Nanomolar-potency \u2018co-potentiator\u2019 therapy for cystic fibrosis caused by a defined subset of minimal function CFTR mutants

Puay-Wah Phuan,Amber A Rivera,Peter M Haggie,Lorna Zlock,Alan S Verkman,Joseph-Anthony Tan,Dennis W Nielson,Walter E Finkbeiner

doi:10.1038/s41598-019-54158-2

Abstract

Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K. We previously introduced the concept of ‘co-potentiators’ (combination-potentiators) to rescue CFTR function in some minimal function CFTR mutants. Herein, a screen of ~120,000 drug-like synthetic small molecules identified active co-potentiators of pyrazoloquinoline, piperidine-pyridoindole, tetrahydroquinoline and phenylazepine classes, with EC50 down to ~300 nM following initial structure-activity studies. Increased CFTR chloride conductance by up to 8-fold was observed when a co-potentiator (termed ‘Class II potentiator’) was used with a classical potentiator (‘Class I potentiator’) such as VX-770 or GLPG1837. To investigate the range of CFTR mutations benefitted by co-potentiators, 14 CF-associated CFTR mutations were studied in transfected cell models. Co-potentiator efficacy was found for CFTR missense, deletion and nonsense mutations in nucleotide binding domain-2 (NBD2), including W1282X, N1303K, c.3700A > G and Q1313X (with corrector for some mutations). In contrast, CFTR mutations G85E, R334W, R347P, V520F, R560T, A561E, M1101K and R1162X showed no co-potentiator activity, even with corrector. Co-potentiator efficacy was confirmed in primary human bronchial epithelial cell cultures generated from a N1303K homozygous CF subject. The Class II potentiators identified here may have clinical benefit for CF caused by mutations in the NBD2 domain of CFTR.

Highlights

Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many lossof-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K
ASP-11 (Fig. 1A) was found previously to increase chloride current by ~8-fold over that produced by a high concentration of VX-770 in transfected Fischer rat thyroid (FRT) cells expressing N1303K-CFTR or CFTR128110,11
The nine missense mutants studied are located throughout the CFTR protein and included mutations in MSD1 (G85E, R334W, R347P), NBD1 (S492F, V520F, R560T, A561E) and MSD2 (L1077P, M1101K)

Summary

Introduction

Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many lossof-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K. Non-responsive minimal function CFTR mutations are distributed throughout the CFTR protein and are associated with low CFTR function due to defective channel processing, cell-surface trafficking, and/or channel gating[9] One such CFTR mutation is N1303K, a missense point mutation located in nucleotide binding domain 2 (NBD2), which is the 5th most common CFTR mutation worldwide accounting for ~2.5% of CFTR mutations (www.cftr2.org). This combination-potentiator (co-potentiator) approach was effective as well in increasing the chloride channel function of G551D-CFTR, with ~50% improvement compared with VX-770 alone[11] Building on these initial results, here we explored the potential utility of co-potentiators for additional minimal function CFTR mutants and identified, by high-throughput screening, novel co-potentiator scaffolds with nanomolar potency. We provide evidence to support a new classification system (Class I vs. Class II) for potentiators, which predicts synergy in CFTR activation

Methods

Results

Conclusion