P. Michael Conn - US grants

Studies dealing with the biochemical and molecular mechanism of gonadotropin releasing hormone (GnRH) action have served to (a) identify new human and veterinary uses for GnRH and its analogs, (b) bring these uses to fruition through a rational process based on understanding of the mechanism of hormone action, and (c) provide the means for anticipating, understanding, and ameliorating side-effects of the agents. FDA approvals of GnRH agonists for the treatment of prostate cancer, endometriosis, and precocious puberty, as well as for the use of natural sequence GnRH to induce ovulation and to test the hypothalamic-pituitary-gonadal axis, are examples of the clinical usefulness derived from these fundamental observations. There are multiple advantages to the study of GnRH-stimulation of the gonadotrope that make it facile to collect interpretable data (a) GnRH stimulation of the gonadotrope cell has clearly defined, specific and measurable endpoints release of endocr ine (a nd potentially endocrine) substances (LH, FSH, secretogranin II, ?-subunit of gonadotropin), regulation of target cell sensitivity, regulation of the GnRH receptor, and biosynthesis of released substances. (b) The releasing hormone itself (as well as its agonists and antagonists) can be radioiodinated to high specific activity. (c) Many (>3,000) analogs exist that can be chemically derivatized without loss of biological activity. Antagonists and agonists which bind the receptor with greater affinities than the natural sequence GnRH are available. (d) Virtually all known agonists and antagonists are Apure@ in action and metabolically stable agonists are available. The present project is divided into areas of focus that form the basis of organization of the work. The first will provide information on the structure of the GnRH receptor and early actions following the interaction of the receptor with GnRH and its analogs and should advance our understanding of the receptor in the mec hanism of GnRH action. The second area will provide information on the relationship between the multiple effector mechanisms already implicated in GnRH action with the multiple actions stimulated by the releasing hormone (release of multiple endocrine substances, regulation of target cell responsiveness, biosynthesis, and regulation of receptor number). This is important since considerable confusion remains regarding these relations. The third area involves understanding the molecular sites of action of activin and inhibin in relation to GnRH action and are significant to understand the actions of these agents in vivo. The approaches will take advantage of newly available genetic probes, antisera, and cell lines. FUNDING NIH HD19899 PUBLICATIONS Conn PM, Parker JV. Animal rights reaching the public. Science 282:1417, 1998. Conn PM (editor-in-chief). Clinical Management of Diabetic Neuropathy. Contemporary Endocrinology Vol 7 (A Veves, ed). Totowa, NJ Humana, 347 pp, 1998. Conn PM (editor-in-chief). G Proteins, Receptors, and Disease. Contemporary Endocrinology Vol 6 (A Spiegel, ed). Totowa, NJ Humana, 324 pp, 1998. Conn PM, Jennes J, Janovick JA. GnRH (Gonadotropin-Releasing Hormone). In Encyclopedia of Reproduction (E Knobil, JD Neill, eds). New York, NY Academic Press, pp 464-477, 1998. Conn PM. Make science relevant, human and clear. The Scientist 12:9, 1998. Cornea A, Janovick JA, Stanislaus D, Conn PM. Redistribution of Gq/11? in pituitary gonadotrope in response to a GnRH agonist. Endocrinology 1:397-402, 1998. Lin X, Janovick JA, Brothers S, Blomenrvhr J, Bogerd J, Conn PM. Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellular carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation. Mol Endocrinol 12:161-171, 1998. Lin X, Janovick JA, Conn PM. Mutations at the consensus phosphorylation sites in the third intracellular loop of the rat GnRH receptor effects on receptor ligand binding and signal transduction. Biol Reprod 59:1470-1476, 1998. Lin X, Conn PM. Transcriptional activation of gonadotropin-releasing hormone (GnRH) receptor gene by GnRH and cyclic AMP. Endocrinology 139:3896-3902, 1998. Lin X, Cornea A, Janovick JA, Conn PM. Visualization of unoccupied and occupied gonadotropin-releasing hormone receptor in living cells. Mol Cell Endocrinol 146:27-37, 1998. Stanislaus D, Ponder S, Ji T, Conn PM. GnRH receptor couples to multiple G-proteins in gonadotropes and in GGH3 cells evidence from palmitoylation and overexpression of G-proteins. Biol Reprod 59:579-586, 1998. Stanislaus D, Janovick JA, Ji T, Wilkie T, Offermanns S, Conn PM. Gonadotropin and gonadal steroid release in response to a GnRH agonist in Gq? and G11? knockout mice. Endocrinology 139:2710-2717, 1998. Stanislaus D, Pinter J, Janovick JA, Conn PM. Mechanisms mediating multiple physiological responses to gonadotropin-releasing hormone. Mol Cell Endocrinol 144:1-10, 1998. Ulloa-Aguirre A, Stanislaus D, Arora V, Vddndnen J, Brothers S, Janovick JA, Conn PM. The third intracellular loop of the rat gonadotropin-releasing hormone (GnRH) receptor couples the receptor to Gs- and Gq/11-mediated signal transduction pathways evidence from loop fragment transfection in GGH3 cells. Endocrinology 5:2472-2478, 1998.

DESCRIPTION One of the most investigated areas of biology is the regulation of cellular function by binding of ligands to receptors on the extracellular surface of cells. Understanding how these receptors signal ligand binding to the interior of the cell -- signal transduction -- is key to understanding the establishment and regulation of homeostasis throughout the body. Additionally, pharmacological intervention in disease processes can be achieved by interfering with the ligand/receptor interaction, and so any additional understanding of the mechanisms whereby receptors transduce their signals has obvious importance in discovery and refinement of pharmacotherapies. This proposal specifically targets signal transduction of the human FSH receptor, which is an important hormonal response pathway in the regulation of fertility. Four specific aims are enumerated: (1) determine the role of three intracellular loops and C- terminal tail of the FSH receptor in G protein coupling, (2) determine the role of specific amino acid residues present in each intracellular loop and in a segment from the membrane-proximal portion of the C-terminal tail of the FSH receptor, (3) quantify the turnover of G proteins involved in signal transduction triggered by activation of the FSH receptor, and (4) determine the role of down-regulation of G proteins coupled to the FSH receptor the cellular responses to different molecular forms of human FSH.

This research training program is designed to enable NIH grant recipients of the Oregon Health Sciences University (OHSU) to continue to extend the geographic base of research and training efforts to Mexico and Chile, countries in which the dynamics of population growth negatively impacts public health, the environment and economic progress. This program includes pre-doctoral and sabbatical training opportunities in reproductive sciences. Because of the proximity of Mexico to the United States and the substantial length of the common border, problems in this neighboring country influence not only United States border towns but also general patterns of immigration into the U.S. and the politics of American entitlement programs. The advent of the North American Free Trade Agreement (NAFTA) agreement in 1994 and severe economic programs in Mexico makes this program particularly appropriate. A training interaction with Mexico is especially timely now since training opportunities for Mexican nationals are severely limited due to the devaluation of the peso against the dollar to nearly the lowest level on record, and on a political situations that closed the doors of the National University, UNAM, for nearly one year. Likewise, due to the fall of a military dictatorship, Chile is enjoying a period of economic and political stability. Chile offers a unique opportunity due to the significant level of training of its scientists and the recent indication of the Chilean federal government's commitment to science and infrastructure development. We have received letters of cooperation from Mexican and Chilean institutions and will continue to have access to trainees selected from over 300 hospitals and 200 research institutions. The proposed program dove-tails with existing expertise, funding, and the presence of proven mentors and a working program in the area of reproductive sciences. Our previous funding period was extremely productive and consistent with the goal of the program.

This research training program is designed to enable NIH grant recipients of the Oregon Health Sciences University (OHSU) to continue to extend the geographic base of research and training efforts to Mexico and Chile, countries in which the dynamics of population growth negatively impacts public health, the environment and economic progress. This program includes pre-doctoral and sabbatical training opportunities in reproductive sciences. Because of the proximity of Mexico to the United States and the substantial length of the common border, problems in this neighboring country influence not only United States border towns but also general patterns of immigration into the U.S. and the politics of American entitlement programs. The advent of the North American Free Trade Agreement (NAFTA) agreement in 1994 and severe economic programs in Mexico makes this program particularly appropriate. A training interaction with Mexico is especially timely now since training opportunities for Mexican nationals are severely limited due to the devaluation of the peso against the dollar to nearly the lowest level on record, and on a political situations that closed the doors of the National University, UNAM, for nearly one year. Likewise, due to the fall of a military dictatorship, Chile is enjoying a period of economic and political stability. Chile offers a unique opportunity due to the significant level of training of its scientists and the recent indication of the Chilean federal government's commitment to science and infrastructure development. We have received letters of cooperation from Mexican and Chilean institutions and will continue to have access to trainees selected from over 300 hospitals and 200 research institutions. The proposed program dove-tails with existing expertise, funding, and the presence of proven mentors and a working program in the area of reproductive sciences. Our previous funding period was extremely productive and consistent with the goal of the program.

