Development of Assays for High-Throughput Drug Screening (R01)
The GCOB staff has participated in managing cancer-related grant applications submitted in response to Program Announcements, PA-04-068, PA-07-054, and PA-07-320 entitled Development of Assays for High- Throughput Drug Screening (R01). The purpose of this initiative is to support 3-year R01 grants for the development of innovative assays that may ultimately be adapted for automated screening of thousands of compounds. The assay should aim to identify new tools for basic research or promising new avenues for therapeutics development, especially in areas related to the missions of the participating Institutes, NCI, NIAID, NIDDK, and NIMH. As of December 2008, the NCI has made the following seventeen awards:
R01 CA113473
DAN CANAANI, TEL AVIV UNIVERSITY
Synthetic Lethality Based Assay for HTS Drug Discovery
DESCRIPTION (provided by applicant): About one-third of breast cancer patients have estrogen receptor alpha-negative disease. Another group of breast cancer patients lose expression of their tumor's receptor during the course of disease progression. Compared to patients with estrogen receptor positive tumors, these patients do not respond to endocrine therapy, and tend to have more aggressive disease with earlier recurrence, resulting in less favorable prognosis. We have recently shown that by applying to cultured human breast cancer cells a methodology for chemical synthetic lethality screening, previously established in our laboratory in human and mouse cells, one can retrieve compounds that are synergistic lethal with ERalpha-deficiency. It is the subject of this application, to develop the methodology and the tools to a level that would enable the HTS of chemicals and the identification of synthetic lethal compounds in this medically important system. The specific aims of the project are: (i) To determine the dose response for three chemicals mediating ERa-episomal plasmid retention, (ii) To determine the false negative rate of these compounds, (iii) To test the false positive rate under conditions of medium alone, as compared to medium supplemented with unrelated bioactive molecules, (iv) To develop the statistical procedures for the HTS. (v) To screen by this method a diverse collection of approximately 750 bioactive molecules, (vi) To perform a counter screen to rule out artifacts, (vii) To evaluate the "hit" compounds by an independent assay, testing for differential toxicity as related to the genetic milieu. The poor prognosis of breast cancer patients with estrogen receptor a-negative disease calls for development of a selective, non-toxic adjuvant systemic therapy for this group. If successful, this novel approach will allow the screening of thousands of compounds derived from synthetic or natural resources, to identify chemicals selectively lethal to ERalpha-deficient tumor cells. This test case could then stimulate the dissemination of this methodology, resulting in wider spectrum of gene-related drugs towards other human diseases as well. Moreover, it should enable the implementation of the multigene shRNA expression libraries to this system, leading to the identification of gene targets synthetic lethal with ERa-deficiency in breast carcinomas.
R01 CA113656
WEI CHEN, DUKE UNIVERSITY
Innovative Assays for Oncogenic Hedgehog Signaling
DESCRIPTION (provided by applicant): Innovative assays for oncogenic hedgehog signaling Basal Cell Carcinoma (BCC) along with pancreatic cancer (PC) and medulloblastoma (Med) have been associated with mutations in Smoothened (Smo), or with dysregulation of the Smo modulators Patched (Ptc) and Hedgehog (Hg). Intracellular signals generated from the interplay among these three proteins are fundamental to normal organ development and tissue patterning, and suggest that abnormalities in their regulation may augment or underlie the metastatic potential observed in many cancerous tumors. In contrast to the classical G protein-coupled receptors (GPCRs), Smo signaling has not been conclusively demonstrated to occur through conventional G protein mediated pathways. However, Smo activation does result in arrestin recruitment, a feature also exhibited by GPCRs when they desensitize after agonist activation. Thus, changes in arrestin recruitment to Smo can serve as a measure of its activation or deactivation by ligands, or as a consequence of the pharmacological modulation of its upstream regulatory partners, Ptc and Hg. We propose to construct cell-based assay systems composed of Smo receptors and arrestin-fluorescent proteins or Smo-fluorescent protein chimeras that can be used in the high-throughput screening of large compound libraries. The specific aims of this proposal are (I) Identification and pharmacological targeting of Smo/Ptc/Hg signaling components underlying malignancies including BCC, PC, and Med. A. To establish an assay protocol using cell lines with each permanently expressing components of the Hg pathway including Smo and a fluorescent arrestin, or a chimeric fluorescent Smo in which the efficacy of compounds for affecting Smo activity can be evaluated. B. To screen known upstream signaling partners such as Ptc for their effects on Smo activity. (II) Identification of a putative endogenous ligand for Smo by the screening of a Natural Compound Library of Small Molecules. (III) To study the effects of the screened compounds antagonizing Smo activity on the growth inhibition of BCC, PC, and Med cell lines. Relevance to Public Health: The availability of a cell-based model in which large numbers of compounds can be quickly screened for their efficacy in modulating Smo/Ptc/Hg signaling would make an important contribution in both developmental research and cancer therapeutics. In particular it should be possible to identify critical proteins modulating altered signaling pathways in pancreatic adenocarcinoma and design pharmacologic strategies to target them for therapeutic benefit.
