Project Title:

Small-molecule Inhibitors of Wee1 Degradation and Mitotic Entry

PI:

AYAD, NAGI G

Institution:

SCRIPPS FLORIDA RESEARCH INSTITUTE

Grant #:

1R21NS056991-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Our long term objectives are to understand the basic machinery and mechanism of mitotic entry and cell proliferation. Understanding cell proliferation is essential in generating cancer therapy since many cancer cells replicate uncontrollably. An essential feature of cell proliferation is the irreversible and controlled nature of its cell cycle transitions. Integral to these transitions are ubiquitin mediated proteolytic pathways that target substrates for proteasomal degradation. Proteolytic pathways contain E1, E2, and E3 enzymes that regulate both the timing and fidelity of degradation events. While we have identified many components of these pathways, we still have an incomplete understanding of how proteins are targeted for degradation. Both the timing and regulation of proteasomal targeting is not understood. This is especially true in the case of the mitotic entry. One of the proteins involved in inhibiting mitosis during the S and G2 phases of the cell cycle, weel, is degraded to initiate mitotic entry. Our goals are to elucidate how this particular protein is turned over to initiate mitosis. We will use a biochemical and cell biological approach to understand weel degradation and mitotic entry. We will develop a high through put assay to measure weel degradation in cells. We will determine if the compounds we attain after screening are specific for weel. Furthermore, we will determine if the same compounds also inhibit mitotic entry. The importance of known components affecting weel degradation is underscored by the finding that they are overexpressed in certain cancers. Relevance to public health: The elucidation of cell proliferation pathways is required for generating novel cancer therapeutics. An inhibitor of the proteasome degradation pathway is currently being used to treat certain cancers including multiple myeloma.-

Project Title:

Development of a high content cell based screen for inhibitors of the mTOR signal

PI:

BLENIS, JOHN

Institution:

HARVARD MEDICAL SCHOOL DEPARTMENT OF CELL BIOLOGY

Grant #:

1-R21-NS059428-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Mammalian target of rapamycin (mTOR) is a critical regulator of cell growth and proliferation, serving as the central integration point for multiple homeostatic inputs, including growth factor availability, energy levels and amino-acid sufficiency. Constitutive, unregulated mTOR kinase activity is a nearly universal feature of cancer cells, and defects in the mTOR signaling network have also been implicated in metabolic disorders and benign tumor syndromes. For these reasons there has been intense interest in the clinical application of derivatives of the natural product rapamycin which inhibits some aspects of mTOR-mediated signal transduction. While rapamycin has shown promise as an anti-cancer agent in clinical trials, the potential contribution of rapamycin-insensitive aspects of mTOR signaling to cancer progression, including the recently identified feedback loop involving the direct activation of Akt by mTOR, points to the critical need for additional therapeutic avenues in the treatment of diseases associated with deregulation of the mTOR signaling network. In order facilitate the identification of additional small molecules targeting mTOR, we will develop a high throughput, cell-based assay for the quantitative detection of rpS6 phosphorylation as a measure of the activation state of the mTOR pathway. Using this assay, we will perform a series of pilot studies, starting with known inhibitors of mTOR signaling, such as rapamycin and wortmannin, and culminating in pilot screens of a library comprising 200 commercially available kinase inhibitors, as well as a larger library of chemically diverse bioactive compounds. In addition, we will develop a series of secondary assays, combining high content imaging, biochemical assays, and cell based functional readouts, to evaluate targets identified in our high throughput assay for their ability to modulate mTOR signaling. Particular emphasis will be placed on identifying compounds that are able to act as ‘broad spectrum’ mTOR inhibitors that block the activity of both mTORC1 and mTORC2. Specific inhibitors identified through this work will not only serve as valuable research tools but may advance the treatment of diseases involving hyperactive mTOR signaling. In many cancers, the defective regulation of a key protein in the cell known as mTOR directly contributes to the uncontrolled growth of malignant cells. Rapamycin is a drug that blocks some, but not all, cellular functions of mTOR and has shown some promise in cancer treatment, but it is not always effective. Our work is aimed at finding new drugs to specifically inhibit all aspects of mTOR activity, with the goal of improving treatments for cancers involving mis-regulation of mTOR signaling. -

Project Title:

Fluorogenic PTP Assay for Use in HTS of Novel LYP Inhibitors

PI:

Bottini, Nunzio

Institution:

University of Southern California

Grant #:

1 R21 NS056945-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): A missense single-nucleotide polymorphism, C1858T in the PTPN22 gene is primarily associated with a wide range of human autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, Graves’ disease, and juvenile idiopathic arthritis. The PTPN22 gene encodes the lymphoid tyrosine phosphatase LYP, which is expressed only in white blood cells and acts as a gatekeeper of T lymphocyte activation. The molecular mechanism by which LYP tempers T lymphocyte activation involves the formation of a complex between LYP and the negative regulatory kinase Csk. We reported that the autoimmune-predisposing LYP-W620 variant cannot bind Csk, and more recently we found that the same variant is a gain-of-function form of the phosphatase. The increased inhibition of TCR signaling by LYP- W620 may lead to weaker signaling and therefore a failure to delete autoreactive T cells during thymic selection and/or insufficient activity of regulatory T cells. We hypothesize that a specific small-molecule inhibitor of LYP might be useful to revert the effects of LYP-W620 at the central and/or peripheral level, and prevent the emergence, or reappearance, of autoreactive T cells. Such an inhibitor would be widely applicable, both in helping to elucidate the biological role(s) of LYP and as a therapeutic in the treatment of many human autoimmune disorders. Unfortunately, there is currently no LYP assay available that is highly sensitive, continuous, and amenable to HTS. In the present proposal we will carry out all the preliminary work needed to begin a screening of NIH molecular libraries for inhibitors of LYP, and in particular we will (1) set up a reliable assay of LYP activity and (2) optimize it for HTS. -

Project Title:

Development of a High Throughput Screening Galanin 3 Receptor Assay

PI:

BROWN, STEVEN J

Institution:

SCRIPPS RESEARCH INSTITUTE

Grant #:

1R21NS057101-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): The major purpose of this Assay Development Proposal is to provide a key step in a fully integrated effort towards the goal of finding small molecule pharmacological tools for the Galanin 3 Receptor. The Scripps Research Institute (TSRI) is a center of excellence in chemistry and biology and its investigators have a strong record of success in identifying and characterizing small molecule pharmacological tools. The Scripps Molecular Libraries Screening Center (MLSC) is taking an active role supporting the NIH Roadmap’s effort to identify useful molecular tools. To create excellent small molecule discovery opportunities for eventual submission to the MLSC Network through the X01 application mechanism, we would like to enhance critical basic receptor tools for a clinically important neuroscience target, and ready this system for high throughput biology. 1. Develop a GALR3 beta-lactamase reporter assay and counter screens for high throughput screening. 2. Format and validate the GALR3 antagonist assay for HTS 3. Define the pathway for evaluating HTS derived compound leads in vitro and in vivo Galanin is a neuropeptide with three GPCRs (GALR1-3) that mediates its effects in the brain and peripheral nervous system. GALR3 represents a novel target for antidepressant drug action and there is a great medical need for antidepressants with new mechanism of action. Potent, specific and bioavailable GALR3 antagonists are needed to further validate this target for the treatment of anxiety and depression. Compounds with the desired profile of high potency, selectivity and ability to cross the blood-brain barrier (BBB) will be evaluated in animal models of anxiety and depression. Proof of concept (POC) studies with Galanin receptor agonists and antagonists may ultimately lead to improved therapeutic modalities for several neurological diseases. -

Project Title:

Development of small molecule inhibitors of Mcl-1 for cancer treament

PI:

Cardone, Michael H

Institution:

EUTROPICS PHARMACEUTICALS, INC.

Grant #:

1 R43 CA135915-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): The Bcl-2 family proteins are key effectors of cancers. Recent studies indicate that Myeloma cell factor-1(Mcl-1), a member of this protein family, is a key regulator of lymphoid cancers including B-cell lymphoma, multiple myeloma, and chronic lymphocytic leukemia. The Bcl-2 family proteins are regulated by distinct protein- protein interactions. Manipulation of these interactions using compounds that mimic the crucial binding domain, the BH3 domain, is a valid approach to developing oncology drugs. Efforts by commercial and academic groups have yielded several classes of compounds that target certain members of the Bcl-2 family. To date however, there has been very little effort in developing BH3 mimetics that selectively target the Mcl-1 function, though there is an identified need for such compounds. Here we propose an effort to do this using a medicinal chemistry approach that will be bolstered by two novel technologies. Our starting compound, EU-517 was discovered at Harvard Medical School in a small molecule screen for inhibitors of BH3 peptide binding to Bcl-xL. More recently derivatives that have preferential activity against Mcl-1 were made. These will be used as the starting point for developing an anti-Mcl-1 lead compound. An innovative flexible docking model that comes from the Gerhard Wagner lab at Harvard Medical School will be used to guide lead optimization. A novel cell based assay that replicates the Mcl-1 “oncogene addicted” state is licensed from Dana Farber and will be used to select appropriate anti-Mcl-1 activity. This effort will receive support from academic consultants Gerhard Wagner and Anthony Letai, the inventors of the two technologies. The medicinal chemistry plan is in place and will be carried out by MedChem Partners Inc. in Medford MA. PUBLIC HEALTH RELEVANCE:Eutropics has assembled a strong science team to develop anti-cancer therapeutics called BH3 mimetics [4]. Recently we found anti- tumor activity of a BH3 mimetic compound that Eutropics has now licensed from Harvard Medical School. This compound and now its derivatives have activity in inhibiting the myeloid factor-1(Mcl-1) protein in in vitro and cell based assays. Mcl-1 has been shown to be causal in certain blood cancers [9,10,11] and is considered a viable drug target. Here we propose utilizing two novel technologies to bolster a medicinal chemistry effort to develop selective Mcl-1 inhibiting compounds. -

Project Title:

Chemical Approaches for Activity Based Proteomics
2 R01 CA087660-05
Dual: GM
IRG: BNP
Chemical Approaches for Activity Based Proteomics

PI:

Cravatt, Benjamin F.

