Rational and Experimental Approaches for Protein Degradation or Amino Acid Biosynthesis

Proteins are the workhorses of the cell, performing vital roles in structure, function, and regulation through extensive protein–protein interactions (PPIs). Abnormal expression of specific protein in cancer can cause dysregulated signaling due to undesired interactions with protein partners, leading to tumorigenesis, dedifferentiation, proliferation, and metastasis. These aberrant proteins have been classically targeted with small molecule inhibitors, carefully designed to bind with high specificity to reduce target activity, requiring a pocket or active site for small molecule binding. However, many proteins interact through surface interactions over a large surface area and often lack deep binding pockets for potential favorable interactions with small molecules. These proteins, such as membrane scaffold proteins and transcription factors, are thus deemed undruggable and require other modes to inhibit undesired activity.

A novel method to target undruggable proteins is through harnessing native protein degradation machinery. The ubiquitin–proteosome system (UPS) maintains protein quality and homeostasis by ubiquitin–tagging protein substrates of the UPS, marking them for degradation by the proteosome. The polyubiquitination of the substrate protein occurs through a multienzyme cascade, in which a ubiquitin ligase enzyme (E3) binds the substrate in proximity to the ubiquitin–conjugating enzyme (E2), catalyzing a covalent attachment of ubiquitin to its substrate. It has recently been discovered that small molecules, termed molecular glues, can induce an interaction between an E3 ligase and a neo–substrate, which can be pharmacologically targeted for degradation. This work targeted degradation of DHX9, an RNA helicase contributing to cancer proliferation. An E3 ligase was found using AlphaFold2 to form favorable interactions with DHX9. Using previously developed pocket-opener and pocket-finder software, gaps in the DHX9-E3 interface were identified as potential glueable surfaces. The protein complex was screened with thousands of small molecules to select candidate molecular glues which stabilize the DHX9–E3 interaction to induce DHX9 proteolysis.

In this poster, I will also briefly describe my undergraduate research related to optimization of the biosynthesis of nonstandard amino acids.