Abstract
Selective degradation of proteins by redirecting the living cell's inherent protein destruction mechanism represents a promising new therapeutic avenue, currently already being pursued in clinical settings. This strategy often employs bispecific molecules, capable of simultaneously interacting with target protein and components of the ubiquitin-proteasome system, eventually resulting in target ubiquitination and subsequent proteasomal degradation. By engaging in multiple rounds of target destruction, and thereby showing catalytic behavior, such degraders can surpass the 1:1 stoichiometry limitation of traditional drugs. However, due to the mechanistic complexity of this approach, it is often difficult to pin-point reasons for unsuccessful designs with low efficacy, thereby precluding insight into design improvements and therefore true engineering of degraders.
This thesis’ first aim is to develop and implement a new method to unambiguously and precisely determine degradation rates in the living cell. For this purpose, we have created a novel method combining microinjection with live-cell fluorescence microscopy. Validation thereof resulted in a number of general insights on microinjection procedures, based on the precise monitoring of in vivo analyte concentrations over time. While initially limited to proteins with inherent fluorescence such as GFP, we have extended the usability of this method by proving that site-specifically conjugated dyes can serve as reporters for protein degradation, their time windows governed by their cell-permeability, which we have shown to be influenced mostly by their lipophilicity.
This method was then used to engineer DARPin-based degraders, focusing on independent pillars of successful design: First, the identification of biochemical characteristics causing the so far unidentified turnover differences in DARPins alone with or without target. Second, the influence of the first N-terminal amino acid in the target, as well as presence of peptide tags, until now often considered bystanders not influencing protein turnover. Third, the choice of the mechanism of engagement with the highly complex ubiquitin-proteasome system, including partial or complete E3-ligase fusion, small-molecule E3 inhibitor conjugation, and novel degraders inspired by pox-virus proteins.
Our insights served as the basis for three manuscripts, the first focusing on method development and two others focusing separately on the engineering of the target-binding moiety, and mechanisms to engage with the protein's degradation machinery.
Vukovic, David