R01 CA113677
LYNNE J. REGAN, YALE UNIVERSITY
HTP Assays of Inhibitors of Protein-Protein Interactions
DESCRIPTION (provided by applicant): Heat shock protein 90 (Hsp90) is a "molecular chaperone" whose activity is essential for the folding and cellular stability of a number of mutated, chimeric or over-expressed proteins that promote the survival andproliferation of cancer cells. Examples of proteins whose activity is Hsp90-dependent include mutated p53, Brc-Abl, Raf-1, Src, and ErbB2. Inhibition of HspQO activity is a potential route to broad range anti-cancer agents. Geldanamycin, a small molecule that inhibits Hsp90 activity, is currently in clinical trials as a potential anti-cancer agent. Hsp90 does not function in isolation, but rather is part of a complex machinery that involves several other proteins and "co-chaperpnes". Two chaperones, Hsp70 and Hsp90, act sequentially on certain client proteins to generate the mature, active, form of the proteins. Hsp Organizing Protein (HOP) has independent binding sites for Hsp70 and Hsp90, and by binding these chaperones simultaneously, it functions to bring them into physical proximity. The interaction of HOP with Hsp70 and Hsp90 is very well characterized. HOP has two independent tetratricopeptide repeats (TRP). TPR1 binds specifically to the C-terminal peptide of Hsp70 and TPR2A binds specifically to the C-terminal peptide ofHsp90. We propose to develop HTP assays to identify specific inhibitors of the interaction of Hsp 90 withTPR2A. Inhibition of this interaction will prevent the Hsp70-Hsp90 dependent folding sequence, a nd thus lead to oncogene degradation. It has already been demonstrated in mammalian cells that preventing its interaction with HOP inhibits Hsp90 activity. We will develop fluorescent-based assays, in vitro and in vivo to identify small molecules that specifically disrupt the Hsp90-HOP interaction. We will follow-up potential leads by assaying their ability to reverse the oncogenic phenotype in HTP morphology assays. Promising compounds will go forward into trials in collaboration with other researchers.
R01 CA114115
JOSEPH A. MONFORTE, ALTHEA TECHNOLOGIES, INC.
High Throughput PCR-Based Gene Expression Screening
DESCRIPTION (provided by applicant): The exponential growth in our knowledgebase of cancer genetics has led to the identification of a large array of genes, proteins and pathways that potentially play a central role in the carcinogenesis and may be potential targets for therapeutic intervention. The challenge now is to delve into how different compounds and compound classes' influence these genes. An enormous number of different cellular activities can be tracked at the gene expression level, including the ability to track in detail the response of cells and tissues to the introduction of a pharmacological agent. For this reason, gene expression analysis can be used to provide detailed and broad insight into the on and off-target mechanistic activities of existing and potential therapeutics. Proposed here is the demonstration and development of a new production-oriented, high throughput, PCR-based approach to gene expression analysis. The approach builds off of the fundamental strengths of PCR and adds a high level of multiplexing, 15-35 genes per PCR reaction. The principal goal of this proposal is to develop and demonstrate the value of a multiplexed PCR approach for use as a compound library gene expression screen. In order to achieve this goal we propose to (a) develop and validate 3 multiplex assays (genotoxicity, nephrotoxicity and HDAC-targeting antitumor activity), (b) develop protocols for high throughput screening, and (c) demonstrate the utility by screening the Gen- Plus and NCI chemical diversity set and performing a lead optimization study on HDAC-targeting candidates. The protocols and assays developed and the data from these demonstration screening studies will be made publicly available.
