Identification, Structure-Activity Relationship, and Biological Characterization of 2,3,4,5-Tetrahydro-1H-pyrido[4,3-b]indoles as a Novel Class of CFTR Potentiators.

Nicoletta Brindani,Francesca Giacomina,Tiziano Bandiera,Loretta Ferrera,Ilaria Penna,Nicoletta Pedemonte,Debora Russo,Simone Giovani,Paolo Di Fruscia,Luca Goldoni,Giuliana Ottonello,Fabio Bertozzi,Ambra Gianotti,Sine Mandrup Bertozzi,Maria Summa,Federico Sorana,Rosalia Bertorelli,Emanuela Pesce,Elvira Sondo,Luis J V Galietta

doi:10.1021/acs.jmedchem.0c01050

Abstract

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease, caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR modulators have been reported to address the basic defects associated with CF-causing mutations, partially restoring the CFTR function in terms of protein processing and/or channel gating. Small-molecule compounds, called potentiators, are known to ameliorate the gating defect. In this study, we describe the identification of the 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole core as a novel chemotype of potentiators. In-depth structure–activity relationship studies led to the discovery of enantiomerically pure 39 endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising in vitro druglike profile. The in vivo characterization of γ-carboline 39 showed considerable exposure levels and good oral bioavailability, with detectable distribution to the lungs after oral administration to rats. Overall, these findings may represent an encouraging starting point to further expand this chemical class, adding a new chemotype to the existing classes of CFTR potentiators.

Highlights

A series of more and more stringent filters were applied in order to discard compounds with suboptimal druglike properties, and those containing chemically reactive moieties, unstable and known cytotoxic groups, and screening performance with the Z′ method gave a score of 0.6, which can be considered optimal for this type of assay
The compounds were tested in the low micromolar range, in the presence of forskolin (10 μM), on F508del-CFTR Fischer rat thyroid (FRT) cells following rescue of the trafficking defect by low-temperature incubation, as done for the primary screening
CFTR modulators have been reported to address the basic defects caused by Cystic fibrosis (CF) mutations restoring, at least partially, the CFTR function

Summary

■ INTRODUCTION

Cystic fibrosis (CF) is the most frequent life-threatening autosomal recessive disease in Caucasians, caused by loss-offunction mutations in the CF transmembrane conductance regulator (cf tr) gene, encoding for CFTR protein. CFTR is a cAMP-regulated chloride channel expressed at the apical membrane of epithelial cells, where it provides a route for electrogenic anion flux, regulating the composition and volume of epithelial secretions. CF is a multiorgan disease, affecting the lungs, pancreas, liver, and other organs. More than 2000 mutations have been described in the cftr gene; the pathogenicity has been demonstrated only for approximately 300 mutations. CF mutations cause the loss of function of CFTR protein by affecting its synthesis, trafficking, or its function as an anion channel. According to the mechanism causing CFTR dysfunction, CF mutations have been grouped into seven different classes: mutations introducing a premature stop codon (class I), mutations causing protein misfolding (class II), mutations causing defective channel gating (class III), mutations causing defective channel conductance (class IV), mutations leading to aberrant mRNA splicing (class V), mutations causing reduced stability at the plasma membrane (class VI), and mutations resulting in no mRNA expression (class VII). Despite this clear classification, the majority of CF mutations cause CFTR dysfunction by multiple mechanisms, as Received: June 30, 2020 Published: September 18, 2020. According to the mechanism causing CFTR dysfunction, CF mutations have been grouped into seven different classes: mutations introducing a premature stop codon (class I), mutations causing protein misfolding (class II), mutations causing defective channel gating (class III), mutations causing defective channel conductance (class IV), mutations leading to aberrant mRNA splicing (class V), mutations causing reduced stability at the plasma membrane (class VI), and mutations resulting in no mRNA expression (class VII).. According to the mechanism causing CFTR dysfunction, CF mutations have been grouped into seven different classes: mutations introducing a premature stop codon (class I), mutations causing protein misfolding (class II), mutations causing defective channel gating (class III), mutations causing defective channel conductance (class IV), mutations leading to aberrant mRNA splicing (class V), mutations causing reduced stability at the plasma membrane (class VI), and mutations resulting in no mRNA expression (class VII).7 Despite this clear classification, the majority of CF mutations cause CFTR dysfunction by multiple mechanisms, as Received: June 30, 2020 Published: September 18, 2020. We disclose the identification and an extended structure−activity relationship (SAR) study of tetrahydro-γcarboline derivatives, which led to the discovery of novel CFTR potentiators, characterized by a nanomolar activity

■ RESULTS AND DISCUSSION

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES