Abstract

Proteolysis
Targeting Chimeras (PROTACs) are heterobifunctional molecules that facilitate
degradation of targeted proteins in the cell via the ubiquitin proteasome
system. The catalytic mechanism offers a novel means to inhibit protein
activity, while protein degradation offers a means to inhibit proteins with
scaffold or other structural functions.  PROTAC
molecules are comprised of a ligand for the protein of interest, and a ligand
for an E3 ligase (typically VHL or Cereblon) bound together by a linker. The
molecule facilitates the recruitment of an E3 ligase to the protein of interest,
enabling the formation of a ternary complex, which allows the E3 ligase to polyubiquitinate
the protein of interest on lysine residues. This causes the protein to be recognised
by cellular machinery resulting in its targeting to the proteasome for degradation.
The ligand for the target protein as well as the linker and E3 ligase ligand
are key in determining the ability of this complex to form.

We
have synthesised multiple PROTACs that are effective at knocking-down two
proteins of interest, utilising VHL or Cereblon.  By using commercially available proteasome
inhibitors we have verified that degradation occurs through the proteasome. Experiments
have shown that degradation is concentration and time dependent, beginning at
around 4 hours with maximal effects observed by 24-48hrs. For one target we have observed a ‘hook’ effect, whereby
the PROTAC becomes saturating at higher concentrations, allowing the compound
to exclusively bind to either the E3 ligase or the protein of interest. This
saturation effect results in only partial protein degradation being observed,
with greatest degradation at lower concentrations (0.625μM).

These
findings present PROTACs as a potential future therapeutic that could be used
to degrade a wide range of proteins, previously deemed ‘undruggable’. These
molecules could therefore be applied to a variety of disease areas.