High-throughput Assay to Identify CFTR Inhibitors

Maitra, Rangan

Abstract

The overall goal of this proposal is to create and validate a microplate-based fluorescent membrane potential assay to identify inhibitors of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). While originally named after its role in Cystic Fibrosis (CF), the CFTR protein plays an important role in other important disorders and biological processes - secretory diarrhea, autosomal dominant polycystic kidney disease (ADPKD) and sperm capacitation in males. Secretory diarrhea kills over two million children under the age of 5 annually and ADPKD is the third most common single cause of end-stage renal failure worldwide. A common feature of both diseases is increased levels of intracellular cAMP. In secretory diarrhea, bacterial exotoxins increase intracellular cAMP and cGMP, which are both CFTR agonists. Increased accumulation of cAMP through altered homeostasis of intracellular Ca2+ is a dominant feature of ADPKD. Stimulation of CFTR by its classical agonist cAMP contributes towards fluid loss in diarrhea and cystogenesis in ADPKD by driving CFTR-dependent active chloride transport and passive water secretion. CFTR in sperm is involved in the transport of HCO3-, which is important for sperm capacitation. Thus, inhibitors of CFTR could be useful as therapeutics in secretory diarrhea, and as probes to elucidate the role of CFTR in ADPKD and sperm capacitation. These compounds could also find use in the development of in vitro and in vivo models of CF.
Two specific aims are proposed for the project. Through aim 1, we propose to develop and optimize a membrane potential-based HTS assay to monitor CFTR activity. We have engineered and characterized a cell line that stably expresses functional wild-type CFTR. We present preliminary data to demonstrate that agonist- mediated activation of CFTR leads to an increase in membrane potential, which is detected using a fluorescent membrane potential dye in an automated microplate assay. The ability of a compound to interfere with agonist-stimulated CFTR activity forms the basis of the proposed HTS assay. Through aim 2, we propose to validate the membrane potential HTS assay by screening a library of pharmacologically active compounds. Additionally, we also delineate our approach to hit validation through secondary assays and our overall compound prioritization strategy.

Public Health Relevance

The goal of the project is to develop a method to measure the activity of a protein called CFTR, which is involved in the transport of certain ions and water in the body. This experimental method can be used to identify compounds that inhibit the function of CFTR. Such compounds may be useful to treat two important diseases - secretory diarrhea and autosomal polycystic kidney disease - that together kill over 2 million people each year across the world.