CFTR activators

The mechanism of action proposed is based on a direct binding to the channel and the following partial block of the ATP-binding pocket of CFTR (French et al., 1997), a mechanism similar to that used by genistein to inhibit the activity of other ATP-utilizing enzymes such as protein kinases and topoisomerase II (Polkowski and Mazurek, 2000 and refs therein). The selection of flavonoid compounds or the development of synthetic drugs reasonably selective for CFTR activation might be an area for future clinical trials. 

Caci E, Folli C, Zegarra-Moran O, Ma T, Springsteel MF, Sammelson RE, Nantz MH, Kurth MJ, Yerkman AS, Galietta LJ. 2003. CFTR activation in human bronchial epithelial cells by novel benzoflavone and benzimidazolone compounds. Am J Physiol... 

CF-subjects with mild or severe lung disease and CBAVD subjects with one or two CFTR mutations (Fig. 6). To measure mutant CFTR function in vivo, nasal potential difference (NPD) recording techniques are used. This in vivo electrophysiology assay measures ion transport across the nasal membrane of human subjects. CFTR activity is isolated by addition of Cl" free media and /J-adrcncrgic agonists to increase cAMP signaling. The non-CF subjects respond with a robust increase in wild-type CFTR activity. In contrast, the response in subjects with no, mild and severe lung disease was 68 9% (44-88%), 9 6% (0-33%), and <1% of that observed for wild-type CFTR, respectively (Fig. 6 Table 4). 

Another approach to determine the amount of CFTR activity that could be beneficial is to measure the amount of wild-type CFTR mRNA in patients with missense mutations and mutations in introns that regulate mRNA splicing (Table 5) [87-93]. In these patients, a small amount of wild-type CFTR mRNA is produced. Patients with 4-10% wild-type CFTR mRNA typically exhibit a more moderate decline in lung function, whereas patients with >30% wild-type CFTR mRNA have mild to no lung disease. 

Taken together, these studies suggest that a pharmacological agent producing 10% wild-type CFTR activity may decrease the rate of lung function decline in severe patients to levels observed in patients with a more moderate decline in lung function, which may result in decreased morbidity and mortality. 

To monitor CFTR activity in HBE cultures, Ussing chamber recording techniques are used (Fig. 7). Like the nasal potential difference recording technique used in vivo, the Ussing chamber technique is an extracellular recording of the membrane potential or short circuit current (Isc) due to ion flux through channels and transporters. To isolate CFTR activity, a basolateral to apical Cl" gradient is established and amiloride is added to block the ep-... 

In non-CF HBE, forskolin stimulated a large, biphasic increase in Cl" secretion, whereas the response in AF508-HBE was monophasic and <5% of the wild-type response. The forskolin response in non-CF and AF508-HBE is blocked by CFTR inhibitors but not the Ca2+-activated Cl" channel blocker, DIDS, indicating that the response is due to CFTR activity in the apical membrane. Residual AF508-CFTR activity in the apical membrane of CF airway... 

Fig. 6 Disease severity in CF patients is determined by the amount of mutant CFTR activity. A In vivo data were pooled from multiple published studies using NPD techniques to measure CFTR-mediated Cl- secretion in subjects with pancreatic insufficiency (PI), pancreatic sufficiency (PS), or congenital bilateral absence of the vas deferens (CBAVD) and obligate heterozygotes (carriers). CFTR-mediated Cl- secretion in each study was normalized to the non-CF control subject and expressed as % wild-type (wt)-CFTR. B The severity of lung disease in each group was determined from the longitudinal decline in FEVi and classified as severe (red 3%/year), mild (yellow l%/year), or no (green) lung disease...

Table 5 Severity of lung disease and predicted CFTR activity in different patient genotypes ...

Fig. 7 Rescue of AF508-CFTR in HBE isolated from AF508-homozygous CF patients. A Representative short circuit current in AF508-HBE pre-treated for 48-hours with DMSO- (blue line) or 6.7 pM VRT-325 (red line). B Dose response to VRT-325 in FSK-stimulated AF508-HBE in the presence (filled circles) and absence (open circles) of the CFTR potentiator, VRT-532 (n = 5). C Maximum response to 10 iM FSK in AF508-HBE pre-treated with 1500 iM 4-PBA-, 6.7 iM VRT-422- (compoimd 1), 6.7 iM VRT-325-(compound 2), or 1 pM Corr-4a (compound 4), as well as 27°C-treated AF508-HBE. The peak response to forskolin was normalized to that in wild-type-HBE isolated from non-CF subjects (% wild-type CFTR). The dashed line indicates the level of residual (untreated) AF508-CFTR activity in AF508-HBE. D Cumulative data for uncorrected and VRT-325-corrected AF508-HBE with and without addition of the CFTR potentiator, VRT-532. Single asterisk = p<0.05 compared to un-treated controls double asterisk = p<0.01 compared to un-treated controls...

Bulteau L, Derand R, Mettey Y et al (2000) Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells. Am J Physiol Cell Physiol 279(6) C1925-C1937... 

Two major but conflicting hypotheses have been proposed to explain the connection between abnormal CFTR activity in cystic fibrosis and the chronic neutrophil-dominated pulmonary inflammation and colonization with common bacteria. These hypotheses have been referred to as the high-salt hypothesis proposed by Michael Welsh and his colleagues at the University of Iowa, and the reduced pericellular volume hypothesis proposed by Richard Boucher and colleagues at the University of North Carolina. 

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