Development of Non-conventional PROTACs - Ubiquitin (Ub) signaling regulates not only proteasomal protein degradation, but also protein trafficking, receptor-
mediated signal transduction, and control of mitophagy and autophagy. Ub conjugating (E3 ligases) and
deconjugating (deubiquitinases) enzymes and the proteasome have been targets of drug discovery focusing on
degradation or preservation of disease-associated proteins, with some success in the approval of proteasome
inhibitors and three drugs (thalidomide, pomalidomide, and lenalidomide) that target the E3 ligase Cereblon for
the treatment of multiple myeloma or mantle cell lymphoma. The non-degradative events of Ub conjugation and
deconjugation have not been widely exploited for therapeutic application, although therapeutic hypotheses lend
themselves to these functions. Currently, a novel approach to target and dispose of disease-associated proteins
is gaining interest and known as targeted protein degradation (TPD). This emerging technology uses bifunctional
molecules known as PROTACs (Proteolysis targeting chimeras) that bind to a protein of interest while
simultaneously tagging it for degradation via an E3 Ub ligase. What’s unique about this technology is that the
target protein doesn’t necessarily need to by a physiological substrate for the E3 ubiquitin ligase. This opens the
door for the “undruggable” proteome including signaling adaptors and transcription factors. Although the
therapeutic application of PROTACs has heretofore been considered protein degradation, the chemistry involved
could have non-degradative applications, including subcellular localization or altering the activity of candidate
substrates to generate a desired pharmacologic effect. The goal of this proposal is to develop a new application
of PROTAC technology which we term “LOTAC” for localization targeting chimeras that hijacks the E3 Ub ligase
TRAF6, which is known to catalyze K63-linked polyubiquitination. This goal will be addressed by achieving
milestones including: 1) reconstituting active TRAF6 from E. coli and showing that specific TRAF6-binding
compounds can function as “LOTAC” ligands; and 2) demonstrating that TRAF6 mediated polyubiquitination of
FOXP3 causes its cellular localization from the cytosol to the nucleus. In Phase II, TRAF6 LOTACs will be
designed using selective, high affinity ligands that bind to TRAF6 and alter the subcellular location of various
substrates via K63-linked ubiquitination. This technology will assist medicinal chemists in developing LOTAC
molecules that work by changing the subcellular location and/or activity of therapeutic target proteins rather than
by eliminating them by proteasomal degradation. Successful development of such LOTACs will expand the utility
of this novel form of ubiquitin-based therapy.
