Abstract

Targeted protein degradation, commonly referred to as PROTACs (PROteolysis TArgeting Chimeras) is becoming more prominent in drug discovery as an approach to
selectively decrease protein expression levels. Unlike conventional small molecules that predominantly inhibit protein function, such as kinase inhibitors, PROTACs harness the cells Ubiquitin Proteasome System (UPS) to destroy undesired proteins. Besides improving potency and efficacy, PROTAC strategy is expected to unlock parts of the proteome that have traditionally been considered “undruggable”. PROTACs are comprised of two ligands joint by a linker; the Warhead selectively binds to the targeted protein and another ligand that binds an E3 ubiquitin ligase. PROTACs act by bringing the target of interest into proximity with an E3 ubiquitin ligase, promoting target ubiquitination and subsequent proteasomal destruction. PROTAC molecules can also be recycled to destroy other newly synthesized proteins. AlphaLISA and HTRF homogenous technologies offer simple, fast, sensitive, and robust solutions to drug discovery research. Using BTK and PROTAC MT-802 we’ve demonstrated AlphaLISA and HTRF biochemical assays can be reliable and valuable technologies for investigating PROTAC mechanisms. Competitive binding of MT-802 to both BTK with the BTK inhibitor Ibrutinib warhead to displace Dasatinib–Red ligand and to E3 ligase cereblon by the Pomalidomide portion of MT-802 to displace Thalidomide-Red ligand. Conversely, dBrd9 and VH032 had no effect in these assays, respectively. In HTRF assays, ternary complexes were formed between MT-802, the targeted BTK protein and the E3 ligase Cereblon. AlphaLISA SureFire Ultra cell-based assays used here to assess the permeability and induction of BTK proteasomal degradation of PROTAC MT-802. These and
HTRF assays measured MT-802 efficacy to induce BTK degradation in a cellular environment using Ramos cell line.