[unreadable] DESCRIPTION (provided by applicant): This research training program is designed to enable NIH grant recipients at the Oregon Health Sciences University (OHSU) and its Institute, the Oregon National Primate Research Center (ONPRC) to continue to provide international research and training in the area of reproduction and population research. Our objective is to provide short-, medium-, and long-term advanced training opportunities with a view toward strengthening intellectual infrastructure. All countries with which we previously interacted (Mexico, Argentina, Chile) or contemplate interaction (Mexico, Brazil) are low- and middle-income (defined by the World Bank standard) and benefit by increasing research capacity in reproductive processes and mechanisms. Reuters estimates (September 26, 2005) that the economies of "Brazil and Mexico are expected to grow by between 3 percent and 6 percent for 2005," making this an appropriate time to interact. It is of special interest with regard to the goals of this program that Latin America is one of the world's areas that will disproportionately contribute to world population. Accordingly we have chosen to interact with countries that have some research infrastructure but have not yet developed the critical mass capacity needed to function optimally. In the present application, we seek to add Brazil to the countries that we serve in order to extend our reach and continue to participate in Mexico. We expect to phase out our activities in Chile and Argentina, consistent with the new programmatic requirement of this award limiting us to two countries of interaction. We provide evidence of a high quality training program that has functioned effectively in returning trainees to their home country where they have developed active and funded research programs that have resulted in significant contributions to knowledge in reproduction. [unreadable] [unreadable] [unreadable]

Access to Information; Aging; Animals; Artificial Insemination; Assay; Assisted Reproduction Technology; Assisted Reproductive Technology; Bacteriophages; Bioassay; Biologic Assays; Biological Assay; CRISP; Cell Isolation; Cell Line; Cell Lines, Strains; Cell Segregation; Cell Separation; Cell Separation Technology; CellLine; Chemotherapy-Hormones/Steroids; Clone Cells; Computer Retrieval of Information on Scientific Projects Database; Confocal Microscopy; Consultations; Cytofluorometry, Flow; Endocrine; Endocrine Gland Secretion; Equipment; Eutelegenesis; Event; Fee-for-Service Plans; Fees for Service; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Funding; Generations; Grant; Head; Health Care Research; Health Services Evaluation; Health Services Research; Healthcare Research; Hormones; Human Resources; ICSI; Image; Image Analyses; Image Analysis; Infectious Agent; Institution; International; Intracytoplasmic Sperm Injections; Investigators; Large-Scale Sequencing; Macaca mulatta; Mammals, Primates; Manpower; Medical Care Research; Medical Illustration; Methods and Techniques; Methods, Other; Microfluorometry, Flow; Microscopy, Confocal; Modeling; Molecular and Cellular Biology; Monkeys; Morphology; Mother Cells; NIH; National Institutes of Health; National Institutes of Health (U.S.); PCR; Phages; Photography; Plasmids; Polymerase Chain Reaction; Preparation; Primates; Procedures; Production; Progenitor Cells; Programs (PT); Programs [Publication Type]; Protocol; Protocols documentation; R01 Mechanism; R01 Program; ROC Analysis; RPG; Rate; Reagent; Recovery; Reporting; Research; Research Grants; Research Personnel; Research Project Grants; Research Projects; Research Projects, R-Series; Research Resources; Researchers; Resource Informatics; Resources; Rhesus; Rhesus Macaque; Rhesus Monkey; Science of Virology; Scientist; Seminal; Senescence; Services; Source; Sperm Injections, Intracytoplasmic; Spottings; Stem cells; Techniques; Technology, Assisted Reproductive; Therapeutic Hormone; Time; Training; Transfection; United States National Institutes of Health; Virology; bacterial virus; cell sorting; cost; cultured cell line; flow cytophotometry; genetic resource; image evaluation; imaging; infectious organism; light microscopy; nuclear transfer; nuclear transplantation; outreach to information; personnel; programs; senescent; services research; virology

DESCRIPTION (provided by applicant): This proposal is a response to PA-07-320, "Development of Assays for High-throughput Drug Screening (HTS) (R01)." The Program Announcement is a component of the Molecular Libraries Initiative, part of the NIH Roadmap for Medical Research, and supports "the development of innovative assays that may ultimately be adapted for automated screening" of molecular libraries. Our specific aim is to produce and validate screens for identification of drugs from molecular libraries which exert their actions by a newly appreciated therapeutic approach-namely, control of post-translational protein folding and, as a consequence, cellular trafficking and rescue of mutants. G protein coupled receptors (GPCRs) are frequently targeted in library screening, yet this approach generally relies on screens that identify agonists or antagonists and would have missed many of the drugs that will be identified in the proposed screens. Our approach identifies drugs with a significant degree of novelty in therapeutic approach, relying on cellular mechanisms that are not currently represented in the Molecular Libraries assay pipeline;this offers an untapped opportunity for use of the HTS approach. This proposal addresses one of the specific areas that the PA identifies as relevant, "assays for molecular chaperones or molecules that improve the post- translational targeting, folding or assembly of proteins, especially involving mutant proteins responsible for inborn errors of metabolism... (or) rare diseases." In the planned studies we will develop and characterize assays for pharmacological chaperones that improve the post-translational folding and targeting of two mutant GPCRs which cause human disease. The products of the proposed HTS assays will be useful for treatment of two rare diseases, nephrogenic diabetes insipidus and hypogonadotropic hypogonadism, each caused by an inborn error. These screens will also serve as prototypes for identification of other therapeutic molecules, especially those involving GPCRs. Many diseases are now understood to be caused by protein misfolding. Development of such assays is important and novel since the extensive use of agonist/antagonist screens alone means that useful chemical structures with the ability to control trafficking (without receptor agonism or antagonism) may already be present in existing libraries, but have not been identified using existing methods. PUBLIC HEALTH RELEVANCE: We will develop and characterize assays for pharmacological chaperones that improve the post- translational folding and targeting of two mutant receptors which cause human diseases, including nephrogenic diabetes insipidus and hypogonadotropic hypogonadism, each caused by an inborn genetic error. These screens will also serve as prototypes for identification of other therapeutic molecules, and are important because existing screens would have missed potentially valuable drugs already present in chemical libraries.

DESCRIPTION (provided by applicant): This project is designed to develop and characterize two prototypic genetically modified mouse models for human diseases of protein folding. These models are required to bring a novel class of target-specific drugs, pharmacoperones, to human (and animal) use. Misfolded mutant proteins are detected by the cellular quality control system (QCS) and are typically retained in the endoplasmic reticulum (ER) for either reprocessing or degradation; frequently, these mutants result in disease. Studies in cell cultures indicate that these mutants can be rescued by target-specific small molecules (pharmacoperones) which enter cells, serve as templates that refold the mutants, and permit their passage to the plasma membrane. Many mutants retain or regain their fundamental properties as ion channels, enzymes or receptors when re-routed correctly. Diseases caused by misfolding (which may benefit from this approach) include cystic fibrosis, hypogonadotropic hypogonadism, nephrogenic diabetes insipidus, retinitis pigmentosa, hypercholesterolemia, cataracts, neurodegenerative diseases (Huntington's, Alzheimer's and Parkinson's), cancers and digestive disorders. It is fair to say that virtually every person will be affected by protein folding diseases during his or her lifetime, either directly or due to the illness of a loved one. In spite of this, there are few model systems, and none in small laboratory animals, that allow the translation of available in vitro data or the testing of hits from high throughput screening on protein rescue into in vivo systems. To date, therapeutic approaches in humans have relied on a small number of studies in end-of-life patients, using drugs that have never been fully characterized in animal models. Such models are needed to address drug safety, the pattern of drug administration required to optimize therapeutic effectiveness, and serve as a test model for new drugs in preclinical studies. The pattern (route, dose and frequency) is particularly important as the persistence of these drugs frequently inhibits the desired activity, once rescue has occurred, so they must be removed. Moreover a convenient laboratory model for these diseases is needed if the use of pharmacoperones is to translate to human well-being, since US law requires animal testing prior to normal human volunteers. The present study will characterize prototypic mouse models of misfolding, relying on an unusually well-characterized mutant of a physiologically important GPCR (i.e. the gonadotropin releasing hormone receptor). There is much information available on the mechanism of activation of the gonadotropin releasing hormone receptor (GnRHR) and on the biochemical mechanism by which the mutant E90K is believed to cause the disease state. This information has been helpful in guiding our choices of mutant E90K and will contribute to the success of this project. The two models to be used are available and have the predicted genotype and necessary phenotype. PUBLIC HEALTH RELEVANCE: This project is designed to develop, characterize, compare and contrast two models for human diseases of protein folding. Diseases caused by misfolding include cystic fibrosis, hypogonadotropic hypogonadism, nephrogenic diabetes insipidus, retinitis pigmentosa, hypercholesterolemia, cataracts, neurodegenerative diseases (Huntington's, Alzheimer's and Parkinson's), particular cancers and a number of digestive disorders resulting from enzyme mutation. A convenient laboratory model for these diseases is needed if the use of pharmacoperones is to translate to human well-being, since US law requires animal testing prior to normal human volunteers.