R01 CA114116
MARK T. BEDFORD, UNIVERSITY OF TEXAS MD ANDERSON CANCER CENTER
Small Molecule Regulators of Arginine Methyltransferases
DESCRIPTION (provided by applicant): Arginine methylation is a common posttranslational modification that can regulate protein function. The main pool of proteins that are arginine methylated possess RNA binding properties. In addition, enzymes that facilitate histone acetylation (CBP/p300) and histones themselves are arginine methylated - thus implicating this posttranslational modification in chromatin remodeling and transcriptional regulation. Seven protein arginine N-methyltransferases (PRMTs) have been identified in mammalian cells: PRMT1, PRMT2, PRMTS, PRMT4/CARM1, PRMT5/JBP1, PRMT6 and PRMT7. Using a chemical library and in vitro experimental approaches, we propose to develop pilot screens to identify small molecules that will perturb arginine methylation in mammalian cells. The screens will be developed in such a way that they can be automated for subsequent high throughput chemical screening (HTS) by the planned NIH-funded HTS screening centers. The PRMTs are novel drug targets that regulate protein interactions and transcription/translation. Thus, the identification of small molecule regulators of arginine methylation will provide lead compounds for future drug development, targeting cancer and possibly other disease states. Most importantly, the PRMTs are a newly identified family of enzymes that have not yet been tapped as "drugable" targets.
R01 CA114393
BRIAN S. J. BLAGG, UNIVERSITY OF KANSAS LAWRENCE
HTS Assays for Hsp90 Inhibitors
DESCRIPTION (provided by applicant): Hsp90 is a molecular chaperone responsible for folding nascent polypeptides into their biologically active, three-dimensional conformations. Disruption of the Hsp90 protein folding process results in the simultaneous inhibition of multiple enzymes that are essential for malignant cell growth. In fact, proteins represented in all six hallmarks of cancer are dependent on Hsp90 for conformational activation, and several of these Hsp90 client proteins are individually sought after cancer chemotherapeutic targets. Consequently, Hsp90 inhibition offers a promising new target for the development of anticancer chemotherapeutic agents because multiple signaling pathways can be simultaneously inhibited by disruption of the Hsp90 protein folding machinery. This application aims to develop new high-throughput assays for the identification of new lead compounds that inhibit Hsp90. A cell lysate assay will be developed to screen for inhibitors that prevent the renaturation of a well-studied Hsp90 client protein that has excellent bio-luminescence properties. It is proposed that molecules capable of inhibiting this renaturation process will be subsequently analyzed for their method of Hsp90 inhibition by two independent assays aimed at identifying both N- and C-terminal ATP binding site inhibitors. Alternatively, each of these assays should be independently capable of identifying new inhibitors of Hsp90 in a complimentary manner. At the completion of this project, we expect to provide three independent assays for high-throughput detection and evaluation of inhibitors that disrupt the Hsp90 protein folding process. The therapeutic potential of these compounds could be immense, as subsequent modification of these lead compounds should lead to the development of novel high-affinity Hsp90 antagonists for the treatment of cancer.
R01 CA114475
JERRY PELLETIER, MCGILL UNIVERSITY
High Throughput Assays Targeting Ribosome Recruitment
DESCRIPTION (provided by applicant): Translation is an essential cellular process whose deregulation is associated with alterations in cell growth regulation, cell cycle progression, and apoptotic responses. There is much evidence supporting the notion that aberrant control of protein translation contributes to neoplastic transformation. Signaling pathways (e.g. - ras and Akt) that regulate the translational machinery are activated in many human cells, over-expression of certain translation factors can lead to malignant transformation, and several components of the translational apparatus are over-expressed in human cancers. Indeed, elevated expression levels of elF4E (the mRNA cap binding protein involved in ribosome recruitment) leads to transformation in murine cancer model and is implicated in chemoresistance. Rapamycin, an inhibitor of the ribosome recruitment step, shows significant anti-cancer activity and is currently being tested in clinical trials. In this grant application, we propose to develop a series of HTS assays that will form the foundation of a chemical biology program aimed at better understanding the mechanism of translation, as well as the role that deregulation of this process plays in tumor progression. The HTS assays will target translation initiation factors involved in eukaryotic ribosome recruitment and will be used to identify compounds that can specifically inhibit this process. Our specific aims are to: i) target the interaction between elF4E and the mRNA cap structure for HTS assay design; ii) develop an HTS assay that monitors the helicase activity of elF4A, an ATP-dependent RNA helicase that unwinds mRNA secondary structure in the 5' UTR of mRNAs; iii) develop an HTS assay for inhibitors of elF4B activity, an RNA binding factor that functions in conjunction with elF4A to facilitate mRNA/ribosome binding; and iv) develop an HTS assay to prevent formation of the elF4E/4E-BP inhibitory complex, a heterodimer whose formation is stabilized by rapamycin.
