NOVEL FLUORESCENT ESTROGEN RECEPTOR LIGAND BINDING ASSAY

Robert G Lowery, President
Panvera Corporation
545 Science Dr
madison, Wi 53711

Grant 3R43DK054619-01S1 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZRG2

Abstract: The recent increased focus on estrogen receptor (ER) as a therapeutic target and the growing interest in the impact of environmental estrogens on human health has created an important need for improved assay methods to identify estrogen receptor ligands, particularly assay methods suited to high throughput screening formats. To address this need, PanVera will investigate development of homogenous in vitro fluorescence polarization assays for two human estrogen receptors, ERalpha and ERbeta. As a first step towards this goal, Phase I studies will develop fluorescent ligands for estrogen receptors by 1) synthesizing estrogen-fluorophore conjugates and characterizing their structure and chemical and photophysical properties, 2) evaluating the binding and fluorescence characteristics of the new conjugates with purified ERalpha and Erbeta, and optimizing fluorescence polarization assay conditions for those conjugates with the best properties, 3) validating the assay for high throughput screening applications by screening a panel of known ER ligands and test compounds from a pharmaceutical chemical collection, and comparing the relative binding affinities with those obtained using standard assay methods. Phase II studies will extend this technology to additional steroid hormone receptors. In Phase III, PanVera will commercialize this technology through its domestic and international distribution network, and pharmaceutical alliances. PROPOSED COMMERCIAL APPLICATION The Phase I SBIR studies will result in development of a proprietary, homogenous fluorescence polarization assay for high throughput screening of estrogen receptor ligands. PanVera will commercialize these assays immediately through its domestic and international research products distribution network. Phase II studies will result in similar assays for other steroid hormone receptors, including androgen receptors and progesterone receptors. In Phase III, PanVera will continue and expand commercialization of each assay through its comprehensive distribution network and pharmaceutical alliances

Keywords: chemical conjugate, chemical synthesis, estrogen receptor, fluorescence polarization, fluorescent dye /probe, ligand, method development chemical binding, chemical stability, chemical substitution, estradiol, estrogen inhibitor, receptor binding, solubility bioengineering /biomedical engineering, fluorescein, fluorescence spectrometry, ionophore

Project start date: 1998-09-15

Project end date: 1999-11-30

3R43DK054619-01S1 (1999): $75000

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P450 FUSION PROTEINS FOR DRUG METABOLISM STUDIES

Robert G Lowery, President
Panvera Corporation 545 Science Dr Madison, Wi 53711

Grant 1R43CA068848-01 from National Cancer Institute, IRG: ZRG7

Abstract: With the increased understanding of the role of specific cytochrome P450 isoforms in drug metabolism, there is a growing need for improved in vitro methodology using purified P450s. The major drawbacks to current methods include interference from contaminating enzymes present in heterogenous assay systems and difficulties in reconstituting the purified P450s with there associated electron transfer proteins. PanVera proposes to extend technology developed by Dr. Ronald Estabrook for the production of self-sufficient catalytic units composed of P450s linked to NADPH-P450 reductase and to incorporate the fusion proteins into a multi-well format. The goal of the proposed research would be to develop 1) a panel of P450-reductase fusion proteins, 2) assay methods for absorbance-based detection of substrate metabolism by the fusion proteins. To demonstrate the feasibility of achieving these results, Phase I specific aims will be 1. Produce and characterize an active recombinant fusion protein of human P450 3A4 linked to human NADPH-P450 reductase. 2. Compare the metabolic profile of the fusion protein with that of the native enzyme system. 3. Examine the extent of coupling between NADPH oxidation and substrate metabolism with the fusion protein. PROPOSED COMMERCIAL APPLICATION New commercial applications from this research will include 1) the ability to screen large numbers of test compounds as substrates for specific P450 isoforms, 2) regio and stereospecific synthesis of large amounts of compounds for toxicological, pharmacological and prodrug studies.

