Discovery of a selective, state-independent inhibitor of NaV1.7 by modification of guanidinium toxins.

The voltage-gated sodium channel isoform NaV1.7 is highly expressed in dorsal root ganglion neurons and is obligatory for nociceptive signal transmission. Genetic gain-of-function and loss-of-function NaV1.7 mutations have been identified in select individuals, and are associated with episodic extreme pain disorders and insensitivity to pain, respectively. These findings implicate NaV1.7 as a key pharmacotherapeutic target for the treatment of pain. While several small molecules targeting NaV1.7 have been advanced to clinical development, no NaV1.7-selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a NaV1.7 inhibitor that blocks the extracellular vestibule of the channel with an IC50 of 72 nM and greater than 200-fold selectivity over off-target sodium channel isoforms, NaV1.1-1.6 and NaV1.8. In contrast to other NaV1.7 inhibitors that preferentially inhibit the inactivated state of the channel, ST-2262 is equipotent in a protocol that favors the resting state of the channel, a protocol that favors the inactivated state, and a high frequency protocol. In a non-human primate study, animals treated with ST-2262 exhibited reduced sensitivity to noxious heat. These findings establish the extracellular vestibule of the sodium channel as a viable receptor site for the design of selective ligands targeting NaV1.7.

Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion.

The guanosine triphosphatase Rab1 regulates the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus through interaction with effector molecules, but the molecular mechanisms by which this occurs are unknown. Here, the tethering factor p115 was shown to be a Rab1 effector that binds directly to activated Rab1. Rab1 recruited p115 to coat protein complex II (COPII) vesicles during budding from the endoplasmic reticulum, where it interacted with a select set of COPII vesicle-associated SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) to form a cis-SNARE complex that promotes targeting to the Golgi apparatus. We propose that Rab1-regulated assembly of functional effector-SNARE complexes defines a conserved molecular mechanism to coordinate recognition between subcellular compartments.

A PDZ-interacting domain in CFTR is an apical membrane polarization signal.

Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain-containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain-containing proteins play a key role in the apical polarization of ion channels in epithelial cells.

Discovery and hit-to-lead evaluation of piperazine amides as selective, state-dependent NaV1.7 inhibitors.

NaV1.7 is a particularly compelling target for the treatment of pain. Herein, we report the discovery and evaluation of a series of piperazine amides that exhibit state-dependent inhibition of NaV1.7. After demonstrating significant pharmacodynamic activity with early lead compound 14 in a NaV1.7-dependent behavioural mouse model, we systematically established SAR trends throughout each sector of the scaffold. The information gleaned from this modular analysis was then applied additively to quickly access analogues that encompass an optimal balance of properties, including NaV1.7 potency, selectivity over NaV1.5, aqueous solubility, and microsomal stability.

A new frontier in pharmacology: the endoplasmic reticulum as a regulated export pathway in health and disease.

The endoplasmic reticulum (ER), the first secretory compartment of eukaryotic cells, co-ordinates the biogenesis and export of all membrane-bound and soluble cargo molecules to the cell surface. ER function is now recognised to have unprecedented links with signalling pathways regulating cell growth and differentiation and host physiology. Misfolding and aggregation of newly synthesised proteins in the ER or alterations in ER processing of cargo mediated by pathogens is responsible for a broad range of diseases including cystic fibrosis, emphysema and neuropathies such as Alzheimer's disease. The central, integrative role of the ER in determining cell physiology in health and disease represents an untapped area for pharmacological intervention. This review focuses on the potential use of pharmacological agents to modulate cargo selection, folding and degradation in the ER with the goal of alleviating ER export disease. In addition, implementation of novel technologies that utilise normal ER function to store and release biologically active substances of therapeutic relevance are presented as a new frontier in drug delivery.

Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering.

Members of the Rab family of small molecular weight GTPases regulate the fusion of transport intermediates to target membranes along the biosynthetic and endocytic pathways. We recently demonstrated that Rab1 recruitment of the tethering factor p115 into a cis-SNARE complex programs coat protein II vesicles budding from the endoplasmic reticulum (donor compartment) for fusion with the Golgi apparatus (acceptor compartment) (Allan BB, Moyer BD, Balch WE. Science 2000; 289: 444-448). However, the molecular mechanism(s) of Rab regulation of Golgi acceptor compartment function in endoplasmic reticulum to Golgi transport are unknown. Here, we demonstrate that the cis-Golgi tethering protein GM130, complexed with GRASP65 and other proteins, forms a novel Rab1 effector complex that interacts with activated Rab1-GTP in a p115-independent manner and is required for coat protein II vesicle targeting/fusion with the cis-Golgi. We propose a 'homing hypothesis' in which the same Rab interacts with distinct tethering factors at donor and acceptor membranes to program heterotypic membrane fusion events between transport intermediates and their target compartments.

