A Chemical Biology Approach to Model Pontocerebellar Hypoplasia Type 1B (PCH1B)

Francois-Moutal, L., Jahanbakhsh, S., Nelson, A., Ray, D., Scott, D.D., Hennefarth, M., Moutal, A., Perez-Miller, S., Ambrose, A.J., Al-Shamari, A., Coursodon, P., Meechoovet, B., Reiman, R., Lyons, E., Beilstein, M., Chapman, E., Morris, Q.D., Van Keuren-Jensen, K., Hughes, T.R., Khanna, R., Koehler, C., Jen, J., Gokhale, V., Khanna, M.
ACS Chemical Biology   13(10): 3000-3010 (Journal)
Registered Authors
Koehler, Carla
MeSH Terms
  • Animals
  • Atrophy
  • Cerebellum/pathology
  • Disease Models, Animal*
  • Down-Regulation
  • Exosome Multienzyme Ribonuclease Complex/chemistry
  • Exosome Multienzyme Ribonuclease Complex/genetics
  • Exosome Multienzyme Ribonuclease Complex/metabolism*
  • Gene Knockdown Techniques
  • Humans
  • Isoquinolines/metabolism
  • Isoquinolines/pharmacology*
  • Isoquinolines/toxicity*
  • Molecular Docking Simulation
  • Mutation
  • Olivopontocerebellar Atrophies/chemically induced
  • Olivopontocerebellar Atrophies/genetics*
  • Olivopontocerebellar Atrophies/pathology
  • Phenotype
  • Protein Binding
  • Protein Domains
  • RNA/metabolism*
  • RNA-Binding Proteins/chemistry
  • RNA-Binding Proteins/genetics
  • RNA-Binding Proteins/metabolism*
  • Spinal Curvatures/chemically induced
  • Transcriptome/drug effects
  • Up-Regulation
  • Zebrafish/abnormalities*
30141626 Full text @ ACS Chem. Biol.
Mutations of EXOSC3 have been linked to the rare neurological disorder known as Pontocerebellar Hypoplasia type 1B (PCH1B). EXOSC3 is one of three putative RNA-binding structural cap proteins that guide RNA into the RNA exosome, the cellular machinery that degrades RNA. Using RNAcompete, we identified a G-rich RNA motif binding to EXOSC3. Surface Plasmon Resonance (SPR) and Microscale Thermophoresis (MST) indicated an affinity in the low micromolar range of EXOSC3 for long and short G-rich RNA sequences. Although several PCH1B-causing mutations in EXOSC3 did not engage a specific RNA motif as shown by RNA compete they exhibited lower binding affinity to G-rich RNA as demonstrated by MST. To test the hypothesis that modification of the RNA-protein interface in EXOSC3 mutants may be phenocopied by small molecules, we performed an in-silico screen of 50,000 small molecules and used enzyme-linked immunosorbant assays (ELISAs) and MST to assess the ability of the molecules to inhibit RNA-binding by EXOSC3. We identified a small molecule, EXOSC3-RNA disrupting (ERD) compound 3 (ERD03), which: (i) bound specifically to EXOSC3 in saturation transfer difference nuclear magnetic resonance (STD NMR); (ii) disrupted the EXOSC3-RNA interaction in a concentration-dependent manner; (iii) produced a PCH1B-like phenotype with a 50% reduction in the cerebellum and an abnormally curved spine in zebrafish embryos. This compound also induced modification of zebrafish RNA expression levels similar to that observed with a morpholino against EXOSC3. To our knowledge, this is the first example of a small molecule obtained by rational design that models the abnormal developmental effects of a neurodegenerative disease in a whole organism.
Genes / Markers
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Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes