Treatment of Acute Lymphoblastic Leukemia from Traditional Chinese Medicine

Acute lymphoblastic leukemia (ALL) is a cancer that immature white blood cells continuously overproduce in the bone marrow. These cells crowd out normal cells in the bone marrow bringing damage and death. Methotrexate (MTX) is a drug used in the treatment of various cancer and autoimmune diseases. In particular, for the treatment of childhood acute lymphoblastic leukemia, it had significant effect. MTX competitively inhibits dihydrofolate reductase (DHFR), an enzyme that participates in the tetrahydrofolate synthesis so as to inhibit purine synthesis. In addition, its downstream metabolite methotrexate polyglutamates (MTX-PGs) inhibit the thymidylate synthase (TS). Therefore, MTX can inhibit the synthesis of DNA. However, MTX has cytotoxicity and neurotoxin may cause multiple organ injury and is potentially lethal. Thus, the lower toxicity drugs are necessary to be developed. Recently, diseases treatments with Traditional Chinese Medicine (TCM) as complements are getting more and more attention. In this study, we attempted to discover the compounds with drug-like potential for ALL treatment from the components in TCM. We applied virtual screen and QSAR models based on structure-based and ligand-based studies to identify the potential TCM component compounds. Our results show that the TCM compounds adenosine triphosphate, manninotriose, raffinose, and stachyose could have potential to improve the side effects of MTX for ALL treatment.


Introduction
Dihydrofolate reductase (DHFR) is essential in cellular metabolism and cell growth. It catalyzes the conversion of dihydrofolate into tetrahydrofolate which is a carrier for the methyl group. The methyl group carried by tetrahydrofolate is required for de novo synthesis of varieties of essential metabolites including amino acids, lipids, pyrimidines, and purines. Methotrexate (MTX), a folate antagonist, arrests cell growth by competitively binding to DHFR, thereby, blocking de novo synthesis of nucleotide precursors and inhibiting DNA synthesis [1]. MTX has been found to be useful as an antineoplastic and immunosuppressive agent because it inhibits the proliferation of rapidly dividing malignant [2].
MTX tightly binding on DHFR is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia [3]. In addition, its folate analogue is widely used in the treatment of acute lymphoblastic leukemia (ALL) [4], ovarian cancer [5], osteosarcoma [6], rheumatoid arthritis [7], psoriasis [8], and inflammatory bowel disease [9] and for prevention of graftversus-host disease after transplantation [10].
In the cells, MTX acts by inhibiting two enzymes. First, as an analog of folate, MTX is a powerful competitive inhibitor with 1000-fold more potent than the natural substrate of DHFR. DHFR is responsible for converting dihydrofolate (FH2) to their active form tetrahydrofolate (FH4), which is a substrate of thymidylate synthase (TS). Second, MTX is converted to active methotrexate polyglutamates (MTX-PGs) by folylpolyglutamate synthase [11,12]. The polyglutamated forms of MTX inhibit TS directly. Due to these inhibitions,   The primary action of MTX is inhibition of the enzyme DHFR, which converts dihydrofolate (FH2) to tetrahydrofolate (FH4) [11,14]. MTX-PGs exert a stronger inhibition of DHFR and TS [15][16][17]. Thus, through direct inhibition by MTX and due to lack of FH4 and accumulation of FH2, deoxythymidine monophosphate synthesis and purine de novo synthesis is blocked, which eventually lead to leukemic cell death, bone marrow suppression, gastrointestinal mucositis, liver toxicity, and, rarely, alopecia [14,15,18,19]. In fact, both MTX and natural folates undergo polyglutamylation catalyzed by the enzyme folylpolyglutamyl synthase. The MTX-PGs ensure intracellular retention and, furthermore, increase the affinity for the MTX-sensitive enzymes [16,18,20] (Figure 1).

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Evidence-Based Complementary and Alternative Medicine lead compounds against tumors [50][51][52][53][54], stroke [55][56][57][58], viral infection [59][60][61][62][63], metabolic syndrome [64][65][66], diabetes [67], inflammation [62], and other diseases [68,69]. For this trend, we attempted to discover the compounds with druglike potential and lower toxicity for ALL treatment from the components in traditional Chinese medicine.   Evidence-Based Complementary and Alternative Medicine We adopted the traditional Chinese medicine formulas that treat acute lymphoblastic leukemia from database "Shanghai Innovative Research Center of Traditional Chinese Medicine" (http://www.sirc-tcm.sh.cn/en/index.html) [72]. The component compounds of these formulas were integrated with the herbs data from the TCM Database@Taiwan [73] and became the ALL disease-specific compound library. Virtual screening of candidates from the compound library was conducted using the LigandFit Module of DS 2.5 under the Chemistry at HARvard Macromolecular Mechanics (CHARMm) force field. DockScore was selected as output values. Candidates were ranked according to DockScore and pharmacokinetic characteristics including absorption, solubility, blood brain barrier (BBB), and plasma protein binding (PPB) were predicted by ADMET protocols for each candidate.  to construct 2D-QSAR models. The training set was used to construct multiple linear regression (MLR), support vector machine (SVM), and Bayesian network (BN) models. The test set was used to test the accuracy of these models.

Multiple Linear Regression (MLR) Model.
Multiple linear regression [75] attempts to model the relationship between two or more explanatory variables and a response where represents the th molecular descriptor and is its fitting coefficient. The generated MLR model was validated with test dataset. The square correlation coefficients ( 2 ) between predicted and actual pIC50 of the training set was used to verify accuracy of the model. This building model was applied to predict the pIC50 values of the TCM candidates.

Support Vector Machine (SVM)
Model. SVM implement classification or regression analysis with linear or nonlinear algorithms [76]. The algorithm identifies a maximummargin hyper-plane to discriminate two class training samples. Samples on the margin are called the support vectors. Lagrange multipliers and kernels were introduced to form the final pattern separating regression model. In this study, LibSVM [77][78][79] package was selected to build our regression SVM model. The selected kernel was the Gaussian radial basis function kernel equation: Cross-validation of the SVM model was also conducted following the default settings in LibSVM [80]. The generated regression SVM model was validated with test dataset. The square correlation coefficients ( 2 ) between predicted and actual pIC50 of the training set was used to verify accuracy of the model. This building model was applied to predict the pIC50 values of the TCM candidates.

Bayesian Network Model.
We used the Bayes Net Toolbox (BNT) in Matlab (https://code.google.com/p/bnt) to create Bayesian network model [81] by the training data set. After data discretization, we applied linear regression analysis for each pIC50 category in the training dataset. For the th pIC50 category with candidates, let and represent the pIC50 value and the th descriptor value in the th ligand, respectively. The regression model of the data sets { , 1 , . . . , } =1 is formulated as and and are the regression coefficients and error term in the th pIC50 category. We used ordinary least squares to estimate the unknown regression coefficient : The Banjo (Bayesian network inference with Java objects) is software for structure learning of static Bayesian networks (BN) [82]. It is implemented in Java. We used training dataset to discover the relationships in the BN structure among the descriptors and the pIC50 by the Banjo package. After that, we used test data to assess the accuracy of our algorithm. For the test data , the pIC50 category ( ) is predicted by the following formula: where represented the th category of pIC50 and represented the total number of the pIC50 categories. The marginal probability ( | ) can be calculated by BNT module. Finally, the pIC50 value is calculated as follows: pIC50 =̂.
8  The square correlation coefficients ( 2 ) between predicted and actual pIC50 of the training set were used to verify accuracy of the model. This building model was applied to predict the pIC50 values of the TCM candidates.

3D-Quantitative Structure Activity Relationship (3D-QSAR) Models.
Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed by Sybyl-X 1.1.1 (Tripos Inc., St. Louis, MO, USA) for DHFR inhibitors. Lennard-Jones potential and Coulomb potential were employed to calculate steric and electrostatic interaction energies. The two 3D-QSAR models were further evaluated by crossvalidated correlation coefficient ( 2 ) and non-cross-validated correlation coefficient ( 2 ). The correlation between the force field and biological activities was calculated by partial least squares (PLSs) method.
The flowchart for the entire experimental procedure for TCM candidates screening is illustrated in Figure 3.  The compounds from our library were docked into the two receptors. In this protocol, the receptors were fixed, and the ligands that complement the binding sites were flexible in energy minimization process. The control compound used in this study was MTX which contains aromatic and heterocyclic rings (Figure 4).

