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1.  Prediction of Cytochrome P450 Profiles of Environmental Chemicals with QSAR Models Built from Drug-like Molecules 
Molecular informatics  2012;31(11-12):783-792.
The human cytochrome P450 (CYP) enzyme family is involved in the biotransformation of many xenobiotics. As part of the U.S. Tox21 Phase I effort, we profiled the CYP activity of approximately three thousand compounds, primarily those of environmental concern, against human CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 isoforms in a quantitative high throughput screening (qHTS) format. In order to evaluate the extent to which computational models built from a drug-like library screened in these five CYP assays under the same conditions can accurately predict the outcome of an environmental compound library, five support vector machines (SVM) models built from over 17,000 drug-like compounds were challenged to predict the CYP activities of the Tox21 compound collection. Although a large fraction of the test compounds fall outside of the applicability domain (AD) of the models, as measured by k-nearest neighbor (k-NN) similarities, the predictions were largely accurate for CYP1A2, CYP2C9, and CYP3A4 ioszymes with area under the receiver operator characteristic curves (AUC-ROC) ranging between 0.82 and 0.84. The lower predictive power of the CYP2C19 model (AUC-ROC = 0.76) is caused by experimental errors and that of the CYP2D6 model (AUC-ROC = 0.76) can be rescued by rebalancing the training data. Our results demonstrate that decomposing molecules into atom types enhanced the coverage of the AD and that computational models built from drug-like molecules can be used to predict the ability of non-drug like compounds to interact with these CYPs.
doi:10.1002/minf.201200065
PMCID: PMC3583379  PMID: 23459712
Human CYPs; QSAR models; Predictive Capacity; SVM; Predictive Toxicology
2.  Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis 
Nature chemical biology  2012;8(10):839-847.
Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism.
doi:10.1038/nchembio.1060
PMCID: PMC3711671  PMID: 22922757
3.  Predictive Models for Cytochrome P450 Isozymes Based on Quantitative High Throughput Screening Data 
The human cytochrome P450 (CYP450) isozymes are the most important enzymes in the body to metabolize many endogenous and exogenous substances including environmental toxins and therapeutic drugs. Any unnecessary interactions between a small molecule and CYP450 isozymes may raise a potential to disarm the integrity of the protection. Accurately predicting the potential interactions between a small molecule and CYP450 isozymes is highly desirable for assessing the metabolic stability and toxicity of the molecule. The National Institutes of Health Chemical Genomics Center (NCGC) has screened a collection of over seventeen thousand compounds against the five major isozymes of CYP450 (1A2, 2C9, 2C19, 2D6 and 3A4) in a quantitative high throughput screening (qHTS) format. In this study, we developed support vector classification (SVC) models for these five isozymes using a set of customized generic atom types. The CYP450 datasets were randomly split into equal-sized training and test sets. The optimized SVC models exhibited high predictive power against the test sets for all five CYP450 isozymes with accuracies of 0.93, 0.89, 0.89, 0.85 and 0.87 for 1A2, 2C9, 2C19, 2D6 and 3A4, respectively, as measured by the area under the receiver operating characteristic (ROC) curves. The important atom types and features extracted from the five models are consistent with the structural preferences for different CYP450 substrates reported in the literature. We also identified novel features with significant discerning power to separate CYP450 actives from inactives. These models can be useful in prioritizing compounds in a drug discovery pipeline, or recognizing the toxic potential of environmental chemicals.
doi:10.1021/ci200311w
PMCID: PMC3200453  PMID: 21905670
4.  Monitoring compound integrity with cytochrome P450 assays and qHTS 
Journal of biomolecular screening  2009;14(5):538-546.
We describe how room temperature storage of a 1,120 member compound library prepared in either DMSO or in a hydrated DMSO/water (67/33) mixture affects the reproducibility of potency values as monitored using cytochrome P450 1A2 and 2D6 isozyme assays. The bioluminescent assays showed Z′-factors of 0.71 and 0.62, with 18% and 32% of the library found as active against the CYP 1A2 and 2D6 isozymes respectively. We tested the library using quantitative high-throughput screening to generate potency values for every library member which was measured at seven time intervals spanning 37 weeks. We calculated the minimum significant ratio (MSR) from these potency values at each time interval and we found that for the library stored in DMSO, the CYP 1A2 and 2D6 assay MSRs progressed from approximately 2.0 to 5.0. The hydrated conditions showed similar performance in both MSR progression and analytical QC results. Based on this study we recommend that DMSO samples be stored in 1,536-well plates for < 4 months at room temperature. Further, the study shows the magnitude of potency changes that can occur in a robust bioassay due to compound sample storage.
