Identification of off-targets has potential application in drug repurposing 
and personalized medicine 
. Compared with the similarity ensemble approach 
and the naive Bayesian classifiers approach 
to off-target identification, both of which build new drug-protein connections within the space of the known therapeutic target, the chemical-protein interactome approach is a step towards analyzing the entire human proteome, although the available human protein structrome is limited. Several of the pocket comparison algorithms have also tried to explore the off-target spaces facing the entire human proteome 
, or tried to map the off-targets onto the pathways 
or the metabolic network 
, but our study is the first one examining the system's perturbation in terms of both the off-target identification and the off-system's gene expression change, providing candidates for pharmacogenetic and pharmacogenomic studies, respectively. Further work may combine the off-target and the off-system in elucidating and predicting adverse drug reactions.
In the retrospective studies, the antitheses CPI recalled the accredited susceptible genes for CIA. As a complement to genetic association studies 
, the CPI reveals the possible mechanism of the CIA based on the drug-protein interaction, the primary step in drug reaction. The difference between the interaction conformation and the interaction strength of CLZ and OLZ towards the off-targets could account for the difference in patients' susceptibility to agranulocytosis. Since none of the nonsynonymous SNPs was found around the ligand binding pocket of the four proteins reported to be involved in CIA, we deduced that individual differences in CIA susceptibility could be explained by a variation in the expression level of the protein. In fact, NQO2
was found to have lower expression levels in CIA susceptible patients 
. The lower expression level in this detoxification enzyme could make the patient more sensitive to the drug. It is also reasonable to expect subsequent discoveries (e.g. some genotypes correlated to Hsp70
expression level) supporting the CLZ off-target hypothesis, which could lead to biomarker development at genotype and gene expression level 
in CLZ therapy.
The reactive oxygen hypothesis is one of the major hypotheses of agranulocytosis etiology 
. In our results, CLZ and other drugs causing agranulocytosis tended to affect the oxidoreductases, which play an important role in reactive oxygen clearance. For example, NQO2 protein and myeloperoxidase are key enzymes in the detoxification of active radicals thus protecting the cells from drug-induced oxidative and electrophilic stress 
. Furthermore, alpha-tocopherol transfer protein is a prioritized target of clozapine (). Blocking the transferring of tocopherol, which is a strong endogenous antioxidant 
, may also explain clozapine's impact on the detoxification system. Clozapine can be oxidized to reactive nitrenium ions 
, which preferably reacts with sulfhydryl and is detoxified by glutathione. In our results, glutathione related enzymes were significantly enriched in the CPI, implying that the drug causing agranulocytosis not only affected the detoxification system of oxidoreductases, but might also interfered in the glutathione system, which is essential to the detoxification of the major metabolites of CLZ.
Besides the unexpected drug-protein interactions, the expression change of the off-system may explain CIA etiology. The perturbation of anti-apoptosis genes by CLZ treatment reflects the fact that CLZ disturbs cell death pathways by binding with Hsp70, and the general up regulation of anti-apoptosis genes can be explained as a feedback towards elevated apoptotic stress mediated by Hsp70 and the anti-oxidation system, since the inhibition of oxidoreductases and the perturbation of oxidoreductase system is a well known mediator of apoptosis 
. By breaking the balance of oxidation and reduction, CLZ can stimulate apoptosis via Hsp70 inhibition and enhanced oxidative stress. Along with the CPI results, biological effects of CLZ further support the hypothesis that Hsp70 and oxidoreductases together with their respective system serve as the off-targets(-systems) of CLZ and potentially mediate CIA. Since HL60 is derived from peripheral blood leukocytes, which is a representative cell model for the immune system, the finding of the systems perturbation in HL60 cells but not in MCF7 (breast cancer) and PC3 (prostate cancer) cell lines strengthens the antiapoptosis and the oxidoreductases systems' function in immune related events. In summary, 53% and 34% of prioritized proteins from the CPI are oxidoreductases, and 16% of the proteins are related to gluthathione metabolism. These findings suggest a much higher participation of the detoxification/antioxidant systems in drug-induced agranulocytosis than previously thought and the off-targets/-systems identified in this study can represent candidates for biomarker development in wet-lab experiments and pharmacogenetic/pharmacogenomic screening in the future.
However, the 410 binding pocket set is a limited representation of the entire human proteome. For instance, it does not include any HLA proteins according to our target preparation criteria, which may be involved in agranulocytosis as a mediator of the immune etiology. Drug-HLA interaction was reported to be an important step determining the drug-HLA specificity in IDR 
. In our previous study, we have built the abacavir-HLA-B*5701 interaction models for abacavir-induced hypersensitivity 
. The identification of the drug-HLA interaction at the F-pocket of HLA molecules has been cited by several immunologists 
. Since HLAs have been identified as the key factors in IDRs 
, the drug-HLA interactome will be systematically studied in future.
Identification of the related genes and the systems is the first step towards understanding and more importantly, predicting the IDR. The IDRs were regarded as unpredictable in response to compounds 
. In this study, we argue that the IDRs are predictable, and the challenge of personalized medicine is not to predict adverse reaction for a compound but for a patient. The biomarkers could be either the genetic variations causing a binding affinity change of the drug towards the off-targets 
, the expression level alteration of one gene 
, or the off-systems' perturbation. Our study demonstrates that beside polymorphisms around the binding pocket that alter the drug efficacy via a change in the binding affinity 
, the off-system expression change could also determine individual variability towards the same drug, suggesting a new way of identifying biomarkers or constructing a prediction model for personalized medicine. Such an approach could also be applied to personalized drug repurposing 
, where the off-targets and the off-systems accounting for the new therapeutic area could also be patient specific.
Adverse drug reaction and the new indication are two ‘off-effects’ of the drug towards human being. So this study will also illuminate the drug repositioning by, 1) helping explain the mode-of-action of the serendipitous repositioned drugs via identifying their off-targets/-systems; 2) predicting the new use for existing drugs based on their interaction profiles with the off-targets and their perturbations on the off-systems. For example, one can recruit the case and the control molecular set for a particular indication. After identifying the off-targets/-systems using the methodology in this study, one can predict the indication of a new compound based on its impact on these newly identified off-targets/-systems.