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

> **NIH NIH F32** · DANA-FARBER CANCER INST · 2021 · $15,515

## Abstract

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.

## Key facts

- **NIH application ID:** 10313707
- **Project number:** 1F32CA265265-01
- **Recipient organization:** DANA-FARBER CANCER INST
- **Principal Investigator:** Kedar Puvar
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $15,515
- **Award type:** 1
- **Project period:** 2022-01-03 → 2022-02-18

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10313707

## Citation

> US National Institutes of Health, RePORTER application 10313707, Structural and functional investigation into protein degradation by the DCAF16 ubiquitin ligase (1F32CA265265-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10313707. Licensed CC0.

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