Pharmacoperone Identification for Therapeutic Rescue of Misfolded Mutant Proteins

G protein-coupled receptors (GPCRs), which includes the gonadotropin-releasing hormone (GnRH) receptor (GnRHR), comprises the largest family of validated drug targets – more than half of all approved drugs derive their benefits by selective targeting of GPCRs. Most drugs in this class are either agonists or antagonists of GPCRs and high throughput screens have typically been designed and performed with a view toward identification of such compounds as lead drug candidates. This manuscript presents the case that valuable drugs which effect the trafficking of GPCRs may have been overlooked because pharmacoperones have been selected from existing screens that identify agonists and antagonists. A “gain of activity assay” is proposed; this assay relies on the expression of a mutant of the GnRHR that is known to be rescuable by pharmacoperone drugs, and which is restored to activity in their presence. Accordingly, “hits” are identified by the appearance of activity. The gene for the mutant is under control of tetracycline and may be prevented from being expressed. This is a valuable feature since it allows false positives to be identified. Such drugs will show apparent activity whether or not the mutant is expressed. This assay will enable identification of these drugs from chemical libraries and does not rely on their activity as agonists or antagonists

In the Classroom: Strategies for Poor Readers (Mar. \u2790)

All readers need successful learning experiences. Recent research suggests that poor readers benefit from using the same whole language reading and writing strategies as more successful readers. In her introduction to the April 1988 themed issue of Vie Reading Teacher on what works with poor readers. Irene West Gaskins stated: The research I was doing supported the current view that reading is a process in which an active and strategic reader gains meaning through an interaction between background knowledge and information in a text. Since I have adopted this definition, the way I teach has changed and students in the classrooms in which I teach seem to be benefitting — especially the hard-to-teach students. I no longer believe that I am successful as a teacher when most of my students are learning- Unless I have reached the hard-to-teach. I may not have taught at all. The challenge is for classroom teachers to accept responsibility for providing successful learning experiences for all children in their classrooms, including the poor readers (p. 749)

In the Classroom: Reading and Writing in the Content Areas (Dec. \u2789)

Students comprehend content material by reading, discussing, writing, questioning, investigating, exploring, and organizing. Reading and writing in the content areas relates prior knowledge, classroom interaction, cooperative learning, vocabulary instruction, and questioning techniques. Children practice research skills by organizing information in a meaningful and practical manner. This month\u27s In the Classroom column presents ways in which teachers can enhance their students\u27 comprehension of content area topics by involving them in various classroom activities. Additional resources for content area reading and writing activities follow : Dupuis, M.M. (1983). Reading in the content areas: Research for teachers. Newark, DE: International Reading Association. Graves, D.H. (1989). Investigate nonfiction. Portsmouth, NH: Heinemann. Heimlich, J.E. and Pittelman, S.D. (1986). Semnntic mapping: Classroom applications. Newark, DE: International Reading Association. Marzano, R.J, and Marzano, J.S. (1988). A cluster approach to elementary vocabulary instruction. Newark, DE: International Reading Association. Thelen, J.N. (1984). Improving reading in science. Newark, DE: International Reading Association

In the Classroom: Vocabulary (April \u2790)

Vocabulary knowledge is one of the most critical elements of comprehension. Helping students to expand their own vocabularies and to become more interested in words is a challenge for all teachers. The following techniques provide teachers various ways to enhance students\u27 vocabulary development. To read more about vocabulary instruction, see: Heimlich, J.E., & Pittelman, S.D. (1986). Semantic mapping: Classroom applications. Newark, DE: International Reading Association. Johnson, D.D. (Ed.) (1986). Vocabulary [Special issuel. Journal of Reading, 29 (7). Johnson, D.D., & Pearson, PD. (1984). Teaching reading vocabulary (2nd ed.). New York: Holt, Rinehart and Winston. Marzano, R.J., & Marzano, J.S. (1988). A cluster approach to elementary vocabulary instruction. Nevrark, DE: International Reading Association

In the Classroom: Using Children\u27s Literature (Oct. \u2789)