Project start date: 1995-08-07

Project end date: 1996-07-31

1R43CA068848-01 (1995): $100000

Fluorescent HTS Assays For Human Sulfotransferases

Robert G Lowery, President
Bellbrook Labs, Llc Suite 250 Madison, Wi 53711

Grant 5R44GM069258-03 from National Institute Of General Medical Sciences, IRG: ZRG1

Abstract: This Phase II proposal is for a collaborative effort between Dr. Richard Weinshilboum of the Mayo Institute and BellBrook Labs, LLC to translate basic pharmacogenetics research on conjugative drug metabolizing enzymes into molecular screening assays that can be applied in preclinical drug development. Drug metabolism problems such as drug interactions and production of toxic metabolites cause many of the adverse reactions that contribute to the extremely high failure rate of clinical trials. The most promising approach for effectively managing individual differences in drug response is the implementation of pharmacogenetic strategies for predicting drug metabolism in each patient. However, critical information on which enzymes are metabolizing drug candidates and how enzyme function is affected by genetic variation is lacking because in vitro assays for profiling compound metabolism with individual drug metabolizing enzymes are not available. Sulfation is a major route of drug metabolism carried out by a family of 12 enzymes in humans, and recently an additional 25 sulotransferase allozymes were identified, many with functional phenotypes. In Phase I, we developed a homogenous, fluorescent high throughput screening (HTS) method called donor product fluorescence polarization immunoassay (DP-FPIA) that provides a universal screening platform for measuring sulfation of any compound by any SUIT isoform. In Phase II, we will optimize the performance of the assay for pharma HTS isolating a highly selective monoclonal antibody against a nucleotide and synthesizing conjugates of red fluors-to  the same nucleotide. In addition a full panel of 20 human sulfotransferases - including several important genetic variants - will be expressed, and purified in parallel using automated, multfwell methods. We will then validate the assay for high throughput screening and kinetic analysis using prototypic substrates, drugs, and hormones screened against the full panel of sulfotransferases. The sulfotransferase DP-FPIA platform will be marketed in a kit format to pharmacuetical companies for preclinical metabolism studies and lead optimization. The unique capabilities of the DP-FPIA and the availability of a panel of recombinant sulfotransferases will enable a systematic delineation of human drug sulfation capacity and how it is affected by genetic variation.

Keywords: drug screening /evaluation, fluorescence polarization, high throughput technology, immunologic assay /test, monoclonal antibody, sulfotransferase, technology /technique development, combinatorial chemistry, drug discovery /isolation, drug metabolism, fluorescent dye /probe, sulfation, biotechnology, protein purification

Project start date: 2003-07-01

Project end date: 2007-05-31

5R44GM069258-03 (2006): $475995

2R44GM069258-02A1 (2005): $545659

Generic Fluorescent HTS Assay For Oxidoreductases

Robert G Lowery, President
Bellbrook Labs, Llc
suite 250
madison, Wi 53711

Grant 1R43GM083478-01 from National Institute Of General Medical Sciences, IRG: ZRG1

Abstract: There is a high level of pharmaceutical interest in targeting enzymes that catalyze oxidation and reduction reactions for therapeutic intervention. Validated redox enzyme targets include HMG-CoA reductase, the target of statin drugs like Lipitor that are used to treat hypercholesterolemia, and 51-reductase, the target of drugs used to treat benign prostatic hyperdysplasia. Emerging targets in this class include glycolytic dehydrogenases in tumor cells growing under hypoxic conditions and in microbial and parasitic pathogens. However, the identification of novel inhibitors for redox enzymes is being slowed by the lack of robust, generic enzyme assay methods that are suitable for use in pharmaceutical high throughput screening (HTS) laboratories. To remove this technical hurdle, we are proposing to develop methods for specific, fluorescent detection of oxidized and reduced pyridine nucleotides, NAD(H) and NADP(H). Methods will be developed for specific chemical modification of pyridine nucleotides based on their oxidation state. Comcomitantly, antibodies and fluorescent tracers will be developed for homogenous immunodetection of unmodified pyridine nucleotides. These novel reagents and methods will be validated for homogenous detection of dehydrogenase and reductase enzyme activity in a fluorescence polarization immunoassay format and commercialized as HTS assay kits. The availability of robust, fluorescence based assay methods applicable to any redox enzyme that utilizes a pyridine nucleotide cofactor will accelerate the exploration of this validated target class for a broad range of diseases and disorders. Enzymes that catalyze oxidation and reduction reactions constitute a major class of proteins that have been successfully exploited as drug targets. We are proposing to develop methods and reagents that will enable pharmaceutical researchers to accelerate drug discovery targeting these enzymes for a broad range of diseases including cancer, cardiovascular disease, and diabetes

