Quantitative Proteomic Analysis Identifies Targets and Pathways of a 2‑Aminobenzamide HDAC Inhibitor in Friedreich’s Ataxia Patient iPSC-Derived Neural Stem Cells

Members
of the 2-aminobenzamide class of histone deacetylase (HDAC) inhibitors
show promise as therapeutics for the neurodegenerative diseases Friedreich’s
ataxia (FRDA) and Huntington’s disease (HD). While it is clear
that HDAC3 is one of the important targets of the 2-aminobenzamide
HDAC inhibitors, inhibition of other class I HDACs (HDACs 1 and 2)
may also be involved in the beneficial effects of these compounds
in FRDA and HD, and other HDAC interacting proteins may be impacted
by the compound. To this end, we synthesized activity-based profiling
probe (ABPP) versions of one of our HDAC inhibitors (compound 106),
and in the present study we used a quantitative proteomic method coupled
with multidimensional protein identification technology (MudPIT) to
identify the proteins captured by the ABPP 106 probe. Nuclear proteins
were extracted from FRDA patient iPSC-derived neural stem cells, and
then were reacted with control and ABPP 106 probe. After reaction,
the bound proteins were digested on the beads, and the peptides were
modified using stable isotope-labeled formaldehyde to form dimethyl
amine. The selectively bound proteins determined by mass spectrometry
were subjected to functional and pathway analysis. Our findings suggest
that the targets of compound 106 are involved not only in transcriptional
regulation but also in posttranscriptional processing of mRNA.