Recent Publications:

Bioorganic & Medicinal Chemistry Letters	Identification of a potent new chemotype for the selective inhibition of PDE4. Skoumbourdis AP, Huang R, Southall N, Leister W, Guo V, Cho MH, Inglese J, Nirenberg M, Austin CP, Xia M, Thomas CJ. A series of substituted 3,6-diphenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines were prepared and analyzed as inhibitors of phosphodiesterase 4 (PDE4). Synthesis, structure–activity relationships, and the selectivity of a highly potent analogue against related phosphodiesterase isoforms are presented.
Chemical Research Toxicology	Characterization of Diversity in Toxicity Mechanism Using In Vitro Cytotoxicity Assays in Quantitative High Throughput Screening. Huang R, Southall N, Cho MH, Xia M, Inglese J, Austin CP. Assessing the potential health risks of environmental chemical compounds is an expensive undertaking which has motivated the development of new alternatives to traditional in vivo toxicological testing. One approach is to stage the evaluation, beginning with less expensive and higher throughput in vitro testing before progressing to more definitive trials. In vitro testing can be used to generate a hypothesis about a compound's mechanism of action, which can then be used to design an appropriate in vivo experiment. Here we begin to address the question of how to design such a battery of in vitro cell-based assays by combining data from two different types of assays, cell viability and caspase activation, with the aim of elucidating mechanism of action. Because caspase activation is a transient event during apoptosis, it is not possible to design a single end-point assay protocol that would identify all instances of compound-induced caspase activation. Nevertheless, useful information about compound mechanism of action can be obtained from these assays in combination with cell viability data. Unsupervised clustering in combination with Dunn's cluster validity index is a robust method for identifying mechanisms of action without requiring any a priori knowledge about mechanisms of toxicity. The performance of this clustering method is evaluated by comparing the clustering results against literature annotations of compound mechanisms.
Toxicology in Vitro	A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker. Cho MH, Niles A, Huang R, Inglese J, Austin CP, Riss T, Xia M. Measurement of cell membrane integrity has been widely used to assess chemical cytotoxity. Several assays are available for determining cell membrane integrity including differential labeling techniques using neutral red and trypan blue dyes or fluorescent compounds such as propidium iodide. Other common methods for assessing cytotoxicity are enzymatic "release" assays which measure the extra-cellular activities of lactate dehydrogenase (LDH), adenylate kinase (AK), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in culture medium. However, all these assays suffer from several practical limitations, including multiple reagent additions, scalability, low sensitivity, poor linearity, or requisite washes and medium exchanges. We have developed a new cytotoxicity assay which measures the activity of released intracellular proteases as a result of cell membrane impairment. It allows for a homogenous, one-step addition assay with a luminescent readout. We have optimized and miniaturized this assay into a 1536-well format, and validated it by screening a library of known compounds from the National Toxicology Program (NTP) using HEK 293 and human renal mesangial cells by quantitative high-throughput screening (qHTS). Several known and novel membrane disrupters were identified from the library, which indicates that the assay is robust and suitable for large scale library screening. This cytotoxicity assay, combined with the qHTS platform, allowed us to quickly and efficiently evaluate compound toxicities related to cell membrane integrity.
Journal of Medicinal Chemistry	Fluorescence Spectroscopic Profiling of Compound Libraries. Simeonov S, Jadhav A, Thomas CJ, Wang Y, Huang R, Southall N, Shinn P, Smith J, Austin CP, Inglese J. Chromo/fluorophoric properties often accompany the conjugated, aromatic and heterocyclic features of many of the scaffolds and impurities that make up library samples used for high throughput screening (HTS). These properties impart highly variable effects on assay outputs employing optical detection, thus complicating the interpretation of data and leading to false positives and negatives. Here, we report the comprehensive fluorescence profile of >70,000 samples across multiple spectral regions commonly utilized in HTS assays. The quantitative HTS (qHTS) paradigm was utilized to test each sample at seven or more concentration points over a 4-log concentration range in 1536-well format, with raw fluorescence response collected using a CCD-based imager. The resulting output was compared with fluorophore standards to compute a normalized fluorescence response (termed fluorophore-equivalent concentration, FEC) for each sample, concentration, and relevant spectral region. The greatest fraction of fluorescent compounds appeared in the UV-end of the light spectrum, where over 5% of library members matched or exceeded 10 nM FEC of 4-methylumbelliferone and AlexaFluor 350, while approximately 1.8% of the library matched or exceeded 100 nM FEC of these standards. Red-shifting the spectral window by as little as 100 nm was accompanied by a dramatic decrease in autofluorescence. Native compound fluorescence, scaffold overlap with known fluorophores, fluorescent impurities, novel fluorescent compounds, and the ability to discriminate generalities of fluorescent interferences and devise strategies to identify them are discussed.