Institution:

The Scripps Research Institute

Grant #:

1 R01 CA087660-05

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Enzymes are central components of nearly all metabolic and signaling pathways in cells and tissues. The dysregulation of enzymes and their endogenous inhibitory proteins contributes to the development of several diseases, including cancer. Nonetheless, efforts to elucidate the function of specific enzymes in cancer have, to date, suffered from a lack of techniques that can assess the activity of these proteins in complex biological systems. To address this problem, we have introduced a chemical proteomic strategy termed activity-based protein profiling (ABPP) that utilizes active site-directed probes to measure changes in enzyme activity directly in native proteomes. To date, we have generated activity-based probes that target numerous enzyme classes, including proteases, lipases, histone deacetylases, and cytochrome P450s. In the previous funding period, we applied ABPP to identify several enzyme activities dysregulated in aggressive human cancer cells, including the serine proteases uPA and tPA, and the previously uncharacterized transmembrane hydrolase KIAA1363. We have shown that these enzymes play important roles in supporting the malignant properties of cancer cells, although the biochemical mechanisms for these effects remain to be fully elucidated. We have also introduced advanced “tag-free” versions of ABPP that exploit the versatility of click chemistry and resolving power of mass spectrometry to enable high-content profiling of small molecule-protein interactions in living systems. Finally, we have developed a complementary proteomic platform for globally mapping the endogenous substrates of proteases termed PROTOMAP (PROtein TOpography and Migration Analysis Platform). In this competitive renewal application, we will apply our suite of ABPP and PROTOMAP technologies to test three major hypotheses of high significance to the fields of cancer and chemical biology: 1) KIAA1363 promotes cancer aggressiveness through regulation of an ether lipid signaling network, 2) dysregulated proteases contribute to cancer pathogenicity by activating and/or inactivating key signaling pathways, and 3) tag-free ABPP will offer a general and quantitative technology to map small molecule-protein interactions in living systems. We anticipate that these studies will define key enzymatic pathways that support cancer malignancy and contain new biomarkers and therapeutic targets, as well as produce methodological advances that greatly expand the scope and utility of the ABPP and PROTOMAP technologies. PUBLIC HEALTH RELEVANCE: A large fraction of human enzymes remain uncharacterized in terms of their function in health and disease. We have developed advanced chemical technologies to functionally characterize enzymes directly in native biological systems. The goal of this application is to further develop and apply these technologies to identify enzymes that play important roles in cancer, which may serve as valuable new biomarkers and therapeutic targets. -

Project Title:

High Throughput/Content Screens for Dynein Inhibitors

PI:

DAY, BILLY W

Institution:

UNIVERSITY OF PITTSBURGH

Grant #:

1R21NS057026-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Cytoplasmic dynein is the major retrograde molecular motor responsible for transport of membranous organelles, viruses, mRNA and proteins with nuclear localization signals. It is also involved in chromosome movement, mitotic checkpoint inactivation and spindle orientation, and many other basic cellular processes. Cytoplasmic dynein is a large (ca. 2 MDa) complex of proteins. Its movement along microtubules is powered by ATP hydrolysis by its 380 kDA motor domains, which are associated with the >500 kDa heavy chain subunits. Other than weak and nonspecific redox perturbers and mimics of ATP or phosphate anion, only one small molecule inhibitor of dynein, the natural product purealin, which we have recently synthesized and examined, is known. It is, however, only weakly active in cells. New inhibitors with potency and specificity for dynein and subsets of dynein function would be invaluable cell biology tools. The goal is to develop a refined suite of high throughput cell and biochemical assays for chemical library screening find inhibitors of cellular cytoplasmic dynein. Aim 1 is to develop a cell-based phenotypic multiparameter fluorescence screen to detect dynein inhibition in interphase cells, as well as to detect possible mitotic block due to inhibition of dynein. Preliminary data provides support of three cellular events, transport from the cytoplasm to the nucleus of: (i) p53 after mild DNA damage; (ii) stably expressed green fluorescent protein-labeled glucocorticoid receptor after binding with an agonist; and (iii) a fluorescent adenovirus. These will be examined and the most useful selected. As some phenotypes could be due to interaction of the small molecules with off-target proteins, Aim 2 is to implement high throughput biochemical screens to confirm that a small molecule’s molecular target is indeed cytoplasmic dynein. These include microtiter plate-based colorimetric, turbidimetric and fluorescence polarization analyses of the direct action of library chemicals on recombinant dynein heavy chain, glucocorticoid receptor ligand binding domain, HSP70 and HSP90, and on isolated myosin and tubulin. The investigators, a team of experts in these areas, will design and perform the assays as well as interpret results, and have previous direct or related experience with most of the assays. The product of this work will be a refined, streamlined set of rapid cell and biochemical screens for dynein inhibitors that can be implemented in molecular library screening centers. -

Project Title:

A cell-based screen for inhibitors of intracellular Abeta aggregation

PI:

DELISA, MATTHEW P

Institution:

CORNELL UNIVERSITY ITHACA

Grant #:

1R21NS056911-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): There is an emerging consensus that non-fibrillar intracellular Abeta aggregates, rather than insoluble fibrils, are the most deleterious Abeta species and may play a central role in Alzheimer’s disease (AD) pathogenesis. Thus, an attractive therapeutic approach to AD would be to seletively reduce the levels of potentially synaptotoxic Abeta aggregates by either stabilizing intracellular Abeta in its monomeric form or destabilizing the oligomeric structure. Low molecular weight drugs represent the most attractive therapeutics for inhibiting Abeta aggregation as many small molecules are capable of permeating the blood-brain barrier (BBB) and crossing cell membranes. Historically, however, protein aggregation has been an extremely difficult target to address with synthetic drug-like molecules, owing in part to the large surface area generally covered by two interacting proteins and to the large, flat binding surfaces between the proteins. Another challenge is that while new types of organic compounds may be extremely potent when tested against isolated targets in the laboratory, they may cross-react with cellular components other than the desired target. Small molecules found in nature, often called ‘natural products’, typically have spent time inside of a cell during the course of evolution and are less likely to interact in a manner that damages cellular components such as membranes or DMA. In addition, it has been shown recently that many natural products are quite effective at inhibiting a diverse array of protein-protein interactions. Thus, an important question that we are exploring is whether natural products or natural product-like molecules can be isolated that effectively inhibit Abeta aggregation and, at the same time, be tolerated by living cells. The long-term goal of this research is to identify natural product-like inhibitors of intracellular Abeta aggregation that have potential as therapeutic agents for treating AD. Towards this goal, we have generated a cell-based assay for directly monitoring Abeta folding in the intracellular environment. This particular application seeks to: (1) configure our novel cell-based folding assay for high-throughput screening of combinatorial small-molecule libraries; and (2) isolate natural product-like compounds from diversity-oriented synthesis libraries that are capable of antagonizing Abeta aggregation. Such compounds will serve as leads for AD therapy and for biological studies that illuminate the physiological role of Abeta folding in mediating neurotoxicity. -

Project Title:

Modulators of CL-dependent Transport Processes

PI:

DELPIRE, ERIC J

Institution:

VANDERBILT UNIVERSITY

Grant #:

1R21NS053658-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Coupled movement of Na+, K+, and Cl-through cell membranes occurs primarily through Na-K 2CI and K-CI co-transporters, which belong to the Slc12 family of membrane transporters. Until the identification of the multiple genes that constitute this family, the physiology of cation-chloride co-transporters was dominated by the critical role that some of these co-transporters play in water and ion homeostasis in the kidney. In fact, the co-transporters are best known through their inhibitors: the loop and thiazide diuretics which are widely used in clinical medicine to volume deplete patients. The cloning of the human and mouse genomes identified 9-10 members of the Slc12 family of solute carriers. Among them, 7 are functionally well characterized: there are 1 Na-CI co-transporters, 2 Na-K-2CI co-transporters, and 4 K-CI co-transporters. These transporters participate in a wide variety of function which range from fluid secretion/absorption in multiple epithelia, modulation of synaptic transmission, cell volume control and regulation, cell proliferation. Mutations in cation-chloride co-transporters are responsible for diseases such as salt wasting disorders and peripheral nerve degeneration. They are also possibly involved in hypertension, age-related loss of hearing, neurological and psychiatric disorders. Whereas loop diuretics such as furosemide and bumetanide inhibit most cation-chloride co-transporters, these drugs are rather unspecific and not very potent. Thus, there is a critical need for the development of new compounds targeting cation-chloride co-transporter function. The development of novel fluorescent-based reagents, sensitive to anions and cations, now permits the development of better methods for High Throughput Screening (HTS). We propose to 1) Develop a fluorescence-based assay to monitor the activity of cation-chloride co-transporters. This will be achieved through heterologous expression of NKCC1 and KCC2 together with anion-sensitive fluorescent protein in HEK-293 cells. Based on studies showing that K+ channels can carry thallium, we will also examine the feasibility of use of a thallium-sensitive indicator dyes. 2) Provide a validation the assay for High Throughput Screening. This will be achieved through a detailed analysis of effects of a known inhibitor (e.g. bumetanide) on fluorescent signals generated by the movement of the surrogate anion or cation through the cell membrane and a through a test screening of a library consisting of 10,000 compounds. The development of a High Throughput fluorescent method to measure cation-chloride co-transporter function will allow us to transit to actual HT screens to identify novel compounds targeting the function of cation-chloride co-transporters. -

Project Title:

Identification of novel modulators of ROMK K+ channel activity

PI:

DENTON, JEROD S

Institution:

VANDERBILT UNIVERSITY

Grant #:

1R21NS057041-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): The Renal Outer Medullary K+ channel, ROMK (Kir1.1, KCNJ1), plays key physiological roles in maintenance of systemic electrolyte and water homeostasis by the kidney. ROMK function is critical for salt re-absorption and is thought to form the dominant pathway for K+ secretion in the kidney nephron. The importance of ROMK is underscored by the identification of heritable loss-of-function ROMK mutations in patients with type Bartter’s syndrome, a severe kidney tubule disorder characterized by salt and water wasting and acid-base disturbances. Given the pivotal role of ROMK in kidney function and disease, the identification of novel ROMK modulators is highly desirable and could provide valuable tools for basic research and the treatment of numerous kidney-related disorders. However, currently available assays of ROMK function are slow, labor intensive and therefore not amenable to high-throughput screening (HTS) of chemical libraries. Furthermore, a significant barrier to the development of high-throughput (HT)-compatible assays of ROMK function has been the inability to express sufficient ROMK in the plasma membrane of transfected cells. To circumvent this limitation, we have engineered a single point mutation (S44D) into the cytoplasmic N- terminus of ROMK that allows expression of robust K+ currents in transfected HEK-293 cells. Importantly, we further show that ROMK-S44D activity can be monitored in 384-well format using a novel fluorescence- based assay of channel function. In this grant application, we propose to 1) develop a fluorescence-based assay of ROMK activity using ROMK-S44D as a surrogate channel. A detailed characterization of ROMK- S44D function will be performed to test its utility as a surrogate for the wild type channel. We will also test the effects of the known ROMK inhibitor tertiapin-Q on fluorescence signals generated by the movement of thallium (Tl+) through ROMK-S44D expressed in HEK-293 cells loaded with the Tl+-sensitive dye BTC. 2) Validate the fluorescence assay for use in HTS. This will be done by performing a small scale, 10,000 compound validation screen to identify lead compounds for secondary HT screens. Lay Summary: The development of high throughput assays for ROMK function holds significant potential for the identification of novel compounds that could be used in basic science discovery as well as clinical medicine for the management of hypertension, edema and other kidney disease-related disorders. -

Project Title:

Chemical Probes for Uncovering Differential Regulators of Apoptosis in Cells

PI:

DESHMUKH, MOHANISH

Institution:

UNIVERSITY OF NORTH CAROLINA CHAPEL HILL

Grant #:

1R03NS057037-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Although studies in cell lines have identified the main components of the mammalian apoptotic pathway, the differences in the regulation of apoptosis among various primary cells remain largely unexplored. For example, biochemical and molecular studies have identified the caspase proteases as central executioners of apoptosis and the release of mitochondrial holocytochrome c (heme-attached cytochrome c) as a critical trigger that activates caspases during apoptosis. However, despite the central importance of cytochrome c- mediated caspase activation in mammalian apoptosis, our understanding of how this pathway is regulated in primary cells is very limited. Our recent results point to a potentially fundamental difference in the regulation of caspase activation in primary mitotic versus postmitotic cells. We find that whereas holocytochrome c alone is necessary and sufficient to activate caspases and induce apoptosis in mitotic cells, it is necessary but not sufficient to induce apoptosis in neurons, cardiomyocytes, and myotubes. This difference is striking and is indicative of novel, differential mechanisms of apoptosis regulation in mitotic versus postmitotic cells. Unfortunately however, our understanding of the molecular basis for this difference is limited because the only current method for activating apoptosis at the point of cytochrome c is via single cell microinjection of holocytochrome c. Thus, these experiments are restricted to only those cells that can be microinjected and they are not compatible with large scale biochemical analysis. To overcome these limitations and to develop novel probes for studying apoptosis regulation, we will adapt a cell-free caspase activation assay for high throughput compatibility to screen for small-molecule compounds that trigger apoptosis after the point of mitochondrial cytochrome c release in cells. The development of these chemical probes is significant in two important aspects: a) They are essential tools for discovering the molecular mechanism of the differential regulation of apoptosis in mitotic and postmitotic cells, and b) These probes are predicted to have therapeutic value as reagents that induce apoptosis only in selective cells. -

Project Title:

Assay to Screen Ligands:Activate Opioid Receptor

PI:

DEVI, LAKSHMI A

Institution:

MOUNT SINAI SCHOOL OF MEDICINE OF NYU

Grant #:

1 R03 NS053751-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): G-protein coupled receptors constitute the single largest family of cell surface receptors that mediate physiological responses to a wide variety of stimuli. These receptors are characterized by seven (7) transmembrane domains and interact with heterotrimeric G-proteins to transducer cellular signals. For a number of years our studies have focused on understanding the molecular mechanisms that modulate opioid receptors; these receptors are activated by binding to classic opiates such as morphine. Opioid receptor function is modulated by multiple mechanisms; we have focused on receptor dimerization as a novel mechanism to regulate opioid receptor function. We have characterized the properties of heterodimers between opioid receptor types (i.e. mu and delta opioid receptors) using biochemical, biophysical and pharmacological techniques. We have found that the mu receptor-mediated signaling in cells and analgesia in mice can be enhanced by a delta receptor antagonist. These results suggest that heterodimerization between mu and delta receptors leads modulation of the extent of signaling. In order to address the physiological consequences of mu delta heterodimerization, ligands that selectively activate or inactivate mu-delta heterodimers (without affecting the activities of mu or delta receptors) are needed. In this application we propose to develop a high throughput screening (HTS) assay to screen for ligands that selectively bind and activate mu-delta heterodimers. These studies will enable HTS screening and identification of small molecules that would be critical tools to probe the role of mu-delta heterodimers in vivo.-

Project Title:

Prostanoid modulators that reduce brain injury and inflammation after seizures

PI:

DINGLEDINE, RAYMOND J

Institution:

EMORY UNIVERSITY SCHOOL OF MEDICINE

Grant #:

1U01NS058158-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Injury of the brain is a major cause of death and morbidity after exposure to nerve agents that activate the muscarinic receptor system, which often causes long term disability with unusually high accompanying social and medical costs. Current countermeasures such as midazolam and atropine are of doubtful utility for civilian populations because they must be administered within minutes of an attack to be effective. Therapies that can be administered hours to days after the attack are needed. Prolonged status epilepticus (SE) induced by the muscarinic agonist, pilocarpine, triggers a similar series of molecular and cellular events in the rodent brain. Cyclooxygenase-2 (cox-2) is induced strongly in principal hippocampal neurons after a seizure. Our overarching goal is to develop small molecules that act on specific prostanoid receptors to oppose seizure-induced neurodegeneration, neuroinflammation and functional deficits in adults and children subject to chemical attack by nerve agents. Specific aims 1 and 2 are designed to answer several key questions in order to better understand the basis of these neuropathologies: Which prostaglandin receptors are involved in neuronal injury and neuroinflammation after SE, and in delayed sensorimotor deficits? Which cell type(s) release PGE2 and PGD2? We will test the specific hypotheses that an allosteric EP2 receptor activator is neuroprotective after pilocarpine-induced SE, and a DP2 receptor blocker blunts the neuroinflammatory response. In aims 3 and 4 we identify novel small molecule modulators of selected prostanoid receptors that are brain permeant and can be delivered after the period of acute convulsions to blunt SE-induced delayed neurodegeneration, neuroinflammation and cognitive deficits. Successful completion of these studies is an essential step to translate our research findings into a useful countermeasure to a prominent class of chemical threats, and will prepare the ground for subsequent validation in animals exposed to sarin-type nerve agents themselves, followed by GLP preclinical pharmacology studies, IND submission, and Phase I clinical safety trials. -

Project Title:

A cell-based HTS for delayed death inhibitors of the malarial parasite plastid

PI:

FIDOCK, DAVID Armand

Institution:

Dept. of Microbiology Columbia University

Grant #:

1-R21-NS059500-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Plasmodium falciparum, an Apicomplexan parasite and the causal agent of severe malaria, causes disease in over 500 million individuals and kills over one million children yearly. Existing drugs suffer from parasite resistance, high cost or toxicity, and the pre-clinical pipeline is woefully thin. Recent antimalarial drug discovery efforts have focused on the apicoplast, an essential organelle phylogenetically related to cyanobacterial plastids. This organelle is the site of fatty acid, isoprenoid and heme biosynthesis, and contains an estimated 400 nuclear-encoded proteins, many of unknown function. Detailed studies with antibiotics that target apicoplast ribosomes (such as azithromycin) reveal that apicoplast inhibitors manifest a unique delayed death phenotype, whereby only the progeny of drug-treated parasites die. Why the apicoplast is essential for P. falciparum and what factors mediate its critical biological functions are intriguing questions that can be experimentally addressed with novel apicoplast-specific probes. We propose to develop assays that identify inhibitors of apicoplast development by taking advantage of this delayed death phenotype. Our primary screen will expose cultured P. falciparum to compounds for one or two generations (48 or 96 hr). Parasite growth will be measured using the dsDNA-intercalating SYBR Green dye. Compounds will be tested first in 96 hr assays, and only active compounds will be retested in 48 hr assays. Compounds that manifest increased potency only in the second generation will be retained for further screening. This will include repeat assays, dose-response assays to identify well- behaved inhibitors, and counterscreens to select against generally cytotoxic inhibitors. One secondary screen will use [3H]-hypoxanthine as an alternative measure of growth inhibition. Another, based on whole cell imaging, will use a transgenic line expressing GFP-labeled apicoplasts to visually identify inhibitors of apicoplast development. Assay development will be guided using the reference compounds azithromycin, chloroquine and isoniazid (representing delayed death, fast-acting and inactive compounds respectively) and configured for HTS using a library of 400 FDA-approved drugs. Later studies will include optimization on the Tecan HTS automation platform at the Molecular Libraries Screening Center Network Center at Columbia University, in collaboration with members of this center. In addition to their therapeutic potential, apicoplast inhibitors will be invaluable in allowing us to directly address questions about key apicoplast processes and proteins. We propose to develop a high throughput assay to screen chemical libraries for compounds that inhibit the development of a compartment called the apicoplast in Plasmodium falciparum malaria parasites. Inhibition of the apicoplast is characterized by a type of delayed death whereby compounds only kill the progeny of drug-treated parasites inside infected red blood cells. We intend to identify new inhibitors that can be used to understand apicoplast biology and the reasons for this delayed death, and that have potential for the development of new therapeutics. -

Project Title:

High-Throughput Screening for Human Immunodeficiency Virus Fusion Inhibitors

PI:

GOCHIN, MIRIAM DR

Institution:

TOURO UNIVERSITY CALIFORNIA

Grant #:

1 R21 NS059403-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): This proposal describes the development of a high throughput screening assay for small molecules effective against Human Immunodeficiency Virus Type 1 (HIV-1) fusion. Preventing HIV-1 fusion would inhibit the entry of virus into human host cells, effectively protecting uninfected cells and improving the available treatment options for HIV infected patients. Currently there is a single fusion inhibitor, a peptide Enfuvirtide(r), which is expensive and available only by intravenous administration. Drugs which can be taken orally are low molecular weight compounds which usually can be manufactured at lower cost and be more widely distributed. The screening assay will be capable of automated testing of thousands of low molecular weight compounds available in academic, government and pharmaceutical facilities. Compounds selected by the initial screen would be subject to further testing and modification to improve potency. The assay described involves the simple addition of two peptides derived from HIV-1 gp41, the viral fusion protein, to wells of plated library compounds. The peptides are modified with a fluorophore and metal-ligated dye complex, which enables their micromolar association to be followed by a simple fluorescence intensity readout. Compounds from a library are assessed for activity by their ability to competitively inhibit the peptide association, with a concomitant fluorescence increase. A positive result indicates that a compound is capable of fusion inhibition. The intensity of the fluorescence signal is directly correlated to the compound’s potency. This is a biochemical assay, using inexpensive and non-hazardous components. We will show that it is highly specific for the viral target, extremely robust and sensitive, and an excellent indicator of a compound’s ability to inhibit fusion in cell culture. In this study, we will optimize the assay for maximum sensitivity, broaden the selection of small molecules to include fragments that could be tethered to create more powerful inhibitors, and explore NMR and fluorescence methods for facilitating the optimization process of newly discovered hits from a library. This project will enable systematic screening of compound libraries for HIV-1 fusion inhibitors. The screen will specifically identify molecules that bind to the gp41 fusion protein in such as way as to prevent the conformational change required for effective viral fusion. Newly discovered small molecule candidates may be developed into entry inhibitors effective in controlling HIV-1 infection, as part of a multi-drug treatment strategy. -

Project Title:

HTS for Novel Activators of Adipocyte and Myocyte Lipolysis

PI:

GRANNEMAN, JAMES G

Institution:

WAYNE STATE UNIVERSITY

Grant #:

1-R21-NS061634-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): The storage and mobilization of lipid are fundamental cellular processes, and its dysregulation contributes to numerous diseases including diabetes, atherosclerosis and cardiomyopathy. Recent work indicates that the rate-limiting step for the mobilization of intracellular triglyceride is the release of Abhd5, a lipase co-activator, from specific lipid droplet scaffold proteins. We hypothesize that compounds that disrupt the association of Abhd5 with the scaffold proteins perilipin (Plin) and muscle lipid droplet protein (Mldp) will promote clearance of lipid from fat and muscle cells, respectively. We have developed a highly-sensitive assay based on protein complementation that can monitor the interaction of Abhd5 with Plin and Mldp in mammalian cells. This proposal seeks to adapt this assay to a cell-free system for high throughput screening. Active compounds will be characterized in secondary screens to demonstrate specificity, potency and efficacy. Compounds meeting criteria will be used as novel chemical probes to test specific mechanisms of cellular lipolysis in fat and muscle cells. Our long term goal is to identify lead compounds that will reduce lipid accumulation specifically in fat and muscle and thus would be potential novel treatments of obesity-related disorders including diabetes and cardiovascular disease. -

Project Title:

Developing HTS Assays for Discovery of Small Molecule Regulators of Integrins

PI:

GUPTA, VINEET

Institution:

MASSACHUSETTS GENERAL HOSPITAL

Grant #:

1R03NS053659-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): The leukocyte specific beta2 integrins are central to the biological function of these cells. These cellular receptors mediate the divalent metal ion dependent adhesion of leukocytes including homing, firm adhesion, migration, respiratory burst and clearance of pathogens through phagocytosis and cell mediated killing. The CD11b/CD18 (alpha-M-beta2) heterodimer is the predominant beta2 integrin receptor in neutrophils and macrophages and mediates the pro-inflammatory functions in these cells. CD11b/CD18 recognizes a wide variety of ligands, including the complement fragment iC3b, fibrinogen, blood clotting factor X and CD54 (ICAM-1). Physiologic binding of CD11b/CD18 receptors to its various ligands is tightly regulated, as defects, its loss or inappropriate activation lead to severe pathological conditions and also contribute to injury in many non-infectious diseases, such as renal failure, allograft rejection, heart attacks, strokes and autoimmune diabetic complications. Thus, CD11b/CD18 represents an important target for small molecule drugs. Although ligand-mimetic antagonists that block CD11b/CD18 ligand binding, such as anti-CD11b/CD18 monoclonal antibodies (mAbs), have been shown to be effective in certain diseases in animals, small molecule regulators, especially allosteric regulators, are pharmacologically more desirable for human therapeutic applications. There is an inadequate array of selective and potent small molecule beta2 integrin modulators available to the public, and there are no CD11b/CD18 selective small molecule antagonists, especially allosteric inhibitors, currently available. Additionally, no CD11b/CD18 specific agonists are available at this time. A number of established functional assays, including some developed in our laboratory, can be modified and adapted for screening of small molecule regulators of CD11b/CD18. The major goals of our current proposal are to optimize and automate these available assays for a high throughput screening (HTS) environment and use them in a pilot screen to discover small molecule effectors of CD11b/CD18 (Aim 1). Furthermore, we have recently discovered a novel allosteric regulatory site, the beta tail Domain (3TD), in integrins. In this proposal, we would also like to develop HTS assays for identifying small molecules that bind at this site and thus, allosterically regulate integrin activation (Aim 2). We believe that unique molecules discovered in our screen will not only be useful as therapeutic leads against CD11b/CD18, but may also serve as novel probes for structural and mechanistic studies and as integrin conformation sensors. Such studies will undoubtedly shed new light into the mechanism of integrin activation as well. Given the central role of beta2 integrins in regulating leukocyte function, we hope that this research will provide important new tools and further guide future experimentation. -

Project Title:

Novel Inhibitors of Nuclear Receptor Function

PI:

Guy, Rodney K.

Institution:

ST. JUDE CHILDREN’S RESEARCH HOSPITAL

Grant #:

2 R01 DK058080-05

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): The broad, long-term objectives of this proposal are the development of novel “drug-like” inhibitors that specifically prevent the interaction of nuclear receptors with their coregulating proteins through their NR box binding site and functionally block transcriptional activation by nuclear receptors. The Specific Aims of this proposal are 1) to determine which chemical scaffolds can afford small molecule inhibitors of the interaction of co-regulators with the thyroid receptor (TR), glucocorticoid receptor (GR), androgen receptor (AR), and peroxisome proliferator activated receptor y (PPARy) and which scaffolds exhibit selectivity for individual receptors; 2) to understand the molecular mechanism of inhibitor function by determining the modes of binding of active inhibitors and the structure activity relationships of scaffolds with individual receptors and among receptors; and 3) to determine how molecular selectivity of action against a particular receptor relates to functional changes in signaling by the targeted receptor and overall changes in transcriptional regulation in the cellular environment. The health relatedness of this project is that the new method of inhibiting nuclear receptor function has the potential to provide new therapies for diseases mediated by nuclear receptors which include cardiovascular disease, diabetes, and osteoporosis-diseases currently treated with drugs based upon hormone structure. The research design is the use of knowledge based structure, chemistry, and cell biology to rationally produce novel small molecule inhibitors of the targeted protein interaction. The methods to be used are high throughput screening, parallel chemistry, medicinal chemistry, high throughput X-ray crystallography (structural genomics), cell biology, genomics, and pharmacology to develop inhibitors. -

Project Title:

Development of a small molecule screen for PhoP regulon inhibitors in Salmonella

PI:

HARRIS, JASON B

Institution:

MASSACHUSETTS GENERAL HOSPITAL

Grant #:

1 R21 NS059429-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Although high-throughput technologies have enhanced our understanding of bacterial virulence gene regulation, the development of antibiotics that target the regulatory systems that are essential for bacterial pathogenesis has not been extensively pursued. We hypothesize that small molecules inhibiting the conserved PhoP virulence regulon may constitute effective antibiotics. The PhoP regulon is an essential regulator of the genes required for intracellular survival and virulence of a number of pathogens and has been best characterized in the model organism of Salmonella enterica serovar Typhimurium. Here, we propose to develop a high throughput molecular screening (HTS) assay to identify chemical inhibitors of the PhoP regulon. In particular, our strategy will be designed to screen for small molecules that simultaneously inhibit the expression of reporter genes from PhoP-activated promoters, while increasing the expression of genes from PhoP-repressed promoters. In Specific Aim 1, we will develop a series of recombinant PhoP-activated and PhoP-repressed promoter-reporter fusions, and quantify the expression of these reporters in serovar Typhimurium grown in PhoP-inducing and non-inducing conditions. In Specific Aim 2, we will configure the assay for HTS by selecting a single PhoP-activated and a single PhoP-repressed promoter with the highest signal-to- background ratios and piloting the assay in 96 and 384 well formats. In this proposal we also outline a detailed sequential strategy for the secondary evaluation and prioritization of active compounds identified in the initial HTS screen. Multi-drug resistant bacteria are important causes of global morbidity and mortality and have the capacity to overcome our current biodefense, all of which necessitate the development of novel antibiotic classes. The proposed assay may identify small molecules with unique antibiotic properties and a novel mechanism of action against intracellular pathogens including the drug- resistant cause of typhoid fever, a major cause of morbidity and mortality worldwide, as well the causative agent of the plague, Yersinia pestis, a significant bioterrorist threat. In future studies, compounds identified using the proposed assays may be developed into a new class of drugs targeted for the treatment of many bacterial infections, including several category A and B priority pathogens. -

Project Title:

HTS assays for discovery of novel beta-lactamase inhibitors via click chemistry

PI:

Hodder, Peter Simmons

Institution:

Scripps Research Institute
5353 Parkside DR, RF-1

Grant #:

1-R21-NS059451-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Beta-lactamase mediated bacterial resistance represents one of the most persistent challenges to antibiotic chemotherapy. In anticipation of a clinical threat from metallo- beta-lactamases, there is need for potent inhibitors of the clinically relevant class B1 metallo-beta-lactamases (MBL). Current MBL inhibitors have only been tested and developed for one clinically relevant class B1 MBL, IMP-1, or less clinically relevant MBLs. Most important to our research endeavor is that an array of potent inhibitors has not been developed or described for VIM-2. This class B1 MBL is second only to IMP-1 in its clinical relevance. The preliminary goal of this effort is to establish a novel FRET-based HTS assay to identify inhibitors of VIM-2. Using a traditional HTS research operating plan, we will use a nitrocefin-based assay will be used to identify compounds that may be artifact in the FRET-based assay. To probe whether a compound active in the VIM-2 assay has potential utility as a broad-spectrum MBL inhibitor, an IMP-1 assay will also be developed & implemented. We feel that our HTS-based approach will not only rapidly discover probes, but also help triage those probes before proceeding to antibiotic potentiation assays. Our long term objective is to design MBL class B1 chemical probes. We will begin this endeavor by screening our in-house “click-chemistry” library, from Professor Barry Sharpless’ research laboratory, against VIM-2. The inhibitors found in this assay will have “spring-loaded” moieties that promote straightforward and rapid SAR studies. Through screening of larger compound libraries, e.g. the MLSCN compound collection, the results of both activities should have an impact on VIM-2 related research by providing publicly available or easily synthesized VIM-2 probes. By modifying the probes to have broad spectrum inhibition, we plan to further advance the clinical relevance of our research. The diversity of infectious bacteria continues to outpace the development of novel drugs to treat them. Often bacteria appear to be resistant to traditional antibiotics. Beta lactamases are bacterial enzymes that are responsible for resistance to many antibiotics. Our goal is to develop several high throughput screening assays suitable for rapid identification of novel drugs that modulate the activity of beta-lactamases. In consort with Prof. Barry Sharpless (TSRI: Dept of Chemistry), we will screen “click-chemistry” libraries for a synergistic and novel approach to the rapid synthesis and discovery of beta-lactamase inhibitors. -

Project Title:

A Cell-based Screen for Small Molecule Modulators of the miRNA Pathway

PI:

HUANG, QIHONG

Institution:

WISTAR INSTITUTE

Grant #:

1 R21 NS059478-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are single-stranded noncoding RNAs and represent a novel class of recently discovered gene regulators. It is estimated that each miRNA controls hundreds of gene targets and that miRNAs are involved in the regulation of about 30% of all genes and almost every genetic pathway. Evidence suggests that they play important roles in developmental processes and viral infection, and they can function as oncogenes and tumor suppressors. Hence, it has been suggested that miRNAs and the components of the miRNA pathway can serve as targets for the discovery of novel therapeutic agents. Although the miRNA pathway is important in physiological and pathological processes, such as cancer development, to our knowledge, no small molecule inhibitor of this pathway has been identified. To fill this gap, a high-throughput cell-based assay will be developed for the screening of small molecule modulators of the miRNA pathway. This goal will be achieved through the following two specific aims: 1) a reporter system consisting of a luciferase gene fused to the binding site of human miRNA-21 (miR- 21) will be introduced into human HeLa cancer cells. Inhibition of the miRNA pathway by small molecules will lead to an increase of a luciferase reporter signal and thus provide an advantageous positive read-out. This assay will be used to screen small molecule libraries (>1000 compounds) in a high throughput fashion to discover molecules that interfere with the miRNA pathway. 2) Three secondary assays will be developed to validate and characterize the compound hits from the primary screen. These assays will exclude non-specific small molecule hits and deliver a more detailed picture of the miRNA pathway steps that are targeted by active compounds. Positive hits will be further investigated and improved through structure-activity relationship studies, via synthesis of second generation compound arrays. The information gained will ultimately lead to the elucidation of a specific target-small molecule interaction. Our long term goal is to develop chemical tools to better understand the molecular mechanisms of the miRNA biogenesis, the functions of specific miRNAs, and to assess the global impact of miRNAs on various cellular processes and pathways. Small molecules discovered in our screen are expected to have a broad impact on human health, due to the involvement of miRNAs in several human diseases (including cancer and viral infection) and the increasing interest in the miRNA pathway as a drug target. These molecules will be promising lead compounds for the development of new biological tools and new therapeutic agents. For example, chemical inhibitors against particular miRNAs, such as the clinically relevant miR-21, have the potential to be developed into novel anticancer therapeutics. Small molecules discovered in our screen are expected to have a broad impact on human health, due to the critical roles that miRNAs play in several human diseases such as cancer and viral infection. These molecules will be promising lead compounds for the development of new chemical tools in biomedical research and new therapeutic agents. Small molecule inhibitors against particular miRNAs, such as the clinically relevant miR-21, have the potential to be developed into novel cancer therapeutics. -

Project Title:

HTS for Small Molecule Modulators of EB Virus Protein-Protein Interactions

PI:

JARDETZKY, THEODORE S

Institution:

NORTHWESTERN UNIVERSITY

Grant #:

1R21NS059415-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) is a gamma-herpesvirus that has infected the majority of the post-adolescent human population. EBV is the causative agent in infectious mononucleosis and is also associated with a number of tumors of lymphoid and epithelial origin, including B and T cell lymphomas and cancers of the upper throat. EBV infects both epithelial cells and B cells, requiring the fusion of viral and cellular membranes. The membrane fusion process needs four EBV proteins (gp42, gH, gL and gB) when B cells are targeted but only three for epithelial cells (gH, gL and gB). The gp42 protein controls the specificity of infection. EBV gp42 binds to a receptor to trigger entry into B cells, but gp42 inhibits membrane fusion with epithelial cells. The gp42 protein also binds tightly to the EBV gH/gL complex, interacting with gH/gL through its N-terminal region. The goal of this project is to develop a high throughput screening assay for the identification of inhibitors of EBV protein interactions, focusing on the gp42 interaction with gH/gL. This interaction is essential for virus- mediated membrane fusion and virus infection and small molecule inhibitors of the interaction will enable the development of antiviral therapeutics as well as provide new tools to dissect the mechanism of herpesvirus entry. Epstein-Barr virus (EBV) is associated with a number of human cancers, especially in individuals whose immune system is weakened because of disease or transplantation. Current work is focused on understanding the initial steps in virus infection, which require the merging of the viral envelope with that of the cell. The proposed research will allow for the identification of inhibitors of the virus entry into cells, potentially opening up new therapeutic routes to preventing EBV infection and transformation of susceptible human cells. -

Project Title:

High-Throughput Screen for Beta-Cell Replication

PI:

LEVINE, FRED

Institution:

Burnham Institute

Grant #:

1-R21-NS-057001-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Human beta-cells have an extremely low mitotic index, even compared with rodent beta-cells. Furthermore, efforts to stimulate proliferation in vitro result in the rapid shut off of insulin gene expression. The molecular mechanism that mediates the inverse relationship between growth and maintenance of a fully differentiated state is poorly understood, but if clarified could be exploited to induce beta-cell replication for diabetes therapy. Cyclin-dependent kinase inhibitors (CDKIs) play key roles in controlling cell cycle entry. In the human islet, microarray analysis has found that only two CDKIs, p57Kip2 and P21Cip1, are expressed at high levels. P57Kip2 is specific to beta-cells in the adult human pancreas and mutational loss of p57Kip2 expression in human patients results in beta-cell hyperplasia. Thus, p57Kip2 is an excellent target for interventions designed to promote beta-cell replication. In this proposal, we will take advantage of human pancreatic endocrine cell lines that inducibly express the cyclin dependent kinase inhibitor p57Kip2 to develop a high-throughput screen for compounds that modulate cell cycle entry and p57Kip2 transcription. Small molecule hits will serve as probes to study p57Kip2 regulation and potentially as lead candidates for development of diabetes therapeutics. By furthering our understanding of the mechanisms that control cell cycle entry in human beta-cells, we should be in an improved position to induce beta-cell regeneration in vivo and/or in vitro. -

Project Title:

Tissue Lysate Arrays for Molecular Screening

PI:

LU, YILING

Institution:

UNIVERSITY OF TEXAS MD ANDERSON CAN CTR

Grant #:

1 R03 NS050840-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Molecular therapeutics targeting the underlying defects in signaling pathways that contribute to cancer initiation and progression is a rapidly emerging avenue for patient therapy. The success of Imatinib Mesylate (Gleevec) has galvanized the field to identify and implement additional targeted therapeutics. A large number of inhibitors against specific kinases are currently in development. Most screening approaches analyze one or at most two criteria usually with purified enzymes or with a phenotypic assay. Many of these inhibitors demonstrated unexpected effects in intact cells, which reflect off target responses and the robust characteristics of signaling networks. There is an urgent need for function-based assays to prioritize and validate candidate targeting reagents. We propose the study of “Tissue lysate array as a high throughput assay for molecular screening”. The assay can rapidly assess over 100 different characteristics of functional proteomics, pathways and networks in intact cells, which greatly assists in selection of molecules for chemical genomics or lead compounds for pharmaceutical development. The assay is based on lysis of drug treated cells under stringent conditions followed by arraying on a solid matrix. The matrix can then be probed with pairs of antibodies identifying activation state and total amount of the protein. Based on this technology, we are able to gather multiple information from each compound and integrated into a “fingerprint” database to allow rapid assessment of on and off target activity. We propose: Specific Aim 1) To develop feasible and robust approaches to adapt tissue lysate arrays to a 96-well format, which will be applied to screen cellular responses to pharmacological standard. Aim 2) To develop multiplex technology to improve the cost, efficacy and robustness of the tissue lysate array. Aim 3) To develop and validate cell lines appropriate for high throughput screening by tissue lysate array. Aim 4) To validate the technology in a synthetic lethality analysis by combinations of screening molecule with drug of known function. -

Project Title:

Glucosamine-1-phosphate and serine acetylases: HTS assays and configurations

PI:

MCNEIL, MICHAEL R

Institution:

COLORADO STATE UNIVERSITY

Grant #:

1R21NS056921-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): We propose to develop and configure assays for high through put screening (HTS) for two acetyl transferases. The first, glucosamine-1-phosphate acetyl transferase (GAT) is an undeveloped target in peptidoglycan synthesis. It is essential for eubacterial growth, and its lack of function results in cell lysis. We propose to develop this assay using M. tuberculosis GAT enzyme (which is part of the bi-functional GlmU protein). Inhibitors of TB GAT are especially needed because there are no good peptidoglycan targeting antibiotics available for TB. Also new drugs against TB are needed because of the magnitude of the disease world wide and the emergence of drug resistant strains. We also propose to develop and configure assays for HTS of serine acetyl transferase (SAT) again using TB SAT. SAT is required for the synthesis of cysteine and has a role in bacterial extracellular signaling. It is not found in humans but is found in bacteria and several lower eukaryotic pathogens. Thus targeting SAT takes a novel approach to new drug development by hypothesizing that many pathogens, if unable to make cysteine, will not get adequate supplies from the host. Inhibitors of this enzyme, as can be found by HTS, are needed to validate this hypothesis. Both enzymes use acetyl-CoA to acetylate their substrate and the assay for both enzymes relies on the detection of free CoA produced by them. CoA is detected via its free SH group by forming a fluorescent adduct. We have preliminary data that this approach works for GAT and propose to develop it for GAT and extend it to SAT. After each assay is developed we propose to configure them for HTS by minimization of background, minimization of costs, demonstration of acceptable statistical parameters, and automation via a robotic liquid handler. We then propose to screen between 5 and 20 thousand compounds to test each assay. Future research will include submission of the assays to the NIH roadmap initiative for screening of large numbers of compounds to allow refinement of hits to candidates for preclinical development of new drugs. Relevance to Public Health: New drugs against bacteria and parasites are needed because drug resistance is resulting in current drugs not working. The work proposed herein will aid in the development of such new drugs by working with NIH to find chemical candidates that can be developed into new drugs. -

Project Title:

Multiplexed flow cytometry screens for RGS inhibitors

PI:

NEUBIG, RICHARD R

Institution:

UNIVERSITY OF MICHIGAN AT ANN ARBOR

Grant #:

1R21NS057014-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Signal transduction processes are major targets of drug discovery with G protein-coupled receptors being a primary site of action of many current therapeutic agents. Recent work, however, has shown that signaling pathways are not just linear chains of information but are webs of interacting regulatory molecules in which protein scaffolding, intracellular proximity, and inhibitory control are major determinants of signaling efficacy and specificity. The twenty Regulator of G protein Signaling (RGS) protein family members which inhibit G protein signaling represent a novel site of pharmacologic intervention but: 1) their physiological functions remain incompletely understood and 2) there are no reported small molecule inhibitors of RGS function. The identification of selective RGS inhibitors would provide both: 1) tools for the study of RGS function in cells and in vivo and 2) a starting point for therapeutic drug development. A flow cytometry method for quantitating RGS/Ga interactions will be adapted for multiplexed high-throughput screening for RGS inhibitors. The binding of fluorescently labeled Ga protein subunits to purified RGS proteins on beads will be optimized to permit multiple simultaneous assays of Ga binding to RGS 4, 6, 7, 8, and 9. This multiplexed method will both speed the throughput of screens and will provide immediate information on specificity of the chemicals identified in primary screens. Secondary analyses to demonstrate functional activity and effects in cell culture models are described. The ultimate aim of this project is the identification of selective small molecule inhibitors of RGS action. This will provide important chemical tools and accelerate the development of novel therapeutics. -

Project Title:

Discovery and characterization of novel small molecule inhibitors of Mcl-1

PI:

NIKOLOVSKA-COLESKA, ZANETA

Institution:

UNIVERSITY OF MICHIGAN AT ANN ARBOR

Grant #:

1R21NS056915-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Myeloid cell leukemia-1 (Mcl-1) is a potent anti-apoptotic molecule and a member of the Bcl-2 family of proteins, which are central regulators of apoptosis. Although Mcl-1 is a homologous protein related to other anti-apoptotic proteins such as Bcl-2 and Bcl-xL, it has a distinctly different structure and specificity for its binding to other pro-death Bcl-2 members. Cancer cells that are highly resistant to small-molecule inhibitors of Bcl-2/Bcl-xL can become highly sensitive when Mcl-1 activity is eliminated by sRNAi, indicating the critical importance of Mcl-1 in maintaining cell survival. Mcl-1 has been to found to be overexpressed in both solid and non-solid tumor cell lines and human cancer tissues. Consistent with its anti-apoptotic function, overexpression of Mcl-1 has been associated with tumor initiation, progression and resistance to current anticancer therapies. Targeting Mcl-1 is therefore an important strategy for the development of new class of anticancer drugs for the treatment of cancer by overcoming apoptosis resistance. In this R21 grant, we propose to employ an experimental high throughout screening (HTS) approach for the discovery of novel, small-molecule inhibitors of Mcl-1. Identified hits will be evaluated in a series of complementary biochemical, biophysical, functional and cellular assays to rule out any potential false positives and to determine their specificity and mechanism of action. We will pursue the following specific Aims: (1) HTS using FP and HTRF based assay; (2) Conformation of the identified hits and their selectivity and specificity in binding by using different biochemical and biophysical assays (ELISA, FP and NMR); (3) Characterization of their: i) effect on cytochrome c release from isolated mitochondria; ii) ability to disrupt the dimerization of Mcl-1 with pro- apoptotic Bcl-2 members in live cells; iii) their activity and selectivity in cell-growth inhibition of cancer cells and normal cells; iv) potency in induction of apoptosis in cancer cells and model cells. Successfully carried out, our studies will provide a number of novel, small-molecule inhibitors of Mcl-1 with detailed characterization of their binding affinity to Mcl-1 and specificity over other proteins, their cellular activity and function. Such well-characterized small-molecule inhibitors of Mcl-1 will not only serve as promising lead compounds for further optimization toward developing a new class of anticancer drugs, but may also be used as a set of much needed pharmacological tools for further elucidation of the functions of Mcl-1. -

Project Title:

Assay for HTS of Gi/Go-linked GPCRs: mGluR8 as the Prototype

PI:

NISWENDER, COLLEEN M

Institution:

VANDERBILT UNIVERSITY

Grant #:

