Bicyclic Picomolar OGA Inhibitors Enable Chemoproteomic Mapping of Its Endogenous Post-translational Modifications

Owing
to its roles in human health and disease, the modification
of nuclear, cytoplasmic, and mitochondrial proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) has emerged as a
topic of great interest. Despite the presence of O-GlcNAc on hundreds
of proteins within cells, only two enzymes regulate this modification.
One of these enzymes is O-GlcNAcase (OGA), a dimeric glycoside hydrolase
that has a deep active site cleft in which diverse substrates are
accommodated. Chemical tools to control OGA are emerging as essential
resources for helping to decode the biochemical and cellular functions
of the O-GlcNAc pathway. Here we describe rationally designed bicyclic
thiazolidine inhibitors that exhibit superb selectivity and picomolar
inhibition of human OGA. Structures of these inhibitors in complex
with human OGA reveal the basis for their exceptional potency and
show that they extend out of the enzyme active site cleft. Leveraging
this structure, we create a high affinity chemoproteomic probe that
enables simple one-step purification of endogenous OGA from brain
and targeted proteomic mapping of its post-translational modifications.
These data uncover a range of new modifications, including some that
are less-known, such as O-ubiquitination and N-formylation. We expect
that these inhibitors and chemoproteomics probes will prove useful
as fundamental tools to decipher the mechanisms by which OGA is regulated
and directed to its diverse cellular substrates. Moreover, the inhibitors
and structures described here lay out a blueprint that will enable
the creation of chemical probes and tools to interrogate OGA and other
carbohydrate active enzymes.