Biol. Pharm. Bull. 30(3) 502—507 (2007)

in the pathogenesis of secretory diarrhea. It is a passive process driven by the active secretion of ion, predominantly chloride. The cellular transport mechanism of intestinal chloride secretion is well defined. Chloride is taken up across the basolateral membrane via a Na /K /2Cl cotransporter (NKCC1) and exits across the apical membrane via cAMP-sensitive (CFTR) and calcium-sensitive (CaCC) chloride channels. Basolateral potassium channels support chloride secretion by allowing for potassium recycling, whereas the energy for the process is supplied by the activity of a basolateral Na -K ATPase. An increase in chloride secretion is usually mediated by elevated intracellular levels of second messengers, such as cAMP and calcium. Secretory responses to these two second messengers are distinct in that responses to cAMP agonists are sustained, whereas those to Ca -mediated agonists are transient. cAMP was found to be a primary second messenger involved in the pathogenesis of diarrhea in cholera, whereas calcium was thought to mediate enhanced intestinal chloride secretion in diarrhea caused by rotavirus infection and diarrhea that is associated with the use of aspartyl protease inhibitors, an antiviral drug for human immunodeficiency virus-infected patients. Cholera is life-threatening secretory diarrhea resulting from intestinal infection with gram negative bacteria Vibrio cholera. Cholera toxin, an enterotoxin produced by Vibrio cholera, acts on enterocytes to increase intracellular cAMP, which in turn leads to massive CFTR-dependent intestinal chloride and, secondarily, fluid secretion. The volume of secreted fluid far exceeds the intestinal absorptive capacity, resulting in severe intestinal fluid loss and associated morbidity and mortality in affected individuals. Therefore, there are considerable efforts focused on the developing of antisecretory agents for patients suffering from cholera. On the basis of both in vitro and in vivo studies, one of the proposed targets to relief the symptom is an inhibition of CFTR, a cAMP-activated chloride channel that provides the apical route for intestinal chloride secretion. At present, two classes of potent CFTR inhibitors (thiazolidinone and glycine hydrazide) have been identified by highthroughput screening. Both are effective in reducing cholera toxin-induced intestinal fluid secretion in mice. The lead thiazolidinone compound, CFTRinh-172, reversibly inhibited CFTR with Ki 0.3 mM, presumably by binding to the CFTR’s nucleotide binding domain 1. It has no effect on other chloride channels and multidrug resistance transporter proteins, but also has poor water solubility ( 20 mM). In contrast, the lead glycine hydrazide compound, GlyH-101, has much greater water solubility ( 1 mM) and CFTR inhibition with Ki of 5 mM. However, its inhibitory effect on calcium-activated chloride channels was also observed at a concentration (50 mM) that completely inhibits CFTR activity. Recently, highly polar, water soluble and cell-impermeable glycine hydrazide-based CFTR inhibitors were synthesized and shown to inhibit CFTR activity with Ki 2—5 mM. In spite of the above mentioned favorable properties of current lead compounds, efforts to develop them as antidiarrheal drugs may be hampered by such drawbacks as poor water solubility and potential off-target effects. Therefore, this study was aimed at identifying new classes of CFTR inhibitors with equal or better properties as additional compounds of choice. We performed high-throughput screening of 50,000 chemically diverse drug-like small molecules and identified two new classes of CFTR inhibitors. They were tested for their inhibitory potency, reversibility and speci502 Vol. 30, No. 3