R01 CA118498-01
RANDAL T. PETERSON, MASSACHUSETTS GENERAL HOSPITAL
Chemical Genetic Modifiers of Acute Myelogenous Leukemia
DESCRIPTION (provided by applicant): The long-term objective of this application is the discovery of small molecule therapies for acute myelogenous leukemia (AML). Therapies that target the underlying molecular causes of cancer represent a new frontier in cancer chemotherapy. At present, only a small number of targeted therapies have been successfully developed, primarily because molecular targets capable of reversing disease progression are difficult to identify. Nevertheless, even when valid therapeutic targets have not been identified, it may still be possible to discover targeted therapies by high-throughput screening for small molecules that promote phenotypic rescue in an appropriate disease model. AML is the most common type of leukemia and is frequently caused by the chromosomal translocation product, AML1-ETO. We have developed a transgenic zebrafish model of AML by expressing human AML1- ETO from an inducible promoter in zebrafish. Induction of AML1-ETO expression in zebrafish produces a reproducible phenotype that exhibits many of the hallmarks of AML. This AML1-ETO phenotype can be detected readily in the intact zebrafish embryo by a variety of automated screening techniques. Using this unique model of AML, we propose to develop assays for automated, high-throughput screens that can be used to identify therapeutic targets and small molecules capable of reversing the oncogenic effects of AML1- ETO expression. We propose to: 1) Test the effects of perturbing putative AML1-ETO effectors on the zebrafish AML1-ETO phenotype. 2) Develop automated assays for detecting suppression of the AML1-ETO phenotype in intact zebrafish. 3) Validate the AML1-ETO assays with pilot screens of 2000 small molecules with known bioactivities. Because zebrafish allow small molecule screening to be performed in an intact vertebrate organism, the proposed assays have the potential to discover small molecules that function through a variety of novel mechanisms, and hits are selected not only for efficacy but also for suitable ADME/toxicity properties. Therefore, the assays are innovative in that they combine the scale and throughput of high- throughput screens with the physiological relevance of testing in animal models. Relevance - This application proposes to develop simple, robust assays that will allow thousands of chemical compounds to be tested for their ability to reverse the effects of a leukemia-causing genetic defect. Compounds discovered using these tests will provide important insights into how this cancer progresses and may be useful tools for treating leukemia.
R01 CA118562
BRIAN T. CUNNINGHAM, UNIVERSITY OF ILLINOIS URBANA-CHAMPAIGN
Label-Free Screening Using Photonic Crystal Sensors for the Apoptotic Pathway
DESCRIPTION (provided by applicant): In this proposal we describe a novel label-free assay that can be used to rapidly screen protein-small molecule interactions that are not easily screened by any other method. The label-free assay is based upon an optical biosensor technology called a "photonic crystal" structure that is inexpensively manufactured from continuous sheets of plastic film and incorporated into standard 96- and 384-well plates. By eliminating the need for a label, the assay is less susceptible to errors and artifacts caused by conformational change or blocking of active binding epitopes. Once fully developed, it is envisioned that the technology will be used in the context of a primary screen of a chemical library and/or as a secondary screen for measuring dose- response characteristics of a protein-small molecule combination. The proposed project develops a new type of label-free assay, called the "Spot Protocol," in which the volume of target protein required is <0.1 mu l/well, and error sources due to bulk refractive index variability and nonspecific binding are automatically eliminated. The new assay utilizes an image-based method to enable high sensitivity detection of small- molecule binding signals, but incorporates a novel image-processing algorithm to provide a simple numerical output that is a direct measurement of the binding interaction between protein and analyte. The successful completion of the proposed work would represent the first application of the photonic crystal assay technology to a high-throughput screening (HTS) application. By focusing the initial demonstrations on proteins that do not function as enzymes and are thus difficult to target through more standard HTS processes, the benefits of this technology towards the targeting of proteins traditionally viewed as "nondrugable" will be obvious. Thus, our goal is to develop this assay technology to the point where any researcher could use this label-free detection method to identify a small molecule ligand to a protein of interest, even if that protein in not an enzyme and thus has no conveniently-monitored activity-based readout. To make the community aware of the potential of such a method and thus facilitate its transfer into other laboratories, we have targeted proteins in the apoptotic pathway. By focusing on biochemical pathways that regulate apoptosis, the proposed project aims to make an impact on understanding the fundamental processes involved in programmed cell death, and on how misregulation of such processes are result in cancer and neurodegenerative disorders. The selection and identification of chemical compounds with the ability to selectively induce apoptosis of cancer cells, or to prevent apoptosis of healthy cells is one key to the development of new drug therapies for many diseases.
