The unifying hypothesis for this grant application is that LPA2 agonists have the potential for therapy of cholera and other types of enterotoxin-induced secretory diarrhea. Thus in this renewal application we propose to develop a physiologically relevant human intestinal stem cells (enteroids) to study CFTR-dependent fluid secretion and develop assays to demonstrate and identify LPA2 receptor specific small molecule agonists. LPA2 dependent inhibition of CFTR requires adenylate cyclase 6 (AC6) suggesting that AC6 is part of the CFTR-NHERF2-LPA2 complex. Two hypotheses will be tested:
Specific Aim 1 : To test the hypothesis that LPA2 agonists attenuate CTX-induced and CFTR- dependent fluid secretion in human intestinal stem cells (spheroids and enteroids), and mitigate diarrheal symptom Specific Aim 2: To test the hypothesis that adenylate cyclase 6 (AC6) exists in the macromolecular complex of CFTR-NHERF2-LPA2 in human intestinal epithelial cells and that AC6 plays an important role in modulating LPA2-dependent inhibition of CFTR channel function that can be therapeutically relevant in targeting LPA2 in controlling secretory diarrheas. The proposed study is highly significant because (a) it addresses a deadly disease; (b) it has clinical relevance and implications; (c) it is a multidisciplinary project coves basic biomedical studies, high throughput assay development, and sets a stage for future drug discovery.

Public Health Relevance

According to the World Health Organization (WHO) reports, cholera is still a major epidemic in several countries. In this renewal application we propose to develop a robust method of isolation of human intestinal stem cells (enteroids) to study macromolecular complexes of LPA2, identify potent LPA2 receptor specific small molecule agonists that can inhibit cholera-induced diarrhea. In addition we will test if LPA2 dependent inhibition of CFTR requires adenylate cyclase 6 (AC6) and demonstrate that AC6 is part of the CFTR-NHERF2-LPA2 macromolecular complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK093045-09
Application #
9513525
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Perrin, Peter J
Project Start
2011-07-15
Project End
2020-06-30
Budget Start
2018-07-01
Budget End
2019-06-30
Support Year
9
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Cincinnati Children's Hospital Medical Center
Department
Type
DUNS #
071284913
City
Cincinnati
State
OH
Country
United States
Zip Code
45229
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Thomas, Andrew; Ramananda, Yashaswini; Mun, KyuShik et al. (2018) AC6 is the major adenylate cyclase forming a diarrheagenic protein complex with cystic fibrosis transmembrane conductance regulator in cholera. J Biol Chem 293:12949-12959
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