1R21NS053536-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Abstract: Pharmacological agents targeting receptors coupled to GTP binding proteins represent promising clinical agents spanning a multitude of human diseases. Within the G-protein coupled receptor (GPCR) superfamily, it has been most challenging to develop high throughput screening strategies to identify drugs modulating Gi/Go-linked receptors. Previous HTS methods examining these receptors have been indirect, cumbersome, and expensive, relying upon either an inhibition of drug-stimulated cAMP accumulation or the use of chimeric G-proteins. We propose to develop a screening strategy for Gi/o-linked receptors by measuring thallium flux through G protein regulated Inwardly Rectifying Potassium (K+) (GIRK) channels. Using this technique in HEK cells stably expressing GIRK 1 and 2 channels, we have generated preliminary data using muscarinic agonists and antagonists and have observed dose-dependent regulation of these channels by M2 and M4selective agents. Using a 384 well plate format, we have generated preliminary Z’ values of >0.5, indicating that this assay is amenable to HTS. We plan to use this technique to perform a small scale, 8000 compound, directed HTS screen for drugs interacting with the metabotropic glutamate receptor 7 (mGluR7). This receptor, based upon its cellular location and functional activity, is known to couple to GIRK in in vitro systems and is predicted to serve as a novel target for neurological and psychiatric disorders. Finally, we will perform secondary screens to validate potential hits and determine specificity for mGluR7 versus other mGluRs. It is anticipated that these studies will open new avenues for Gi/Go-linked receptor screening as well as generate valuable tools and drug leads for mGluR7. Lay summary: G-protein coupled receptors (GPCRs) represent accessible therapeutic targets in human disease. Within the GPCR family, it has been challenging to develop technically direct, time-efficient, sensitive, and cost effective assays to identify drugs targeting receptors coupled to Gi/Go GTP binding proteins. We propose to develop and characterize a new high throughput screening technique for receptors that signal through Gi/o to regulate the G protein regulated Inwardly Rectifying Potassium (K+) channel. Using the metabotropic glutamate receptor 7 (mGluR7) as an initial prototype Gi/o-coupled receptor, we will screen a small, targeted library to develop new tools and potential lead compounds for agents modulating mGluR7, a protein for which limited pharmacological agents are available and which represents a novel drug target in neurological and psychiatric diseases such as epilepsy and schizophrenia. -

Project Title:

Novel Chemical and Immunological Approaches to Influenza Therapy

PI:

Oldstone, Michael BA

Institution:

SCRIPPS RESEARCH INSTITUTE

Grant #:

1 U01 AI074564-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Influenza virus remains a major public health concern for the USA and the world. To best insure that a pandemic like the 1918-1919 episode does not reoccur, national and international surveillance, effective vaccine and novel and effective antiviral therapy are required. Here we propose a multi-institute cooperative research project focused on discovery and use of novel chemicals that selectively manipulate the immunologic response in the lung as a therapeutic approach to better control influenza viral infection. We and others have evidence in animal models that the immune response (immunopathology) to influenza contributes significantly to morbidity and mortality. We have preliminary but impressive data that low doses of sphingosine analogs administered as a single dose intratracheally but not systemically acts locally in the lung to specifically suppress antiviral T cell proliferative responses. We propose that: 1) single aerosol exposure of the lungs to a sphingosine analog will inhibit antiviral proliferation thus modulating the resultant immunopathologic injury; 2) this mechanism will synergize with classic antiviral protective therapy; 3) combination of these therapeutic approaches will protect the host during H5N1 infection. To test this proposal Hugh Rosen, with expertise in immunology, medicinal chemistry and human therapeutics, will continue the development of sphingosine compounds and their testing with Michael Oldstone, experienced in viral-immunobiology and use of the WSN recombinant virus expressing immunodominant CDS and CD4 T cell epitopes of LCMV. WSN recombinant infection is preceded with transfer of GFP, RFP or Th1.1 congenically labeled CD4 and CDS T cells to these LCMV epitopes allowing quantitation of flu-specific T cells in the lung. The LCMV recombinant as well as GFP-labeled WSN virus used has been prepared by the third partner in this enterprise, Yoshihiro Kawaoka, an expert in influenza viruses. Kawaoka will test novel sphingosine analogs against H5N1 influenza virus. -

Project Title:

Allosteric, Small-Molecule Inhibitors of Actin Nucleation by the Formin mDia1

PI:

PETERSON, JEFFREY R

Institution:

INSTITUTE FOR CANCER RESEARCH Fox Chase Cancer Center

Grant #:

1-R21-NS059359-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): The goal of this proposal is to develop a high-throughput assay to identify allosteric, small-molecule inhibitors of the formin protein mDia1 and to validate the assay by screening a collection of 10,000 structurally diverse compounds. A second aim is to develop assays to be used to characterize mDia1 inhibitors in terms of their specificity, mechanism of action, and efficacy in live cells. Formin proteins have emerged as key regulators of actin filament nucleation required for cytokinesis, cell polarity, and development. Despite their importance, there is little detailed information on cellular functions for any of the 15 mammalian formin isoforms. In addition to the formins, two other major actin nucleation factors, Arp2/3 complex and Spir, have been identified and the relative contribution of specific nucleators to particular actin structures in cells is a major open question. Here we focus on the ubiquitously expressed formin mDia1. Current strategies to conduct loss-of-function studies on mDia1 in mammalian cells are limited by poor efficacy (RNAi) and compensatory up-regulation of other mDia isoforms (mouse knock- out). Cell-permeable inhibitors that rapidly inactivate mDia1 would mitigate these challenges and greatly facilitate the elucidation of its unique functions and could have potential therapeutic utility in cancer. mDia1 is regulated by autoinhibition and we have proposed that autoinhibited proteins may be susceptible to allosteric inhibitors that allosterically stabilize the inactive conformation. Such inhibitors should exhibit greater target selectivity than those directly targeting the highly conserved, catalytic FH2 domain, an hypothesis to be directly tested in Aim 2. We propose two screens to be conducted in parallel using purified, recombinant proteins that together will identify allosteric, small-molecule inhibitors of mDia1. This dual screen will also eliminate the major anticipated classes of false positives, caused by compounds that target actin directly or perturb proteins non-specifically. Future work will apply this screen to a much larger compound collection and we envisage applying a similar strategy to develop inhibitors for other formins. -

Project Title:

Assay Development and Screening for Notch Agonists

PI:

PETRIE, HOWARD T.

Institution:

Scripps Research Institute

Grant #:

1-R21-NS061649-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Cell surface receptors in the Notch family (Notch1 through Notch4) are expressed on numerous types of cells, including many types of stem cells and undifferentiated progenitor cells, either in the embryo or in self-renewing tissues after birth. A signal is induced when Notch binds to its specific ligands (Delta or Jagged in vertebrates), leading to transcriptional changes that subsequently specify lineage fate. In addition to this critical biological function, Notch signaling has also been implicated in a variety of human diseases, including autoimmunity, a heritable form of strokes, and multiple forms of cancer. Although much is known about the biochemistry and consequences of Notch signaling, a large number of questions remain. Identifying and/or developing small molecules (chemical compounds) that activate Notch signaling in stem/progenitor cells would greatly facilitate further study Notch function in both health and disease. Further, such Notch agonist compounds will provide a powerful platform with which to subsequently identify compounds that antagonize this signal, and thus have potential therapeutic uses. In the proposed project, we will exploit the fact that T lymphocyte progenitors have an absolute requirement for Notch signaling, and respond to Notch signaling by proliferation. We will use T progenitors to screen a library of 300,000 chemical compounds to identify those that activate Notch. The goals are to optimize conditions for an existing positive control assay for T progenitor proliferation, and to miniaturize this control assay into a format that is physically and economically compatible with a high-throughput screen. Subsequently, suitability of the miniaturized proliferation assay for compound screening will be validated using a smaller library of 1280 compounds with known pharmacologic activity (LOPAC), including compounds that either inhibit or activate proliferation. Once the assay is validated, we will screen the library of 300,000 compounds to identify those that induce proliferation in T lymphocyte progenitors. Since some compounds will almost certainly act as non-specific mitogens, the final step will be to validate activation of the Notch pathway in those compounds that induce proliferation in T progenitor cells. Ultimately, Notch agonists identified by this screen will be used as tools to study the mechanisms and effects of Notch signaling. In addition, these compounds will be used in future projects to re-screen compound libraries to identify Notch antagonists, which may be further developed for the treatment of Notch-associated diseases. -

Project Title:

Development of the HTS Assay for Agonists of the Orphan Nuclear Receptor NR2E3

PI:

Petrukhin, Konstantin

Institution:

COLUMBIA UNIVERSITY HEALTH SCIENCES

Grant #:

1 R21 NS061718-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is the most common cause of legal blindness in the US. There is no effective treatment for the most prevalent atrophic (dry) form of AMD. The loss of vision in atrophic AMD is caused by degeneration of photoreceptor cells (rods and cones) in the central part of the retina called macula. Preservation of macular rods and cones is the ultimate goal of AMD treatment. NR2E3 is an orphan nuclear receptor expressed exclusively in photoreceptor cells of the retina where it is involved in photoreceptor maintenance and differentiation. Our overall objective is to identify a small molecule NR2E3 agonist suitable for performing proof-of-concept photoreceptor protection experiments in animal models of retinal degeneration. Identification of NR2E3 agonists will also provide an important pharmacological tool for probing basic biologic mechanisms of photoreceptor development and differentiation. The studies outlined in this proposal seek to develop an HTRF assay for agonist-induced release of corepressor NCOR from the NR2E3-NCOR complex (Specific Aim 1) and to adapt this assay to the HTS format suitable for screening of a compound collection at one of the NIH MLSCN screening centers (Specific Aim 2). -

Project Title:

MLSN Screen of the PD Alpha Synuclein 5′UTR

PI:

ROGERS, JACK T

Institution:

MASSACHUSETTS GENERAL HOSPITAL
DEPARTMENT OF PSYCHIATRY

Grant #:

1-R21-NS059434-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): The 5′untranslated region of the mRNA for the Parkinson’s a-synuclein (ASYN 5′UTR) is a key translational regulatory element that contributes to setting the amount of alpha-synuclein production in neural cells, particularly in response to Fe influx (iron accumulation is one of the key hallmarks of PD in the Substantia nigra). Our preliminary data showed ASYN gene expression is controlled by iron at the level of message translation without change to transcription or steady state levels of ASYN mRNA in neuroblastoma (SH-SY5Y cells). This regulation is similar to another metalloprotein, the Amyloid Precursor Protein of Alzheimer’s disease, implying that neurodegenerative disease genes may be linked to the mis-metabolism of iron and/or associated oxidative events. These findings, and our experience with the regulation of APP mRNA translation, suggested the ASYN 5′UTR would be an excellent drug target. We already generated both ASYN and APP 5′UTR constructs and stable cell lines available for screening small molecules from the MLSCN molecular library of 500,000 compounds. Our screening strategy to identify APP 5′UTR drug hits was previously conducted with FDA and LDDN molecular libraries, and has now been adapted to screen the alpha-synuclein 5′UTR for this NIMH solicitation. As prior validation of our approach to screen the 5′UTRs of neurodegenerative disease transcripts, our APP 5′UTR screens generated drug selectivity since paroxetine was an APP 5′UTR directed FDA inhibitor that did not change APLP-1 and APLP-2 expression in SH-SY5Y cells (Payton et al., 2003). Paroxetine subsequently reduced the amyloid burden in a transgenic mouse model for AD (TgCRND8 mice) (Tucker et al., (2005/2006). The aim of this grant application is to collaborate with the MLSCN to screen for 5′UTR directed small molecule inhibitors of translation of alpha synuclein mRNA for PD therapeutics. Our lab has stable cell lines that express luciferase reporter genes translationally driven by the 5′UTRs of target neurodegenerative proteins including ASYN (PD), APP (AD) and Prion protein (PrP, Creutzfeldt-Jacob disease). Dr. Ippolita Cantuti-Castelvetri (coPI, MIND, MGH) will provide the correct reagents, cell lines and experience to successfully ensure ASYN 5′UTR specific leads maintain neuronal viability and are sufficiently specific to inhibit toxic alpha-synuclein expression but leave compensatory beta-synuclein and gamma- synuclein expression unchanged. The data to be accumulated through this R21 molecular libraries screening cooperative mechanism will permit a high throughput transfection based screen of an important RNA target, the 5′untranslated region of the Parkinson’s alpha synuclein (ASYN) transcript. This 5′UTR is an attractive therapeutic target for Parkinson’s disease (PD), and newly identified ASYN 5′UTR specific leads may be developed by medicinal chemistry potentially to limit neurotoxic ASYN production in dopaminergic neurons. Use of 5′UTR specific MLSCN hits will probe the mechanism of translation of ASYN mRNA relevant to PD. Compounds directed to 5′UTRs of other mRNAs will probe mechanism of translation of the Abeta-amyloid precursor protein mRNA in Alzheimer’s disease (SOD-1 mRNA in ALS, and PrP mRNA in Cruetzsfeld- Jacob Syndrome). -

