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

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 RNAcompete,
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, and (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.