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Novel Approaches to PROTAC Drug Discovery

Authors: Dahmane Ouazia, Haley O'Malley, Karteek Kadimisetty

Proteolysis targeting chimeras (PROTAC) is a very promising strategy to ubiquitylate and degrade target proteins, especially undruggable targets. Despite initial euphoria over this novel approach to knock down a protein function, meaningful progress has not been made.  Nearly all the companies working to develop PROTAC drugs are focused on known ligases, such as cereblon, VHL or HDM ligases as vehicles. This approach may be flawed, however, in that nature does not employ the “one ligase fits all” target approach. In addition, major hurdles hinder current screening approaches, which include western blotting to monitor degradation of protein bands or reporter-based cellular assays such as that developed by Promega. These methods require multiple steps and are time-consuming, irreproducible, subject to human error, and not amenable to high throughput screening. Cell reporter assays require the generation of stable cell lines and are prone to artifacts. Moreover, current PROTAC screens do not provide any information regarding the poly-ubiquitination state of the target protein, a modification required for degradation. These problems have frustrated medicinal chemists and biochemists alike. We have developed a high-throughput microtiter plate-based approach, the UbiQuant Ultra, that evaluates protein ubiquitination, a true measure of a PROTAC molecule under physiological conditions. We have demonstrated the efficiency of a bromodomain PROTAC and a promiscuous kinase inhibitor that subsequently degrade their corresponding target proteins. We have established poly-ubiquitination and degradation of BRD3 in response to bromodomain PROTAC in a microplate environment. These data correlate directly with gel-based analysis of PROTACs, and the method itself rapidly provides reproducible data, requires less tissue sample, and is easily translated to a plate-based environment, thereby remarkably enhancing PROTAC drug discovery.  The application of unique affinity matrices -- our tandem ubiquitin binding entities (TUBE)-based mass spec proteomics platform -- allowed us to confirm the selectivity and degradation status of the target proteins in response to treatment with various PROTACs. For the first time, one can determine if a PROTAC ubiquitinates a target protein without degrading it, thus allowing chemists to design selective PROTACs. The goal of this study is to implement efficient methods to analyze PROTAC functions in a high-throughput fashion and accelerate development of novel PROTAC drugs.



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