Project start date: 2008-02-01

Project end date: 2009-01-31

Generic Fluorescent HTS Assay For Kinases And ATPases

Robert G Lowery, President
Bellbrook Labs, Llc Suite 250 Madison, Wi 53711

Grant 5R44CA110535-03 from National Cancer Institute, IRG: ZRG1

Abstract: There are over 500 protein kinases in the human genome and several hundred other enzymes that utilize ATP as a phosphate donor or energy source. Protein kinases are the second most intensively screened target class, with links to all of the major therapeutic areas, most notably cancer. Important disease links for cancer and anti-infectives also are emerging for lipid kinases, carbohydrate kinases and ATPases. Despite their high pharmaceutical relevance, the rapid movement of new ATP- dependent enzyme targets into drug discovery is being hampered by a lack of flexible high throughput screening (HTS) assay methods. This problem extends into secondary screening efforts for selectivity profiling and lead optimization as well. To address this need, BellBrook Labs has developed a novel fluorescence-based immunodetection method for ADP, the invariant product of all kinase and ATPase reactions. In Phase I, we developed a monoclonal antibody and fluorescent tracer that enable detection of ADP with 100-fold selectivity over ATP using fluorescence polarization as a readout. We commercialized the ADP detection platform as the Transcreener Kinase Assay kit in November, 2005, and it has since generated revenue approaching $250,000. In Phase II we propose to 1) enhance Transcreener Kinase Assay performance by developing a more selective ADP antibody, 2) extend the platform to additional target families, including GPCRs, by developing a similar immunodetection method for GDP, 3) increase HTS market penetration by formatting the assay for a second commonly used fluorescent detection mode, TR-FRET, 4) develop bioreactor hybridoma culture methods to scale up monoclonal antibody production and reduce animal usage, 5) use the unique capabilities of the Transcreener Kinase Assay to test a) the importance of using physiological protein substrates rather than widely used peptide acceptors for kinase screening and b) the potential for off target effects of kinase inhibitors with other types of ATP-dependent enzymes. By providing pharma with the tools and strategies to implement more physiological kinase screening methods, we hope to accelerate the discovery of improved therapies for cancer and other debilitating diseases. There are over 500 distinct protein kinase proteins encoded in the human genome and they have attracted intense interest from pharmaceutical companies because of their links with numerous types of cancer, inflammatory disorders such as arthritis, and other debilitating diseases. To accelerate the discovery of improved therapies for these diseases, we are developing an enabling screening method for finding drug molecules that will correct the aberrant behavior of malfunctioning kinase proteins.

