NIH Chemical Genomics Center Research Articles

RARE-29. AZD8055 ENHANCES IN VIVO EFFICACY OF AFATINIB IN CHORDOMAS

Abstract INTRODUCTION Chordomas are rare, locally aggressive bone tumors that arise in cranial base, mobile spine, and sacrum. Currently, there are no FDA-approved therapies for chordoma patients, thus there is a high unmet need to develop effective treatments. In this study, we aim to evaluate the anti-tumor efficacy of small molecule inhibitors that target crucial oncogenic pathways in chordoma, as single agents or in combination, to identify novel therapies with the greatest translation potential. METHODS A panel of small molecule compounds that had exhibited in vitro efficacy against human chordoma cell lines or target known chordoma drivers was screened in vivo against patient-derived xenograft (PDX) models of chordoma, and their efficacy was further evaluated using chordoma cell lines and xenograft models. RESULTS The in vivo activity of compounds identified in a NIH Chemical Genomics Center screen utilizing chordoma cell lines, together with inhibitors of c-MET and PDGFR, were evaluated in PDX models of chordoma that were previously described or recently established for this study. Inhibitors of EGFR (BIBX1382, erlotinib and afatinib), c-MET (crizotinib) or mTOR (AZD8055) significantly inhibited tumor growth in vivo but did not induce tumor regression. Co-inhibition of EGFR and c-MET using erlotinib and crizotinib synergistically reduced cell viability in chordoma cell lines but did not result in enhanced in vivo activity. Co-inhibition of EGFR and mTOR pathways using afatinib and AZD8055 synergistically reduced cell viability in chordoma cell lines. Importantly, co-inhibition of EGFR and mTOR also synergistically suppressed tumor growth in vivo, showing improved disease control. CONCLUSION Single inhibition of EGFR, c-MET or mTOR suppresses chordoma growth both in vitro and in vivo. Co-inhibition of EGFR and mTOR synergistically inhibits chordoma growth in a range of preclinical models. The insights gained from our study provide a novel combination therapeutic strategy for patients with chordoma.

Advances in high-throughput screening technology for toxicology

The US Tox21 collaboration utilises a quantitative high-throughput screening (qHTS) platform to efficiently profile a large number of environmental chemicals. qHTS combined with informatics facilitates chemical prioritisation for further in-depth toxicity testing and development of computational models to predict chemical toxicity. The NIH Chemical Genomics Center (NCGC), now part of the National Center for Advancing Translational Sciences (NCATS), is a key contributor during all phases of the Tox21 collaboration, from assay development and compound screening to data analysis and model building. Since 2011, the Tox21/NCGC has been profiling the phase 2 Tox21 library of approximately 10,000 (10K) environmental chemicals and drugs. The advances in HTS assays and qHTS screens against the Tox21's and other chemical libraries are described in this review.

