Selective Inhibition of ADAR1 Using 8-Azanebularine-Modified RNA Duplexes

Abstract

Adenosine deaminases acting on RNA (ADARs) are RNA editing enzymes that catalyze the hydrolytic deamination of adenosine (A) to inosine (I) in dsRNA. In humans, two catalytically active ADARs, ADAR1 and ADAR2, perform this A-to-I editing event. The growing field of nucleotide base editing has highlighted ADARs as promising therapeutic agents while multiple studies have also identified ADAR1’s role in cancer progression. However, the potential for site-directed RNA editing as well as the rational design of inhibitors is being hindered by the lack of detailed molecular understanding of RNA recognition by ADAR1. Here, we designed short RNA duplexes containing the nucleoside analog, 8-azanebularine (8-azaN), to gain insight into molecular recognition by the human ADAR1 catalytic domain. From gel shift and in vitro deamination experiments, we validate ADAR1 catalytic domain’s duplex secondary structure requirement and present a minimum duplex length for binding (14 bp, with 5 bp 5′ and 8 bp 3′ to editing site). These findings concur with predicted RNA-binding contacts from a previous structural model of the ADAR1 catalytic domain. Finally, we establish that neither 8-azaN as a free nucleoside nor a ssRNA bearing 8-azaN inhibits ADAR1 and demonstrate that the 8-azaN-modified RNA duplexes selectively inhibit ADAR1 and not the closely related ADAR2 enzyme.

Chemical Modifications in RNA: Elucidating the Chemistry of dsRNA-Specific Adenosine Deaminases (ADARs)

“…The growing evidence linking ADAR1 to cancer progression has triggered the search for potent and targeted inhibitors of this enzyme. ,, Unfortunately, the lack of high-resolution structures for ADAR1 has hampered these research endeavors. Our more detailed understanding of ADAR2 can be attributed to the successful utilization of chemically modified substrates, with the particular use of 8-azaN. ,, We then sought to determine whether we could similarly employ 8-azaN-modified RNA duplexes for probing ADAR1-RNA interactions.…”

“…Aza substitution at C8 of nebularine increases the susceptibility of hydration across N1–C6, while the absence of a good leaving group at C6 allows for the mechanistic trapping of the covalent hydrate . Therefore, 8-azaN-modified RNA duplexes facilitate the formation of active site-directed and high-affinity ADAR-RNA complexes suitable for biochemical and biophysical investigations with ADARs. ,, …”

“…Importantly, mutating K594 to alanine (K594A) appreciably reduced ADAR2-D’s editing activity, suggesting the significance of this contact for substrate recognition and editing . A lysine residue is also conserved at this position in ADAR1 (K1120) and is predicted to make a similar hydrogen bonding contact in the same location of the RNA duplex based on a Rosetta homology model of the ADAR1 deaminase domain (ADAR1-D, Figure A). , Indeed, experiments using known ADAR1 and ADAR2 substrates with shortened duplex 3′ to the editing site (≤6 bp) displayed a substantial loss in binding or editing activity for both enzymes. , These observations imply that both ADARs require an RNA duplex with at least 8 bp in the 3′ direction relative to the edited base for efficient editing.…”

“…These enzymes are composed of a deaminase domain and dsRNA-binding domains (dsRBDs), typical of dsRNA binding or modifying proteins (Figure C) . However, existing experimental data support that the deaminase domains of both enzymes also require a duplex RNA structure for substrate binding. , ADAR1 also contains an additional nucleic acid binding domain for Z-DNA/RNA binding (Zα and Zß) and exists primarily as the two isoforms, p110 and p150 (Figure C) . A nuclear localization signal (NLS) near the dsRBD3 of ADAR1 and at the N-terminus of ADAR2 allows these proteins to localize in the nucleus.…”

“…These structural changes can impact RNA stability and its interaction with other molecules, such as miRNAs and proteins involved in RNA degradation and immune responses (Figure D). ,, Mutations in the ADAR genes as well as aberrant ADAR-mediated A-to-I editing have been associated with various diseases, including neurological disorders and cancer, , highlighting the functional significance of this tightly regulated RNA modification process. Moreover, the growing list of ADAR’s diverse roles in various cancer types has led to an increasing interest in the development of ADAR inhibitors as novel anticancer drugs. ,, …”