8-Azaadenosine and 8-Chloroadenosine are not Selective Inhibitors of ADAR

Abstract

The RNA editing enzyme ADAR is an attractive therapeutic target for multiple cancers. Through its deaminase activity, ADAR edits adenosine to inosine in double-stranded RNAs. Loss of ADAR in some cancer cell lines causes activation of the type I IFN pathway and the PKR translational repressor, leading to inhibition of proliferation and stimulation of cell death. As such, inhibition of ADAR function is a viable therapeutic strategy for many cancers. However, there are no FDA-approved inhibitors of ADAR. Two small molecules have been previously shown to inhibit ADAR or reduce its expression: 8-azaadenosine and 8-chloroadenosine. Here we show that neither molecule is a selective inhibitor of ADAR. Both 8-azaadenosine and 8-chloroadenosine show similar toxicity to ADAR-dependent and -independent cancer cell lines. Furthermore, the toxicity of both small molecules is comparable between cell lines with either knockdown or overexpression of ADAR, and cells with unperturbed ADAR expression. Treatment with neither molecule causes activation of PKR. Finally, treatment with either molecule has no effect on A-to-I editing of multiple ADAR substrates. Together, these data show that 8-azaadenosine and 8-chloroadenosine are not suitable small molecules for therapies that require selective inhibition of ADAR, and neither should be used in preclinical studies as ADAR inhibitors. Significance: ADAR is a good therapeutic target for multiple cancers; neither 8-chloroadenosine nor 8-azaadenosine are selective inhibitors of ADAR.

Structurally Specific Z-DNA Proteolysis Targeting Chimera Enables Targeted Degradation of Adenosine Deaminase Acting on RNA 1

“…To comprehensively assess the antiproliferative effects of Z-PROTAC across diverse cancer cell lines, we conducted a series of assays, including cell growth curves, CCK-8 assays, and colony formation assays, as shown in Figure C–F. Upon transfection of different cell lines with 5 μg/mL Z-DNA ODN and 9c , our findings revealed a potent inhibitory effect of 9c on cancer cells in comparison to the reported ADAR1 inhibitors 8-Aza − , and Z-DNA ODN, as evident from the cell growth curve depicted in Figure C. Notably, while normal cells (MRC5 and LF1) exhibited some degree of inhibition, this effect was relatively weaker than that observed in cancer cells (Figure S13).…”

“…11−14 However, the inhibitor has not been fully demonstrated to selectively inhibit ADAR1's biological functions, in part due to the presence of various isoforms containing the same A to I deaminase catalytic domain. 13 Given ADAR's multifaceted domain structure, the reported inhibitor only targets the catalytic editing-dependent domain, leaving the other editing-independent functionalities untouched within the cellular milieu that compromises its potential therapeutic capacity. 1 Thus, a relatively more compelling strategy has emerged in the form of targeting ADAR1 for degradation, potentially eradicating the presence of the protein rather than merely inhibiting it.…”

Inhibiting DNA methylation and RNA editing upregulates immunogenic RNA to transform the tumor microenvironment and prolong survival in ovarian cancer

“…There are several ADAR1 inhibitors currently in development for use against cancer. Although there are no known, selective ADAR1 inhibitors commercially available at the moment, 84 future directions include testing these inhibitors as they become available.…”

RNA epigenetic modifications in ovarian cancer: The changes, chances, and challenges

“…Virtual screening and in vitro enzymatic assay identified compound 17 as a PUS7 inhibitor which blocks the growth of glioblastoma stem cells (GSC; Cui et al, 2021). For A‐to‐I editing, 8‐azaadenosine and 8‐chloroadenosine have been suggested as ADAR1 inhibitors, but they are not considered for clinical use due to low specificity (Cottrell et al, 2021; Goncharov et al, 2022). As our understanding of RNA modification regulators improves, more targeted inhibitors with clinical potential can be identified.…”