Inhibiting SOX11-DNA Interaction in Mantle Cell Lymphoma

Mantle Cell Lymphoma (MCL) is a fatal subtype of Non-Hodgkins Lymphoma. MCL is characterized by cell cycle dysregulation due to Cyclin D1 (CCND1) overexpression. Murine models overexpressing CCND1 do not develop B-cell proliferation. The SOX11 transcription factor is overexpressed in the majority of nodal human MCL and has been implicated in MCL pathogenesis. We have developed a transgenic Eµ-SOX11-EGFP mouse model that overexpresses SOX11 with aberrant B cell proliferation, lymphadenopathy, spenomegaly and hepatomegaly phenocopying the characteristics of patients with MCL.Using Mass Cytometry (CyTOF), we further characterized this B cell population to be CD23-, CD21/35 dim, CD138-, high surface IgM, and variable IgD expression, an immunophenotype identical to human MCL. SOX11 overexpression drives B cell receptor (BCR) signaling in murine MCL cells, assayed by phosphorylation of p-BTK and p-PLC gamma.To further the study of the role of SOX11 in MCL, compounds inhibiting the binding interaction of SOX11 and DNA are highly desirable and we aim at their discovery by using structure-based virtual screening predictions and experimental validations. No small-molecule inhibitor of SOX11-DNA binding has been developed to date and there is an unmet need in MCL therapeutics for novel and successful treatments.Transcription factors have been considered "undruggable" but since SOX11 binds the minor groove in DNA, we hypothesized that there may be pockets created by SOX11-DNA binding that could be inhibited by small molecules. We therefore built a human SOX11 homology model using the crystal structure of murine SOX4 (98% homology). We first docked molecules from the NIH Chemical Genomics Center (NCGC) Pharmaceutical Collection (NPC). From this virtual screening, 70 compounds were purchased for experimental testing. Using a fluorescence anisotropy assay, Flavitan was identified as a potential inhibitor of the SOX11-DNA interaction. Based on inferences from the predicted binding mode of Flavitan on SOX11, we performed another virtual screening of ~12 million molecules from the ZINC database followed by assaying 26 top-scoring compounds, as well as, 16 derivatives of one of them, for SOX11 binding. We thus discovered two compounds, R and T, with binding constants in the low micromolar range. Importantly, compounds R and T were found not to intercalate DNA in a Topoisomerase I based assay.To assess the biological activity of these molecules, we tested whether BCR signaling in MCL cell lines expressing SOX11 would be inhibited by treatment with these compounds. Compounds R and T significantly inhibited p-BTK as compared to vehicle control. Ibrutinib, an FDA approved BTK inhibitor, was used as a positive control in these experiments.To determine whether BCR pathway inhibition through compounds R and T would be cytotoxic in SOX11 positive MCL cell lines, we treated Z138 and JEKO cells with these small-molecules and assayed Annexin-V and PI staining 48 hours later by flow-cytometry. Our results indicate that compound R has greater single agent cytotoxic activity than compound T and Ibrutinib. Compounds R and T also showed synergistic cytotoxicity in combination with Ibrutinib in SOX11 expressing Z138 and JEKO cells, but not in JVM2, a SOX11 negative MCL cell line.In summary, through a combination of in silico predictions and experimental validation, we have identified small-molecule inhibitors of SOX11-DNA binding as candidate leads for optimization for anti-MCL therapy. DisclosuresNo relevant conflicts of interest to declare.

Predictive Modeling of Estrogen Receptor Binding Agents Using Advanced Cheminformatics Tools and Massive Public Data

Estrogen receptors (ERα) are a critical target for drug design as well as a potential source of toxicity when activated unintentionally. Thus, evaluating potential ERα binding agents is critical in both drug discovery and chemical toxicity areas. Using computational tools, e.g., Quantitative Structure-Activity Relationship (QSAR) models, can predict potential ERα binding agents before chemical synthesis. The purpose of this project was to develop enhanced predictive models of ERα binding agents by utilizing advanced cheminformatics tools that can integrate publicly available bioassay data. The initial ERα binding agent data set, consisting of 446 binders and 8307 non-binders, was obtained from the Tox21 Challenge project organized by the NIH Chemical Genomics Center (NCGC). After removing the duplicates and inorganic compounds, this data set was used to create a training set (259 binders and 259 non-binders). This training set was used to develop QSAR models using chemical descriptors. The resulting models were then used to predict the binding activity of 264 external compounds, which were available to us after the models were developed. The cross-validation results of training set [Correct Classification Rate (CCR) = 0.72] were much higher than the external predictivity of the unknown compounds (CCR = 0.59). To improve the conventional QSAR models, all compounds in the training set were used to search PubChem and generate a profile of their biological responses across thousands of bioassays. The most important bioassays were prioritized to generate a similarity index that was used to calculate the biosimilarity score between each two compounds. The nearest neighbors for each compound within the set were then identified and its ERα binding potential was predicted by its nearest neighbors in the training set. The hybrid model performance (CCR = 0.94 for cross validation; CCR = 0.68 for external prediction) showed significant improvement over the original QSAR models, particularly for the activity cliffs that induce prediction errors. The results of this study indicate that the response profile of chemicals from public data provides useful information for modeling and evaluation purposes. The public big data resources should be considered along with chemical structure information when predicting new compounds, such as unknown ERα binding agents.

Quantitative high-throughput identification of drugs as modulators of human constitutive androstane receptor.

