Next generation APOBEC3 inhibitors: optimally designed for potency and nuclease stability.

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 cellular stability and potency. We have enhanced both potency and nuclease stability of 2'-deoxyzebularine (dZ) substrate-based oligonucleotide inhibitors targeting two critical A3s: A3A and A3G. While replacing the phosphate backbone with phosphorothioate (PS) linkages increased nuclease stability, fully PS-modified inhibitors lost potency (up to three-fold) due to the structural constraints of the active site. For both enzymes, mixed PO/PS backbones enhanced potency (up to nine-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 locked nucleic acid (LNA) sugar modifications, we characterize the first single-digit nanomolar inhibitor targeting A3A. These extremely potent A3A inhibitors were highly resistant to nuclease degradation and crucially, restricted A3A deamination in cellulo. Overall, our optimally designed A3 oligonucleotide inhibitors show improved potency and stability compared to previous inhibitors targeting these critical enzymes, toward realizing the therapeutic potential of A3 inhibition.