Structural and functional investigation into protein degradation by the DCAF16 ubiquitin ligase

Project Summary (Abstract) A broad and diverse family of proteins called E3 ligases recognize the target protein that is to be ubiquitinated, which marks it for proteasomal degradation. In effect, they are a key factor in determining the fate of proteins.

Consistent with the vast diversity of the proteome, over 600 E3 ligases have been identified, with much of them remaining enigmatic in their mode of substrate recognition.

An emerging concept in drug discovery has been the use of small molecule ligands to recruit a therapeutically relevant protein to an E3 ligase, leading to the ubiquitination and degradation of the recruited protein. This strategy offers unique advantages over simple inhibition.

However, a major limitation of targeted protein degradation has been the relative dearth of E3s that can be used for substrate recruitment, and the large size of most degradative molecules.

Therefore, advances in these areas will significantly expand the possibilities of degrader design and improve the chances of generating a therapeutically effective molecule while reducing the risk of harmful off-target effects.

The long-term goal of this proposal is to decrypt structural principles by which new E3s can be induced to recruit substrates via small molecules.

The objective of this application is to gain molecular insights into how the E3 ligase DCAF16 can be recruited by small molecules and how that affects its normal cellular function.

The central hypothesis is that the previously reported compound GNE-0011 utilizes DCAF16 as the E3 ligase to degrade the oncogenic protein BRD4, and that it may be utilized to recruit DCAF16 to other protein targets, including transcription factors that are difficult to drug.

To address this hypothesis, Aim 1 will seek to structurally and biochemically characterize the interactions between GNE-0011, DCAF16, and BRD4. An atomic-level picture will provide a basis for substrate recruitment, validated by mutational experiments. These interactions will be compared with KB02, the only reported DCAF16-binding compound.

Aim 2 will investigate the broader physiological function of the little-studied DCAF16.

While BRD4 is degraded by GNE-0011, several proteins are upregulated, possibly representing the original targets of DCAF16.

Investigating these targets, combined with unbiased pulldown experiments, will provide a better understanding of this E3 ligase?s targets and activity, including possible roles in disease.

Together, these two aims will contribute to our knowledge of E3 ligase structure and function while greatly broadening the prospects for the design of new targeted protein degrader molecules.

The Fischer Lab at the Dana-Farber Cancer Institute will provide an optimal environment to conduct this research and this work will provide training in cell biology, drug design, and proteomics that will greatly enhance and round out my existing skillset.