The constitutive androstane receptor (CAR, NR1I3) plays a key role in governing the transcription of numerous hepatic genes that involve xenobiotic metabolism/clearance, energy homeostasis, and cell proliferation. Thus, identification of novel human CAR (hCAR) modulators may not only enhance early prediction of drug-drug interactions but also offer potentially novel therapeutics for diseases such as metabolic disorders and cancer. In this study, we have generated a double stable cell line expressing both hCAR and a CYP2B6-driven luciferase reporter for quantitative high-throughput screening (qHTS) of hCAR modulators. Approximately 2800 compounds from the NIH Chemical Genomics Center Pharmaceutical Collection were screened employing both the activation and deactivation modes of the qHTS. Activators (115) and deactivators (152) of hCAR were identified from the primary qHTS, among which 10 agonists and 10 antagonists were further validated in the physiologically relevant human primary hepatocytes for compound-mediated hCAR nuclear translocation and target gene expression. Collectively, our results reveal that hCAR modulators can be efficiently identified through this newly established qHTS assay. Profiling drug collections for hCAR activity would facilitate the prediction of metabolism-based drug-drug interactions, and may lead to the identification of potential novel therapeutics.

Identification of known drugs targeting the endoplasmic reticulum stress response.

The endoplasmic reticulum (ER), a multifunctional organelle, plays a central role in cellular signaling, development, and stress response. Dysregulation of ER homeostasis has been associated with human diseases, such as cancer, inflammation, and diabetes. A broad spectrum of stressful stimuli including hypoxia as well as a variety of pharmacological agents can lead to the ER stress response. In this study, we have developed a stable ER stress reporter cell line that stably expresses a β-lactamase reporter gene under the control of the ER stress response element (ESRE) present in the glucose-regulated protein, 78kDa (GRP78) gene promoter. This assay has been optimized and miniaturized into a 1536-well plate format. In order to identify clinically used drugs that induce ER stress response, we screened approximately 2800 drugs from the NIH Chemical Genomics Center Pharmaceutical Collection (NPC library) using a quantitative high-throughput screening (qHTS) platform. From this study, we have identified several known ER stress inducers, such as 17-AAG (via HSP90 inhibition), as well as several novel ER stress inducers such as AMI-193 and spiperone. The confirmed drugs were further studied for their effects on the phosphorylation of eukaryotic initiation factor 2α (eIF2α), the X-box-binding protein (XBP1) splicing, and GRP78 gene expression. These results suggest that the ER stress inducers identified from the NPC library using the qHTS approach could shed new lights on the potential therapeutic targets of these drugs.

Abstract 5361: Development of targeted inhibitors against RecQ1 helicase

Abstract Background: RecQ helicases are a ubiquitous family of DNA unwinding enzymes involved in the maintenance of chromosome stability. RecQ1 helicase genomic variations have an important prognostic role and represents a therapeutic target in pancreatic cancer [JCO 2006; 24 (11) 1720-1728]. Pubchem data indicated potential RecQ1 activity >600 compounds using a qHTS RecQ1 assay (NIH Chemical Genomics Center). Methods: We applied a set of filters including molecular weight, potency and Hill curve fitting and also removed those interfering with biological assay to identify sixteen compounds for studying RecQ1 inhibition. Two compounds showed strong inhibition of RecQ1 helicase activity at 50 µM and at IC50 < 5 µM, respectively. From a large library of compounds, eight potential inhibitors were synthesized in three simple steps. First, sulfonylation of various amines with 3-Nitrobenzenesulfonyl chloride was performed followed by reduction of the nitro group into an amine. Finally, acylation with variety of acid chlorides resulted in the final library of RecQ1 analogs. These agents were studied for RecQ1 helicase activity assay with the radiolabeled partial duplex substrate in vitro. The RecQ1 inhibitor candidate compounds were investigated in pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, PANC-1, and Mia-Paca2) for anti-tumor efficacy using cell proliferation assay with CellTiter-Glo® luminescence. Combination studies with test compounds and poly (ADP ribose) polymerase (PARP) inhibitor olaparib and topoisomerase Inhibitor SN38 were performed. Results: Though none of the compounds showed the same level of activity of the parent compounds, we were able to demonstrate a modest level of helicase activity with several. Two compounds displayed strong inhibition of pancreatic cancer cell proliferation. Two other compounds displayed synergistic inhibition with SN38 or olaparib of pancreatic cancer cell proliferation and induced DNA damage. Conclusions: Synthesis of RecQ1 inhibitors is feasible and shows potential for anti-tumor efficacy. Note: This abstract was not presented at the meeting. Citation Format: Mingxin Zuo, David Maxwell, Basvoju A. Bhanu Prasad, Zhenghong Peng, William Bornmann, Milind M. Javle. Development of targeted inhibitors against RecQ1 helicase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5361. doi:10.1158/1538-7445.AM2014-5361