Lists children\u27s literature in reading instruction. Children\u27s Literature in the Reading Program ; Literature-based Reading Programs at Work ; Children\u27s Choices: Teaching With Books Children Like ; Transitions: From Literature to Literacy

An Introduction to the SMARTRISK Heroes Program: Positive Social Marketing for Adolescent Injury Prevention

This article provides background on the SMARTRISK Heroes Program, a mobile stage production that introduces young people to the prevalence of unintentional injury for their age group and presents them with a series of strategies that will reduce the likelihood that they will be unintentionally injured or killed. The program logic is consistent with theoretical work from the area of health promotion including the Protection Motivation Theory and the Transtheoretical Model of Stages of Change. The SMARTRISK Heroes Program has been the subject of a number of past evaluations that are briefly described. The program logic model was included in this article. Additional information on the program and its evaluations can be found at http://www.smartrisk.ca by selecting SMARTRISK Heroes, under the “Youth Tab.

Receptor antagonism/agonism can be uncoupled from pharmacoperone activity

Pharmacoperones rescue misrouted mutants of the vasopressin receptor type 2 (V2R) and enable them to traffic to the correct biological locus where they function. Previously, a library of nearly 645,000 structures was interrogated with a high throughput screen; pharmacoperones were identified for V2R mutants with a view toward correcting the underlying mutational defects in nephrogenic diabetes insipidus. In the present study, an orthologous assay was used to evaluate hits from the earlier study. We found no consistent relation between antagonism or agonism and pharmacoperone activity. Active pharmacoperones were identified which had minimal antagonistic activity. This increases the therapeutic reach of these drugs, since virtually all pharmacoperone drugs reported to date were selected from peptidomimetic antagonists. Such mixed-activity drugs have a complex pharmacology limiting their therapeutic utility and requiring their removal prior to stimulation of the receptor with agonist

Gonadotropin-releasing hormone receptor activates GTPase RhoA and inhibits cell invasion in the breast cancer cell line MDA-MB-231

BACKGROUND: Gonadotropin-releasing hormone (GnRH) and its receptor (GnRHR) are both expressed by a number of malignant tumors, including those of the breast. In the latter, both behave as potent inhibitors of invasion. Nevertheless, the signaling pathways whereby the activated GnRH/GnRHR system exerts this effect have not been clearly established. In this study, we provide experimental evidence that describes components of the mechanism(s) whereby GnRH inhibits breast cancer cell invasion. METHODS: Actin polymerization and substrate adhesion was measured in the highly invasive cell line, MDA-MB-231 transiently expressing the wild-type or mutant DesK191 GnRHR by fluorometry, flow cytometric analysis, and confocal microscopy, in the absence or presence of GnRH agonist. The effect of RhoA-GTP on stress fiber formation and focal adhesion assembly was measured in MDA-MB-231 cells co-expressing the GnRHRs and the GAP domain of human p190Rho GAP-A or the dominant negative mutant GAP-Y1284D. Cell invasion was determined by the transwell migration assay. RESULTS: Agonist-stimulated activation of the wild-type GnRHR and the highly plasma membrane expressed mutant GnRHR-DesK191 transiently transfected to MDA-MB-231 cells, favored F-actin polymerization and substrate adhesion. Confocal imaging allowed detection of an association between F-actin levels and the increase in stress fibers promoted by exposure to GnRH. Pull-down assays showed that the effects observed on actin cytoskeleton resulted from GnRH-stimulated activation of RhoA GTPase. Activation of this small G protein favored the marked increase in both cell adhesion to Collagen-I and number of focal adhesion complexes leading to inhibition of the invasion capacity of MDA-MB-231 cells as disclosed by assays in Transwell Chambers. CONCLUSIONS: We here show that GnRH inhibits invasion of highly invasive breast cancer-derived MDA-MB-231 cells. This effect is mediated through an increase in substrate adhesion promoted by activation of RhoA GTPase and formation of stress fibers and focal adhesions. These observations offer new insights into the molecular mechanisms whereby activation of overexpressed GnRHRs affects cell invasion potential of this malignant cell line, and provide opportunities for designing mechanism-based adjuvant therapies for breast cancer