Date of Award
Open Access Dissertation
Chemistry and Biochemistry
Direct post-translational control over a protein of interest (POI) can be exploited by manipulating the cells protein quality-control systems responsible for either intracellular turnover through ubiquitination (POI half-life) or repairing and degrading misfolded proteins.1 In an effort to target and degrade a high value POI we selected the enzyme human thymidylate synthase (TS) for post-translational degradation through ligand-directed ubiquitination and hydrophobic tagging. Thymidylate synthase catalyzes the reductive methylation of 2’-deoxyuridine monophosphate (dUMP) to 2’-deoxythymidine monophosphate (dTMP). As the de novo source of dTMP the regulation of intracellular TS is strictly controlled by ubiquitinindependent proteasomal degradation mediated by an internal TS-degron sequence.2,3 Using conventional peptide synthesis and the TS-directed antifolate, raltitrexed (RTX), we designed a series of a high affinity RTX-directed conjugates that specifically recruited TS to the von Hippel Linadau (VHL) E3 ligase in order to facilitate ubiquitin-dependent proteasomal degradation. These protein suppression conjugates are known as PROteolysis TArgeting Chimera’s (PROTAC). These conjugates were challenged against TS and TS overexpressing cell lines with their inhibition and ubiquitination events monitored by half-life and Western blot analyses. However, no evidence of ubiquitinmediated proteasomal degradation was found.
Contrary to the reliance of ubiquitination by the PROTAC’s system, we sought to simplify our approach and “tag” TS with a hydrophobic probe. The seminal driving force behind protein folding and stabilization is the burial of core hydrophobic residues.4 While these residues are critical to maintaining the native folding state, partial denaturation results in their exposure, leading to incomplete folding events and proteasomal degradation.5,6 In an effort to mimic this endogenous event and promote active TS proteasomal degradation, we generated a small library of RTX linked hydrophobic tags using the Kenner safety-catch handle under solid phase peptide synthesis. In this study we found that the general length of both the linkage and surface area of the hydrophobic tag were essential to growth inhibition of TS bearing cells. The implications of these findings, with regards to both the synthetic endeavors and their respective biological results, are discussed.
Menasco, D. J.(2014). Toward the Combinatorial Development of Hydrophobic-Tagged Antifolate-Directed Degradation of Human Thymidylate Synthase. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/2973