Jerod S Denton
Vanderbilt University
Project start date: 2010-09-27
Project end date: 2012-08-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Jerod S Denton
Identification Of Novel Modulators Of ROMK K+ Channel Activity
Jerod S Denton
Vanderbilt University Medical Center Nashville, Tn 372036869
Grant 1R21NS057041-01 from National Institute Of Neurological Disorders And Stroke IRG: ZNS1
Abstract: 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.
Keywords: drug discovery /isolation, potassium channel, protein structure function, technology /technique development, electrolyte balance, gene deletion mutation, kidney function, NIH Roadmap Initiative tag, fluorescent dye /probe, high throughput technology
Project start date: 2006-07-01
Project end date: 2007-06-30
1R21NS057041-01 (2006): $76500
Regulation Of A CIC Channel By An Ste20-like Kinase
Jerod S Denton
Vanderbilt University Medical Center Nashville, Tn 372036869
Grant 1F32GM067424-01 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: Members of the CIC superfamily of voltage gated Cl- channels have been identified in plants, yeast, eubacteria, archaebacteria, and various invertebrate and vertebrate animals. The function and regulation of most ClC channels are not well understood, but the existence of disease-causing mutations and presence of ClC genes in widely divergent organisms indicate that they play important and fundamental physiological roles. C. elegans offers significant experimental advantages for defining the molecular mechanisms and signaling pathways involved in regulation of ClC channels. Our laboratory has identified an inwardly rectifying Cl- channel, CLH-3, expressed in oocytes that has biophysical properties virtually identical to those of mammalian ClC-2. CLH-3 is activated by hypotonic cell swelling and resumption of the meiotic cell cycle and functions to regulate timing of ovulatory sheath cell contractions. CLH-3 activity is regulated by protein phosphorylation events. Interestingly, we identified in yeast two-hybrid studies an Ste20-related kinase that interacts with the C-terminus of CLH-3. Ste20-1ike kinases are key regulators of cell cycle progression and osmotic adaptation in yeast and mammals. We therefore postulate that the Ste20-like kinase regulates CLH-3 activity. The central goal of my proposal is to combine molecular biological and protein chemistry techniques with genome-assisted mass spectrometry protein microsequencing and patch clamp electrophysiology to examine the role of the Ste20-related kinase in regulation of CLH-3 activity. Specifically, I will assess further the interaction of the Ste20-1ike kinase with CLH-3 under more physiological conditions and use patch clamp techniques to test the functional relevance of this interaction. I will also determine if CLH-3 activity is regulated by direct protein phosphorylation. The experiments outlined in this proposal represent the starting point for developing a detailed of the cell cycle-dependent regulation of CLH-3. These studies will continue to broaden our understanding of the molecular bases of regulation of ClC channels specifically, and of ion channels in general.
Keywords: chloride channel, protein kinase, voltage gated channel, cell morphology, phosphorylation, protein protein interaction, Caenorhabditis elegans, SDS polyacrylamide gel electrophoresis, autoradiography, immunoprecipitation, mass spectrometry, site directed mutagenesis, voltage /patch clamp, yeast two hybrid system
1F32GM067424-01 (2003): $41608
MOLECULAR PHARMACOLOGY AND PHYSIOLOGY OF KIDNEY POTASSIUM TRANSPORT
Jerod S Denton
Vanderbilt University, Medical Center, Nashville, Tn 37203-6869
Grant 1R01DK082884-01A2 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: Inwardly rectifying potassium (Kir) channels are key regulators of diverse physiological processes and may represent novel drug targets for diseases. Their therapeutic potential has not been tested directly, however, due to the lack of drug-like compounds targeting inward rectifiers. The lack of selective "probes" has also hindered efforts to define the physiological functions of some Kir channels. To overcome this formidable barrier and create new opportunities for studying inward rectifier physiology, the investigators performed a high- throughput screen (HTS) of more than 200,000 compounds for small-molecule modulators of ROMK (Kir1.1), a putative target for a novel class