Expression And Functional Characterization Of AQP1 Water Channel And CFTR Chloride Channel In Gastrointestinal System

The cyctic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed extensively in mammalian epithelium involving secretion and absorption of body fluid. CFTR-mediated Cl" secretion in small intestine is the final pathway of various types of secretary diarrhea, thus high-affinity CFTR inhibitors will be efficient antidiarrheal drugs. In addition, mutations in CFTR gene cause the hereditary lethal disease cystic fibrosis (CF). A large animal CF model is still lacking to investigate the mechanisms by which defective CFTR produces airway disease, which is the major cause of morbidity and mortality in CF patients. High-affinity CFTR-specific inhibitors will be useful to create pharmacologically the CF phenotype in large animals to provide such in vivo models.A thiazolidinone CFTR inhibitor, CFTRjnh-172, was identified by high throughput screening of combinatorial chemical library using a cell-based fluorescent assay. Previous studies demonstrated that CFTRjnh-172 is a high-affinity (Kd=300 nM), nontoxic, membrane-permeable, reversible, and CFTR-specific inhibitor in cell-based fluorescence and electrophysiological assays. CFTRinh-172 inhibited cholera toxin-induced intestinal fluid secretion to the same extent as seen in the CFTR deficient mice. These studies indicated that CFTRinh-m efficiently and specifically blocked CFTR-mediated Cl" transport both in vitro and in vivo. Therefore CFTRinh-m is suitable for creating CF phenotype in large animals and has the potential to become an antidiarrheal agent.In this study, we developed a simple three-step method to synthesize high-quality and large amount of the thiazolidinone CFTR inhibitor. The final product 2-thioxo-3-(3-trifluoromethylphenyl)-5-(4-carboxyphenylmethylene) -4-thiazolidinone is purified by recrystallization and characterized structurally by NMR and functionally by cell-based fluorescence and short-circuit current assays. The synthesized CFTRjnh-m was used for in-field antidiarrheal studies and for creation of CF models in pigs.The results demonstrated that CFTRjnh-i72 is a very effective antidiarrheal agent in pig, which prevented 80% death of severe secretary diarrhea caused by unknown pathogens. However, the efforts in producing cystic fibrosis phenotypes in pigs using CFTRinh-m did not reach the pathological point in the lung by administration of the inhibitor for 55 consecutive days. Further studies are needed to create large animal CF models by using CFTR inhibitors.