Environmental Health Perspectives	Compound Cytotoxicity Profiling Using Quantitative High-Throughput Screening. Xia M, Huang R, Witt KL, Southall N, Fostel J, Cho MH, Jadhav A, Smith CS, Inglese J, Portier CJ, Tice RR, Austin CP. Background: The propensity of compounds to produce adverse health effects in humans is generally evaluated using animal-based test methods. Such methods can be relatively expensive, low-throughput, and associated with pain suffered by the treated animals. In addition, differences in species biology may confound extrapolation to human health effects. Objective: The U.S. National Toxicology Program and the NIH Chemical Genomics Center are collaborating to identify a battery of cell-based screens to prioritize compounds for further toxicological evaluation. Methods: 1,408 compounds previously tested in one or more traditional toxicological assays were profiled for cytotoxicity using quantitative high-throughput screening (qHTS) in 13 human and rodent cell types derived from six common targets of xenobiotic toxicity (liver, blood, kidney, nerve, lung, skin). Selected cytotoxicants were further tested to define response kinetics. Results: qHTS of these compounds produced robust and reproducible results which allowed cross-compound, cross-cell type, and cross-species comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, while others exhibited species- or cell typespecific cytotoxicity. Closely related cell types and analogous cell types in human and rodent frequently showed different patterns of cytotoxicity. Some compounds inducing similar levels of cytotoxicity showed distinct time-dependence in kinetic studies, consistent with known mechanisms of toxicity. Conclusions: The generation of high-quality cytotoxicity data on this large library of known compounds using qHTS demonstrates the potential of this methodology to profile a much broader array of assays and compounds, which, in aggregate, may be valuable for prioritizing compounds for further toxicological evaluation, identifying compounds with particular mechanisms of action, and potentially predicting in vivo biological response
Molecular Cancer Therapeutics	Anticancer medicines in development: assessment of bioactivity profiles within the National Cancer Institute anticancer screening data. Covell DG, Huang R, Wallqvist A. We present an analysis of current anticancer compounds that are in phase I, II, or III clinical trials and their structural analogues that have been screened in the National Cancer Institute (NCI) anticancer screening program. Bioactivity profiles, measured across the NCI 60 cell lines, were examined for a correspondence between the type of cancer proposed for clinical testing and selective sensitivity to appropriately matched tumor subpanels in the NCI screen. These results find strongest support for using the NCI anticancer screen to select analogue compounds with selective sensitivity to the leukemia, colon, central nervous system, melanoma, and ovarian panels, but not for renal, prostate, and breast panels. These results are extended to applications of two-dimensional structural features to further refine compound selections based on tumor panel sensitivity obtained from tumor screening results.
Journal of Chemical Information and Modeling	Chemoinformatic analysis of NCI preclinical tumor data: evaluating compound efficacy from mouse xenograft data, NCI-60 screening data, and compound descriptors Wallqvist A, Huang R, Covell DG. We provide a chemoinformatic examination of the NCI public human tumor xenograft data to explore relationships between small molecules, treatment modality, efficacy, and toxicity. Efficacy endpoints of tumor weight reduction (TW) and survival time increase (ST) compared to tumor bearing control mice were augmented by a toxicity measure, defined as the survival advantage of treated versus control animals (TX). These endpoints were used to define two independent therapeutic indices (TIs) as the ratio of efficacy (TW or ST) to toxicity (TX). Linear models predictive of xenograft endpoints were successfully constructed (0.67 < r(2) < or = 0.74)(observed_versus_predicted) using a model comprised of variables in treatment modality, chemoinformatic descriptors, and in vitro cell growth inhibition in the NCI 60-cell assay. Cross-validation analysis based on randomly chosen training subsets found these predictive correlations to be robust. Model-based sensitivity analysis found chemistry and growth inhibition to provide the best, and treatment modality the worst, indicators of xenograft endpoint. The poor predictive power derived from treatment alone appears to be of less importance to xenograft outcome for compounds having strongly similar chemical and biological features. ROC-based model validation found a 70% positive predictive value for distinguishing FDA approved oncology agents from available xenograft tested compounds. Additional chemoinformatic applications are provided that relate xenograft outcome to biological pathways and putative mechanism of compound action. These results find a strong relationship between xenograft efficacy and pathways comprised of genes having highly correlated mRNA expressions. Our analysis demonstrates that chemoinformatic studies utilizing a combination of xenograft data and in vitro preclinical testing offer an effective means to identify compound classes with superior efficacy and reduced toxicity.