of diuretic. One compound, termed VU590, inhibits ROMK at nanomolar concentrations and Kir7.1 in the low micromolar range, making it the first small-molecule inhibitor of both channels. The investigators went on to use medicinal chemistry to rationally design a nanomolar-affinity probe, termed VU591, which is highly selective for ROMK over more than 60 potential off targets, including inward rectifiers and BK channels. In Aim 1, the investigators will employ state-of-the-art molecular modeling techniques, atomic structure-guided mutagenesis and electrophysiology to define the VU590/591 binding sites in ROMK and Kir7.1. VU591 is remarkably selective for ROMK and therefore represents a promising candidate for further development for use in animal studies. In Aim 2, the investigators will first determine if VU591 is active in the native tissue by assessing its effects on K and Na transport in isolated perfused cortical collecting ducts under low- and high-flow conditions. The investigators also discovered a nanomolar-affinity inhibitor of a G-protein regulated inward rectifier (GIRK), a putative therapeutic target for atrial fibrillation. In Aim 3, the investigators will use medicinal chemistry, structure-guided mutagenesis and electrophysiology to define the molecular binding sites for this novel compound termed VU592. These studies will provide important new insights into the atomic structures of inward rectifiers and generate critically needed probes with which to define the integrative physiology and therapeutic potential of these channels. Lay summary The investigators will combine medicinal chemistry, advanced computational techniques and classical physiological methods to develop drug-like compounds targeting potassium channels that could be therapeutic targets for hypertension, edema and cardiac arrhythmia. Inward rectifying potassium (Kir) channels play key physiological roles in diverse cellular functions and may represent novel drug targets. However, the lack of selective pharmacological "probes" has hindered efforts to explore the integrative physiology and therapeutic potential of most Kir channels. Here we propose to employ medicinal chemistry, atomic structure-guided mutagenesis, kidney tubule microperfusion and electrophysiology to develop the small-molecule pharmacology for Kir1.1, Kir7.1 and Kir3 channels
Keywords: Affinity; Amino Acids; Animals; Architecture; Arrhythmia; Arts; Atrial Fibrillation; Auricular Fibrillation; BK channels; Big K channels; Binding; Binding (Molecular Function); Binding Sites; Blood Coagulation Factor IV; Blood Pressure, High; Body Tissues; Ca++ element; Calcium; Cardiac; Cardiac Arrhythmia; Cations; Cell Function; Cell Process; Cell physiology; Cellular Function; Cellular Physiology; Cellular Process; Chemistry, Pharmaceutical; Coagulation Factor IV; Combining Site; Computational Technique; Development; Disease; Disorder; Distal; Diuresis; Diuretics; Dropsy; Drug Delivery; Drug Delivery Systems; Drug Targeting; Drug Targetings; Drugs; Duct; Duct (organ) structure; Edema; Electrophysiology; Electrophysiology (science); Engineering / Architecture; Factor IV; Follow-Up Studies; Followup Studies; G-Proteins; GTP-Binding Proteins; GTP-Regulatory Proteins; Genetics-Mutagenesis; Guanine Nucleotide Coupling Protein; Guanine Nucleotide Regulatory Proteins; Heart Arrhythmias; High Throughput Assay; Hydrops; Hypertension; Investigators; Ion Channels, Potassium; K channel; K element; KCNJ1 gene; Kidney; Kidney Tubules; Lead; Libraries; Location; Macromolecular Structure; MaxiK channels; Medication; Medicinal Chemistry; Methods; Methods and Techniques; Methods, Other; Molecular; Molecular Biology, Mutagenesis; Molecular Interaction; Molecular Models; Molecular Probes; Molecular Structure; Mutagenesis; Na element; Nephrons; Nervous System Physiology; Neurologic function; Neurological function; Neurophysiology / Electrophysiology; Nucleic Acid Biochemistry, Molecular Modeling; Organism-Level Process; Organismal Process; Pb element; Pharmaceutic Chemistry; Pharmaceutic Preparations; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Physiologic; Physiologic Processes; Physiological; Physiological Processes; Physiology; Play; Potassium; Potassium Channel; Protein/Amino Acid Biochemistry, Molecular Modeling; ROMK; Reactive Site; Renal tubule structure; Research Personnel; Researchers; Role; Series; Sodium; Structure; Structure-Activity