Before small-molecule screening, the available inhibitors of CFTR Cl− conductance included diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropyl-amino)benzoate, and glibenclamide (), all of which are nonspecific in their action and have low potency. High-throughput screening of small-molecule collections has yielded several new chemical classes of CFTR inhibitors. Screening has been carried out in epithelial cells expressing human CFTR and a genetically encoded I−-sensing fluorescent protein, in which cAMP-induced CFTR activity is deduced from the kinetics of intracellular fluorescence following the extracellular addition of I−.2
Figure 2 Chemical structures of small-molecule CFTR inhibitors. Top: original (pre-high–throughput screening) CFTR inhibitors. Center: absorbable CFTR inhibitors acting at the CFTR cytoplasmic surface. Bottom: externally acting hydrazide-class CFTR inhibitors. (more ...)
The original class of CFTR inhibitors includes the thiazolidinone CFTRinh
-172 (), which has been widely used in cystic fibrosis research to investigate the involvement of CFTR in various cellular processes. Patch-clamp and mutagenesis studies indicate that CFTRinh
-172 stabilizes the CFTR channel closed-state by binding at or near arginine-347 on the CFTR cytoplasmic surface. The IC50
for inhibition of CFTR Cl−
current by CFTRinh
-172 is ~300 nmol/l, although this value rises to several μmol/l in epithelial cells because of their strongly negative interior membrane potential. CFTRinh
-172 has low toxicity and is excreted mainly by the kidney with minimal metabolism. CFTRinh
-172 appears to accumulate in the intestine by enterohepatic recirculation, as shown by the high concentrations in bile. Studies in mouse models of cholera and heat-stable enterotoxin–induced intestinal fluid secretion have demonstrated the efficacy of CFTRinh
Structure–activity studies have identified thiazolidinone CFTR inhibitors with greater water solubility as compared with CFTRinh
-172. These include an analog containing a 4-tetrazolophenyl in place of the 4-carboxyphenyl in CFTRinh
-172. The tetrazolo analog reduced kidney cyst progression in mouse models of polycystic kidney disease, in which fluid secretion into cysts is CFTR-dependent.
Additional screening identified a second class of absorbable CFTR inhibitors—the PPQ/BPO inhibitors—that have a cytoplasmic site of action (). PPQ-102 inhibits CFTR Cl−
conductance, with an IC50
of ~ 90 nmol/l. Structure–activity analysis and follow-on medicinal chemistry efforts yielded the improved compound BPO-27, with structural changes that greatly increase its metabolic stability, inhibitory potency, and aqueous solubility.4
for CFTR inhibition by (racemic) BPO-27 is ~8 nmol/l. We recently separated and determined the crystal structure of BPO-27 enantiomers; one enantiomer had an IC50
of ~ 4 nmol/l and the other was inactive. PPQ-102 and BPO-27 have shown efficacy in models of polycystic kidney disease but have not yet been tested in models of diarrhea.
The hydrazides () are an interesting class of CFTR inhibitors. They were discovered using a screen biased to identify compounds that act at the external surface of CFTR. Patch-clamp analysis of glycine hydrazide GlyH-101 showed an altered CFTR current–voltage relationship that changed from linear to inwardly rectifying, as well as rapid single-channel flicker. These findings, together with additional biophysical data, suggested an external pore-blocking inhibition mechanism. The lumen-facing site of action of the hydrazides provided an interesting opportunity to develop nonabsorbable compounds for antisecretory therapy. An inhibitor that is in early-phase clinical trials is iOWH032, an analog of GlyH-101(). It has been shown to inhibit CFTR with an IC50 of ~ 8 μmol/l. However, several concerns raise doubts about the utility of small-molecule hydrazide inhibitors. These include (i) predicted rapid washout (by convection) of an externally bound inhibitor, (ii) multiple off-target effects of hydrazides, and (iii) poor inhibitory potency of hydrazides at interior-negative membrane potentials. The first concern is particularly critical; we have computed that, because of fluid convection across the luminal surface during secretion, the intestinal concentration of an externally acting inhibitor needs to be >100-fold its IC50 value to maintain inhibitory efficacy.
In attempting to overcome these concerns, we discovered from structure–activity studies that substitutions on the glycyl methylene group of the glycine hydrazide scaffold were tolerated, allowing the synthesis of nonabsorbable polyethylene glycol conjugates containing a malonic acid hydrazide (MalH) CFTR-inhibiting moiety. These macromolecular conjugates fully inhibited CFTR when added at the extracellular surfaces of cells. Various MalH–macromolecular conjugates were synthesized, including a MalH–lectin conjugate (), the goal being to improve CFTR inhibitory potency and resist dissociation from the luminal surface of CFTR. MalH–lectin conjugates were generated with IC50
values lowered to 50 nmol/l for inhibition of CFTR Cl−
conductance; these remained bound to CFTR for many hours, as compared with only a few seconds in the case of small-molecule hydrazides.5
The improved potency of the MalH–lectin conjugate and its resistance to washout is probably due to trapping in the enterocyte glycocalyx. Despite the advantages they possess as compared with the hydrazides, MalH–lectins are probably not practical development candidates for antisecretory therapy in developing countries because, as protein-containing conjugates, they are relatively costly to synthesize and may have limited stability during storage.
New pharmacologic therapies aimed at reducing the volume and duration of diarrheal episodes should prove valuable in reducing the mortality and morbidity associated with infectious diarrheas. The burden of diarrheal disease lies predominantly in the developing world, and therefore new antidiarrheal drugs should be very inexpensive to manufacture and distribute. To be widely available and not cost-prohibitive at the point of care, the development of antisecretory drugs will probably require the support of international health organizations and charities, as well as of governments and the pharmaceutical industry. CFTR is an attractive target for the development of drugs aimed at reducing secretion, although inhibition of CaCC may also be required in treating some diarrheas. At present, because of concerns about the potentially low in vivo efficacy of the externally acting hydrazides due to convective washout, the absorbable CFTR inhibitors of the thiazolidinone and PPQ/BPO classes are the most promising candidates for antisecretory therapy.