Development of Inhibitors and Degraders of ADAR1 | Development of Proximity Capture Probes Targeting Transcription Factors

Abstract

Adenosine deaminase acting on RNA (ADAR1) is an enzyme that edits double stranded RNA (dsRNA) by deaminating adenosine residues to inosine. ADAR1’s major role is regulating the immune response to dsRNA in a cell, and dysregulation of this enzyme can lead to cancer formation. ADAR1 is a significant target in drug discovery campaigns, but all published attempts at inhibition or degradation of this target have been unsuccessful thus far. Several modes of targeting ADAR1 were pursued to inhibit the activity of ADAR1.

C75-based Small Molecule Inhibition: C75 is a fatty-acid synthase inhibitor that was used to identify new biological targets like ADAR1 in a cell with click-based in situ profiling. Our group first validated binding to ADAR1, then discovered the mechanism of action was through non-specific Michael addition to the nucleophilic residues on ADAR1. Several attempts were made to reduce the electrophilicity of C75, but no significant improvements in specificity were made while maintaining binding. [chapter 2]

Imidazotriazine-based Small Molecule Inhibition: An orthosteric scaffold was designed to inhibit the catalytic activity of ADAR1 by mimicking the high energy intermediate of adenosine to inosine conversion. Derivatives of this scaffold were tested in several in vitro and in cellulo assays that could measure out ADAR1 inhibition. Several compounds showed micromolar inhibition of ADAR1 both in vitro and in cellulo. However, a key in vitro assay was found to contain a contaminating enzyme, ADA, that stopped further development. [chapter 3]

Small Molecule PROTAC Degradation of ADAR1: A DNA-encoded library (DEL) screen was performed to identify new small molecule binders of ADAR1. Several compounds were identified in the initial screen, and one selected compound was confirmed to bind to ADAR1 via SPR. Several PROTACs were synthesized by attaching linkers containing pomalidomide to the hit compound. Some evidence of degradation was found in PROTACs containing long alkyl linkers, but the mechanism was not through the ubiquitin proteasome system. [chapter 4]

RNA-PROTAC Degradation of ADAR1: A piece of short dsRNA was designed to bind to ADAR1 for use in an RNA-PROTAC. The RNA was first confirmed to bind to ADAR1 through fluorescence polarization. The RNA-PROTAC was then synthesized by attaching PEG linkers containing pomalidomide to the RNA. Several RNA-PROTACs were then delivered into human cells, and one showed nanomolar degradation of ADAR1 through the ubiquitin proteasome system. Subsequent attempts were unable to replicate degradation, and therefore this approach to degradation was pursued no further. [chapter 5]

Transcription factors are a family of proteins that regulate the expression of all proteins in a cell through their binding to DNA. Dysregulation in the activity of transcription factors is required for the survival and growth of all human cancers. Transcription factors are difficult proteins to target with small molecules because most lack small molecule binding sites. This paper describes an alternative approach to drugging transcription factors using a proximity labeling system.

Drugging Transcription Factors with Proximity Covalent Capture: Several chromatin associated proteins (CAPs) were identified that were proximal to transcription factors and already had known small molecule inhibitors and degraders. The small molecule inhibitors of these CAPs were then attached with linkers containing an alkyne handle to an electrophilic warhead, making probes that could label proximal transcription factors. These are called proximity covalent capture (PCC) probes. Several of these probes were tested for labeling in human cells and found to label proteins with low efficiency. Work continues to further develop these probes to better target oncogenic transcription factors. [chapter 6]