Project start date: 2005-07-01

Project end date: 2008-09-29

5R44CA110535-03 (2007): $257753

2R44CA110535-02 (2006): $390536

Generic Fluorescent HTS Assay For Protein Kinases

Robert G Lowery, President
Bellbrook Labs, Llc Suite 250 Madison, Wi 53711

Grant 1R43CA110535-01A1 from National Cancer Institute, IRG: ZRG1

Abstract: There are more than 400 distinct protein kinases encoded in the human genome; elucidating their role in disease and identifying selective inhibitors is a major pharma initiative. Kinase malfunction has been linked to all of the most important therapeutic areas, including cancer, cardiovascular diseases, inflammation, neurodegenerative diseases, and metabolic disorders. Moreover, clinical validation of kinases as drug targets has recently been shown in the cases of Herceptin and Gleevec, which inhibit aberrant tyrosine kinases that contribute to breast cancer and leukemia, respectively. High throughput screening (HTS) - the parallel testing of many thousands of compounds for interaction with a drug target - has become the dominant mode of drug discovery. The total market for HTS assay reagents in 2003 was over $500M, and approximately 20% of screening was done on protein kinases. Despite the high level of interest, shortcomings with the current assay methods are hampering efforts to incorporate new kinases into HTS programs and to compare inhibitor profiles for different kinases. To overcome these technical hurdles we propose to develop a universal kinase assay method based on BellBrook Labs  proprietary Transcreener(tm) platform. The assay relies on a competitive fluorescence polarization-based immunoassay for detection of adenosine diphosphate (ADP), a product of all kinase reactions. The Transcreener(tm) kinase assay will accelerate efforts to define kinase substrate specificity and to identify novel inhibitors by providing a universal catalytic assay that can be used with any kinase and any acceptor substrate.

Project start date: 2005-07-01

Project end date: 2006-06-30

1R43CA110535-01A1 (2005): $173113

HTS ASSAYS FOR SELECTIVE STEROID RECEPTOR MODULATORS

Robert G Lowery, President
Panvera Corporation
545 Science Dr
madison, Wi 53711

Grant 5R44DK054619-03 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZRG1

Abstract: Recent progress in the steroid hormone receptor (SHR) field coupled with advances in combinatorial chemistry and high throughput screening (HTS) instrumentation have set the stage for development of more selective therapies for a broad range of cancers, metabolic diseases, and chronic conditions. To enable the drug discovery opportunities provided by these advances, PanVera is developing the robust molecular screening assays needed for identification and characterization of novel SHR ligands. In Phase I, PanVera developed fluorescence polarization (FP)-based competitive ligand binding assays for estrogen receptors alpha and beta that have been rapidly adapted for high throughput drug discovery programs at several pharmaceutical companies. In Phase II, we will develop similar FP- based ligand binding assays for additional SHRs, including wild type and mutant androgen and progesterone receptors and additional estrogen receptor isoforms. In addition, we will develop secondary FP-based assays, based on fluorescent peptides that mimic the agonist-dependent binding of transcriptional coactivators to SHRs. These secondary assays will allow pharmaceutical researchers to predict the in vivo tissue specificity and transcriptional activity of novel SHR ligands. In combination, these two HTS assay methods will provide the molecular tools needed to accelerate discovery of highly selective SHR modulators. PROPOSED COMMERCIAL APPLICATIONS The Phase II SBIR studies will result in development of proprietary fluorescent steroids and FP-based HTS assays for identification and characterization of novel ligands for estrogen, androgen, and progesterone receptors. PanVera will commercialize these assays immediately as part of their CoreHTS drug discovery product line through their domestic and international drug discovery products distribution network

Keywords: chemical conjugate, chemical synthesis, fluorescence polarization, fluorescent dye /probe, high throughput technology, ligand, method development, steroid hormone receptor chemical binding, chemical stability, chemical substitution, estradiol, estrogen inhibitor, estrogen receptor, genetic transcription, receptor binding, solubility fluorescein, fluorescence spectrometry, ionophore

Project start date: 1998-09-15

Project end date: 2002-02-28

5R44DK054619-03 (2001): $456056

2R44DK054619-02 (2000): $583144

Fluorescent HTS Assay For Methyltransferases In Neurodegenerative Diseases

Robert G Lowery, President
Bellbrook Labs, Llc Suite 250 Madison, Wi 53711

Grant 1R43GM073290-01A1 from National Institute Of General Medical Sciences, IRG: ZRG1

Abstract: Project Summary/ Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids and lipids. More than 50 distinct methyltransferase (MTs) enzymes are present in humans, and they are being targeted for a broad range of diseases, but their involvement in neurodegenerative disease pathways is of special relevance. Modulation of neurotransmitter methylation is an emerging therapeutic strategy for the treatment of several neurodegenerative diseases, most notably Parkinson s and Alzheimer s diseases, and DNA and protein MTs are also being targeted for neurodegenerative diseases and cancers of the CNS. Moreover, the involvement of some MT family members in disease pathways is intertwined with their role in metabolizing commonly prescribed drugs. The development of highly selective MT modulators is clearly a compelling medical priority. However, efforts to achieve this are being hampered by a lack of flexible enzyme assay methods adaptable to high throughput screening (HTS). The current use of isozyme-specific assay methods that rely on radioactivity and cumbersome post reaction processing steps is preventing facile incorporation of diverse MT enzymes into pharmaceutical drug discovery programs. To accelerate efforts to identify selective MT inhibitors we propose to develop a universal catalytic assay that will enable screening of all members of the MT family using a single set of detection reagents. The method is based on detection of the invariable MT reaction product S-adenosyl homocysteine-using a competitive fluorescence polarization immunoassay. The Phase I work includes development of antibodies and fluorescent nucleotide conjugates that enable highly selective detection of S-adenosyl homocysteine in the presence of S-adenosyl methionine, which differs only in the addition of a methyl group. Project Narrative Aberrant behavior by several proteins in the methyltransferase family is a possible cause of debilitating neurodegenerative disorders like Alzheimer s disease and Parkinson s disease. To accelerate the discovery of improved therapies for these diseases, we are developing screening kits to find drug molecules that correct the malfunctioning methyltransferase proteins.

Keywords: enzyme activity, enzyme inhibitor, high throughput technology, immunologic assay /test, method development, methylation, methyltransferase, neurotransmitter, S adenosylmethionine, catechol methyltransferase, homocysteine, neural degeneration, pathologic process, Escherichia coli, antibody, bioengineering /biomedical engineering, fluorescence polarization, laboratory rabbit, microorganism culture, radiotracer

Project start date: 2006-09-15

Project end date: 2007-06-30

1R43GM073290-01A1 (2006): $163177

HIGH THROUGHPUT SCREENING ASSAYS FOR P450 METABOLISM

Robert G Lowery, President
Panvera Corporation
545 Science Dr
madison, Wi 53711

Grant 5R44CA068848-03 from National Cancer Institute, IRG: ZRG3

Abstract: Poor metabolism and toxicity cause over 50% of all clinical failures of new drug candidates. While the P450 enzymes play a critical role in metabolism of many drugs by catalyzing the first step in their biotransformation, there is no effective technology to screen for drug candidates´ interaction with purified human P450 isozymes. Such capability would provide early notice to drug discovery teams of potential pharmacokinetic problems, drug-drug interactions, and pharmacogenetic complications before entering into more expensive testing in animals or humans. To address the strong commercial interest in such technology,Phase II studies will focus on creating an absorbance-based, universally applicable assay for the six major P450s that allow prediction of in vivo pharmacokinetic parameters. PanVera made a significant technical breakthrough towards this goal in Phase I by developing the RECO(TM) assay, a novel in vitro system that overcomes the historical problems of using purified proteins for screening P450 enzymes. In Phase II, PanVera will use the RECO system to incorporate the key human P450 isozymes into a high throughput screening assay for rapid analysis of P450-dependent metabolism of large, diverse compound collections. Phase III efforts will focus on commercializing the assays through PanVera´s extensive drug discovery research customer base. PROPOSED COMMERCIAL APPLICATIONS New commercial applications from this research include 1) non- radioactive, homogeneous, high throughput P450 screening assays to aid in drug discovery and 2) production of large amounts of purified P450 proteins for structure-function studies and synthesis of metabolites

Keywords: bioassay, biotransformation, cytochrome P450, drug metabolism, drug screening /evaluation, isozyme, technology /technique development combinatorial chemistry, drug discovery /isolation, pharmacokinetics

Project start date: 1995-08-07

Project end date: 2001-11-30

5R44CA068848-03 (2000): $358921

2R44CA068848-02A1 (1999): $460926

HTS Assays For Modulators Of GPCR Signaling

Robert G Lowery, President
Bellbrook Labs, Llc
suite 250
madison, Wi 53711

Grant 1R43NS059082-01 from National Institute Of Neurological Disorders And Stroke, IRG: ZRG1