R01 CA119001
GABRIELA CHIOSIS, SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH
Assays for Rapid Identification of Hsp70 Modulators
DESCRIPTION (provided by applicant): Hsp70 molecular chaperones bind and release polypeptide substrates concomitant with ATP dissociation and association. Based on this attribute, members of this abundant and conserved family of chaperones catalyze a diverse number of essential processes, including protein maturation and degradation, and prevention of protein aggregation. Because aberrations in these processes may lead to human diseases, there is much interest in modulating HspTO function and/or expression. Specifically, Hsp70s are known to inhibit apoptosis and maintain the transformed state in a wide range of malignancies. Moreover, Hsp70s help "correct" the conformations of mis-folded proteins and destroy potentially cytotoxic unfolded polypeptides. Unfortunately, few Hsp70 modulators have been identified, in part due to the lack of tools amenable to high throughput screens (HTS). Thus, the goal of the current effort is to create and optimize assays to identify both inactivators and activators of the inducible Hsp70. These tools can be used to conduct HTS and will be made available to the scientific community. The compounds identified from such efforts will represent important leads to gain insight into the rational translation of Hsp70 modulators in the treatment of cancers, neurodegenerative diseases, inflammation, ischemia and viral infections. To these ends, two aims are proposed in this grant application. First, a phenotypic cell-based assay will be developed, optimized and validated to identify modulators of inducible Hsp70 expression. The assay employs a miniaturized, cell-based immunodetection system amenable to HTS. Second, a fluorescence polarization assay will be developed, optimized and validated to identify modulators of Hsp70 function. This assay is based on the competition of a fluorescently (FITC or red-shifted) labeled peptide to bind recombinant Hsp70. Overall, given the importance of Hsp70 function and activation in human disease, the time is ripe to design and perform a large-scale screening regimen to identify Hsp70 modulators. Surprisingly, this has not yet been accomplished. To develop the requisite tools to screen for Hsp70 modulators, this proposal co-opts the combined expertise of the collaborating Pis in chaperone biology, biochemistry and drug discovery, and their abilities and proven track-records in the design and development of screening regimens for small molecule chaperone modulators.
R01 CA120227
MICHAEL R. MATTERN, PROGENRA, INC.
Ubiquitin E3 Ligases and Apoptosis in Cancer Drug Discovery
DESCRIPTION (provided by applicant): CARP2 is an anti-apoptotic RING-domain protein that specifically binds to and negatively regulates death domain (DED) caspases 8 and 10 by acting as an E3 ubiquitin ligase, causing ubiquitination and ultimately proteasomal degradation of the target caspase. This ligase is over-expressed in many tumors, permitting them to escape apoptosis and survive. Inhibitors of CARP2 would thus be expected to have intrinsic antitumor activity in addition to facilitating antitumor activity of agents such as TRAIL, which act via the death receptor pathway. This proposal describes the development for high-throughput screening of a yeast-based assay in which human CARP2, its p53-fused substrate, caspase 8, and a p53 reporter (a-galactosidase) are expressed in S. cerevisiae. When all of these components are functional, CARP2, in collaboration with the endogenous yeast ubiquitin- proteasomal pathway elements (E1, E2, proteasomes), keeps the level of Caspase 8-p53 at a minimum, resulting in no reporter activity. Inhibition of CARP2 permits Caspase-fused p53 to bind to its response element and activate the reporter, producing a positive signal (fluorescence). The assay will be configured, following cloning and construction of the necessary components, by transformation of CARP2, its substrate caspase 8 fused to p53, and a p53-responsive reporter, in yeast. After adjusting assay conditions to produce an acceptable signal: noise ratio and other parameters of high-throughput screening, limited numbers of compounds and natural product extracts will be screened to evaluate the suitability of the assay for full scale high-throughput screening for anticancer drug development. In addition, secondary screening assays, including a yeast based assay for CARP1, will be developed to filter primary hits for progression to preclinical development. The ultimate goal of the proposed work is to translate the assay to high- throughput screening for a drug that is active as a single agent or a component of combination therapy against refractory cancers.
