(1065-B) Plate-based screening approaches and mechanistic insights to inform optimization of PROTAC® degraders for the treatment of neurodegenerative disease
Monday, February 5, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: PROteolysis Targeting Chimeras (PROTAC) are small molecules that employ the ubiquitin-proteosome system (UPS) to achieve degradation of a target protein of interest (PoI). PROTAC degraders are heterobifunctional molecules targeting a PoI via two distinct recognition elements joined together by a linker moiety: one end of the PROTAC incorporates a ligand to bind the PoI, while the other end of the molecule incorporates an E3 ubiquitin ligase binding ligand. While several E3-ligases have been utilized for targeted protein degradation (TPD), Cereblon (CRBN)- based PROTAC degraders have entered and are progressing through the clinic for the potential treatment of cancer and inflammatory conditions. Neurodegenerative diseases represent an area of high unmet clinical need where PROTAC degrader molecules could have distinct advantages over other therapeutic modalities, including the opportunity for oral administration to remove toxic gain of function proteins from the central nervous system. Two neurodegenerative proteins we describe here are Leucine-rich repeat kinase 2 (LRRK2), a kinase whose increased activity is seen in Parkinson’s disease (PD), with mutations such as G2019S that are linked to familial PD; and mutant Huntingtin (mHTT), a protein whose polyglutamate expansion leads to Huntington’s disease after exceeding a threshold polyQ length.
Optimization of PROTAC molecules presents several challenges, including low aqueous solubility and, for CRBN-based PROTAC degraders, recruitment and degradation of neomorphic protein substrates such as GSPT1, CK1α, SALL4, and other potentially cytotoxic profiles. To assess activity on their PoI, PROTAC degraders are often tested in cell-based degradation assays where poor compound solubility and general toxicity can confound structure activity relationships (SAR) and lead to misinterpretation of data. Here we present a testing workflow to efficiently generate PROTAC Structure-Activity and Liability Relationships (SAR/SLR) that addresses neomorphic degradation for CRBN-engaging PROTAC molecules, cellular toxicity, and aqueous solubility. Furthermore, we present in vitro screening data for both mHTT- and LRRK2-targeting PROTAC degraders engaging different E3 ligases that show high potency and no measurable toxicity utilizing mHTT- or LRRK2-expressing cell lines. We demonstrate that our mHTT-targeting PROTAC molecules also exhibit allelic selectivity, binding mHTT over the wild-type protein. Lastly, utilizing competition experiments with either an E3 binding ligand or PoI binding competitor, we demonstrate that both our LRRK2- and mHTT-targeting PROTAC molecules are functioning on mechanism, binding the correct PoI, form appropriate ternary complexes, and subsequently degrade the PoI via the UPS.