Today's modern techniques and methods have helped delineate the role of proteins as drug targets. However, regulation of their translational fate within the milieu of the proteome remains challenging even for �druggable� protein targets. Nonetheless, exploitation of the protein quality-control system responsible for repairing misfolded proteins may provide a means to clandestinely assert control over disease-inducing proteins. The perturbation of a protein's native folding-state through partial denaturation results in hydrophobic-residue exposure, leading to incomplete folding events and marking the protein for degradation. Inducing degradation by mimicking protein destabilization through hydrophobic exposure may systematically target �hydrophobic-tagged� proteins for proteasomal digestion. A potential target for this strategy is the evolutionarily conserved and de novo source of thymidine, human Thymidylate Synthase (hTS). Recently, our lab has synthesized and evaluated hydrophobic conjugates of the hTS inhibitor Raltitrexed (RTX) necessary to maintain target avidity and strong inhibitory characteristics. We observed maximum binding could be achieved through the regiospecific conjugation of two gamma-linked glutamic acid residues to RTX: RTX-EE. In vitro analyses confirmed that the RTX-EE-conjugate was both specific for hTS and retained the potency and avidity of free RTX at nanomolar levels. We further investigated the potential of RTX-EE to induce an aberrant folding event by appending a hydrophobic moiety to its terminal carboxylate using safety-catch solid phase peptide synthesis. Evaluation of multiple hydrophobic-tagged RTX-EE-conjugates demonstrated moderate increase cell mortality with varying hydrophobicity's (logP) of each tag. Taken together, these results highlight the potential of a noncovalent hydrophobic tagging-system to effectively promote protein knockdown.

Antifolate hydrophobic-tagged degradation of human thymidylate synthase

248th National Meeting (2014)

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