Molecular Cancer Therapeutics	Targeting changes in cancer: assessing pathway stability by comparing pathway gene expression coherence levels in tumor and normal tissues. Huang R, Wallqvist A, Covell DG. The purpose of this study is to examine gene expression changes occurring in cancer from a pathway perspective by analyzing the level of pathway coherence in tumor tissues in comparison with their normal counterparts. Instability in pathway regulation patterns can be considered either as a result of or as a contributing factor to genetic instability and possibly cancer. Our analysis has identified pathways that show a significant change in their coherence level in tumor tissues, some of which are tumor type specific, indicating novel targets for cancer type-specific therapies. Pathways are found to have a general tendency to lose their gene expression coherence in tumor tissues when compared with normal tissues, especially for signaling pathways. The selective growth advantage of cancer cells over normal cells seems to originate from their preserved control over vital pathways to ensure survival and altered signaling, allowing excessive proliferation. We have additionally investigated the tissue-related instability of pathways, providing valuable clues to the cellular processes underlying the tumorigenesis and/or growth of specific cancer types. Pathways that contain known cancer genes (i.e., "cancer pathways") show significantly greater instability and are more likely to become incoherent in tumor tissues. Finally, we have proposed strategies to target instability (i.e., pathways that are prone to changes) by identifying compound groups that show selective activity against pathways with a detectable coherence change in cancer. These results can serve as guidelines for selecting novel agents that have the potential to specifically target a particular pathway that has relevance in cancer.
Journal of Medicinal Chemistry	Targeting changes in cancer: assessing pathway stability by comparing pathway gene expression coherence levels in tumor and normal tissues. Huang R, Wallqvist A, Covell DG. This paper examines two biological models of anticancer activity, cytotoxicity and hollow fiber (HF) activity, for chemotherapeutic agents evaluated as part of the National Cancer Institute's (NCI's) drug screening effort. Our analysis proposes strategies to globally assess compounds tested in the NCI's 60-cell (NCI60) in vitro anticancer screen in terms of structural features, biological activity, target specificity, and mechanism of action by data integration via our self-organizing maps of structural and biological response patterns. We have built statistical models to predict compound potency and HF activity based on physicochemical properties. Our results find that it is the combination of different structural properties that determines a compound's biological activity. A direct correlation is also found between compound potency and specificity, indicating that specific targeting, rather than promiscuous poisoning, gives rise to potency. Finally, we offer a strategy to exploit this relationship for future mining of novel anticancer candidates.
Journal of Chemical Information and Modeling	Evaluating Chemical Structure Similarity as an Indicator of Cellular Growth Inhibition Wallqvist A, Huang R, Thanki N, Covell DG. Chemical variations of small compounds are commonly used to probe biological systems and potentially discover lead-like compounds with selective target activity. Molecular probes are either generated by synthesis or acquired through directed searches of commercially available compound libraries. The data generated when testing the probes in various biological systems constitutes a structure/activity analysis. The ability to detect variations and classify biological responses requires the analysis of a compound in multiple assays. While the concept of a structure/activity relationship is straightforward, its implementation can vary considerably depending on the biological system under study and the probe library selected for testing. The analysis presented here will focus on the accumulated compound library used to screen for growth inhibition across the National Cancer Institute's panel of 60 tumor cells. The considerable chemical and biological diversity inherent in these data offers an opportunity to establish a quantifiable connection between chemical structure and biological activity. We find that the connection between structure and biological response is not symmetric, with biological response better at predicting chemical structure than vice versa. Structurally and functionally similar compounds can have distinguishable biological responses reflecting different mechanisms of action.
