APOBEC3 enzymes form part of the innate immune system by deaminating cytosine to uracil in single-stranded DNA (ssDNA) and thereby preventing the spread of pathogenic genetic information. However, APOBEC mutagenesis is also exploited by viruses and cancer cells to increase rates of evolution, escape adaptive immune responses, and resist drugs. This raises the possibility of APOBEC3 inhibition as a strategy for augmenting existing antiviral and anticancer therapies. Here we show that, upon incorporation into short ssDNAs, the cytidine nucleoside analogue 2′-deoxyzebularine (dZ) becomes capable of inhibiting the catalytic activity of selected APOBEC variants derived from APOBEC3A, APOBEC3B, and APOBEC3G, supporting a mechanism in which ssDNA delivers dZ to the active site. Multiple experimental approaches, including isothermal titration calorimetry, fluorescence polarization, protein thermal shift, and nuclear magnetic resonance spectroscopy assays, demonstrate nanomolar dissociation constants and low micromolar inhibition constants. These dZ-containing ssDNAs constitute the first substrate-like APOBEC3 inhibitors and, together, comprise a platform for developing nucleic acid-based inhibitors with cellular activity.
Bibliographical noteFunding Information:
*E-mail: firstname.lastname@example.org. *E-mail: email@example.com. *E-mail: firstname.lastname@example.org. ORCID Hideki Aihara: 0000-0001-7508-6230 Vyacheslav V. Filichev: 0000-0002-7383-3025 Daniel A. Harki: 0000-0001-5950-931X Elena Harjes: 0000-0002-3643-9432 Author Contributions #M.V.K., F.M.B., S.H., and H.A.M.S. contributed equally to this work. Funding V.V.F., E.H., G.B.J., M.V.K., and S.H. are grateful for the financial support provided by Worldwide Cancer Research (Grant 16-1197), the Massey University Research Fund (MURF 2015, 7003), and the Institute of Fundamental Sciences of Massey University. This work was also supported by the National Institutes of Health (R01-GM110129 to D.A.H. and R.S.H. and R01-GM118000 to R.S.H., H.A., and D.A.H.), the University of Minnesota (UMN) Masonic Cancer Center (SPORE-Program Project planning seed grant to D.A.H., R.S.H., and H.A.), the UMN College of Biological Sciences, and the Prospect Creek Foundation (award to D.A.H. and R.S.H.). F.M.B. is a recipient of the graduate assistance Ph.D. scholarship awarded by the Institute of Fundamental Sciences, Massey University. The UMN Office of the Vice President for Research is gratefully acknowledged (Equipment Grant-in-Aid for purchase of a BioAutomation MerMade DNA synthesizer). R.S.H. is the Margaret Harvey Schering Land Grant Chair for Cancer Research, a Distinguished University McKnight Professor, and an Investigator of the Howard Hughes Medical Institute. The authors thank the International Mobility Fund from the Royal Society of New Zealand (IMF-Mau140) for sponsoring the visit of R.S.H. to Massey University to start the collaboration as well as Massey University Research Funding (international visitor, 2016) for sponsoring the visit of D.A.H. to Massey University to design this research. Notes The authors declare the following competing financial interest(s): D.A.H. and R.S.H. are co-founders, shareholders, and consultants of ApoGen Biotechnologies, Inc. H.A. is a consultant of ApoGen Biotechnologies, Inc.