Discovery of Clinically Approved Agents That Promote Suppression of Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations.
Premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Several agents are known to suppress PTCs but are poorly efficacious or toxic.



To determine whether there are clinically available agents that elicit translational readthrough and improve CFTR function sufficient to confer therapeutic benefit to patients with CF with PTCs.



Two independent screens, firefly luciferase and CFTR-mediated transepithelial chloride conductance assay, were performed on a library of 1,600 clinically approved compounds using fisher rat thyroid cells stably transfected with stop codons. Select agents were further evaluated using secondary screening assays including short circuit current analysis on primary cells from patients with CF. In addition, the effect of CFTR modulators (ivacaftor) was tested in combination with the most efficacious agents.



From the primary screen, 48 agents were selected as potentially active. Following confirmatory tests in the transepithelial chloride conductance assay and prioritizing agents based on favorable pharmacologic properties, eight agents were advanced for secondary screening. Ivacaftor significantly increased short circuit current following forskolin stimulation in cells treated with pyranoradine tetraphosphate, potassium p-aminobenzoate, and escin as compared with vehicle control. Escin, an herbal agent, consistently induced readthrough activity as demonstrated by enhanced CFTR expression and function in vitro.



Clinically approved drugs identified as potential readthrough agents, in combination with ivacaftor, may induce nonsense suppression to restore therapeutic levels of CFTR function. One or more agents may be suitable to advance to human testing.
1 Department of Medicine.
Venkateshwar
V
Mutyam
2 Department of Microbiology.
Ming
M
Du
2 Department of Microbiology.
Xiaojiao
X
Xue
2 Department of Microbiology.
Kim M
KM
Keeling
3 Southern Research, Birmingham, Alabama; and.
E Lucile
EL
White
3 Southern Research, Birmingham, Alabama; and.
J Robert
JR
Bostwick
3 Southern Research, Birmingham, Alabama; and.
Lynn
L
Rasmussen
1 Department of Medicine.
Bo
B
Liu
4 Gregory Fleming James Cystic Fibrosis Research Center.
Marina
M
Mazur
4 Gregory Fleming James Cystic Fibrosis Research Center.|5 Department of Cell Developmental and Integrative Biology, and.
Jeong S
JS
Hong
4 Gregory Fleming James Cystic Fibrosis Research Center.
Emily
E
Falk Libby
6 Cystic Fibrosis Foundation Therapeutics, Boston, Massachusetts.
Feng
F
Liang
6 Cystic Fibrosis Foundation Therapeutics, Boston, Massachusetts.
Haibo
H
Shang
6 Cystic Fibrosis Foundation Therapeutics, Boston, Massachusetts.
Martin
M
Mense
3 Southern Research, Birmingham, Alabama; and.
Mark J
MJ
Suto
2 Department of Microbiology.|4 Gregory Fleming James Cystic Fibrosis Research Center.
David M
DM
Bedwell
1 Department of Medicine.|4 Gregory Fleming James Cystic Fibrosis Research Center.|5 Department of Cell Developmental and Integrative Biology, and.|7 Department of Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama.
Steven M
0000-0001-9045-0133
SM
Rowe
D000818:Animals; D002460:Cell Line; D018389:Codon, Nonsense / Q000187:drug effects* / Q000235:genetics; D003550:Cystic Fibrosis / Q000188:drug therapy* / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000235:genetics*; D055808:Drug Discovery / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D011916:Rats, Inbred F344; D060888:Real-Time Polymerase Chain Reaction
C505032:CFTR protein, human; D018389:Codon, Nonsense; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D008156:Luciferases
clinical agents; cystic fibrosis transmembrane conductance regulator; premature termination codon mutations; readthrough