Rational Design of Dumbbell PROTACs for Targeted Degradation of the Genomic Mutator APOBEC3A

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Degree type
Doctor of Philosophy (PhD)
Graduate group
Biochemistry and Molecular Biophysics
Discipline
Biochemistry, Biophysics, and Structural Biology
Subject
Base editing
Chemical biology
Deaminase
DNA damage
Inhibitor
Molecular mimicry
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Copyright date
2022
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Author
Serrano, Juan, C
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Abstract

DNA deaminases of the AID/APOBEC family are powerful DNA and RNA mutators that perform essential biological functions ranging from antibody diversification to viral restriction. Robust editing capabilities come at a cost, however, as certain members suchas APOBEC3A (A3A) can transiently target the host genome and promote tumor evolution through sublethal mutation, generating mutational signatures in >50% of cancers. The pathological dysregulation of these DNA deaminases also affects their utility as cytosine base editors due to safety concerns from significant off-target genomic and transcriptomic editing. To better control and understand deaminase-driven mutation of host DNA and RNA, we need molecular tools that can selectively probe these enzymes, whether through inhibition or quantification of activity. Here, we demonstrate how a deeper understanding of structure-guided substrate selection by AID/APOBEC members can be leveraged to develop both targeted degraders of APOBEC3A and sensitive assays to track its deamination activities in live cells. Genome sequencing studies of tumor samples and in vitro enzymology has revealed A3A preferentially targets cytosine found in hairpin DNA and RNA. We used this insight to construct DNA dumbbells that mimic the natural stem loop structure and incorporated the inhibitory base methylzebularine (mZ) to develop more potent nanomolar inhibitors of A3A. Structure-activity relationship studies using a panel of structurally diverse inhibitors further highlighted which features of the dumbbell scaffold were the main drivers of the increased potency observed. Finally, we demonstrated dumbbell inhibitors are specific for A3A compared to a closely-related family member. We used our knowledge of A3A’s mutagenic activity on the wider transcriptome to develop three assays that quantify RNA editing in live cells by tracking deamination at RNA hairpin hotspots. In our first two assays, editing at a DDOST or CYFIP1 site in mRNA is measured through sequencing- and restriction digest-based endpoint analysis, facilitating RNA editing quantification through conventional molecular biology techniques. Our third assay to track deaminase activity uses the generation of a stop codon upon A3A RNA editing of a hairpin hotspot situated between an mCherry-d2GFP fusion protein. Measuring GFPoff cell populations by flow cytometry can then be used for sensitive, high-throughput assessment of deaminase off-target RNA editing activity. Finally, we translate our DNA inhibitors into PROTAC degraders of A3A through structure-guided functionalization of the dumbbell loop. Our PROTAC dumbbells are capable of targeted deaminase degradation in a dose-dependent manner when transfected into HepaRG cells expressing dox-inducible A3A. Moreover, preliminary studies show our PROTACs can degrade endogenous A3A in cancer cell lines induced by pro-inflammatory stimuli. Using E3 ligase ligand controls and a free ligand competition assay, we are starting to see hints that the mechanism of degradation is dependent on E3 ligase recruitment and stable ternary complex formation. Altogether, my results add to the chemical toolbox with which to probe DNA deaminases and are the first demonstration of targeted degradation of an AID/APOBEC member by a DNA PROTAC.

Advisor
Kohli, Rahul, M
Date of degree
2022
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