R01 CA121689
NICHOLAS COWAN, NEW YORK UNIVERSITY SCHOOL OF MEDICINE
Inhibition of the Tubulin Folding Pathway as a Novel Therapy for Cancer
DESCRIPTION (provided by applicant): Microtubules are dynamic polymers that play an important role in many vital cellular functions. They are assembled from heterodimers consisting of one alpha and one beta-tubulin polypeptide. The participation of microtubules in cell division as an essential component of the mitotic spindle has made these structures attractive targets for cancer chemotherapy: several drugs that interfere with normal microtubule dynamics are currently in clinical use and many other such compounds are currently undergoing clinical trials. Microtubules are thus well established as a validated and highly successful anti-cancer target. All of the currently known compounds that interfere with microtubule dynamics do so by binding to tubulin, but none are known that interfere with the pathway leading to the de novo assembly of the tubulin heterodimer. This pathway involves interaction of newly synthesized tubulin polypeptides with a series of chaperone proteins, beginning with the cytosolic chaperonin CCT. Quasi-native subunits released from CCT interact with several tubulin-specific chaperones (known as cofactors A-E) in a reaction that leads to release of newly generated heterodimers following GTP hydrolysis by cofactor-bound beta-tubulin. Cofactors C, D and E also function as a GTPase activating protein (GAP) for tubulin; this reaction is distinct from the GTP hydrolysis that accompanies microtubule polymerization in that it occurs at a much lower tubulin concentration. Because cofactors C, D and E are essential for tubulin heterodimer formation, they represent unique and novel potential targets for interfering with the generation of productively folded tubulin heterodimers. Experiments using systematic siRNA knockdown and our recent analysis of a human genetic disorder (HRD) involving cofactor E provide proof-of-concept and further functional validation for this approach. The experiments we propose are intended to lay the groundwork for a search for compounds that interfere with de novo tubulin heterodimer formation. We will 1) Develop the tubulin GAP assay for application to a high throughput format; 2) Devise methods for the optimization of cofactor production for use in high throughput assays; 3) Develop methods for the elucidation of the mechanism of inhibition in tubulin GAP assays in order to eliminate artifacts and prioritize compounds for further study; and 4) Perform pilot high throughput screens in order to establish appropriate conditions, optimize our assays, and define thresholds and hits.
R01 CA122608
JONATHAN M. HIGGINS, BRIGHAM AND WOMEN'S HOSPITAL
Development of a Haspin Kinase Assay for High Throughput Drug Screening
DESCRIPTION (provided by applicant): Proper control of mitosis is critical to maintain the stability of the genome during cell proliferation, and genomic instability may contribute directly to the generation of cancer. Study of mechanisms that regulate mitosis, therefore, is critical to understand how cancer develops, and to discover new ways to prevent and treat the disease. Mitosis is also an important target for cancer therapy. Recently, new selective anti-mitotic drugs such as aurora kinase inhibitors have shown great promise in pre-clinical experiments, and there is now immense interest in identifying new drug targets in mitosis. We have recently discovered a novel mitotic histone kinase, haspin, that has homologs in diverse eukaryotes. Human haspin mRNA is expressed in proliferating but not non-proliferating cells. During mitosis, haspin associates with condensed chromosomes, particularly at centromeres, and is responsible for phosphorylation of Thr-3 in histone H3. Haspin is also found at mitotic centrosomes. Importantly, haspin RNA interference causes misalignment of metaphase chromosomes and spindle defects, preventing completion of normal mitosis. These studies add haspin to the select group of kinases that regulate mitotic chromosome dynamics and spindle activity and provide the first indication that haspin, like the aurora kinases, might be a suitable target for cancer therapy. Further study of haspin action in mitosis and validation of haspin as a cancer drug target are currently limited, however, by the lack of specific small molecule inhibitors of the kinase. To identify small molecule inhibitors of haspin, we will develop an in vitro haspin kinase assay suitable for high-throughput screening of chemical libraries. In Aim 1 we will produce, in E. coli or the baculovirus system, functional full-length recombinant haspin for use in screening assays. In Aim 2 we will develop and optimize a homogenous time-resolved fluorescence kinase assay for haspin. An alternative strategy using a separation-based approach is also described. In Aim 3, we outline assays to confirm hits from the screening process and develop secondary screens to assess the inhibitory properties and functional effects of these compounds in vitro and in cells. Haspin inhibitors will provide a new approach to investigate the basic biology of cell division and will yield insights that cannot be obtained using existing technology. Furthermore, such inhibitors will provide an excellent way to validate haspin as a target for cancer treatment, and they might find direct application as chemotherapeutic drugs.