Relationship; Subcellular Process; Techniques; Testing; Therapeutic; Tissues; Urinary System, Kidney; Uriniferous Tube; V (voltage); Vascular Hypertensive Disease; Vascular Hypertensive Disorder; Work; aminoacid; analog; cell type; chemical structure function; combinatorial chemistry; counterscreen; design; designing; disease/disorder; drug/agent; experiment; experimental research; experimental study; heavy metal Pb; heavy metal lead; high throughput screening; hyperpiesia; hyperpiesis; hypertensive disease; in vivo; inhibitor; inhibitor/antagonist; insight; large-conductance calcium-activated potassium channels; maxi-K channels; molecular modeling; nervous system function; novel; patch clamp; public health relevance; renal; renal outer medullary potassium channel; renal tubule; research study; response; slowpoke protein; small molecule; social role; structure function relationship; therapeutic target; voltage
Relevance: NARRATIVE Inward rectifying potassium (Kir) channels play key physiological roles in diverse cellular functions and may represent novel drug targets. However, the lack of selective pharmacological "probes" has hindered efforts to explore the integrative physiology and therapeutic potential of most Kir channels. Here we propose to employ medicinal chemistry, atomic structure-guided mutagenesis, kidney tubule microperfusion and electrophysiology to develop the small-molecule pharmacology for Kir1.1, Kir7.1 and Kir3 channels
Project start date: 2010-09-01
Project end date: 2015-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PA-07-070
1R01DK082884-01A2 (2010): $305734
CHEMICAL PROBES OF THE ASTROGLIAL POTASSIUM CHANNEL KIR4.1
Jerod S Denton
Vanderbilt University, Medical Center, Nashville, Tn 37203-6869
Grant 1R21NS073097-01 from Office Of The Director, National Institutes Of Health
Abstract: Inward rectifying potassium (Kir) channels are key regulators of diverse physiological processes and may represent novel drug targets for diseases. In most cases, however, the lack of selective pharmacological probes has hindered progress toward defining their specific cellular functions as well as their drugability and therapeutic potential. Here the investigators propose to develop robust primary and secondary assays to support probe development efforts directed toward the "astroglial" inward rectifier channel Kir4.1. This channel is expressed predominately in glial cells of the nervous system, inner ear and kidney tubule and may be a drug target for glial-cell cancers, disorders of myelination and hypertension. In Aim 1, the investigators will develop a fluorescence-based thallium (Tl+) flux assay of Kir4.1 channel function for high-throughput screening (HTS) in either 384- or 1536- well plates. The assay will be validated for HTS by performing a screen of approximately 3,000 small molecules for modulators of Kir4.1 activity. In Aim 2, the investigators will develop additional high- throughput Tl+ flux and moderate-throughput electrophysiological assays to support their subsequent probe development campaign. Lay summary The long-term objective of this work is to develop novel chemical tools which to probe the structure, function and therapeutic potential of a "potassium channel" expressed in the nervous system and kidney. These studies may lay the foundation for the development of novel drugs to treat cancer, movement disorders and high blood pressure. Inward rectifying potassium (Kir) channels play key physiological roles in diverse cell types and may represent novel therapeutic targets. However, the lack of selective small-molecule probes has hindered efforts to understand the integrative physiology and pharmacology of most Kir channels. Here we propose to develop robust fluorescence and electrophysiological assays to support high-throughput screening (HTS) and probe development efforts directed toward the "astroglial" potassium channel Kir4.1
