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bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.05.611238
preprint
1
Article
Next generation APOBEC3 inhibitors: Optimally designed for potency and nuclease stability
Hedger Adam K. http://orcid.org/0000-0003-1529-2598

Myint Wazo
Lee Jeong Min
Suchenski-Loustaunau Diego
Balachandran Vanivilasini
Shaqra Ala M.
Kurt-Yilmaz Nese http://orcid.org/0000-0002-5036-676X

Watts Jonathan K.
Matsuo Hiroshi
Schiffer Celia A.
06 9 2024
2024.09.05.611238https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.09.05.611238
nihpp-2024.09.05.611238.pdf
ABSTRACT

APOBEC3 (or A3) enzymes have emerged as potential therapeutic targets due to their role in introducing heterogeneity in viruses and cancer, often leading to drug resistance. Inhibiting these enzymes has remained elusive as initial phosphodiester (PO) linked DNA based inhibitors lack stability and potency. We have enhanced both potency and nuclease stability, of 2′-deoxy-zebularine (dZ), substrate-based oligonucleotide inhibitors for two critical A3’s: A3A and A3G. While replacing the phosphate backbone with phosphorothioate (PS) linkages increased nuclease stability, fully PS-modified inhibitors lost potency (1.4-3.7 fold) due to the structural constraints of the active site. For both enzymes, mixed PO/PS backbones enhanced potency (2.3-9.2 fold), while also vastly improving nuclease resistance. We also strategically introduced 2′-fluoro sugar modifications, creating the first nanomolar inhibitor of A3G-CTD2. With hairpin-structured inhibitors containing optimized PS patterns and LNA sugar modifications, we characterize the first single-digit nanomolar inhibitor targeting A3A. These extremely potent A3A inhibitors, were highly resistant to nuclease degradation in serum stability assays. Overall, our optimally designed A3 oligonucleotide inhibitors show improved potency and stability, compared to previous attempts to inhibit these critical enzymes, opening the door to realize the therapeutic potential of A3 inhibition.
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