R01 CA131059
ROYCE MOHAN, UNIVERSITY OF KENTUCKY
Novel Modular Vascular Patterning Assays for HTS
DESCRIPTION (provided by applicant): The vascular endothelial cells lining blood vessels in humans are one of the principle sites that become involved in inflammatory and proliferative responses to a diverse array of human diseases. Microvascular homeostasis is thus a vital component of human health; its inappropriate activation in response to inflammatory and angiogenic stimuli can become a pathogenic component fueling the growth and spread of cancers, and contributing to debilitating arthritis, age-related macular degeneration and multiple organ failure associated with underlying diseases such as diabetes. On the other hand, impaired angiogenesis is also equally pathogenic, and afflicts its victims by slowing down wound healing and contributing to heart diseases and stroke. Collectively, given the complexity of the angiogenesis signaling system, these major burdens of human health that arise from dysregulation of blood vessel growth need to be addressed by a more concerted effort in drug discovery. Biological assays that model the processes of angiogenic diseases can assist the process of drug discovery and disease target identification. However, currently few assays represent the complexity of the diseased microvasculature as they typically focus on one pathogenic mechanism/pathway. With this in mind, we propose to continue the development of a high content high throughput screening (HC-HTS) vascular patterning assay, which we have recently validated as drug screening tool through a previously funded NIH Roadmap Initiative R21 grant. In this R01 proposal, we plan to extend the scale and scope of the three dimensional endothelial cell sprouting assay (3D-ECSA) to promote its adoption for the HTS paradigm. Our innovative approaches bring in 1) modern automated robotic systems that allow us to improve efficiency and standardize production of spheroids, 2) high content image analysis software to use with 3D-ECSA under HTS conditions, and 3) development of a pilot scale chemical library focused on the immunoproteosome as a chemical enabling tool towards validation of the 3D-ECSA. The successful accomplishments of these goals will not only afford us a valuable tool for large scale biology, but will help bring forward a technology advancement to identify new classes of chemical probes of protein function and drug leads for life saving therapeutics.
R01 CA131081
CHARLES S. HEMENWAY, LOYOLA UNIVERSITY CHICAGO
Disrupting the AF4-AF9 Protein Complex in MLL Leukemias
DESCRIPTION (provided by applicant): The ultimate goal of this project is to develop promising new agents for the treatment of acute lymphoblastic leukemia (ALL) characterized by a specific t(4;11) chromosome translocation. t(4;11) leukemia is a relatively common subtype of ALL and is unusually resistant to cytotoxic chemotherapy. The isolation, validation, and optimization of new compounds to treat t(4;11) leukemia is important given the limitations of currently available treatments. We have identified a protein:protein interaction that appears to be critical for the survival of t(4;11) leukemia. More important, small peptides that disrupt the interaction of these two proteins, AF4 and AF9, induce programmed cell death in t(4;11) leukemia cells. Additionally, these peptides do not inhibit the growth and differentiation of normal hematopoietic progenitor cells. These data indicate that the AF4-AF9 protein complex is a promising target for drug development. The NIH Molecular Libraries Screening Center Network offers a unique opportunity to screen an extensive molecular library to identify additional compounds that block AF4-AF9 binding. Here, we propose to develop an assay system that is well suited for high throughput screening of inhibitors of AF4-AF9 binding. Fluorescence polarization (FP) is a powerful technique to measure protein binding in a homogeneous solution. The technique is particularly well suited to measuring AF4-AF9 binding as the molecular complex can be mimicked by a peptide "probe" bound to the C-terminal domain of AF9. Furthermore, FP assays can be easily modified to analyze inhibitors of protein binding. FP competition assays have been used successfully for high throughput screening of inhibitors of other protein:protein interactions. Once optimized, the assay is simple, rapid, and inexpensive. In addition to a primary FP screening assay, we describe secondary assays to analyze "hits". These secondary assays include analyses of the anti-leukemic activity of lead compounds and have already been validated. It is anticipated that, following refinement, compounds identified and verified by this screening process will provide useful new anti-leukemic therapies. Despite improvements in the treatment of leukemia, some forms of the disease remain resistant to therapy. Here, we describe a system to rapidly identify chemicals that may serve as new drugs for the treatment of one of the most resistant forms of acute leukemia. We propose to develop and refine this testing system so that it may be used to isolate promising drug candidates from a large collection of chemicals developed by NIH.
