The dual phosphodiesterase 3/4 inhibitor RPL554 stimulates rare class III and IV CFTR mutants

Abstract

Over 2,000 mutations have been reported in the cystic fibrosis transmembrane conductance regulator ( cftr) gene, many of which cause disease but are rare and have no effective treatment. Thus, there is an unmet need for new, mutation-agnostic therapies for cystic fibrosis (CF). Phosphodiesterase (PDE) inhibitors are one such class of therapeutics that have been shown to elevate intracellular cAMP levels and stimulate CFTR-dependent anion secretion in human airway epithelia; however, the number of people with CF that could be helped by PDE inhibitors remains to be determined. Here we used Fisher rat thyroid (FRT) cells stably transduced with rare human CFTR mutants and studied their responsiveness to the dual phosphodiesterase 3/4 inhibitor RPL554 (Verona Pharma). Through its inhibitory effect on PDE4D, we find that RPL554 can elevate intracellular cAMP leading to a potentiation of forskolin-stimulated current mediated by R334W, T338I, G551D, and S549R mutants of CFTR when used alone or in combination with CFTR modulators. We also were able to reproduce these effects of RPL554 on G551D-CFTR when it was expressed in primary human bronchial epithelial cells, indicating that RPL554 would have stimulatory effects on rare CFTR mutants in human airways and validating FRT cells as a model for PDE inhibitor studies. Furthermore, we provide biochemical evidence that VX-809 causes surprisingly robust correction of several class III and IV CFTR mutants. Together, our findings further support the therapeutic potential of RPL554 for patients with CF with class III/IV mutations and emphasize the potential of PDEs as potential drug targets that could benefit patients with CF.

Whole-Exome Sequencing Identified CFTR Variants in Two Consanguineous Families in China

“…New research showed that phosphodiesterase (PDE) inhibitors RPL554 (Verona Pharma) can elevate intracellular cAMP in R334W Fisher rat thyroid (FRT) cells. This finding supports the therapeutic potential of RPL554 for CF patients with class III/IV variants including R334W (Turner et al, 2020). In addition, gene and molecular therapy for some mutations is also a research hot spot.…”

The Phosphodiesterase Inhibitor Ensifentrine Reduces Production of Proinflammatory Mediators in Well Differentiated Bronchial Epithelial Cells by Inhibiting PDE4

“…For measurements of short circuit current, cells were cultured at the air-liquid interface in ALI medium for 7 days before being washed 3 x with PBS and incubated for 48 h in M-BEGM. I sc measurements were then performed as previously described (Turner et al, 2020).…”

“…Recently, we demonstrated that the PDE inhibitor ensifentrine (RPL554; Verona Pharma) stimulates both wild-type CFTR and the disease causing mutant R117H-CFTR when endogenously expressed in well-differentiated primary human bronchial epithelial cells (Turner et al, 2016). We further observed that ensifentrine activates the CFTR mutants R334W-, T338I-, S549R-and G551D-CFTR when expressed in the Fisher rat thyroid (FRT) cell model (Turner et al, 2020). These data indicate that activation of cAMP/PKA signalling by ensifentrine has therapeutic potential for CFTR mutants with residual trafficking of CFTR to the plasma membrane.…”

Characterization of the Mechanism of Action of RDR01752, a Novel Corrector of F508del-CFTR

“…CFBE cells stably expressing mCherry-Flag-CFTR (WT, F508del, DD/AA variants or carrying G550E, R1070W, 4RK in cis with F508del) were cultured and CFTR expression was induced with doxycycline (Dox) 1 μg/mL as described (Botelho et al, 2015). Fischer rat thyroid (FRT) epithelial cells stably expressing CFTR variants (wt, G85E, R334W, T338I, R347P, F508del, V520F, S549F, G551D, M1101K, N1303K) were cultured as before (Turner et al, 2020). All cell lines were maintained in a humidified incubator at 5% CO 2 and 37°C, except during low temperature experiments, in which cells were incubated at 27°C for 24h.…”

“…Changes in transepithelial voltage (V te ) were continuously recorded and equivalent Fsk/IBMX-stimulated short-circuit currents (I eq-sc ) were calculated by Ohm's law from V te and R te (I eq-sc =V te /R te ). For FRT cell lines, transepithelial voltage was measured at 37°C with continuous stirring by gassing with 95% O 2 and 5% CO 2 as described (Turner et al, 2020).…”