Abstract: More than 50% of drugs on the market target G protein-coupled receptors (GPCRs). Among the most important of these are drugs used to treat neurological disorders, such as pain relievers, antidepressants and anti-psychotics, as well drugs used for neurodegenerative diseases such as Parkinson´s disease and Alzheimer´s disease. The relatively recent discovery of a family of proteins called "regulator of G protein signaling" (RGS), that attenuate GPCR signals by increasing the GTPase activity of associated Ga proteins, has opened up a new avenue for modulating the activity of endogenous and administered GPCR ligands. However, development of inhibitors to RGS proteins has been hampered by the lack of robust assay methods for high throughput screening (HTS). To overcome this technical hurdle, we propose to use fluorescence based immunodetection of GDP to measure RGS-dependent increases in the steady state GTPase activity of modified Ga proteins. Based on the approach used previously at BellBrook Labs for development of an ADP detection assay, a highly specific monoclonal antibody and fluorescent tracer will be developed for detection of GDP. The Ga proteins will be mutated to increase the rate of GDP dissociation relative to GTP hydrolysis so that the former is no longer rate limiting. The availability of these novel HTS assays will accelerate drug discovery focused on RGS proteins and delineation of their roles in GPCR signal transduction. Over half of current drugs exert their effects through a family of proteins called G protein- coupled receptors, and the targeting of these receptors has been especially useful for the development of drugs used to treat neurological disorders such as schizophrenia, depression, and Parkinson´s disease. To accelerate discovery of more selective therapies for these and other diseases, we are developing novel screening assays for a family of proteins, called ´regulator of G-protein signaling´ (RGS) that modulate the effects of GPCR ligands in a tissue-specific manner

Project start date: 2007-09-01

Project end date: 2008-08-31

1R43NS059082-01 (2007): $235189

Fluorescent HTS Assays For Methyltransferases In Neurodegenerative Diseases

Robert G Lowery, President
Bellbrook Labs, Llc

Grant 2R44GM073290-02 from National Institute Of General Medical Sciences, IRG: ZRG1

Abstract: Methylation is a ubiquitous and reversible covalent modification used to control the function of diverse biomolecules including their activity, stability and localization. Like phosphorylation, methylation is used to modulate protein function, but in addition many small molecules are regulated by methylation including hormones, neurotransmitters, xenobiotics, and lipids. More than 50 distinct methyltransferase (MT´s) enzymes are present in humans, and not surprisingly they are emerging as therapeutic targets for a broad range of diseases. Their involvement in neurodegenerative disease pathways is of special relevance because of the central role that methylation plays in regulating neurotransmitter function. The development of highly selective MT modulators is clearly a compelling medical priority. However, efforts to achieve this are being hampered by a lack of robust, flexible enzyme assay methods adaptable to high throughput screening (HTS). To address this need, in Phase I BellBrook Labs developed a novel fluorescence polarization immunoassay for S-adenosylhomocysteine (SAH), the invariant product of all MT reactions. This required development of a highly selective antibody that is able to differentiate between SAH and the substrate S-adenosylmethionine on the basis of a single methyl group. The Transcreener MT assay enables detection of any MT with any acceptor substrate in a homogenous format - and without the use of coupling enzymes - thus it eliminates all of the shortcomings associated with alternative methods. In Phase II we propose to 1) rapidly commercialize a first generation, fluorescence polarization-based Transcreener MT assay based of the polyclonal antibodies developed in Phase I, 2) increase flexibility and ease-of-use for the MT assay by using targeted mutagenesis of recombinant IgG clones to increase antibody selectivity for SAH, 3) increase HTS market penetration by formatting the assay for a second commonly used fluorescent detection mode, TR-FRET, and 4) use the novel MT assay combined with in silico screening to develop isoform selective inhibitors for five small molecule methyltransferases. The availability of robust, generic fluorescent HTS assay methods and selective chemical probes for MT´s should greatly accelerate their validation as drug targets and movement into the pharma drug discovery pipeline

Project start date: 2006-09-15

Project end date: 2010-07-31

Microfluidic Reconstituted Mammary Tissue System

Robert G Lowery, President
Bellbrook Labs, Llc Suite 250 Madison, Wi 53711

Grant 1R43DE017432-01 from National Institute Of Dental And Craniofacial Research, IRG: ZRG1