Genomics	Comprehensive analysis of pathway or functionally related gene expression in the National Cancer Institute's anticancer screen. Huang R, Wallqvist A, Covell DG. We have analyzed the level of gene coregulation, using gene expression patterns measured across the National Cancer Institute's 60 tumor cell panels (NCI(60)), in the context of predefined pathways or functional categories annotated by KEGG (Kyoto Encyclopedia of Genes and Genomes), BioCarta, and GO (Gene Ontology). Statistical methods were used to evaluate the level of gene expression coherence (coordinated expression) by comparing intra- and interpathway gene-gene correlations. Our results show that gene expression in pathways, or groups of functionally related genes, has a significantly higher level of coherence than that of a randomly selected set of genes. Transcriptional-level gene regulation appears to be on a "need to be" basis, such that pathways comprising genes encoding closely interacting proteins and pathways responsible for vital cellular processes or processes that are related to growth or proliferation, specifically in cancer cells, such as those engaged in genetic information processing, cell cycle, energy metabolism, and nucleotide metabolism, tend to be more modular (lower degree of gene sharing) and to have genes significantly more coherently expressed than most signaling and regular metabolic pathways. Hierarchical clustering of pathways based on their differential gene expression in the NCI(60) further revealed interesting interpathway communications or interactions indicative of a higher level of pathway regulation. The knowledge of the nature of gene expression regulation and biological pathways can be applied to understanding the mechanism by which small drug molecules interfere with biological systems.
Molecular Cancer Therapeutics	Drugs aimed at targeting characteristic karyotypic phenotypes of cancer cells. Wallqvist A, Huang R, Covell DG, Roschke AV, Gelhaus KS, Kirsch IR. The karyotypic features of cancer cells have not been a particular focus of anticancer drug targeting either as guidance for treatment or as specific drug targets themselves. Cancer cell lines typically have considerable, characteristic, and variable chromosomal aberrations. Here, we consider small-molecule screening data across the National Cancer Institute's 60 tumor cell line drug screening panel (NCI-60) analyzed for specific association with karyotypic variables (numerical and structural complexity and heterogeneity) determined for these same cell lines. This analysis is carried out with the aid of a self-organizing map allowing for a simultaneous assessment of all screened compounds, revealing an association between karyotypic variables and a unique part of the cytotoxic response space. Thirteen groups of compounds based on related specific chemical structural motifs are identified as possible leads for anticancer drug discovery. These compounds form distinct groups of molecules associated with relatively unexplored regions of the NCI-60 self-organizing map where anticancer agents currently standard in the clinic are not present. We suggest that compounds identified in this study may represent new classes of potential anticancer agents.
Proteins	Linking tumor cell cytotoxicity to mechanism of drug action: an integrated analysis of gene expression, small-molecule screening and structural databases. Covell DG, Wallqvist A, Huang R, Thanki N, Rabow AA, Lu XJ. An integrated, bioinformatic analysis of three databases comprising tumor-cell-based small molecule screening data, gene expression measurements, and PDB (Protein Data Bank) ligand-target structures has been developed for probing mechanism of drug action (MOA). Clustering analysis of GI50 profiles for the NCI's database of compounds screened across a panel of tumor cells (NCI60) was used to select a subset of unique cytotoxic responses for about 4000 small molecules. Drug-gene-PDB relationships for this test set were examined by correlative analysis of cytotoxic response and differential gene expression profiles within the NCI60 and structural comparisons with known ligand-target crystallographic complexes. A survey of molecular features within these compounds finds thirteen conserved Compound Classes, each class exhibiting chemical features important for interactions with a variety of biological targets. Protein targets for an additional twelve Compound Classes could be directly assigned using drug-protein interactions observed in the crystallographic database. Results from the analysis of constitutive gene expressions established a clear connection between chemo-resistance and overexpression of gene families associated with the extracellular matrix, cytoskeletal organization, and xenobiotic metabolism. Conversely, chemo-sensitivity implicated overexpression of gene families involved in homeostatic functions of nucleic acid repair, aryl hydrocarbon metabolism, heat shock response, proteasome degradation and apoptosis. Correlations between chemo-responsiveness and differential gene expressions identified chemotypes with nonselective (i.e., many) molecular targets from those likely to have selective (i.e., few) molecular targets. Applications of data mining strategies that jointly utilize tumor cell screening, genomic, and structural data are presented for hypotheses generation and identifying novel anticancer candidates.
