Targeting the interferon-inducible isoform, p150 of ADAR1 for the treatment of oral squamous cell carcinoma (OSCC)

Oral squamous cell carcinoma (OSCC) is the fourth most common cancer in low- and middle-income countries (LMIC) and is one of the leading causes of cancer deaths in Asia, accounting for nearly three-quarters of global oral cancer mortality. Five-year survival rates remain poor, reflecting both the scarcity of actionable genetic drivers and the absence of Asian patient-derived models in global cancer datasets. This underrepresentation has slowed therapeutic innovation for populations that bear the greatest burden, leaving a significant unmet clinical need. To address this gap, we conducted genome-wide CRISPR/Cas9 dependency screens on a panel of unique Asian-derived OSCC cell lines that reflect the genetic and etiological diversity of these patients to identify vulnerabilities for therapeutic development. We have identified Adenosine deaminase acting on RNA (ADAR1) as an essential gene for OSCC survival. ADAR1 is an RNA-editing enzyme, whereby its dysregulation has been associated with tumor progression in various cancers. There are two isoforms of ADAR1 (p110 and p150), which are identical except that p150 has a unique Zalpha domain, which binds Z-DNA and offers a unique targeting opportunity to treat this deadly OSCC. To take this forward, we applied a proprietary computer-aided drug discovery pipeline, termed the Multi-layer Ultra-large Screening and Exploration (M.U.S.E), to identify potential Zalpha-targeting ADAR1 inhibitors. Approximately six billion compounds were evaluated through integrated filters for solubility, drug-likeness, binding affinity, and stability. A total of 51 compounds were shortlisted from the in-silico screening, and their binding affinities and specificities were evaluated. The intrinsic tryptophan fluorescence (ITF) spectroscopy was used to determine the binding affinity, while the electrophoretic mobility shift assay (EMSA) was used to determine the on-target effect of the compounds in disrupting the Zalpha-Z-DNA binding. From ITF analysis, 21 compounds demonstrated strong binding affinity with Stern-Volmer constants (K SV ) exceeding 10,000 M -1 . Using EMSA, we identified eight compounds with robust evidence of disruption of Zalpha-Z-DNA binding complexes. Our selected top hits demonstrated better binding affinity and on-target efficacy in disrupting ADAR1-Zalpha function, compared to other reported experimental candidates of ADAR1 inhibitors. While further effort in the hit-to-lead optimization and validation of its anti-tumor efficacy in OSCC cells are still ongoing, the discovery of these top hits with validated binding affinity and functional efficacy assures a promising outlook of our drug discovery strategy.