Structural Basis for Inactivation of Giardia lamblia Carbamate Kinase by Disulfiram

Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.

A Novel Chordoma Xenograft Allows In Vivo Drug Testing and Reveals the Importance of NF-κB Signaling in Chordoma Biology

Chordoma is a rare primary bone malignancy that arises in the skull base, spine and sacrum and originates from remnants of the notochord. These tumors are typically resistant to conventional chemotherapy, and to date there are no FDA-approved agents to treat chordoma. The lack of in vivo models of chordoma has impeded the development of new therapies for this tumor. Primary tumor from a sacral chordoma was xenografted into NOD/SCID/IL-2R γ-null mice. The xenograft is serially transplantable and was characterized by both gene expression analysis and whole genome SNP genotyping. The NIH Chemical Genomics Center performed high-throughput screening of 2,816 compounds using two established chordoma cell lines, U-CH1 and U-CH2B. The screen yielded several compounds that showed activity and two, sunitinib and bortezomib, were tested in the xenograft. Both agents slowed the growth of the xenograft tumor. Sensitivity to an inhibitor of IκB, as well as inhibition of an NF-κB gene expression signature demonstrated the importance of NF-κB signaling for chordoma growth. This serially transplantable chordoma xenograft is thus a practical model to study chordomas and perform in vivo preclinical drug testing.

Systematic Study of Mitochondrial Toxicity of Environmental Chemicals Using Quantitative High Throughput Screening

A goal of the Tox21 program is to transit toxicity testing from traditional in vivo models to in vitro assays that assess how chemicals affect cellular responses and toxicity pathways. A critical contribution of the NIH Chemical Genomics center (NCGC) to the Tox21 program is the implementation of a quantitative high throughput screening (qHTS) approach, using cell- and biochemical-based assays to generate toxicological profiles for thousands of environmental compounds. Here, we evaluated the effect of chemical compounds on mitochondrial membrane potential in HepG2 cells by screening a library of 1,408 compounds provided by the National Toxicology Program (NTP) in a qHTS platform. Compounds were screened over 14 concentrations, and results showed that 91 and 88 compounds disrupted mitochondrial membrane potential after treatment for 1 or 5 h, respectively. Seventy-six compounds active at both time points were clustered by structural similarity, producing 11 clusters and 23 singletons. Thirty-eight compounds covering most of the active chemical space were more extensively evaluated. Thirty-six of the 38 compounds were confirmed to disrupt mitochondrial membrane potential using a fluorescence plate reader, and 35 were confirmed using a high content imaging approach. Among the 38 compounds, 4 and 6 induced LDH release, a measure of cytotoxicity, at 1 or 5 h, respectively. Compounds were further assessed for mechanism of action (MOA) by measuring changes in oxygen consumption rate, which enabled the identification of 20 compounds as uncouplers. This comprehensive approach allows for the evaluation of thousands of environmental chemicals for mitochondrial toxicity and identification of possible MOAs.