Keywords: 1-Propanamine, 3-(10, 11-dihydro-5H-dibenzo(a, d)cyclohepten-5-ylidene)-N-methyl-; Assay; Bioassay; Biologic Assays; Biological Assay; Blood Pressure, High; Cancers; Cell Function; Cell Line; Cell Lines, Strains; Cell Process; Cell physiology; CellLine; Cellular Function; Cellular Physiology; Cellular Process; Chemicals; Chemistry; Desitriptyline; Desmethylamitriptylin; Development; Disease; Disorder; Diuretics; Drug Delivery; Drug Delivery Systems; Drug Targeting; Drug Targetings; Drugs; Dyskinesia Syndromes; Ear, Internal; Embryo; Embryonic; Exhibits; Family; Fluorescence; Fluorescent Probes; Fluoxetin; Fluoxetine; Foundations; Genes; Glia; Glial Cells; Goals; High Throughput Assay; Human; Human, General; Hypertension; Investigators; Ion Channels, Potassium; K channel; K element; KCNJ1 gene; Kidney; Kidney Tubules; Kolliker`s reticulum; Labyrinth; Lead; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Medication; Movement; Movement Disorder Syndromes; Movement Disorders; N-Methyl-gamma-(4-(trifluoromethyl)phenoxy)benzenepropanamine; NRVS-SYS; Nervous System; Nervous system structure; Neuroglia; Neuroglial Cells; Neurologic Body System; Neurologic Organ System; Non-neuronal cell; Nortriptyline; Organism-Level Process; Organismal Process; Pb element; Pharmaceutic Preparations; Pharmaceutical Preparations; Pharmacology; Physiologic; Physiologic Processes; Physiological; Physiological Processes; Physiology; Play; Potassium; Potassium Channel; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); ROMK; Renal tubule structure; Research Personnel; Researchers; Role; SSRI; Science of Chemistry; Screening procedure; Selective Serotonin Reuptake Inhibitor; Selective serotonin re-uptake inhibitor; Structure; Subcellular Process; Tetracycline Antibiotic; Tetracyclines; Thallium; Therapeutic; Tl element; Urinary System, Kidney; Vascular Hypertensive Disease; Vascular Hypertensive Disorder; Work; analog; base; body movement; cell type; cost; cultured cell line; disease/disorder; drug/agent; heavy metal Pb; heavy metal lead; high throughput screening; hyperpiesia; hyperpiesis; hypertensive disease; inhibitor; inhibitor/antagonist; inner ear; interest; malignancy; member; miniaturize; myelination; neoplasm/cancer; nerve cement; new therapeutic target; novel; patch clamp; renal; renal outer medullary potassium channel; renal tubule; screening; screenings; serotonin reuptake inhibitor; small molecule; social role; success; tool
Relevance: NARRATIVE Inward rectifying potassium (Kir) channels play key physiological roles in diverse cell types and may represent novel therapeutic targets. However, the lack of selective small-molecule probes has hindered efforts to understand the integrative physiology and pharmacology of most Kir channels. Here we propose to develop robust fluorescence and electrophysiological assays to support high-throughput screening (HTS) and probe development efforts directed toward the "astroglial" potassium channel Kir4.1
Project start date: 2010-09-27
Project end date: 2011-08-31
Budget start date: 27-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: PAR-08-024
1R21NS073097-01 (2010): $155167
REGULATION OF MACROPHAGE ACTIVATION STATE BY INTRACELLULAR TRIGLYCERIDE
Jerod S Denton, Asst Professor
Vanderbilt University, Medical Center, Nashville, Tn 37203-6869
Grant 5R21AI079523-02 from National Institute Of Allergy And Infectious Diseases
Abstract: A growing body of evidence suggests a causal relationship between the accumulation of classically activated macrophages in adipose tissue and development of insulin resistance in obesity. The molecular mechanisms regulating adipose tissue macrophage (ATM) activation state are poorly understood. Recent studies of peroxisome proliferator activated receptor-gamma (PPAR3) knockout mice indicate its importance for development of the ´alternative activation´ state in ATMs. Alternatively activated macrophages exhibit low inflammatory potential, upregulate genes involved in fatty acid catabolism (2-oxidation) and function primarily in tissue repair. During progression to the obese state, however, there is a phenotypic switch from alternative activation to classical activation, in which ATMs produce abundant inflammatory mediators linked to insulin resistance. The mechanisms underlying PPAR3-dependent alternative activation and the phenotypic switch in obesity are largely unknown. Several recent studies have reported that ATMs are filled with cytoplasmic triglyceride (TG). Given that TG stores are continuously metabolized to FFAs and since FFAs are endogenous ligands for PPAR3, we reasoned accumulation of adipose tissue TG might play an important role in PPAR3-dependent alternative activation in ATMs. To test this hypothesis, we developed a novel model system that allows us to induce dose-dependent uptake of adipose tissue TG in cultured macrophages. Our preliminary studies indicate that TG accumulation has