Optimizing stability of engineered anti-RAS bioPROTAC for rapid degradation of undruggable proteins

Shaila S. Rao

doi:10.17918/00011055

Proteins that are commonly mutated and overexpressed in cancers are called oncoproteins and are typical targets for cancer therapies. Cancer treatment faces significant challenges due to the presence of "undruggable" proteins--key drivers of tumor growth and survival that lack suitable binding sites for conventional therapeutics. Undruggable proteins constitute a great percentage of all proteins and have thus been a focus of drug and therapeutic delivery. Due to their structure lacking deep drug-binding sites or having a large smooth surface, traditional inhibitors cannot successfully bind to and block or degrade the protein. Many off-target effects result from this, which can cause unwanted physiological responses while leaving the target protein relatively uninhibited. A key example of undruggable oncoproteins is the Ras family of proteins, a key player in many cancer pathways. Small-molecule inhibitors, degraders, and other biotherapeutics have been developed to target oncoproteins. A powerful small-molecule drug, known as a Proteolysis Targeting Chimera (PROTAC), has been developed. A PROTAC is made of two functional parts: a target protein binding domain and an E3 ubiquitin ligase recruiting domain. PROTACs have superior degradation properties compared to traditional small-molecule degraders due to their structure, which allows them to bring the target protein and an E3 ligase in proximity so that the target protein can be tagged with ubiquitin for Ubiquitin-proteasome degradation. While PROTACs can be designed to degrade undruggable proteins, their application becomes limited due to their size and inability to enter cells. These small-molecule drugs allow for the degradation of druggable proteins. Using a protein-based biologic method, on the other hand, can more effectively target "undruggable" proteins. One of these protein-based degraders, known as a bioPROTAC, was developed. bioPROTACs are heterobifunctional degrader fusion proteins consisting of a target-binding domain, a linker, and an E3 ligase complex. Compared to the PROTAC, bioPROTACs have higher success rates, fewer design constraints, are biodegradable, and can clear up to 95% of the target protein within hours of treatment. An anti-RAS bioPROTAC was developed by the Tsourkas Lab at the University of Pennsylvania. Points of improvement were identified, one of which was to improve the stability of these anti-RAS bioPROTACs. It was found that the bioPROTACS were subject to autoubiquitination, which means the bioPROTAC was being artificially tagged for self-destruction, leading to reduced stability, potency, and specificity. To stabilize the biodegrader, the lysine sites prone to auto-ubiquitination on the wild-type bioPROTAC were identified and substituted with an arginine, which is known as a K to R mutation. Lysine and arginine are both positively charged and have similar structures, but arginine is resistant to auto-ubiquitination. A Q5 Site-Directed Mutagenesis, followed by Minipreps and sequencing, provided plasmid samples to be expressed into proteins in E. coli and purified via the lab's sortase-tag expressed protein ligation (STEPL) protocol. These mutated bioPROTACs were tested for auto-ubiquitination, and their ability to drive KRAS ubiquitination and degradation in vitro. Several protein engineering techniques and machinery were employed, including but not limited to Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), protein expression and purification, lipid nanoparticle (LNP) formulation, and flow cytometry. The performance of the individual mutants informed which K to R mutations were combined into a single gene block plasmid. The same process for individual mutations was followed to develop a single engineered anti-RAS bioPROTAC molecule. This paper delves into the specific methods used and the results from the experiments that led to the successful mutation of the wild-type bioPROTAC, likely resulting in increased stability of the bioPROTAC fusion protein.

Optimizing stability of engineered anti-RAS bioPROTAC for rapid degradation of undruggable proteins

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Optimizing stability of engineered anti-RAS bioPROTAC for rapid degradation of undruggable proteins

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