DESCRIPTION (provided by applicant): This proposal is a response to PAR-12-058, Solicitation of Assays for High Throughput Screening (HTS) to Discover Chemical Probes (R01). This funding opportunity supports collaboration between academic, nonprofit, or commercial HTS screening facilities that have the requisite expertise and experience to implement an HTS-ready assay for the discovery and development of small molecule chemical probes. The work proposed will be conducted by collaboration between the laboratory that developed the concept of pharmacoperones (and the assay) and two other laboratories with extensive experience in HTS and access to large chemical libraries. We will use an existing and well-validated high throughput assay to identify pharmacoperones of the vasopressin type 2 (V2) receptor (V2R). The assay will be applied to screen a library of approximately 640,000 compounds and will identify therapeutic molecules for the treatment of misrouting of the V2R in nephrogenic diabetes insipidus, a rare and debilitating disease for which there are presently no effective drug treatments. Pharmacoperones are target-specific and small molecules that diffuse into cells, rescue misfolded protein mutants and restore them to function. Rescue is based on a newly appreciated mechanism: correcting the cellular routing of mutants that would otherwise be retained in the endoplasmic reticulum and unable to function. Because of the newness of this type of screen, this project will also serve as a prototype for the identification of pharmacoperones present in large chemical libraries. Accordingly, the proposed approach identifies drugs with a significant degree of novelty in therapeutic approach, relying on cellular mechanisms that are not currently represented in the Molecular Libraries assay pipeline; this technique offers an untapped opportunity for use of the HTS approach. Development of such assays is important and novel since useful chemical structures with the ability to control cellular trafficking may already be present in existing libraries, but have not been identified using existing screens. Preliminary data show that the assay was successfully transferred to the HTS facility and early screening results show that the proposed approach is likely to be successful in identification of hits.

Project Summary This proposal is a resubmission in response to PAR-14-284, ?High Throughput Screening (HTS) to Discover Chemical Probes (R01). This funding opportunity supports collaboration between academic, nonprofit, or commercial HTS screening facilities that have the requisite expertise and experience to implement an HTS-ready assay for the discovery and development of small molecule chemical probes. The work proposed will be conducted by collaboration between the Conn laboratory that developed the concept of pharmacoperones (and the assay) and the laboratories at Scripps Research Institute with extensive experience in HTS Drug Discovery, Medicinal Chemistry, and Drug Metabolism and Pharmacokinetics (DMPK). We will use an existing and well-validated high throughput assay to identify pharmacoperones of the gonadotropin releasing hormone (GnRH) receptor (GnRHR). Pharmacoperones are target-specific and small molecules that diffuse into cells, rescue misfolded protein mutants and restore them to function. Rescue is based on a newly appreciated mechanism: correcting the cellular routing of mutants that would otherwise be retained in the endoplasmic reticulum and unable to function. The efficacy of these drugs has been demonstrated both in cell cultures and in vivo. The assay will be applied to screen a library of more than 640,000 compounds and will identify therapeutic molecules for the treatment of misrouting of the GnRHR in diseases of reproduction. One such disease is hypothalamic hypogonadism (HH). HH is a family of disorders, a subset of which is caused by mutations in the GnRHR which result in retention in the endoplasmic reticulum. In addition to increasing the expression of mutants at the plasma membrane, pharmacoperones of this receptor increase plasma membrane expression of WT GnRHR. Only 50% of the synthesized human GnRHR is normally expressed at the plasma membrane and this percent can be increased by pharmacoperones. Accordingly, these drugs may be used in treatment of disorders in which this receptor is sub optimally expressed and cancers in which GnRH is a therapeutic target. Because of the newness of this type of screen, this project will also serve as a prototype for the identification of pharmacoperones present in large chemical libraries. Accordingly, the proposed approach identifies drugs with a significant degree of novelty in therapeutic approach, relying on cellular mechanisms that are not currently represented in the Molecular Libraries assay pipeline; this technique offers an untapped opportunity for use of the HTS approach. Development of such assays is important and novel since useful chemical structures with the ability to control cellular trafficking may already be present in existing libraries, but have not been identified using existing screens. Preliminary data show that the assay was successfully transferred to the HTS facility and early screening results show that the proposed approach is likely to be successful in identification of tractable hits.