Background Adenosine deaminase, RNA specific (ADAR1), catalyzes the deamination of adenosine (A) to inosine (I) in dsRNAs. ADAR1 has two isoforms: p110 in the nucleus and p150 which shuttles between nucleus and cytoplasm. The ADAR1 p150 isoform is expressed from an IFN-response promoter and has a Z-DNA/Z-RNA binding domain and is a key determinant of resistance to immunotherapy by suppressing immunogenic dsRNAs. Mechanistically, depletion of ADAR1 resulted in accumulation of Z-RNA and activation of the Z-RNA sensor ZBP1, culminating in RIPK3-mediated necroptosis. ADAR1 has been implicated in promoting cell survival and resistance to immune response in several cancers. Therefore, inhibition of ADAR1 p150 has the potential to enhance anti-tumor efficacy as monotherapy and in combination with other therapeutic modalities. Herein, we outline the discovery of a potential first-in-class Za targeted ADAR1 p150 inhibitor for cancer immunotherapy.

Methods A class of ADAR1 p150 inhibitors (AVA-ADR) were identified through a high-throughput ADAR1p150 binding assay. The ability of these inhibitors to induce interferons was confirmed in various cell-lines. The on-target mechanisms were confirmed by downstream PKR activation. The anti-tumor efficacy of AVA-ADR-001 was evaluated in B16F10 syngeneic melanoma mice model as monotherapy and in combination with anti-PD-1.

Results We discovered a potential first-in-class small molecule inhibitor of the ADAR1 enzyme with a potent in-vitro IFN response in an MDA5-dependent manner. Direct binding study reveals a family of AVA-ADR compounds with superior affinity (sub-uM) compared to our first-generation inhibitors for the Zα-domain. ADME and Pharmacokinetics analysis demonstrate satisfactory PK profiles and oral bioavailability. Our ADAR1 inhibitors have a micromolar EC₅₀ and demonstrated antitumor efficacy in syngeneic B16F10 melanoma model. In vivo efficacy study resulted in 45% tumor growth inhibition (TGI) compared to 33% TGI in the anti-PD1 group. Interestingly, the TGI of AVA-ADR-001 and anti-PD1 increased significantly to 56%. In addition, several interferon-stimulated genes and T-cell activation markers were significantly increased in tumor samples from the combined group. Cellular target engagement was confirmed with a PROTAC version which demonstrated selective degradation of the ADAR1 p150 protein.

Conclusions To our knowledge, AVA-ADR-001 is the first report of a small molecule inhibitor of ADAR1 p150. 2^nd generation AVA-ADR compounds are stable/orally bioavailable first-in-class ADAR1 inhibitor that demonstrated significant IFN induction in-vitro and in-vivo, demonstrating significant tumor growth inhibition as monotherapy and synergistically in combination with Anti -PD1.

Ethics Approval Ethics approval for all studies were obtained from institutional review boards.