R01 CA131983
SARA A. COURTNEIDGE, BURNHAM INSTITUTE FOR MEDICAL RESEARCH
High Throughput Microscopy Assays to Identify Inhibitors of Metastasis
DESCRIPTION (provided by applicant): When cancer is detected early enough, surgery alone can often be curative. However, by the time of diagnosis, the cancer has frequently spread to other sites in the body, by the process of metastasis. Much of the pain, suffering, and mortality associated with cancer are the result of metastatic disease. We need better ways to diagnose metastatic disease early, and better ways to treat metastatic disease therapeutically. The ultimate goal is to prevent metastasis from occurring, particularly in those who have been diagnosed with pre-invasive conditions such as ductal carcinoma in situ of the breast, and prostate intra-epithelial neoplasia. We will achieve this goal when we are able to define and develop therapeutics against targets present in metastatic cells which are not essentially required for most normal physiology, thus allowing for safe and effective long term treatment. Both metastatic and benign tumors proliferate without regard to the cellular cues that normally control cell division and cell death. But only the metastatic cells have the ability to break free from the organ in which they arise, enter the vasculature, and move into and grow in other organs. These steps are collectively called invasion. It is widely believed that the most common way in which invasion occurs during metastasis is through the action of proteases, which degrade extracellular matrix (ECM) proteins. Since continued ECM degradation is also thought to be required for disseminated tumor growth in secondary organ sites, inhibiting this process may provide a means to control metastasis. Cancer cells frequently over-express a number of proteases that are capable of degrading ECM. Some of these proteases are secreted, while others remain associated with the plasma membrane. In recent years, it has become increasingly clear that specialized protrusions of the ventral membrane of cancer cells called podosomes (also known as invadopodia) are the concentrated sites of action of these proteases. For example, using a model system of invasion, mouse fibroblasts transformed by an activated version of the Src oncogene, we have shown that podosomes are required for the invasiveness that typifies Src transformation, both in vitro and in vivo. Cells lacking podosomes still secrete proteases, but they are unable to degrade ECM efficiently. Furthermore, human cancer cells also require podosomes/invadopodia for invasion. These results are consistent with a model in which podosomes/invadopodia are required for the coordinated activity of proteases. Further, these data support our hypothesis that small molecules that prevent the formation of podosomes would be effective agents to limit the growth of tumor metastases. Here, we propose to develop and utilize cell-based, high throughput screening systems to identify chemical entities that inhibit podosome formation and invasion. Metastasis is the leading cause of cancer mortality in this country. We need better ways to diagnose metastatic disease early, and better ways to treat metastatic disease therapeutically. The proposed research aims to set develop high throughput screening assays to identify inhibitors of metastasis, and therefore represents the first step to developing anti-metastatic drugs with a new mechanism of action.
R01 CA133791
LONGQUIN HU, RUTGERS THE ST UNIV OF NJ NEW BRUNSWICK
Homogenous HTS Assays to Screen for Inhibitors of Keap1-Nrf2 Interaction
DESCRIPTION (provided by applicant): Keap1-Nrf2-antioxidant response element (ARE) system regulates cellular defense mechanisms that protect cells from oxidative stress. It has been demonstrated that Nrf2 knockout mice are more sensitive to toxicological effects of carcinogens, drugs, and inflammatory stresses while Keap1 knockout mice exhibited high levels of Nrf2, high constitutive expression of cytoprotective enzymes and striking resistance to the environmental stresses. Keap1-Nrf2 interaction plays a key role in cancer chemoprevention by many chemicals like sulforaphane, curcumin, and epigallocatechin gallate derived from natural sources such as fruits, vegetables, and tea products. Modification, by these natural electrophiles, of sensitive cysteine residues found in the redox "sensor" protein Keap1 is believed to be responsible for causing the translocation of Nrf2 to the nucleus and subsequent upregulation of anti-oxidative stress cytoprotective enzymes. One approach to address concerns over the general use of purified natural thiol-reactive compounds as chemopreventive agents is to use high throughput screening assays of chemical libraries to discover and develop novel small molecules as direct inhibitors of Keap1-Nrf2 interaction at the protein-protein interface. These inhibitors will mimic the actions of electrophiles like isothiocyanates and Michael acceptors in the induction of cytoprotective enzymes but will potentially be more selective and specific against the Keap-Nrf2 axis. Two solution fluorescence-based high throughput screening assays will be developed and validated in this new R01 application using fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET). These assays will be applied to the screening of chemical libraries for the discovery of new leads in an interdisciplinary program involving medicinal chemistry, computational chemistry, X-ray crystallography, and cancer biology. These studies will provide important information about the protein-protein interaction between Keap1 and Nrf2 and facilitate the development of more potent and selective Keap1-Nrf2 inhibitors as inducers of oxidative stress response enzymes. PUBLIC HEALTH REVELANCE:HTS assays developed in this project will facilitate the discovery of novel leads for the development of new Keap1-Nrf2 inhibitors. Specific and potent Keap1-Nrf2 inhibitors will be useful as important pharmacological probes for the elucidation of cytoprotective pathways and as potential cancer chemopreventive agents in high risk populations.
Direct inquiries regarding the cancer-targeting assays to:
Minkyung (Min) H. Song, Ph.D.
Developmental Therapeutics Program
Division of Cancer Treatment and Diagnosis
National Cancer Institute
6130 Executive Blvd., Room 8153, MSC-7456
Rockville, MD 20852 (for express/courier service)
Telephone: (301) 496-8783
Fax: (301) 402-5200
Email: songm@mail.nih.gov