Abstract: Discovery of improved therapies for breast cancer will require assay systems that accurately recapitulate the cellular signaling of intact mammary tissue. To reconstitute mammary tissue structure and function faithfully in vitro, we will develop a microfluidic device for co-culturing human mammary epithelial and stromal cells in adjacent, separately addressable compartments that can be directly observed by microscopy. This assay system will enable identification and validation of molecular drug targets in a controlled microenvironment specifically designed to mimic the in vivo tissue context of mammary tumorigenesis. Three dimensional organotypic and heterotypic culture methods using cells imbedded in gel matrices have been developed to model ECM effects and paracrine signaling in the breast, but their reliance on traditional cell culture systems (e.g. tissue culture dishes) limits their accuracy and utility. In Phase I, we will fabricate a microfluidic device designed specifically for reconstructing and monitoring mammary tissue structure and function. Existing methods for 3D culture of epithelial organoids in proximity to stromal cells will be optimized for the device to produce a Microfluidic Reconstituted Mammary Tissue System (uF-rMTS). The scale and fluid control properties of the uF-rMTS will provide a more native microenvironment than the bulk aqueous environment of current tissue culture systems and allow better replication of in vivo signaling phenomena. In Phase II, the microfluidics device will be multiplexed to increase throughput, additional normal and malignant cell lines will be incorporated, and methods will be developed to probe paracrine signaling by growth factors and steroid hormones. Development of a system for probing tumorigenic signaling processes in the context of intact mammary tissue will accelerate the discovery of more effective therapies for breast cancer.

Keywords: biological signal transduction, biomimetics, mammary epithelium, microfluidics, mixed tissue /cell culture, paracrine, stromal cell, technology /technique development, antineoplastic, breast neoplasm, cell cell interaction, cellular polarity, controlled environment, drug screening /evaluation, extracellular matrix, growth factor, neoplasm /cancer chemotherapy, steroid hormone, 3T3 cell, biotechnology, confocal scanning microscopy, fluorescence microscopy, fluorescent dye /probe, immunocytochemistry

Project start date: 2005-09-15

Project end date: 2006-11-30

1R43DE017432-01 (2005): $252748

STRUCTURE BASED PREDICTION OF DRUG GLUCURONIDATION

Robert G Lowery, President
Panvera Corporation
545 Science Dr
madison, Wi 53711

Grant 3R43GM059542-01S1 from National Institute Of General Medical Sciences, IRG: ZRG1

Abstract: adapted from applicant´s ) Drug metabolism problems such as production of toxic metabolites and unfavorable pharmacokinetics cause almost half of all drug candidate failures during clinical trials. Although glucuronidation is one of the most important routes of biotransformation, the broad, overlapping substrate specificity of the hepatic UDP-glucuronosyltransferases (UGTs) that catalyze glucuronidation remains poorly understood. To meet the strong market demand for improved preclinical metabolism assessment and enable more rapid, cost-effective drug design, PanVera will develop a system for structure-based prediction of xenobiotic glucuronidation. To accomplish this, a quantitative structure activity relationship (QSAR) approach will be used to delineate the key properties of chemicals that determine their suitability as substrates for individual UGT isozymes. Phase I studies will focus on developing molecular tools for elucidating UGT substrate specificity using UGT2B7 as a model. Phase II studies will employ these tools to develop QSAR models for the key hepatic UGT isozymes. Both Phase I and Phase II studies will result in valuable commercial products for drug discovery, including the HTS assay method developed in Phase I, and the QSAR database and predictive modeling software developed in Phase II. The goal is to revolutionize drug discovery by providing a computerized solution to one of the most serious bottlenecks in drug discovery. PROPOSED COMMERCIAL APPLICATION NOT AVAILABLE

Keywords: chemical structure /function, drug /agent, enzyme activity, glucuronosyltransferase, liver metabolism, structural biology, technology /technique development enzyme substrate X ray crystallography, nuclear magnetic resonance spectroscopy

Project start date: 1999-05-01

Project end date: 2000-11-30

3R43GM059542-01S1 (2000): $45219

1R43GM059542-01 (1999): $144036