Results and Discussion
The top eighteen results from DHFR docking score are tabulated in Table 2. The TS docking score for the eighteen candidates are also tabulated in Table 2. All the eighteen TCM candidates had higher Dock Scores than the control methotrexate (MTX) and MTX-PGs. Chemical scaffolds of MTX, MTX-PGs, and the eighteen TCM candidates are shown in Figure 4. Adsorption, solubility, hepatotoxicity, and plasma protein binding were assessed to evaluate pharmacokinetic properties of the selected candidates (Table 3). Considering the factor of hepatotoxicity, we selected the TCM compounds adenosine triphosphate, manninotriose, raffinose, and stachyose for advanced study. MTX and TCM candidates had very poor absorption for human intestine. Binding strength of the ligands to carrier proteins in the blood stream is indicated by the plasma protein binding (PPB) value [21]. MTX has more than 90% for PPB but adenosine triphosphate, manninotriose, raffinose, and stachyose were less than 90% for PPB.
Ligand-receptor interactions during docking are shown in Figures 5 and 6. MTX docked on DHFR (Figure 5(a)) through four hydrogen bondings of Glu30, Gln35, Lys68, and Arg70. Adenosne triphosphate formed three H-bonds with Glu30, Gln35, and Arg70 ( Figure 5(b)). Manninotriose formed H-bond with Arg28 ( Figure 5(c)). Raffinose formed H-bonds with Asn64 and NDP ( Figure 5(d)). Stachyose formed H-bonds with Lys63, Asn64, and Lys68 ( Figure 5(e)). MTX-PGs docked on TS ( Figure 5    Analysis of hydrophobic interactions showed that MTX docking on DHFR was more stable than the TCM candidates. Comparing with chemical structures of the TCM candidates, it could be attributed to the larger size for MTX docking on DHFR (Figures 6(a), 6(b), 6(c), 6(d), and 6(e)). However, the TCM candidates docking on TS were more stable than MTX-PGs due to hydrophobic interactions (Figures 6(f), 6(g), 6(h), 6(i), and 6(j)). In CoMFA model, the steric fields were the primary contributing factor. In CoMSIA, various factors were considered and modeled. The optimum CoMSIA models were "EHA model" and "EHDA model" based on high 2 , high 2 , and low SEE values ( Table 4). The "EHA model" was consisting of electrostatic field and hydrophobic and hydrogen bond acceptor. The "EHDA model" was consisting of electrostatic field and hydrophobic and hydrogen bond donor, and hydrogen bond acceptor. The CoMFA model and CoMSIA model of EHDA and of EHA were with ONC of 7, 11, and 12, respectively. Experimental and predicted pIC50 values of 45 DHFR inhibitors using CoMFA and CoMSIA models are shown in Table 5. Residuals calculated from the differences between observed and predicted pIC50 values ranged between −0.3655 and 0.4311 for the CoMFA, between −0.411 and 0.589 for the CoMSIA with "EHA model, " and between −0.431 and 0.569 for CoMSIA with "EHDA model. "

Bioactivity Prediction
The correlations between the predicted and actual bioactivity for DHFR inhibitors are shown in Figure 7. The 2 values are 0.936 for MLR, 0.734 for Bayesian network, 0.884 for SVM, 0.957 for CoMFA, 0.977 for CoMSIA with EHA model, and 0.978 for CoMSIA with EHDA model implicate high correlation. High correlation coefficients validated the reliability of the constructed CoMFA and CoMSIA models. The predicted bioactivity values of TCM candidates by 2D-QSAR and 3D-QSAR models are listed in Table 6. predicted based on the 3D-QSAR contour map, including features in steric field, hydrophobic field, and H-bond donor/acceptor characteristics. MTX and the TCM candidates contoured well to the steric features of the CoMFA in Figure 8. CoMSIA map provides more information with regard to bioactivity differences for "EHA model" and "EHDA model" in Figures 9 and 10, respectively. From the consistent results observed among the 3D-QSAR models validations, we inferred that adenosine triphosphate, manninotriose, raffinose, and stachyose of TCM candidates might have good biological activity for DHFR.
Contour to steric favoring and hydrophobic favoring regions was observed for adenosine triphosphate, manninotriose, raffinose, and stachyose. Consistent with the docking pose contour (Figures 8, 9, and 10), we propose that the four TCM candidates may maintain bioactivity for DHFR under dynamic conditions in physiological environments.

Conclusion
DHFR and TS proteins are key regulators in de novo synthesis of purines and thymidylate. Inhibiton of these proteins has the potential for treating acute lymphoblastic leukemia. In this study, we applied virtual screen and QSAR models based on structure-based and ligand-based methods in order to identify the potential TCM compounds. The TCM compounds adenosine triphosphate, manninotriose, raffinose, and stachyose could bind on DHFR and TS specifically and had low hepatotoxicity. These TCM compounds had potential to improve the side effects of MTX for ALL treatment.