Project Title:

Small molecule effectors of maternal gene expression in C. elegans embryogenesis

PI:

Ryder, Sean Patrick

Institution:

UNIV OF MASSACHUSETTS MED SCH WORCESTER

Grant #:

1-R21-NS059380-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): A cohesive strategy is presented to identify small molecule effectors of maternal gene expression. During oogenesis, the cytoplasm of the egg is loaded with silenced maternal transcripts that are activated after fertilization in response to developmental cues. Initial patterning is achieved before nascent transcription begins by activating translation of maternal transcripts at specific times in defined locations. A handful of RNA-binding proteins are responsible for silencing maternal transcripts during oogenesis and coordinating activation after fertilization. Here, we define a strategy to screen for inhibitors of two such proteins, the tandem zinc finger proteins MEX-5 and POS-1. These proteins are critical for anterior and posterior cell fate determination in the nematode worm Caenorhabditis elegans, and are related to tristetraprolin, a factor critical in the regulation of the inflammation response in humans. A process for identifying, counter-screening, and validating small molecule inhibitors is presented that relies on both in vitro fluorescence assays and live imaging during embryogenesis. Inhibitory compounds identified through this screen could serve as “chemical alleles”, enabling the temporal and spatial dissection of maternal gene patterning beyond the earliest points in embryogenesis. The strategy delineated here is generally useful and can be adapted to any RNA-binding protein, opening the door to screening for inhibitors of potentially therapeutic target RNA-binding proteins including tristetraprolin, which may lead to new therapies for rheumatoid arthritis, psoriasis, and other inflammatory diseases including multiple sclerosis. This proposal describes a method for identification and validation of small chemical inhibitors of proteins that bind to RNA. The strategy will be perfected using two protein targets that are critical to differentiation of embryonic stem cells in a worm model. Once optimized, it may be used for therapeutically relevant RNA-binding proteins, including a related protein that is required for deactivating inflammation. This work will have direct relevance to the search for new and better treatments for rheumatoid arthritis, psoriasis, and other inflammatory diseases. Moreover, the lessons learned from this project will contribute to our understanding of the biological processes that allow stem cells to change into all types of adult cells. -

Project Title:

Real-Time Fluorescence Assay:RGS Domain GAP Activity

PI:

SIDEROVSKI, DAVID P.

Institution:

UNIVERSITY OF NORTH CAROLINA CHAPEL HILL

Grant #:

1R03NS053754-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Members of the “regulator of G-protein signaling” (RGS)-protein superfamily have emerged as critical modulators of specific G-protein-coupled receptor (GPCR) signal transduction pathways. Via their “GTPase accelerating protein” (GAP) activity, RGS proteins deactivate heterotrimeric G-protein alpha subunits and thereby reduce GPCR signal transduction. Combining existing GPCR agonists with specific RGS domain inhibitors should potentiate cellular responses to these drugs. The diversity of RGS proteins with highly localized and dynamically regulated distributions in the human brain, makes them attractive targets for pharmacotherapy of central nervous system disorders such as Parkinson’s disease. Unfortunately, no small molecule inhibitor (or activator) of RGS protein GAP activity is publicly available for study. Therefore, to identify small molecule tools for further advancing knowledge of RGS protein function in specific GPCR signaling pathways, and also to facilitate identification of lead compounds for developing RGS protein directed therapeutics, we will modify and validate novel, real-time, fluorescence-based assays of RGS protein function for automated high throughput molecular screening: a fluorescence resonance energy transfer (FRET)-based binding assay that employs cyan fluorescent protein-labeled G-alpha subunits and yellow fluorescent protein-labeled RGS proteins, a single-turnover GTP hydrolysis assay using a fluorescent sensor for inorganic phosphate production, and an assay of G-alpha nucleotide binding and hydrolysis that employs the fluor-modified nucleotide BODIPY(r) FL 2′-(or-3′)-O-(N-(2-aminoethyl)urethane)guanosine 5′-triphosphate. Many useful drugs act by binding a particular type of protein receptor on the cell’s surface: a G-protein coupled receptor. Our group has discovered a new family of proteins-the RGS proteins-that interfere with these receptors. We wish to create ways to screen for new drug compounds that can stop RGS proteins from interfering and thereby allow existing drugs to act more potently. -

Project Title:

Assay Development Relating to the HTS of the Neuropeptide Y-Y2 Receptor

PI:

WAHLESTEDT, CLAES ROBERT

Institution:

SCRIPPS RESEARCH INSTITUTE

Grant #:

1R21NS056950-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): This application relates to the NIH efforts to develop and adapt biological assays for use in HTS. Our team has extensive experience in drug discovery and pharmaceutical screening campaigns. Our overall aim is to develop assays for HTS of small molecule compounds that display affinity to the neuropeptide Y (NPY) Y2- receptor. Specifically, we seek to identify compounds that are functional antagonists at the human Y2- receptor. Following optimization, also to be pursued at Scripps by individuals with much pharmaceutical industry experience, such a compound might be clinically useful in anxiety, depression and/or alcoholism. No clinically testable compounds that inhibit signaling of NPY through the Y2-receptor are in existence today. The NPY-Y2 receptor was first described by us previously and recent studies in human in particular have made us more convinced than ever that it is an important target in psychiatric disease and addiction. SPECIFIC OBJECTIVES AND AIMS: We will use a modified version of the HitHunter cAMP assay. This assay features high signal to background ratios for monitoring cellular activation of GPCRs, particularly G.i -coupled receptors. It is a homogeneous microtiter plate assay for measuring cellular cAMP from cell lysates using Enzyme Fragment Complementation (EFC). We aim to initially implement the DiscoveRx HitHunter cAMP XS technology and then develop further assays described herein, amenable to HTS (moving from 384- to 1536- well format) to screen a 600,000 compound library utilizing robotics for NPY-Y2 receptor antagonists. We will develop and use HA-Y2/CHO-K1 cells and 50% inhibition of NPY at 10 microM compound, measured at a single point will define hits. Confirmation of hits and potencies will be in 10 point dose response curves. Counter-screening of the NPY-Y1 receptor will help evaluate selectivity and parse down hits. IC50 determination will allow selection of compounds for chemical optimization. A radioactive detection method will be used to eliminate “false positives”. Successful development and implementation of this assay format will pave the way for future G.s and G.i- coupled receptor HTS campaigns. RELEVANCE TO PUBLIC HEALTH: Many lines of evidence from studies on humans (and animals) suggest that the brain NPY-Y2 receptor is involved in affective disorders (depression as well as anxiety) and in alcoholism. Notably, only a few scattered efforts have previously focused on developing a Y2 receptor drug for psychiatric purposes. Our approach is novel, powerful and part of a comprehensive strategy to succeed in developing such a drug to alleviate human suffering. -

Project Title:

Cell-Based Screens for Cancer:Targeting PTEN (RMI)

PI:

WALDMAN, TODD A

Institution:

GEORGETOWN UNIVERSITY

Grant #:

1R03NS050857-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Cancer research has been revolutionized by the realization that inactivating somatic mutations in tumor suppressor genes are a key component that drives the process of tumorigenesis. While the discovery of such tumor suppressor genes have had profound implications for identification of at-risk individuals in rare cancer prone-families and have contributed to a better understanding of the biological mechanisms of tumor progression, thus far they have led to the discovery of few (if any) new drugs. Academic and pharmaceutical company researchers are currently grappling with this difficult question – what are the best ways to translate the discovery of tumor suppressor genes into therapeutics that target them? PTEN is a prototype tumor suppressor gene commonly inactivated by mutations in glioblastoma, endometrial cancer, melanoma, prostate cancer, and other tumor types. We have recently employed human somatic cell gene targeting to create isogenic sets of human HCT116 colon cancer cells that differ only in the presence or absence of their endogenous wild-type PTEN genes. Here we propose to employ this set of cells as the basis of a cell-based screen to identify small molecules that are specifically cytotoxic or cytostatic towards PTEN-deficient cells. Such compounds would be useful probes for study of the PTEN tumor suppressor pathway and might form the basis of therapeutics for treatment of cancers harboring mutations in PTEN. Furthermore, we propose to create additional isogenic sets of PTEN gene-targeted human cancer cells to function as secondary screens for confirmation of the hits obtained in the primary assay. Specific Aim #1: Validate and implement a cell-based screen for the identification of molecules that specifically kill PTEN-deficient human cancer cells. Specific Aim #2: Employ human somatic cell gene targeting to create additional isogenic sets of human cancer cells differing only in the presence or absence of PTEN. Validate the hits obtained in Aim #1 in these new cell lines. -

Project Title:

Development of a robust high throughput assay for the discovery of RAM network in

PI:

WEISS, ERIC LYLE

Institution:

NORTHWESTERN UNIVERSITY

Grant #:

1R21NS056968-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Control of cell architecture is essential for formation and maintenance of functional tissues, and is often deranged in a number of important human diseases including cancer and some immune disorders. Morphology control is also important for cell differentiation: mechanisms that promote asymmetric segregation of cell fate determinants rely upon the cell’s underlying structure to do so. Since pathological states (such as invasive metastatic cancer) often involve inappropriate elevation of motility, compounds that modulate the activity of pathways that control cell architecture are candidate therapeutic agents. The budding yeast RAM network (Regulation of Ace2 and Morphogenesis) is a novel signaling pathway required for asymmetric gene expression and maintenance of polarized growth. This pathway is evidently conserved from yeast to humans; in metazoans, homologs of RAM network proteins are involved in control of cell proliferation, morphogenesis of polarized cells, and organization of dendritic fields. Very little is understood about these proteins’ functions. We have developed a simple and powerful way to find treatments that block the RAM network. In this approach, cells retaining pathway function are killed, while those that lose it can live. In a forward genetic screen this selection-based strategy quickly yielded over 400 new loss-of-function alleles of RAM network genes. We propose to adapt this strategy for high-throughput screening to identify compounds that block the RAM network. We will use advanced NTS equipment at Northwestern to develop this approach and conduct pilot screens, ensuring that the assay sent to the MLSCN is robust and portable. In preliminary assays, selection against growth of cells that retain RAM network function gives a Z’ of approximately 0.72. -

