Development of Inhibitors of ADAR1

Abstract

Adenosine Deaminase Acting on RNA 1 (ADAR1) is an RNA-editing enzyme that deaminates adenosine residues within double stranded RNA, transforming adenosine into inosine. The deregulation of ADAR1 activity has been implicated in a variety of human diseases, including cancer, through mechanisms involving both its catalytic and non-catalytic activity. Drug discovery campaigns against ADAR1 have been stymied by the lack of small molecular scaffolds that bind to ADAR1 and the lack of assays with enough sensitivity to discover these scaffolds. Three methods were attempted to address these obstacles.

C75 as a covalent modifier of ADAR1: The fatty acid synthase inhibitor C75 was shown to covalently modify ADAR1. The mechanism was confirmed to be a non-specific Michael addition by nucleophilic amino acid residues on ADAR1. Analogues were designed and synthesized that tempered the electrophilicity of C75 and curbed the promiscuity of the ligand, but they were ultimately unsuccessful in retaining covalent binding to ADAR1. iv Imidazotriazines as competitive inhibitors of ADAR1: Contrary to previous literature reports, a commercial sample of ADAR1 was observed to slowly deaminate monomeric adenosine into inosine, leading to an assay that quantified the amount of inosine produced as a proxy for ADAR1 activity. Small molecule probes using the imidazotriazine scaffold were found to competitively inhibit the inosine generating activity. However, further observations with a more potent analogue revealed that the deaminase activity was due to a small impurity of Adenosine Deaminase in the sample of ADAR1 used. DNA-Encoded Library (DEL) Screen to Identify Degraders of ADAR1: Several ligands were identified as possible binding partners to ADAR1 in a DEL screen, but none showed inhibitory activity towards ADAR1. The ligand with the highest binding affinity towards ADAR1 was elaborated into a PROTAC molecule that was shown to decrease the level of ADAR1 in a cellular assay.