Tox21 to Date: Steps toward Modernizing Human Hazard Characterization

A new review in EHP describes the first phase of the federal government’s Tox21 collaboration, which is attempting to establish alternatives to the time-consuming and expensive animal testing used to evaluate chemical toxicity.1 The review details important strides investigators have made in demonstrating the usefulness of high-throughput data for identifying potential hazards and prioritizing chemicals for more extensive testing. In the long run, the program may help “protect people from exposure to a much greater number of harmful chemicals than is possible based on current testing methods,” says lead author Raymond Tice, chief of the Biomolecular Screening Branch of the NIEHS’s National Toxicology Program (NTP) Division. Officially begun five years ago, Tox21 is an outgrowth of U.S. federal interagency collaborations initiated in 2005 to explore the utility of a high-throughput screening program. It includes scientists at NTP and other NIH entities as well as the Environmental Protection Agency’s (EPA) National Center for Computational Toxicology. In 2010 the Food and Drug Administration (FDA) joined Tox21, but the new report focuses mainly on the efforts of the original Tox21 partners. “At the moment, I think Tox21 is clearly the most important effort to revamp toxicology,” says Thomas Hartung, who directs the Center for Alternatives to Animal Testing (CAAT), part of the Johns Hopkins Bloomberg School of Public Health. He considers the number of substances included, the quality assurance applied, and the public availability of data from Tox21 unique. Tox21 mainly uses cell-based and biochemical in vitro tests, although some testing is conducted with lower animals such as Danio rerio (zebrafish) and Caenorhabditis elegans (a nematode species). In the first phase of testing at the NIH Chemical Genomics Center, scientists screened up to 2,800 compounds with about 70 high-throughput assays.1 Tice points out that the number of compounds screened is relatively small from the perspective of the tens of thousands that need screening. But he says this number was adequate to demonstrate the utility of the approach and to identify assay-specific limitations. Phase I focused on evaluating how assays performed and the extent to which they could be optimized. The percentage of compounds classified as active by the assays ranged from 0.07% for an epigenetics cell-based assay to 41% for an assay that tested the ability of compounds to interact with the cytochrome P450 enzyme CYP1A2, which is involved in the metabolism of foreign substances.1 Phase I of Tox21 has provided insight into possible ways of optimizing highthroughput screening. Lorenz Rhomberg, a principal for the environmental consulting firm Gradient, says he believes the review is most important for describing the limitations of Tox21’s first phase, especially the fact that although thousands of chemicals can be screened in a single assay, it is difficult to interpret the patterns of changes in terms of particular pathways of toxicity generation. In addition, Rhomberg says, the chosen assays may not cover all the toxicological outcomes of potential concern that are currently evaluated in whole-animal studies. Problems identified during Phase I are being addressed in Phase II, which is now in full swing. According to the authors, the primary limitation of Tox21’s quantitative high-throughput testing in Phase I was that the biological output of each of the assays currently used was generally limited to one or two “signals” (measurable responses). Investigators have begun developing cell-based assays with hundreds to thousands of signals. And because Tice and his colleagues discovered that some of the compounds they purchased for testing had inaccurate information on their certificates of analysis or were unstable during storage and use, Phase II includes analysis of test chemicals for identity, purity, and stability. Another issue that came to light was what Tice calls “compound carryover”—some compounds can be transferred from one set of plates to another by the pin tools used in the quantitative high-throughput screening system. To compensate for this problem, the investigators are screening each substance at least three times in Phase II, changing the compounds’ well locations in each run. Another important limitation that Tice and his colleagues are trying to remedy for Tox21’s next phase is the inability of most cell lines to mimic in vivo metabolism of chemicals, which can produce intermediates that are more biologically active than the parent compound. Because the liver is the primary site where such reactive intermediates are metabolized, the Tox21 researchers are investigating whether cells with hepatocyte functionality can be used to assess a chemical’s ability to interact with multiple cellular pathways. The investigators stress that they still face many obstacles before they can definitively show that Tox21 will indeed transform toxicity testing. But they nonetheless believe the data and information they are obtaining is “foretelling the future of toxicology.”

