Danijela Dukovski1, Adriana Villella1, Cecilia Bastos1, Randall King2, Daniel Finley2, Jeffery W Kelly3, Richard I Morimoto4, F Ulrich Hartl5, Benito Munoz1, Po-Shun Lee1, Marija Zecevic1, John Preston Miller6. 1. Proteostasis Therapeutics, Inc., 80 Guest St, Suite 500, Boston, MA 02135, United States. 2. Department of Cell Biology, Harvard Medical School, Boston, MA 02115, United States. 3. Departments of Molecular Medicine and Chemistry, The Scripps Research Institute, La Jolla, CA 92037, United States. 4. Department of Molecular Bioscience, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL, United States. 5. Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany. 6. Proteostasis Therapeutics, Inc., 80 Guest St, Suite 500, Boston, MA 02135, United States. Electronic address: john.miller@proteostasis.com.
Abstract
BACKGROUND: Cystic fibrosis (CF) is a recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We previously described a first-in-class CFTR modulator that functions as an amplifier to selectively increase CFTR expression and function. The amplifier mechanism is distinct from and complementary to corrector and potentiator classes of CFTR modulators. Here we characterize the mechanism by which amplifiers increase CFTR mRNA, protein, and activity. METHODS: Biochemical studies elucidated the action of amplifiers on CFTR mRNA abundance and translation and defined the role of an amplifier-binding protein that was identified using chemical proteomics. RESULTS: Amplifiers stabilize CFTR mRNA through a process that requires only the translated sequence of CFTR and involves translational elongation. Amplifiers enrich ER-associated CFTR mRNA and increase its translational efficiency through increasing the fraction of CFTR mRNA associated with polysomes. Pulldowns identified the poly(rC)-binding protein 1 (PCBP1) as directly binding to amplifier. A PCBP1 consensus element was identified within the CFTR open reading frame that binds PCBP1. This sequence proved necessary for amplifier responsiveness. CONCLUSIONS: Small molecule amplifiers co-translationally increase CFTR mRNA stability. They enhance translation through addressing the inherently inefficient membrane targeting of CFTR mRNA. Amplifiers bind directly to PCBP1, show enhanced affinity in the presence of bound RNA, and require a PCBP1 consensus element within CFTR mRNA to elicit translational effects. These modulators represent a promising new and mechanistically novel class of CFTR therapeutic. They may be useful as a monotherapy or in combination with other CFTR modulators.
BACKGROUND:Cystic fibrosis (CF) is a recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We previously described a first-in-class CFTR modulator that functions as an amplifier to selectively increase CFTR expression and function. The amplifier mechanism is distinct from and complementary to corrector and potentiator classes of CFTR modulators. Here we characterize the mechanism by which amplifiers increase CFTR mRNA, protein, and activity. METHODS: Biochemical studies elucidated the action of amplifiers on CFTR mRNA abundance and translation and defined the role of an amplifier-binding protein that was identified using chemical proteomics. RESULTS: Amplifiers stabilize CFTR mRNA through a process that requires only the translated sequence of CFTR and involves translational elongation. Amplifiers enrich ER-associated CFTR mRNA and increase its translational efficiency through increasing the fraction of CFTR mRNA associated with polysomes. Pulldowns identified the poly(rC)-binding protein 1 (PCBP1) as directly binding to amplifier. A PCBP1 consensus element was identified within the CFTR open reading frame that binds PCBP1. This sequence proved necessary for amplifier responsiveness. CONCLUSIONS: Small molecule amplifiers co-translationally increase CFTR mRNA stability. They enhance translation through addressing the inherently inefficient membrane targeting of CFTR mRNA. Amplifiers bind directly to PCBP1, show enhanced affinity in the presence of bound RNA, and require a PCBP1 consensus element within CFTR mRNA to elicit translational effects. These modulators represent a promising new and mechanistically novel class of CFTR therapeutic. They may be useful as a monotherapy or in combination with other CFTR modulators.
Authors: Arianna Venturini; Anna Borrelli; Ilaria Musante; Paolo Scudieri; Valeria Capurro; Mario Renda; Nicoletta Pedemonte; Luis J V Galietta Journal: Int J Mol Sci Date: 2021-11-04 Impact factor: 5.923
Authors: Guido Veit; Ariel Roldan; Mark A Hancock; Dillon F Da Fonte; Haijin Xu; Maytham Hussein; Saul Frenkiel; Elias Matouk; Tony Velkov; Gergely L Lukacs Journal: JCI Insight Date: 2020-09-17