
and Golbraikh and Tropsha14, publication, where they
Roy (2007)55. The parameter R2p, which penalize the model
demonstrated that all of the above-mentioned criteria are
R2 for the difference between squared mean correlation
necessary to adequately assess the predictive ability of a
coefficient (R2r) of the randomized models and squared
QSAR model. Norinder suggest52 that the external test set
correlation coefficient (R2) of the non-randomized model.
must contain at least five compounds, representing the
A value of R2p should be greater than 0.5 may be taken as
whole range of both descriptor and activities of compounds
an indicator of model acceptability.
included into the training set.
Recently the use of internal versus external validation R 2p R 2 * 1 R 2 R 2r
has been a matter of great debate55. One group of QSAR
workers supports internal validation, while the other group The value of r2m (overall) determines whether the range
considers that internal validation is not a sufficient test for of predicted activity values for the whole dataset of
checking robustness of the models and external validation molecules are really close to the observed activity or not
must be done. Hawkins et al., the major group of (best predictive model or not). The value of R2p, on the
supporters of internal validation, are of the opinion that divergent, determines whether the model obtained is really
cross-validation is able to assess the model fit and to check robust or obtained as a result of chance only. Hence it can
whether the predictions will carry over to fresh data not be inferred that a QSAR model can be considered
used in the model fitting exercise. They have argued that acceptable if the values of r2m (overall) and R2p are equal to
when the sample size is small, holding a portion of it back or above 0.5 (or at least near 0.5).
for testing is wasteful and it is much better to use
The selection of robust and well predictive QSAR
“computationally more burdensome” leave-one-out cross-
models on the basis of R2, Q2 and R2pred may mislead the
validation56,57.
search for the ideally predictive model so it may also be
Recent term to check the external predictability of the done on the basis of few other parameters, such as R2CVext,
selected model is r2m, which was proposed by Roy and Paul r2 - r20 / r2, r2 - r’20 / r2, k, k’, r2m (overall) and R2p.
(2008) [58] and it was calculated by the following formula
When can we accept the developed QSAR model as
rm2 r 2
1 r 2
r02 reliable and predictive one?
A developed QSAR model can be accepted generally in
Where r2 is squared correlation coefficient between QSAR (MLR and PLS) studies when it can satisfy the
observed and predicted values and r20 is squared following criterion (The following values are the minimum
correlation coefficient between observed and predicted recommended values for significant QSAR model
Ravichandran Veerasamy, et al : Validation of QSAR Models-Strategies and Importance 517
meanwhile these evaluation measures are depend on the Recently Jaworska et al.60 reviewed the methods and
response measure scale or measure unit): criteria for estimating applicability domain through
If correlation coefficient R 0.8 (for in vivo data). training set interpolation based on range, distance,
geometrical and probability density distribution based
If coefficient of determination R2 0.6
approaches. A cluster-based approach have been proposed
If the standard deviation s is not much larger than by Stanforth et al.61 to modeling the domain of
standard deviation of the biological data. applicability by applying an intelligent version of the K-
If its F value indicate that overall significance level means clustering algorithm, modeling the training set as a
is better than 95%. collection of clusters in the descriptors space, assigning a
If its confidence interval of all individual regression test compound fuzzy membership of each individual
coefficients proves that they are justified at the 95% cluster from which an overall distance may be calculated.
significance level. Guha and Jurs50 have used a classification method that
divides the regression residuals from a previously
If cross-validated R2 (Q2) > 0.5
generated model into a good class and a bad class and
If R2 for external test set, R2pred > 0.6 builds a classifier based on the division. The trained
Randomized R2 value should be as low as to R2. classifier is then used to determine the class of the residual
Randomized Q2 value should be as low as to Q2. of a new compound.
(r2 – r20)/r2 < 0.1 and 0.85 < = k < = 1.15, or (r2 – Dispute on QSAR Validation
r'20) / r2 < 0.1 and 0.85 < = k' < = 1.15 (for test set).
Hawkins et al., the major group of supporters of internal
r2m (overall) and R2p are 0.5 (or at least near 0.5).
validation, are of the opinion that cross-validation is able
In addition, the biological data should cover a range to assess the model fit and to check whether the predictions
of at least one, two or even more logarithmic units: will carry over to fresh data not used in the model fitting
they should be well distributed over whole distance. exercise. They have argued that when the sample size is
Also, physicochemical parameter should be spread small, holding a portion of it back for testing is wasteful
over a certain range and should be more or less and it is much better to use “computationally more
evenly distributed. burdensome” leave-one-out cross-validation56,57.
Equation has to be rejected An inconsistency between internal and external
If the above mentioned statistical measures are not predictivity was reported in a few QSAR studies 51,52,62. It
satisfied was reported that, in general, there is no relationship
If the number of the variables in the regression between internal and external predictivity63,64: high internal
equation is unreasonably large. predictivity may result in low external predictivity and vice
versa. In many cases, comparable models are obtained
If standard deviation is smaller than error in the
where some models show comparatively better internal
biological data.
validation parameters and some other models show
Applicability domain comparatively superior external validation parameters.
This may create a problem in selecting the final model. So
Activity of the entire universe of chemicals can not be it is must to develop some good validation techniques to
predicted even by a robust and validated QSAR model. overcome the entire above mentioned disputes.
The prediction of a modeled response using QSAR is valid
only if the compound being predicted is within the
applicability domain of the model. The applicability Conclusion
domain is a theoretical region of the chemical space, Validation of QSAR models is a very important aspect to
defined by the model descriptors and modeled response understand reliability of model for prediction of a new
and, thus, by the nature of the training set molecules59. It is compound not present in the data set. If we consider 1000
possible to check whether a new chemical lies within reported QSAR models, out of which only 50 to 60 models
applicability domain using the leverage approach. A are really predictive but its not sure that these 60 models
compound will be considered outside the applicability have been obeyed all the conditions and validation
domain when the leverage value is higher than the critical parameters discussed in this articles. Our opinion is, both
value of 3p/n, where p is the number of model variables internal and external validation strategies are important
plus 1 and n is the number of objects used to develop the and, in fact, one should adopt all available validation
model. strategies to check robustness of the model. Only few
518 International Journal of Drug Design and Discovery Volume 2 Issue 3 July – September 2011
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researcher should be followed, also the chemical space of
[17] Aptula, A.O.; Jeliazkova, N.G.; Schultz, T.W.; Cronin,
training and test sets has to be analyzed; real outliers, with
M.T.D. QSAR Comb.Sci. 2005, 24, 385-390.
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