Selective disruption of histone deacetylase complexes by small-molecule inhibitors of protein interactions

This thesis is divided into four chapters, including an introduction, and three research
articles. Chapter one positions this work in the context of the druggability of protein-protein
interactions (PPIs) and inhibition of histone deacetylase (HDAC) complexes.
Chapter two describes a framework to maximize PPI detection by combining a
minimal number of binary interaction assays. It also describes the development of a versatile
assay, NanoLuc 2-Hybrid (N2H), to test PPIs in different experimental conditions to recover
as many interactions as when various assays are combined. This chapter appears as published
in Nature Communications 10:3907 (2019).
Chapter three demonstrates the ability of binary interaction assays to comprehensively
map directly interacting subunits within multiprotein complexes supported by 3D structures.
It also positions binary PPI assays as powerful complementary tools to increase the resolution
of affinity purification-mass-spectrometry (AP-MS)-based maps, by systematically
distinguishing direct interactions from indirect associations within complexes.
Chapter four describes the functional disruption of the yeast S. cerevisiae Rpd3L
histone deacetylase (HDAC) complex by small molecules, and exploits findings from
chapters two and three to test compounds against binary PPIs between subunits of the
complex. In particular, it presents the discovery and validation of a functional inhibitor that
does not affect HDAC enzymatic activity, but rather acts by binding to a large scaffold
protein, ultimately inhibiting its interactions with transcription factors at specific
chromosomal loci. The pieces of evidence presented in this chapter also suggest the mode of
action of the PPI inhibitor is conserved in human cells and in mice since it confers
physiologically-relevant phenotypes (e.g. antipsychotic effect) identical to that of wellestablished
enzymatic inhibitors, or knockouts of the catalytic HDAC component, while
perturbing expression of an order of magnitude fewer genes. Our results highlight the
potential clinical relevance of such higher-selectivity HDAC complex PPI inhibitors.