Enzyme prodrug therapies, in which an innocuous prodrug is introduced and later enzymatically converted to the potent active drug by a prodrug converting enzyme (PCE), is a promising direction for treating cancer. Unlike traditional chemotherapies, which offer no ability to target diseased cells specifically, PCEs can be deployed using a variety of targeted delivery mechanisms, including cell-specific protein uptake through targeting peptides and transcriptional control through gene therapies. These strategies, while demonstrating potential, frequently suffer from “leaky” off-target effects which are amplified through the catalytic ability of PCEs.<br/><br/>Degradation tags are a post-translational mechanism for controlling cellular protein levels with much faster response times and lower background levels than transcriptional control. Herein, we propose a novel method for modulating degradation tag activity in mammalian cells by sterically blocking endogenous cellular proteins from acting upon the tags. By fusing a small peptide proximal to a degradation tag, the peptide’s corresponding protein-interaction partner is able to effectively conceal the degradation tag from the proteasome. This can be done using both N- and C-terminal degradation tags. This system is tunable depending on the inherent strength of both the degradation tag and the protein-peptide pair’s interaction. Furthermore, the use of orthogonal protein-peptide interactions potentially allows for constructing post-transcriptional logic gate architectures such that multiple protein cues are required to increase the cellular concentration of a protein of interest. Since our system operates independently of previously described translational or delivery control mechanisms, it could expand the current complexity of cellular computing devices. Looking forward, the system can be adapted to detect one or more cancer-specific protein concentrations as a method for avoiding off-target PCE activity.

Tunable, post-translational method for controlling prodrug converting enzymes in cancer cells

255th National Meeting (2018)

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