doi:10.1177/1087057109336954
PMCID: PMC3430136  PMID: 19483146
HTS; compound storage; DMSO; quantitative HTS
5.  2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase 
Compared to normal differentiated cells, cancer cells have altered metabolic regulation to support biosynthesis and the expression of the M2 isozyme of pyruvate kinase (PKM2) plays an important role in this anabolic metabolism. While the M1 isoform is a highly active enzyme, the alternatively spliced M2 variant is considerably less active and expressed in tumors. While the exact mechanism by which decreased pyruvate kinase activity contributes to anabolic metabolism remains unclear, it is hypothesized that activation of PKM2 to levels seen with PKM1 may promote a metabolic program that is not conducive to cell proliferation. Here we report the third chemotype in a series of PKM2 activators based on the 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamide scaffold. The synthesis, structure activity relationships, selectivity and notable physiochemical properties are described.
doi:10.1016/j.bmcl.2011.08.114
PMCID: PMC3224553  PMID: 21958545
PKM2; pyruvate kinase; cellular metabolism; anti-cancer strategies; small molecule activators
6.  Evaluation of Thieno[3,2-b]pyrrole[3,2-d]pyridazinones as Activators of the Tumor Cell Specific M2 Isoform of Pyruvate Kinase 
Cancer cells have distinct metabolic needs that are different from normal cells and can be exploited for development of anti-cancer therapeutics. Activation of the tumor specific M2 form of pyruvate kinase (PKM2) is a potential strategy for returning cancer cells to a metabolic state characteristic of normal cells. Here, we describe activators of PKM2 based upon a substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone scaffold. The synthesis of these agents, structure activity relationships, analysis of activity at related targets (PKM1, PKR and PKL) and examination of aqueous solubility are investigated. These agents represent the second reported chemotype for activation of PKM2.
doi:10.1016/j.bmcl.2010.04.015
PMCID: PMC2874658  PMID: 20451379
Warburg effect; pyruvate kinase; cellular metabolism; anti-cancer strategies; small molecule activators
7.  Evaluation of Substituted N,N′-Diarylsulfonamides as Activators of the Tumor Cell Specific M2 Isoform of Pyruvate Kinase 
The metabolism of cancer cells is altered to support rapid proliferation. Pharmacological activators of a tumor cell specific pyruvate kinase isozyme (PKM2) may be an approach for altering the classic Warburg effect characteristic of aberrant metabolism in cancer cells yielding a novel anti-proliferation strategy. In this manuscript we detail the discovery of a series of substituted N,N′-diarylsulfonamides as activators of PKM2. The synthesis of numerous analogues and the evaluation of structure activity relationships are presented as well as assessments of mechanism and selectivity. Several agents are found that have good potencies and appropriate solubility for use as chemical probes of PKM2 including 55 (AC50 = 43 nM, maximum response = 84%; solubility = 7.3 μg/mL), 56 (AC50 = 99 nM, maximum response = 84%; solubility = 5.7 μg/mL) and 58 (AC50 = 38 nM, maximum response = 82%; solubility = 51.2 μg/mL). The small molecules described here represent first-in-class activators of PKM2
doi:10.1021/jm901577g
PMCID: PMC2818804  PMID: 20017496
Warburg effect; pyruvate kinase; cellular metabolism; high-throughput screening; small molecule activators
8.  Comprehensive Characterization of Cytochrome P450 Isozyme Selectivity across Chemical Libraries 
Nature biotechnology  2009;27(11):1050-1055.
The cytochrome P450 (CYP) gene family strongly influences drug development. We determined potency values for 17,143 compounds against recombinant CYP 1A2, 2C9, 2C19, 2D6, and 3A4 enzymes through an in vitro bioluminescent assay. The compound collections included substances from typical libraries and FDA-approved drugs. Cross-library isozyme inhibition (30–78%) was observed with important differences between collections. While only 7% of the typical screening library was inactive against all five isozymes, 33% of FDA-approved drugs were inactive, reflecting the optimized pharmacological properties of the latter. Unexpectedly, drugs exhibited less activity towards the CYP 2C9 and 2C19 isozymes compared to un-optimized collections. We then identified substructures that differentiated between the five isozymes as well as substructures trending towards active or inactive categories. We describe here a pharmacological compendium to further the understanding of CYP isozymes.
doi:10.1038/nbt.1581
PMCID: PMC2783980  PMID: 19855396

Results 1-8 (8)