dose-dependent and striking effects on inflammatory cytokine production. While modest TG accumulation inhibits classical macrophage activation, higher levels of TG loading triggers inflammatory cytokine production. This is the first evidence to our knowledge that adipose tissue TG accumulation may play an important role in regulating the activation state of ATMs. The broad objective of this grant proposal is to begin understanding how TG modulates cell signaling pathways and inflammatory gene expression in macrophages. Specifically, we will 1) test the hypothesis that modest triglyceride accumulation promotes alternative macrophage activation through PPAR3-dependent mechanisms, and 2) test the hypothesis that fatty acid overload promotes classical macrophage activation. Our studies will provide novel insights into this poorly understood, but potentially very important, aspect of macrophage-adipocyte interactions in obesity. This work is significant to understanding the relationship between obesity, inflammation, and the innate immune system, particularly in regards to the role that macrophages play. PUBLIC HEALTH RELEVANCE A growing body of evidence suggests a causal relationship between macrophage accumulation in adipose tissue and insulin resistance in the obese state. We have developed a novel model system that will allow us to study how adipose tissue triglyceride accumulation, as occurs in vivo, modulates inflammatory gene expression in macrophages. Our studies are the first to explore the molecular mechanisms underlying this poorly understood, but potentially very important, niche of adipose tissue macrophage biology
Keywords: Active Oxygen; Adipocytes; Adipose Cell; Adipose tissue; Applications Grants; Biochemical; Biological Models; Biology; Blotting, Western; Catabolism; Cell Communication and Signaling; Cell Signaling; Cytofluorometry, Flow; Development; Dose; EC 2.7.2-; ELISA; Employee Strikes; Endoplasmic Reticulum; Enzyme-Linked Immunosorbent Assay; Ergastoplasm; Exhibits; Extracellular Signal-Regulated Kinases; Fat Cells; Fatty Acids; Fatty Acids, sterified; Fatty Tissue; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Free Fatty Acids; Gene Expression; Gene Expression Monitoring; Gene Expression Pattern Analysis; Gene Expression Profiling; Generations; Genes; Grant Proposals; Grants, Applications; INFLM; Imaging Procedures; Imaging Techniques; Immune system; Inflammation; Inflammation Mediators; Inflammatory; Insulin Resistance; Intracellular Communication and Signaling; Knockout Mice; Knowledge; Ligands; Light; Link; Lipids; Lipocytes; MAP kinase; MAPK; Macrophage Activation; Mature Lipocyte; Mature fat cell; Mediating; Mice, Knock-out; Mice, Knockout; Microfluorometry, Flow; Mitogen-Activated Protein Kinases; Model System; Models, Biologic; Molecular; sterified Fatty Acids; Null Mouse; Obesity; Oxygen Radicals; PPAR gamma; PPARG; PPARG1; PPARG2; PPARgamma; Pathway interactions; Peroxisome Proliferative Activated Receptor Gamma; Peroxisome Proliferator-Activated Receptor gamma; Photoradiation; Play; Pro-Oxidants; Production; Profilings, Gene Expression; Reactive Oxygen Species; Regulation; Reporting; Role; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Strikes; Strikes, Employee; Technics, Imaging; Testing; Thiazolidinedione Receptor; Transcript Expression Analyses; Transcript Expression Analysis; Triacylglycerol; Triglycerides; Western Blotting; Western Blottings; Western Immunoblotting; Work; Wound Healing; Wound Repair; adipose; adiposity; biological signal transduction; body system, allergic/immunologic; corpulence; corpulency; corpulentia; cytokine; endoplasmic reticulum stress; flow cytophotometry; in vivo; insight; insulin resistant; macrophage; novel; obese; obese people; obese person; obese population; organ system, allergic/immunologic; oxidation; pathway; protein blotting; public health relevance; social role; tissue repair; uptake; white adipose tissue; yellow adipose tissue
Project start date: 2009-07-17
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-06-181
5R21AI079523-02 (2010): $193750
1R21AI079523-01 (2009): $193594
Regulation Of A CIC Channel By An Ste20-like Kinase
Jerod S Denton
Vanderbilt University Medical Center Nashville, Tn 372036869
Grant 5F32GM067424-02 from National Institute Of General Medical Sciences IRG: ZRG1
Project start date: 2003-01-14
Project end date: 2005-01-13
5F32GM067424-02 (2004): $47296