PP-2, a src-kinase inhibitor, is a potential corrector for F508del-CFTR in cystic fibrosis

Abstract

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The most common mutation in CF, an in-frame deletion of phenylalanine 508, leads to a trafficking defect and endoplasmic reticulum retention of the protein where it becomes targeted for degradation. Successful clinical deployments of ivacaftor and ivacaftor/lumacaftor combination have been an exciting translational development in treating CF. However, their therapeutic effects are variable between subjects and remain insufficient. We used the Library of Integrated Network-based Cellular Signatures (LINCS) database as our chemical pool to screen for candidates. For in silico screening, we integrated connectivity mapping and CF systems biology to identify candidate therapeutic compounds for CF. Following in silico screening, we validated our candidate compounds with (i) an enteroid-based compound screening assay using CF (ΔF508/ΔF508-CFTR) patient-derived enteroids, (ii) short-circuit current analysis using polarized CF primary human airway epithelial cells and (iii) Western blots to measure F508-del-CFTR protein maturation. We identified 184 candidate compounds with in silico screening and tested 24 of them with enteroid-based forskolin-induced swelling (FIS) assay. The top hit compound was PP2, a known src-kinase inhibitor that induced swelling in enteroid comparable to known CF corrector (lumacaftor). Further validation with Western blot and short-circuit current analysis showed that PP-2 could correct mutant CFTR mis-folding and restore CFTR-mediated transmembrane current. We have identified PP2, a known src-kinase inhibitor, as a novel corrector of ΔF508-CFTR. Based on our studies and previous reports, src kinase inhibition may represent a novel paradigm of multi-action therapeutics – corrector, anti-inflammatory, and anti-infective – in CF.