Exocytosis is not involved in activation of Cl- secretion via CFTR in Calu-3 airway epithelial cells.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, which mediates transepithelial Cl- transport in a variety of epithelia, including airway, intestine, pancreas, and sweat duct. In some but not all epithelial cells, cAMP stimulates Cl- secretion in part by increasing the number of CFTR Cl- channels in the apical plasma membrane. Because the mechanism whereby cAMP stimulates CFTR Cl- secretion is cell-type specific, our goal was to determine whether cAMP elevates CFTR-mediated Cl- secretion across serous airway epithelial cells by stimulating the insertion of CFTR Cl- channels from an intracellular pool into the apical plasma membrane. To this end we studied Calu-3 cells, a human airway cell line with a serous cell phenotype. Serous cells in human airways, such as Calu-3 cells, express high levels of CFTR, secrete antibiotic-rich fluid, and play a critical role in airway function. Moreover, dysregulation of CFTR-mediated Cl- secretion in serous cells is thought to contribute to the pathophysiology of cystic fibrosis lung disease. We report that cAMP activation of CFTR-mediated Cl- secretion across human serous cells involves stimulation of CFTR channels present in the apical plasma membrane and does not involve the recruitment of CFTR from an intracellular pool to the apical plasma membrane.

Adenosine inhibits arginine vasopressin-stimulated chloride secretion in a mouse IMCD cell line (mIMCD-K2).

Previously, we demonstrated that a mouse inner medullary collecting duct cell line (mIMCD-K2) secretes Cl- by an electrogenic mechanism via cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995; D. Vandorpe, N. Kizer, F. Ciampolillo-Bates, B. Moyer, K. Karlson, W. B. Guggino, and B. A. Stanton. Am. J. Physiol. 269 (Cell Physiol. 38): C683-C689, 1995]. The objective of the present study was to determine whether adenosine, and adenosine A1 receptors (A1AR) specifically, regulate electrogenic Cl- secretion (IscCl) in mIMCD-K2 cells. Neither N6-cyclohexyladenosine (CHA), a specific A1AR agonist, nor 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific A1AR antagonist, altered basal, unstimulated IscCl in monolayers of mIMCD-K2 cells mounted in Ussing-type chambers. In contrast, DPCPX increased arginine vasopressin (AVP)-stimulated IscCl, an effect that was reversed by CHA. Adenosine deaminase (ADA), which oxidatively deaminates adenosine to inosine, increased AVP-stimulated IscCl. CHA reversed the stimulatory effect of ADA on AVP-stimulated IscCl. These results suggest that adenosine, via A1AR, inhibits AVP-stimulated IscCl. To identify the source(s) of extracellular adenosine, we examined the effects of dipyridamole, an inhibitor of nucleoside transport, and alpha,beta-methyleneadenosine 5'-diphosphate (AOPCP), an inhibitor of ecto-5'-nucleotidase, on AVP-stimulated IscCl. Both compounds increased AVP-stimulated IscCl. CHA reversed the stimulatory effect of dipyridamole and AOPCP on IscCl. Neither ADA nor CHA had an effect on 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP)-stimulated IscCl. Moreover, U-73122, an inhibitor of phospholipase C, failed to attenuate the increase in AVP-stimulated IscCl elicited by dipyridamole and AOPCP or the decrease in AVP-stimulated IscCl elicited by CHA. We conclude that adenosine, released by a nucleoside transporter and formed extracellularly by the breakdown of AMP, binds to A1AR, and decreases AVP-stimulated IscCl in mIMCD-K2 cells by reducing intracellular cAMP levels.

PBA increases CFTR expression but at high doses inhibits Cl(-) secretion in Calu-3 airway epithelial cells.

Sodium 4-phenylbutyrate (PBA), a short-chain fatty acid, has been approved to treat patients with urea cycle enzyme deficiencies and is being evaluated in the management of sickle cell disease, thalassemia, cancer, and cystic fibrosis (CF). Because relatively little is known about the effects of PBA on the expression and function of the wild-type CF transmembrane conductance regulator (wt CFTR), the goal of this study was to examine the effects of PBA and related compounds on wt CFTR-mediated Cl(-) secretion. To this end, we studied Calu-3 cells, a human airway cell line that expresses endogenous wt CFTR and has a serous cell phenotype. We report that chronic treatment of Calu-3 cells with a high concentration (5 mM) of PBA, sodium butyrate, or sodium valproate but not of sodium acetate reduced basal and 8-(4-chlorophenylthio)-cAMP-stimulated Cl(-) secretion. Paradoxically, PBA enhanced CFTR protein expression 6- to 10-fold and increased the intensity of CFTR staining in the apical plasma membrane. PBA also increased protein expression of Na(+)-K(+)-ATPase. PBA reduced CFTR Cl(-) currents across the apical membrane but had no effect on Na(+)-K(+)-ATPase activity in the basolateral membrane. Thus a high concentration of PBA (5 mM) reduces Cl(-) secretion by inhibiting CFTR Cl(-) currents across the apical membrane. In contrast, lower therapeutic concentrations of PBA (0.05-2 mM) had no effect on cAMP-stimulated Cl(-) secretion across Calu-3 cells. We conclude that PBA concentrations in the therapeutic range are unlikely to have a negative effect on Cl(-) secretion. However, concentrations >5 mM might reduce transepithelial Cl(-) secretion by serous cells in submucosal glands in individuals expressing wt CFTR.