Project Title:

Discovery of Inhibitors of Mycobacterium tuberculosis strain 37Rv, a virulent strain requiring BSL3

PI:

White, Lucile

Institution:

SOUTHERN RESEARCH INSTITUTE

Grant #:

N01-AI-15449

Application Cycle:

FT

Abstract of Grant Proposal:

Project Title:

HTS for Discovery of Inhibitors of STAR Protein-RNA complexes

PI:

WILLIAMSON, JAMES R

Institution:

THE SCRIPPS RESEARCH INSTITUTE DEPT OF MOLECULAR BIOLOGY

Grant #:

1-R21-NS056951-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): This proposal outlines a plan to develop a validated high throughput screen for inhibitors of RNA-protein complex formation by two STAR proteins: GLD-1 and Quaking. The ultimate goal will be to configure these assays for submission to the MLSCN for a large scale screening effort. GLD-1 is a regulatory of germline development in C. elegans, and Quaking is a mouse protein that regulates myelination in the developing nervous system. The STAR protein family is an important class of RNA binding proteins that also includes Sam68 and the SLM proteins that are implicated in a wide variety of processes, and is linked to certain types of cancer. A fluorescence polarization assay will be developed that monitors the change in polarization of a fluorescently labeled RNA upon binding to the protein. Small molecule competitors will be identified by screening for decreases in polarization. Such validated inhibitors would be powerful tools for functional studies in C. elegans and mice, and the assay should be readily extended to inhibitors of Sam68 once suitable RNA targets have been identified. -

Project Title:

Friedreich Ataxia High Throughput Drug Screening Assays

PI:

WILSON, ROBERT B

Institution:

UNIVERSITY OF PENNSYLVANIA

Grant #:

1R21NS045331-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Friedreich’s ataxia (FRDA) is an autosomal recessive, inherited neurodegenerative disorder. The signs and symptoms of FRDA are reminiscent of the mitochondrial encephalomyopathies and include progressive ataxia of all four limbs, dysarthria, areflexia, sensory loss, and muscle weakness. Skeletal deformities and cardiomyopathy are found in most patients, impaired glucose tolerance and diabetes mellitus are found in -30% of patients, and reduced visual acuity and hearing loss are occasionally seen. Onset of symptoms usually occurs around puberty and most patients are confined to a wheelchair by their late 20s. Myocardial failure is the most common cause of premature death. FRDA is the most common hereditary ataxia, with a prevalence of approximately 1 in 40,000 in European populations, and there is currently no proven effective treatment. FRDA is caused by mutations in the FRDA gene, which encodes the protein frataxin. Although encoded in the nucleus, frataxin is imported into the mitochondrial matrix. Studies of yeast and murine frataxin homologues, and of patient material, indicate that mitochondrial dysfunction, caused by oxidative damage and concomitant mitochondrial iron accumulation, underlies the signs and symptoms of FRDA. Preliminary studies using simple measures of mitochondrial function in the yeast model system, and in primary FRDA cells, support the feasibility of using a cell-based approach to high-throughput drug screening for FRDA. Hit compounds from such a screen could then be tested in the recently developed mouse models of the disease. The protein targets of hit compounds could be identified using a chemical genetic approach in yeast, allowing further drug development. The overall goal of the proposed research is to identify potential treatments for FRDA. The Specific Aims are: 1) To develop high-throughput, cell-based drug screening assays for FRDA. Because mitochondrial dysfunction underlies the signs and symptoms of FRDA, our assays will be designed to screen for compounds that improve mitochondrial function. We will use measures of mitochondrial function suitable for a 96-well format, in the yeast model system, and in primary FRDA cells. 2) To develop a chemical genetic screening assay for the identification of drug targets. We will use a colony-color screening technique in the yeast model system to identify the target proteins of hit compounds from our high-throughput drug screening assays for FRDA. -

Project Title:

HTS Assay for Ca 3 T-type Channels Using FLIPR (RMI)

PI:

XIE, XINMIN SIMON

Institution:

SRI international

Grant #:

1R03NS050771-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): T-type Ca2+ channels, encoded by three genes (Cav3.1, 3.2 and 3.3, or alpha1-G, H, and I), are heterogeneously expressed in the brain and many peripheral organs such as the heart and vascular smooth muscle. They play a role as the pacemaker that regulates spontaneous neuronal activity, cardiac rhythm and vascular tone. T-channels have been a drug target for absence epilepsy and hypertension. The conventional anti-absence epilepsy drug ethosuximide produces weak inhibition of the T-channel. The anti-hypertensive drug, mibefradil, potently blocks the T channel but was withdrawn from the market due to drug interaction. Inhibition of T-channels by 619C89 and SB-209712 may contribute to the neuroprotective effects of the compounds; however, these drugs are non-specific and produce many side effects. Phenotypic studies on the Cav3.1 or 3.2 gene knockout mice have shed light on the biological role of T-channels and suggest the T-channel could be a potential target for therapeutic intervention in pathological pain and cardiovascular diseases. There is no doubt that subtype-selective and potent Cav3 modulators should produce more specific pharmacological actions with fewer side effects. High throughput screening (HTS) of a large compound library is the initial step to identify such a novel compound. Standard electrophysiological techniques are not suitable for primary HTS. A recent study using single cell Ca2+ imaging techniques has demonstrated that manipulation of extracellular Ca2+ significantly changes the intracellular Ca2+ ([Ca2+]j) in the HEK293 cells expressing human Cav3.2 channels (Cav3.2-HEK293). Detection of [Ca2+]j indirectly measures Ca2+ entry through spontaneous opening of the Cav3.2 channels (i.e., window currents). This method is amenable to HTS. In a pilot study we tested the feasibility using the Ca2+ sensitive dye fluo-4/AM and measured with a fluorometric imaging plate reader (FLIPRR) in a 96-well format. In this proposal, our specific aims are to (1) develop a 384-well HTS format through optimization of assay conditions and automation of procedures such as the integration of a robotic system and automated data analysis, and (2) validate this fluorometric method by comparison of the potency of a large number of ion channel inhibitors determined using both the FLIPR assay and whole-cell voltage-clamp recording techniques. The proposed fluorometric method will provide a high throughput and quantitative assay for the primary screening of compounds against three subtypes of Cav3 T-type channels. -

Project Title:

Development of HTS Platform for Human Fatty Acid Transport Protein 4 Using FLIPR

PI:

XIE, XINMIN SIMON

Institution:

SRI INTERNATIONAL

Grant #:

1-R21-NS057052-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Dietary long-chain fatty acids (LCFAs) contribute up to 40% of the caloric content in western diets. LCFAs are important metabolites and contribute to many cellular structures and functions, but excessive serum fatty acid levels are linked to obesity, insulin desensitization, and type 2 diabetes. LCFA uptake across cell membranes is mediated principally by fatty acid transport proteins (FATPs). In humans, six members of this transporter family, designated as hsFATP1-6, have been characterized and shown to be differentially expressed throughout the body. The FATP4 subtype constitutes the major FATP in the small intestine and preferentially uptakes long- (>10 carbon atoms) but not short-chain fatty acids. Selective inhibition of this subtype FATP may effectively reduce LCFA absorption. High-throughput screening (HTS) of a large compound library will be the initial step toward identifying novel subtype-selective inhibitors of FATPs. Current methods using radiolabeled fatty acids or fluorescent fatty acid analogs to measure LCFA uptake in murine 3T3-L1 adipocytes are not suitable for HTS to identify subtype-selective inhibitors against human FATPs. In response to RAF-RM-06-004: “Assay Development for High Throughput Molecular Screening,” we propose to develop and configure an HTS platform using a fluorometric imaging plate reader (FLIPRZ) with the newly developed QBT(TM) Fatty Acid Uptake Assay Kit to quality LCFA uptake kinetics by the stable hsFATP4-expressing HEK293 cells. Our specific aims are to: 1. Develop a FLIPR 96- or 384-well format assay against the hsFATP4 for primary HTS. LCFA uptake kinetics in hsFATP4-expressing HEK293 cells will be compared with the vector control HEK293 cells and 3T3-L1 adipocytes to determine signal-to-noise ratios, specificity, and reproducibility for the HTS assay. Through assay optimization, we will configure the assay for primary HTS. 2. Evaluate and validate the HTS assay through an initial screening. A panel of several hundred compounds consisting of natural long- and short-chain fatty acids, synthetic fatty acid derivatives, and small molecules will be screened against the hsFATP4 as an initial evaluation. The HTS assay we develop could be used in the Molecular Library Screening Center Network for primary screens and will serve as a general prototype for subsequent development of assays for other FATP family members. Secondary screens will use the standard radiolabeled method against the same target for hit confirmation and the FLIPR assay against another FATP subtype in counter-screening for selectivity. The identified subtype-selective FATP inhibitors will provide a novel pharmacologic tool for further studies of FATP biology. Furthermore, they may form a basis for developing novel therapeutics for the treatment of metabolic disorders. -

Project Title:

High throughput screening of ligands of TRP channels

PI:

ZHU, MICHAEL X

Institution:

OHIO STATE UNIVERSITY

Grant #:

1R21NS056942-01

Application Cycle:

FT

Abstract of Grant Proposal:

DESCRIPTION (provided by applicant): Transient receptor potential (TRP) channels have emerged as cellular sensors of various internal and external cues. The canonical TRP (TRPC) channels are involved in receptor and store-operated Ca2+ entry, an important component of phospholipase C-activated Ca2+ signaling cascade. Under resting potentials, these channels also conduct Na+ influx, allowing cells to be depolarized in a receptor-operated manner. TRPC channels have been implicated in the regulation of many functions, including smooth muscle and vascular tones in blood vessels, endothelial permeability, fertilization, saliva secretion, synaptic transmission, and neurite outgrowth. Our long-term objective is to understand the mechanism of regulation of Ca2+ signal inside cells and its roles in human health. However, the lack of specific agonists and antagonists has seriously hindered the studies on the characterization of physiological functions as well as the mechanisms of regulation of different TRP channels. The biological research community has an urgent need for pharmacological tools that are specifically targeted at different TRP members. Moreover, several TRP channels have been implicated in human diseases. The development of specific drugs for TRP channels could also have therapeutic implications. The goals of the current proposal are to develop microplate-based assays to monitor TRP channel function and to configure them for high throughput screening. Emphasis will be placed on TRPC channels with the use of fluorescence-based membrane potential assays. A selected number of TRPV and TRPM channels will also be used for counter-screens. Intracellular Ca2+ measurement will be used as secondary screens and whole cell voltage clamp experiments will be used for final verification of the active compounds. Two specific aims are proposed. AIM I will explore conditions that give rise to the best signal-to-noise ratio, high consistency, and optimal reproducibility of receptor-induced activation of TRPC channels stably expressed in HEK293 cells in microplate-based fluorescence membrane potential assays. AIM II will configure the assays for high throughput screening. Pilot screens on TRPC5 and TRPC6 will be performed using analogs of 2-aminoethoxydiphenyl borate and classical L-type Ca2+ channel blockers. The configured assay conditions and cell lines are to be used in the Molecular Libraries Screening Centers Network to screen for ligands of TRPC channels. -