The Pharmacogenomics Journal	Linking pathway gene expressions to the growth inhibition response from the National Cancer Institute's anticancer screen and drug mechanism of action. Huang R, Wallqvist A, Thanki N, Covell DG. Novel strategies are proposed to quantitatively analyze and relate biological pathways to drug responses using gene expression and small-molecule growth inhibition data (GI(50)) derived from the National Cancer Institute's 60 cancer cells (NCI(60)). We have annotated groups of drug GI(50) responses with pathways defined by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and BioCarta, and functional categories defined by Gene Ontology (GO), through correlations between pathway gene expression patterns and drug GI(50) profiles. Drug-gene-pathway relationships may then be utilized to find drug targets or target-specific drugs. Significantly correlated pathways and the gene products involved represent interesting targets for further exploration, whereas drugs that are significantly correlated with only certain pathways are more likely to be target specific. Separate pathway clustering finds that pathways engaged in the same biological process tend to have similar drug correlation patterns. The biological and statistical significances of our method are established by comparison to known small-molecule inhibitor-gene target relationships reported in the literature and by standard randomization procedures. The results of our pathway, gene expression and drug-induced growth inhibition associations, can serve as a basis for proposing testable hypotheses about potential anticancer drugs, their targets, and mechanisms of action.
Biochemical Pharmacology	Anticancer metal compounds in NCI's tumor-screening database: putative mode of action. Huang R, Wallqvist A, Covell DG Clustering analysis of tumor cell cytotoxicity profiles for the National Cancer Institute (NCI)'s open compound repository has been used to catalog over 1100 metal or metalloid containing compounds with potential anticancer activity. The molecular features and corresponding reactivity of these compounds have been analyzed in terms of properties of their metals, their associated organic components (ligands) and their capacity to inhibit tumor cell growth. Cytotoxic responses are influenced by both the identity of the metal and the properties of its coordination ligand, with clear associations between structural similarities and cytotoxicity. Assignments of mechanisms of action (MOAs) for these compounds could be segregated into four broad response classes according to preference for binding to biological sulfhydryl groups, chelation, generation of reactive oxygen species (ROS), and production of lipophilic ions. Correlations between specific cytotoxic responses and differential gene expression profiles within the NCI's tumor cell panel serve as a validation for candidate biological targets and putative MOA classes. In addition, specific sensitivity toward subsets of metal containing agents has been found for certain tumor cell panels. Taken together, our results expand the knowledge base available for evaluating, designing and developing new metal-based anticancer drugs that may provide the basis for target-specific therapeutics.
Anticancer Drugs	Cytotoxicity of RH1: NAD(P)H:quinone acceptor oxidoreductase (NQO1)-independent oxidative stress and apoptosis induction. Tudor G, Alley M, Nelson CM, Huang R, Covell DG, Gutierrez P, Sausville EA. The elevated expression of the flavoprotein NAD(P)H:quinone acceptor oxidoreductase (NQO1) (EC 1.6.99.2) in many human solid tumors, along with its ability to activate quinone-based anticancer agents, makes it an excellent target for enzyme-directed drug development. Previous studies have shown a significant statistical correlation between NQO1 enzymatic activity and the cytotoxicity of certain antitumor quinones. RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], presently in late preclinical and entering early clinical development, has been previously considered to be an excellent substrate for activation by NQO1. In this study we investigate the cytotoxicity of RH1 in cell lines selected from the NCI's 60 tumor cell line panel, expressing varying levels of NQO1 activity. Exposure time- and concentration-dependent cytotoxicity was seen, apparently independent from levels of NQO1 activity in these cells. Furthermore, the NQO1 inhibitor dicoumarol had no impact on the sensitivity profiles of RH1 response. The HL-60 myeloid leukemia cells, which do not have detectable NQO1 activity, were further investigated. RH1 treatment of HL-60 cells generated high levels of free radicals, which was accompanied by robust redox cycling, oxygen consumption and induction of apoptosis. These results are in agreement with previous data suggesting that, in addition to its activation by NQO1, RH1-induced cytotoxicity might involve alternative pathways for activation of this compound. Furthermore, the high cytotoxicity of RH1 in the leukemia/lymphoma subpanel of the NCI in vitro cell line screen would suggest an empirical rationale for the utilization of this compound in the treatment of these malignancies.