(647f) Developing a Highly Specific, Modular Platform for Conditional Protein Degradation

Current approaches to balancing dysregulated protein levels focus on either RNA or protein level control schemes. Targeting RNA acts to turn OFF the production of over-expressed cellular proteins; however, this approach does not address the issue of extant proteins already in a cellular environment. Using a protein level control mechanism can deplete both pre-existing targets and those that will continue to be produced. In nature, intracellular proteolysis is either promiscuous (lysosomal), or selective (ubiquitin-dependent proteasomal targeting). The selective nature of proteasomal degradation makes it an attractive choice for retargeting to synthetic applications.^[2] Recent advances in protein degradation approaches, such as proteolysis-targeting chimeric molecules (PROTACs), have expanded the scope of potential treatments for protein disease targets. PROTACS redirect native ubiquitination machinery to a specific degradation target protein. By destroying targets rather than inhibiting function, previously ineffectual inhibitor molecules could be adapted as targeting modalities for next-generation drug therapies.^[3] This approach is particularly useful in treatments where the survival of diseased cells is not desirable. Unfortunately, many proteins that are essential to cellular survival are poor candidates for current degradation treatment approaches that result in complete protein degradation. Thus, the uncontrolled destruction effected by existing approaches has proven to be a roadblock to their adaption for such use-cases.

To address this challenge, we are developing a new synthetic biology framework to elicit dynamic fine-tuning of target protein levels based on intracellular information (proteins and/or miRNAs) for restoration of basal, healthy protein levels. I have identified several promising systems that utilize highly specific nanobodies to repurpose cellular E3 ligases for substrate ubiquitination. Notably, the Affinity Directed Protein Missile (AdPROM) redirects the Von Hippel Lindau (VHL) domain targeting domain of the CUL2 E3 Ligase and is the backbone of our new framework.^[4] Insertion of the Tobacco Etch Virus protease (TEVp) cleavage site between the nanobody and VHL domain produces a novel turn OFF control mechanism that has similar activity levels to the original AdPROM system, and no significant impact on target protein levels when turned OFF. We demonstrated the feasibility of this approach with a model fluorescent protein target. The addition of the TEVp control layer allows us to more readily adapt the platform to various input mechanisms. Literature has shown the suitability of split TEVp assembly via lock and key devices. ^[5]

A variety of inputs can be used for mechanistic control with this system: small molecules, light activation, or even RNA level markers. For example, we can direct split TEVp assembly with miRNA marker inputs by exploiting CRISPR Cas6 proteins in conjunction with toehold mediated strand displacement of engineered RNA binding partners. We have demonstrated the suitability of our system for modulating target proteins using small molecule and light inputs. Future work includes tuning system responsiveness to various inputs, notably the RNA level markers, and nanobody discovery for novel protein targets to demonstrate our approach’s effectiveness as a highly adaptable tool for protein degradation.

1. Le Quesne JP, et al. J. Pathol. 220 (2): 140-51 (2009).
2. Lecker SH, et al. Am. Soc. Nephrol. 17 (7): 1807-1819 (2006).
3. Cermakova K, Hodges HC. Molecules. 23(8): 1958 (2018).
4. Fulcher LJ, et al. Open Biol. 6(10): 2046-2441. (2016).
5. Fink T, et al. Nat. Chem. Biol. 15: 115-122 (2019).