Original Investigation |

Comparison of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Ciliary Beat Frequency Activation by the CFTR Modulators Genistein, VRT-532, and UCCF-152 in Primary Sinonasal Epithelial Cultures

Bryant T. Conger, Jr, MD1; Shaoyan Zhang, PhD1,2; Daniel Skinner, BS1,2; Stephen B. Hicks, BS1; Eric J. Sorscher, MD2,3; Steven M. Rowe, MD, MSPH2,3; Bradford A. Woodworth, MD1,2
[+] Author Affiliations
1Division of Otolaryngology, Department of Surgery, University of Alabama at Birmingham, Birmingham
2Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham
3Department of Medicine, University of Alabama at Birmingham, Birmingham
JAMA Otolaryngol Head Neck Surg. 2013;139(8):822-827. doi:10.1001/jamaoto.2013.3917.
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Importance  Pharmacologic activation of mucociliary clearance (MCC) represents an emerging therapeutic strategy for patients with chronic rhinosinusitis, even in the absence of congenital mutations of the CFTR gene. Drug discovery efforts have identified small molecules that activate the cystic fibrosis transmembrane conductance regulator (CFTR), including potentiators under development for treatment of cystic fibrosis.

Objective  To evaluate the properties of CFTR modulators and their effects on ciliary beat frequency (CBF) in human sinonasal epithelium (HSNE).

Design  Primary HSNE cultures (wild type and F508del/F508del) were used to compare stimulation of CFTR-mediated Cl conductance and CBF by the CFTR modulators genistein, VRT-532, and UCCF-152.

Main Outcomes and Measures  Increase in CFTR-dependent anion transport and CBF.

Results  HSNE cultures were analyzed using pharmacologic manipulation of ion transport (change in short-circuit current [∆ISC]) and high-speed digital imaging (CBF). Activation of CFTR-dependent anion transport was significantly different among agonists (P < .001), with genistein exerting the greatest effect (mean [SD] ∆ISC, genistein, 23.1 [1.8] μA/cm2 > VRT-532, 8.1 [1.0] μA/cm2 > UCCF-152, 3.4 [1.4] μA/cm2 > control, 0.7 [0.2] μA/cm2; Tukey-Kramer P < .05) in the absence of forskolin. Genistein and UCCF-152 augmented CBF (under submerged conditions) significantly better (Tukey-Kramer P < .05) than cells treated with VRT-532 or dimethyl sulfoxide vehicle control (mean [SD] fold change over baseline, genistein, 1.63 [0.06]; UCCF-152, 1.56 [0.06]; VRT-532, 1.38 [0.08]; control, 1.27 [0.02]). Activation of CBF was blunted in F508del/F508del HSNE cultures.

Conclusions and Relevance  The degree of CBF stimulation was not dependent on the magnitude of Cl secretion, suggesting that different mechanisms of action may underlie MCC activation by these small molecule potentiators. Agents that activate both CFTR-dependent ISC and CBF are particularly attractive as therapeutics because they may address 2 independent pathways that contribute to deficient MCC in chronic rhinosinusitis.

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Figure 1.
Chemical Structure of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulators

Genistein, VRT-532, and UCCF-152 are organic, small molecules (<800 Da) that alter the activity of the CFTR channel protein by either direct effects (eg, potentiation—increasing channel open time) or activation of cell signaling pathways (eg, adenylyl cyclase and intracellular Ca2+).

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Figure 2.
Representative Ussing Chamber Tracings of Modulator Cl Transport Activation in Human Sinonasal Epithelium (HSNE)

Wild-type HSNE cells grown on Transwell permeable supports were mounted in modified Ussing chambers under short-circuit conditions and sequentially exposed to amiloride (100µM), modulators, forskolin (100nM), and CFTRINH-172 (10µM). By convention, a positive deflection in the tracing (ΔISC) represents movement of an anion (ie, Cl) in the serosal to mucosal direction. DMSO, dimethyl sulfoxide.

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Figure 3.
Change in Short-Circuit Current (∆ISC) by Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulators in Human Sinonasal Epithelium

Genistein was a more robust activator of CFTR-mediated anion transport in the absence of forskolin (A) than the potentiators VRT-532 and UCCF-152 (Tukey-Kramer P < .05). Cl transport (measured by short-circuit current [∆ISC]) was significantly greater for each modulator compared with dimethyl sulfoxide vehicle control (Tukey-Kramer P < .05). However, total activation of CFTR (plus forskolin 100nM) was similar between drugs (B). All agonists significantly increased total CFTR-mediated transport compared with forskolin alone (Tukey-Kramer P < .05).

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Figure 4.
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulator Effects on Ciliary Beat Frequency (CBF)

Potentiators were administered apically to wild-type (A) or F508del/F508del (B) human sinonasal epithelium for 15 minutes. Genistein and UCCF-152 increased CBF compared with baseline, and CBF was significantly greater than in cells treated with dimethyl sulfoxide vehicle control (Tukey-Kramer P < .05) in wild-type HSNE. Ciliary beat frequency was not significantly increased with VRT-532 despite significantly greater stimulation of Cl secretion when compared with UCCF-152. Activation of CBF was diminished in cells that lack functional CFTR, but genistein significantly increased CBF compared with controls (Tukey-Kramer P < .05).

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