Structure Based Model for the Prediction of Phospholipidosis Induction Potential of Small Molecules

Drug-induced phospholipidosis (PLD), characterized by an intracellular accumulation of phospholipids and formation of concentric lamellar bodies, has raised concerns in the drug discovery community, due to its potential adverse effects. To evaluate the PLD induction potential, 4,161 nonredundant drug-like molecules from the National Institutes of Health Chemical Genomics Center (NCGC) Pharmaceutical Collection (NPC), the Library of Pharmacologically Active Compounds (LOPAC), and the Tocris Biosciences collection were screened in a quantitative high-throughput screening (qHTS) format. The potential of drug-lipid complex formation can be linked directly to the structures of drug molecules, and many PLD inducing drugs were found to share common structural features. Support vector machine (SVM) models were constructed by using customized atom types or Molecular Operating Environment (MOE) 2D descriptors as structural descriptors. Either the compounds from LOPAC or randomly selected from the entire data set were used as the training set. The impact of training data with biased structural features and the impact of molecule descriptors emphasizing whole-molecule properties or detailed functional groups at the atom level on model performance were analyzed and discussed. Rebalancing strategies were applied to improve the predictive power of the SVM models. Using the undersampling method, the consensus model using one-third of the compounds randomly selected from the data set as the training set achieved high accuracy of 0.90 in predicting the remaining two-thirds of the compounds constituting the test set, as measured by the area under the receiver operator characteristic curve (AUC-ROC).

Establishment of a chordoma xenograft and in vivo testing of compounds identified by high-throughput screening.

10025 Background: Chordoma is a rare primary bone malignancy that arises in the skull base, spine and sacrum and originates from remnants of the notochord. Therapy primarily consists of surgical resection and radiation. These tumors are typically resistant to conventional chemotherapy, and to date there are no FDA-approved agents for chordoma. The lack of in vivo models of chordoma has impeded the development of new therapies for this tumor. Methods: Primary tumor from a classic sacral chordoma was obtained, immediately processed into a single cell suspension and injected in to the parasacral area of a NOD/SCID/IL-2R gamma-null mouse, and tumor grew after 3 months. The NIH Chemical Genomics Center performed high-throughput screening of 2,816 compounds. Two established chordoma cell lines, U-CH1 and UCH2B, were treated and cell viability measured by CellTiter-Glo assay. Cells were incubated for 48 hours with drug concentrations ranging from 0.5nM to 46uM. The screen yielded several compounds that showed activity and two were tested in the xenograft. Results: We have established a xenograft model of dedifferentiated chordoma. High-throughput screening of compounds identified compounds that show activity against chordoma cell lines. In vivo testing of two identified compounds showed a dramatic reduction of tumor growth. Conclusions: We have established a xenograft model of dedifferentiated chordoma. High-throughput screening of compounds identified compounds that show activity against chordoma cell lines. In vivo testing of two identified compounds showed a dramatic reduction of tumor growth.

Abstract LB-110: Establishment of a chordoma xenograft and in vivo testing of compounds identified by high-throughput screening

Abstract Chordoma is a rare primary bone malignancy that arises in the skull base, spine and sacrum and originates from remnants of the notochord. Therapy primarily consists of surgical resection and radiation. These tumors are typically resistant to conventional chemotherapy, and to date there are no FDA-approved agents for chordoma. The lack of in vivo models of chordoma has impeded the development of new therapies for this tumor. We have established a chordoma xenograft growing in NOD/SCID/IL-2R gamma-null mice. The histology of the xenograft is consistent with a dedifferentiated chordoma. The xenograft expresses the chordoma marker T brachyury, which is aberrantly localized in the cytoplasm. SNP array shows loss of heterozygosity at the p16 locus, and gene expression profiling shows high expression of numerous cancer-related genes, such as ROS1 and CPA4 The NIH Chemical Genomics Center performed high-throughput screening of 2,816 compounds. Two established chordoma cell lines, U-CH1 and UCH2B, were treated and cell viability measured by CellTiter-Glo assay. Cells were incubated for 48 hours with drug concentrations ranging from 0.5nM to 46uM. The screen yielded several compounds that showed activity and two were tested in the xenograft. Compared with mice treated with vehicle alone, both drugs showed a dramatic inhibition of tumor growth. In conclusion, we have established a xenograft model of dedifferentiated chordoma. High-throughput screening of compounds identified compounds that show activity against chordoma. In vivo testing of two identified compounds showed a dramatic reduction of tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-110. doi:1538-7445.AM2012-LB-110

Repurposing Approved and Abandoned Drugs for the Treatment and Prevention of Cancer through Public–Private Partnership

