Accelerating PROTAC drug discovery: Establishing a relationship between ubiquitination and target protein degradation.

PROTACs have emerged as a new class of drugs that can target the "undruggable" proteome by hijacking the ubiquitin proteasome system. Despite PROTACs' success, most current PROTACs interface with a limited number of E3 ligases, hindering their expansion to many challenging therapeutic uses. Currently, PROTAC drug discovery relies heavily on traditional Western blotting and reporter gene assays which are insensitive and prone to artifacts, respectively. New reliable methods to monitor true PROTAC function (i.e., ubiquitination and subsequent degradation of targets at physiological expression levels) without external tags are essential to accelerate the PROTAC discovery process and to address many unmet therapeutic areas. In this study, we developed a new high-throughput screening technology using "TUBEs" as ubiquitin-binding entities to monitor PROTAC-mediated poly-ubiquitination of native target proteins with exceptional sensitivity. As a proof of concept, targets including BRD3, Aurora A Kinase, and KRAS were used to demonstrate that ubiquitination kinetics can reliably establish the rank order potencies of PROTAC with variable ligands and linkers. PROTAC-treated cell lysates with the highest levels of endogenous target protein ubiquitination - termed "UbMax" - display excellent correlations with DC50 values obtained from traditional Western blots with the added benefits of being high throughput, providing improved sensitivity, and reducing technical errors.

Tandem Ubiquitin Binding Entities (TUBEs) as Tools to Explore Ubiquitin-Proteasome System and PROTAC Drug Discovery.

The ubiquitin proteasome system (UPS) is a complex pathway that involves multiple enzymes and culminates in the formation of a polyubiquitin chain on a target protein. As its importance is becoming more evident in drug discovery, there is a renewed interest in understanding the role that polyubiquitin chains play. This has been a challenge, mostly due to the lack of experimental tools for detecting the polyubiquitinated forms of a protein of interest (POI). Tandem Ubiquitin Binding Entities (TUBEs) are engineered protein domains that bind specifically to polyubiquitin chains. These polyubiquitin affinity matrices are highly sensitive as they bind to polyubiquitin chains in the nanomolar range. They exist in two forms: pan-selective TUBEs and chain-selective TUBEs. The ability of TUBEs to be conjugated to different entities is truly what makes them unique. TUBEs are used in a wide variety of experiments such as in protein pulldowns to enrich for polyubiquitinated proteins. They are an alternative to ubiquitin antibodies in Western blots. Further, TUBEs are used as capture reagents for immobilizing polyubiquitinated proteins on a microtiter plate. The use of TUBEs as components of in vitro and cell-based assays presents the unique feature of confirming and assessing the polyubiquitination of a POI in response to inhibitors, activators, or PROTAC® molecules. Therefore, TUBEs not only play a big role in studying the UPS but also have a huge potential for speeding up the drug discovery process.