First-in-class Zalpha-domain-targeted ADAR1 p150 inhibitor demonstrates potent antitumor efficacy in high IFN syngeneic melanoma

Background: ADAR1 (Adenosine deaminase acting on RNA) exists in two isoforms: the nuclear p110 and the p150, which shuttles between the nucleus and cytoplasm. ADAR1 p150 promotes immunotherapy resistance by suppressing immunogenic dsRNAs, and by preventing the accumulation of Z-RNA structures. Loss of ADAR1 leads to Z-RNA buildup, activating ZBP1 and triggering necroptosis. Tumors exhibiting high IFN signatures enhance synthetic lethality when ADAR1 is inhibited, while also upregulating PD-L1. This dual effect presents an opportunity for synergistic efficacy for ADAR1 inhibition and checkpoint inhibitors. In this context, we report the discovery of a novel, first-in-class inhibitor that specifically targets the Zalpha domain of ADAR1 p150 for cancer immunotherapy. Additionally, we evaluated the potential of this ADAR1 p150 inhibitor as a novel payload delivered via PD-L1-targeted ADCs. Methods: A series of ADAR1 p150 inhibitors (AVA-ADR) were identified using a high-throughput binding assay, Zalpha domain binding confirmed by FRET assay. The anti-tumor efficacy of AVA-ADR compounds was assessed in a B16F10 syngeneic melanoma mouse model, both as a monotherapy and in combination with anti-PD-1 therapy in a high interferon condition. AVA-ADR compounds were also evaluated as a novel payload for PDL1 targeted ADCs. Results: We identified a novel series of small-molecule inhibitors of ADAR1 p150 that elicits a robust interferon response. Binding studies showed AVA-ADR compounds exhibit submicromolar Zalpha-domain affinity, surpassing prior inhibitors. AVA-ADR inhibitors displayed nanomolar EC50 values and significant anti-tumor activity in the B16F10 melanoma model. AVA-ADR compounds demonstrate additive efficacy in combination with anti-PD1 treatment (~1.5 X vs anti-PD1). AVA-ADR compounds demonstrate strong anti-tumor potency in high interferon environment, >1.5x superior mIFN- beta alone. Tumor samples from the combination group showed significantly elevated expression of interferon-stimulated genes and T-cell activation markers. Conclusion: To our knowledge no selective small molecule inhibitors of ADAR1 p150 targeting the Za domain have been reported so far. Our SAR approach yielded several next generation Zalpha binders with nM potency demonstrating effective and competitive displacement of the tightly bound Z-DNA. Next generation compounds demonstrate low nM interferon dependent toxicity in OE21 cell lines. In vivo efficacy study in B16F10 melanoma model demonstrates synergistic efficacy of AVA-ADR compounds with mIFN-beta. This is the first disclosure of a selective ADAR1 p150 inhibitor that was systemically administered demonstrating strong anti-tumor potency in high IFN tumors. Furthermore, these inhibitors show promise as payloads for antibody-drug conjugates (ADCs), enabling targeted delivery and enhanced therapeutic index in IFN-responsive cancers.