The Learning Collaborative (TLC) is a dedicated collaboration between the NIH Chemical Genomics Center (NCGC) and its Therapeutics for Rare and Neglected Diseases (TRND) program, The Leukemia & Lymphoma Society (LLS), and KUCC to discover and develop new drug therapies for rare blood cancers. TLC is creating a pipeline of new therapies to treat blood cancers using less traditional forms of drug discovery and development. The Learning Collaborative is focused on both the discovery of new blood cancer treatments as well as identifying new uses for approved and abandoned drugs, termed drug repurposing. Repurposing approved and abandoned drugs for cancer represents an opportunity to rapidly advance to patients promising drug therapies by capitalizing on existing data and experience. TLC9s first project, which is also a pilot project of the TRND program, focuses on repurposing an existing small molecule drug, auranofin, initially approved to treat rheumatoid arthritis in the mid-1980s. Auranofin is being evaluated by The Learning Collaborative as a treatment for relapsed chronic lymphocytic leukemia (CLL). In just two years after the identification of auranofin9s activity in CLL cells, this project has entered clinical trials. The Learning Collaborative will engage a for-profit partner to advance auranofin through late stage drug development, registration and commercialization. TLC has the unique ability to de-risk promising drug therapies, like auranofin, using an innovative approach to quickly determine proof-of-concept. The partnership is named The Learning Collaborative because this model is scalable, applicable across a wide range of therapeutic areas, and replicable by organizations committed to innovative models of collaboration. TLC continues to explore solutions to barriers that include addressing regulatory science issues (with particular focus on repurposing off patent and abandoned drugs for rare and neglected diseases) and defining exclusivity path(s) to interest future for-profit partners.

Leveraging the Promise of Chemical Genomics

Leveraging the Promise of Chemical Genomics

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 17,000 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 data sets 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.

Abstract 1378: Identification and characterization of small molecule inhibitors targeting Eya phosphatase activity for anti-breast cancer therapy

Abstract Eya proteins are transcriptional co-activators of the homeobox gene Six1 and are also unique protein tyrosine phosphatases. Six1 and Eya2 are required for normal development but are down-regulated in most adult tissues. However, Six1 and Eya are over-expressed in a large number of breast tumors and play a causal role in the initiation and development of these tumors. The phosphatase activity of Eya was shown to be important for the transformation, migration, invasion, and metastasis of breast cancer cells. Eya's phosphatase activity therefore provides an attractive enzymatic target for anti-breast cancer therapy. In addition, Eyas also direct cells to a DNA-damage-repair pathway instead of an apoptotic pathway upon DNA damage through the dephosphorylation of histone H2AX. Inhibiting Eya phosphatase activity can therefore potentially sensitize tumor tissue to radiation or chemotherapy treatment. We developed an HTS assay to identify small molecule inhibitors of Eya's phosphatase activity. In collaboration with the NIH Chemical Genomics Center, we have screened over 300,000 compounds and identified several promising lead compounds. These hits are currently being evaluated for their potency, specificity, and mechanism of action using biochemical, structural, and cell-based assays. We believe that Eya is a significant target for cancer therapy through sensitizing cancer cells to radiation and chemotherapy and through the inhibition of Six1-mediated tumorigenesis and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1378. doi:10.1158/1538-7445.AM2011-1378

Discovery of new antimalarial chemotypes through chemical methodology and library development

In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC(50)'s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.

The future of toxicity testing: a focus on in vitro methods using a quantitative high-throughput screening platform

The US Tox21 collaborative program represents a paradigm shift in toxicity testing of chemical compounds from traditional in vivo tests to less expensive and higher throughput in vitro methods to prioritize compounds for further study, identify mechanisms of action and ultimately develop predictive models for adverse health effects in humans. The NIH Chemical Genomics Center (NCGC) is an integral component of the Tox21 collaboration owing to its quantitative high-throughput screening (qHTS) paradigm, in which titration-based screening is used to profile hundreds of thousands of compounds per week. Here, we describe the Tox21 collaboration, qHTS-based compound testing and the various Tox21 screening assays that have been validated and tested at the NCGC to date.