CFTR Channel Pharmacology: Novel Pore Blockers Identified by High-throughput Screening

Investigators of anion channels are frequently heard bemoaning the absence of potent, specific inhibitors of their favorite channel. The lack of such blockers has been particularly frustrating for researchers investigating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel, which plays a central role in electrolyte transport across epithelial tissues (Welsh et al., 2001). Perhaps the complaints of CFTR researchers might soon be a thing of the past following the discovery of glycine hydrazides by Chatchai Muanprasat and colleagues, which is reported in this issue of the Journal of General Physiology (Muanprasat et al., 2004). In brief, the authors employ a high-throughput screening (HTS) assay for the identification of CFTR inhibitors to discover glycine hydrazides and then investigate the effects of these agents on CFTR function in experiments that range from single channels to animal models (Muanprasat et al., 2004). In the process, Muanprasat and colleagues find that glycine hydrazides have a novel mechanism of action: occlusion of the extracellular end of the CFTR pore. All in all, the work is a tour de force of CFTR pharmacology. The pharmacology of the CFTR Cl channel has attracted significant interest in recent years. Much of this attention has been fuelled by the search for rational new therapies for diseases caused by CFTR malfunction. Mutations that, in general, abolish the function of CFTR cause the life-threatening genetic disease cystic fibrosis (CF; Welsh et al., 2001). By contrast, some forms of male infertility, disseminated bronchiectasis and chronic pancreatitis are caused by CFTR mutations that are likely to preserve partial CFTR function (Welsh et al., 2001). Other diseases, such as secretory diarrhea and polycystic kidney disease involve unphysiologic activation of the CFTR Cl channel (Sullivan et al., 1998; Al-Awqati, 2002). Besides treatments for disease and perhaps even male contraception, CFTR inhibitors are important in several other respects: (a) as probes to identify CFTR-dependent function and to investigate CFTR structure and function, (b) to study the pathogenesis of lung disease in CF, and (c) to develop animal models with which to evaluate new therapies for CF. A wide spectrum of diverse small molecules, which inhibit the CFTR Cl channel, has been identified. Analysis of these molecules reveals several common themes: they are anions, most are lipophilic, and many are large in size. Moreover they inhibit CFTR by two general mechanisms: open-channel and allosteric block (Cai et al., 2002). A variety of large organic anions (e.g., glibenclamide) are open-channel blockers of the CFTR Cl channel. These agents inhibit CFTR by binding within the deep wide vestibule at the intracellular end of the CFTR pore and preventing Cl flow by occluding the permeation pathway (Fig. 1, A and B; Cai et al., 2002). By contrast, elevated concentrations of several agents that potentiate CFTR Cl currents (e.g., genistein) inhibit CFTR by an allosteric mechanism. These agents inhibit CFTR by interacting with the nucleotide-binding domains (NBDs), which control channel gating, and slowing greatly the rate of channel opening (Fig. 1, A and C; Cai et al., 2002). Because the characteristics of CFTR inhibition by open-channel and allosteric blockers differ, these agents can be distinguished by the effects of (a) voltage, (b) external Cl concentration, and (c) the inorganic phosphate analogue pyrophosphate (PP i ) that disrupts ATP-dependent gating. Inhibition of CFTR by open-channel blockers is voltage dependent and enhanced when the external Cl concentration is reduced, but unaffected by PP i (Cai et al., 2002). In contrast, inhibition of CFTR by allosteric blockers is voltage independent and unaffected by reducing the external Cl concentration, but relieved by PP i and elevated concentrations of ATP (Cai et al., 2002). Despite the plethora of CFTR inhibitors identified using conventional assays of CFTR function, potency and specificity have remained intractable problems. Few agents have been identified that inhibit CFTR with nanomolar affinity. Worse, no specific blockers of the CFTR Cl channel have been identified. Open-channel