Butyrate increases apical membrane CFTR but reduces chloride secretion in MDCK cells.

Sodium butyrate and its derivatives are useful therapeutic agents for the treatment of genetic diseases including urea cycle disorders, sickle cell disease, thalassemias, and possibly cystic fibrosis (CF). Butyrate partially restores cAMP-activated Cl(-) secretion in CF epithelial cells by stimulating DeltaF508 cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) gene expression and increasing the amount of DeltaF508-CFTR in the plasma membrane. Because the effect of butyrate on Cl(-) secretion by renal epithelial cells has not been reported, we examined the effects of chronic butyrate treatment (15-18 h) on the function, expression, and localization of CFTR fused to the green fluorescent protein (GFP-CFTR) in stably transfected MDCK cells. We report that sodium butyrate reduced Cl(-) secretion across MDCK cells, yet increased apical membrane GFP-CFTR expression 25-fold and increased apical membrane Cl(-) currents 30-fold. Although butyrate also increased Na-K-ATPase protein expression twofold, the drug reduced the activity of the Na-K-ATPase by 55%. Our findings suggest that butyrate inhibits cAMP-stimulated Cl(-) secretion across MDCK cells in part by reducing the activity of the Na-K-ATPase.

Functional and molecular characterization of an anion exchanger in airway serous epithelial cells.

Serous cells secrete Cl(-) and HCO(3)(-) and play an important role in airway function. Recent studies suggest that a Cl(-)/HCO(3)(-) anion exchanger (AE) may contribute to Cl(-) secretion by airway epithelial cells. However, the molecular identity, the cellular location, and the contribution of AEs to Cl(-) secretion in serous epithelial cells in tracheal submucosal glands are unknown. The goal of the present study was to determine the molecular identity, the cellular location, and the role of AEs in the function of serous epithelial cells. To this end, Calu-3 cells, a human airway cell line with a serous-cell phenotype, were studied by RT-PCR, immunoblot, and electrophysiological analysis to examine the role of AEs in Cl(-) secretion. In addition, the subcellular location of AE proteins was examined by immunofluorescence microscopy. Calu-3 cells expressed mRNA and protein for AE2 as determined by RT-PCR and Western blot analysis, respectively. Immunofluorescence microscopy identified AE2 in the basolateral membrane of Calu-3 cells in culture and rat tracheal serous cells in situ. In Cl(-)/HCO(3)(-)/Na(+)-containing media, the 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP)-stimulated short-circuit anion current (I(sc)) was reduced by basolateral but not by apical application of 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (50 microM) and 4, 4'-dinitrostilbene-2,2'-disulfonic acid [DNDS (500 microM)], inhibitors of AEs. In the absence of Na(+) in the bath solutions, to eliminate the contributions of the Na(+)/HCO(3)(-) and Na(+)/K(+)/2Cl(-) cotransporters to I(sc), CPT-cAMP stimulated a small DNDS-sensitive I(sc). Taken together with previous studies, these observations suggest that a small component of cAMP-stimulated I(sc) across serous cells may be referable to Cl(-) secretion and that uptake of Cl(-) across the basolateral membrane may be mediated by AE2.

Traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway.

The pathway of transport of the cystic fibrosis transmembrane regulator (CFTR) through the early exocytic pathway has not been examined. In contrast to most membrane proteins that are concentrated during export from the ER and therefore readily detectable at elevated levels in pre-Golgi intermediates and Golgi compartments, wild-type CFTR could not be detected in these compartments using deconvolution immunofluorescence microscopy. To determine the basis for this unusual feature, we analyzed CFTR localization using quantitative immunoelectron microscopy (IEM). We found that wild-type CFTR is present in pre-Golgi compartments and peripheral tubular elements associated with the cis and trans faces of the Golgi stack, albeit at a concentration 2-fold lower than that found in the endoplasmic reticulum (ER). delta F508 CFTR, a mutant form that is not efficiently delivered to the cell surface and the most common mutation in cystic fibrosis, could also be detected at a reduced concentration in pre-Golgi intermediates and peripheral cis Golgi elements, but not in post-Golgi compartments. Our results suggest that the low level of wild-type CFTR in the Golgi region reflects a limiting step in selective recruitment by the ER export machinery, an event that is largely deficient in delta F508. We raise the possibility that novel modes of selective anterograde and retrograde traffic between the ER and the Golgi may serve to regulate CFTR function in the early secretory compartments.

