Journal: iScience

Article Title: Identification of ADAR1i-124: The first effective A-to-I RNA editing inhibitor with promising cancer therapeutic potential

doi: 10.1016/j.isci.2025.114615

Figure Lengend Snippet: A-to-I RNA editing inhibition and direct binding to ADAR1 by ADAR1i-124 (A) ADAR1i-124 was selected from three compounds with ADAR1 inhibitory activity. (B) In vitro editing assay using purified FLAG-ADAR1p150 and a synthetic dsRNA containing six target sites (1–6 position) was conducted in triplicate ( n = 3) with varying concentrations of ADAR1i-124. The sense RNA strand was analyzed by RT-PCR and cDNA sequencing. The efficiency of A-to-I editing (A-to-G cDNA change) was determined by the guanosine peak (black) replacing the adenosine peak (green) in sequencing chromatograms. (C) Inhibition kinetics analysis resulted in IC 50 of 8.8 μM and 6.5 μM at position 2 and 5, respectively. Data: mean ± SD ( n = 3, technical replicates). (D) No editing inhibitory activity was detected for ADAR2 by in vitro editing assay conducted at 300 μM. (E) SPR analysis for binding of ADAR1i-124 to ADAR1p110, ADAR2, and a negative control protein GAPDH. (F) Docking simulation of ADAR1i-124 into the catalytic center pocket of a cryo-EM-resolved ADAR1. ADAR1i-124 fits well within the active site near the Zn 2+ ion, forming close contacts with key residues, including E912 (left). Its nitrile group is nestled within a tight pocket formed by two adjacent loops (middle). Structural alignment with the ADAR1 deaminase domain bound to dsRNA reveals that ADAR1i-124 blocks adenosine access to the active site (right). (G) Docking simulation with the ADAR2 catalytic center shows a conserved binding mode (left). However, structural overlay of ADAR1 (blue) and ADAR2 (green) highlights steric interference from ADAR2 residue R455, which prevents optimal fitting of ADAR1i-124 into the ADAR2 active site (right).

Article Snippet: The aqueous-phase from the TRIzol extraction was then purified using the RNA Clean and Concentrator-5 kit (Zymo Research, R1015) and eluted into 20 μl of elution buffer.

Techniques: Inhibition, Binding Assay, Activity Assay, In Vitro, Purification, Reverse Transcription Polymerase Chain Reaction, Sequencing, Negative Control, Cryo-EM Sample Prep, Residue