Differential effects of mitomycin C and doxorubicin on P-glycoprotein expression.

Previous studies have demonstrated that mitomycin C (MMC) and other DNA cross-linking agents can suppress MDR1 (multidrug resistance 1) gene expression and subsequent functional P-glycoprotein (Pgp) expression, whereas doxorubicin and other anthracyclines increase MDR1 gene expression. In the present study, with stably transfected Madin-Darby canine kidney C7 epithelial cells expressing a human Pgp tagged with green fluorescent protein under the proximal human MDR1 gene promoter, we demonstrated that MMC and doxorubicin have differential effects on Pgp expression and function. Doxorubicin caused a progressive increase in the cell-surface expression of Pgp and function. In contrast, MMC initially increased plasma membrane expression and function at a time when total cellular Pgp was constant and Pgp mRNA expression had been shown to be suppressed. This was followed by a rapid and sustained decrease in cell-surface expression at later times, presumably as a consequence of the initial decrease in mRNA expression. These studies imply that there are at least two independent chemosensitive steps that can alter Pgp biogenesis: one at the level of mRNA transcription and the other at the level of Pgp trafficking. Understanding the combined consequences of these two mechanisms might lead to novel chemotherapeutic approaches to overcoming drug resistance in human cancers by altering either Pgp mRNA expression or trafficking to the membrane.

A PDZ-binding motif is essential but not sufficient to localize the C terminus of CFTR to the apical membrane.

Localization of ion channels and transporters to the correct membrane of polarized epithelia is important for vectorial ion movement. Prior studies have shown that the cytoplasmic carboxyl terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the apical localization of this protein. Here we show that the C-terminal tail alone, or when fused to the green fluorescent protein (GFP), can localize to the apical plasma membrane, despite the absence of transmembrane domains. Co-expression of the C terminus with full-length CFTR results in redistribution of CFTR from apical to basolateral membranes, indicating that both proteins interact with the same target at the apical membrane. Amino acid substitution and deletion analysis confirms the importance of a PDZ-binding motif D-T-R-L> for apical localization. However, two other C-terminal regions, encompassing amino acids 1370-1394 and 1404-1425 of human CFTR, are also required for localizing to the apical plasma membrane. Based on these results, we propose a model of polarized distribution of CFTR, which includes a mechanism of selective retention of this protein in the apical plasma membrane and stresses the requirement for other C-terminal sequences in addition to a PDZ-binding motif.

Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells.

The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl-) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl- secretion by stimulating CFTR Cl- channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patch-clamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl- secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl- secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.

The PDZ-interacting domain of cystic fibrosis transmembrane conductance regulator is required for functional expression in the apical plasma membrane.

Polarization of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel to the apical plasma membrane in epithelial cells is critical for vectorial chloride transport. Previously, we reported that the C terminus of CFTR constitutes a PDZ-interacting domain that is required for CFTR polarization to the apical plasma membrane and interaction with the PDZ domain-containing protein EBP50 (NHERF). PDZ-interacting domains are typically composed of the C-terminal three to five amino acids, which in CFTR are QDTRL. Our goal was to identify the key amino acid(s) in the PDZ-interacting domain of CFTR with regard to its apical polarization, interaction with EBP50, and ability to mediate transepithelial chloride secretion. Point substitution of the C-terminal leucine (Leu at position 0) with alanine abrogated apical polarization of CFTR, interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane, and chloride secretion. Point substitution of the threonine (Thr at position -2) with alanine or valine had no effect on the apical polarization of CFTR, but reduced interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane as well as chloride secretion. By contrast, individual point substitution of the other C-terminal amino acids (Gln at position -4, Asp at position -3 and Arg at position -1) with alanine had no effect on measured parameters. We conclude that the PDZ-interacting domain, in particular the leucine (position 0) and threonine (position -2) residues, are required for the efficient, polarized expression of CFTR in the apical plasma membrane, interaction of CFTR with EBP50, and for the ability of CFTR to mediate chloride secretion. Mutations that delete the C terminus of CFTR may cause cystic fibrosis because CFTR is not polarized, complexed with EBP50, or efficiently expressed in the apical membrane of epithelial cells.