Oral Presentation Crystal36-AXAA Conference 2026

Towards preventing evolution of therapeutic resistance in cancers: design, synthesis, and structural and functional characterisation of nanomolar non-cytotoxic inhibitors of DNA mutator enzymes APOBEC3A and APOBEC3B (144456)

Geoffrey B Jameson 1 , Stefan Harjes 1 , Harikrishnan M Kurup 1 , Amanda C Reiffer 2 , Reuben S Harris 3 , Jana Filitcheva 1 , Tracy K Hale 1 , Elena Harjes 1 , Vyacheslav V Filichev 1
  1. School of Food Technology and Natural Sciences, Massey University, Palmerston North, MANAW, New Zealand
  2. Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota, USA
  3. Department of Biochemistry and Structural Biology, University of Texas Health Science, San Antonio, Texas, USA

Tumour cells develop drug resistance when mutations accumulate in their genome that drive phenotypic diversity. Normally, cytosine deaminases APOBEC3A-H1 are part of antiviral defence, mutating C-to-U on single-stranded DNA. However, many drug-resistant tumours exhibit mutagenic signatures associated with increased activity of specifically APOBEC3A and APOBEC3B (A3A/B), which recognise only a TC-motif in a minimal 4-nucleotide recognition sequence.2 Therefore, inhibition of A3A/B working alongside existing front-line anticancer therapies could potentially extend their efficacy. Within the genome, the loops of DNA-hairpin structures are known hotspots for mutagenesis.3

Here we reveal through X-ray structures of A3A in complex with modified DNA stem-loop (hairpin) inhibitors and substrates the structural basis of potent inhibition of A3A by hairpin-DNA bearing 2’-deoxy-5-fluorozebularine (Ki=7.8 ± 0.8 nM) (and other moieties4). We have recently explored design space around the stem and the loop of the hairpin DNA to (i)  increase hydrophobicity for membrane transfer without transfection reagents, (ii) prevent digestion by cellular nucleases through selective phosphorothiolation of phoshate linkers , (iii) perturb loop dynamics for greater potency, (iv) reduce cytotoxicity to negligible levels, and (v) modify the inhibitor moiety itself to better mimic the transition-state.

Importantly, nuclease-resistant derivatives of these inhibitors maintain in-vitro potency against A3A and potently inhibit mutagenic activity of A3A in cancer.5

  1. Harris, R.S., et al. (2003) DNA deamination mediates innate immunity to retroviral infection. Cell 113: 803-809.
  2. Law, E.K., et al. (2020) APOBEC3A catalyzes mutation and drives carcinogenesis in vivo. J Exp Med 217: e20200261.
  3. Langenbucher, et al. (2021) An extended APOBEC3A mutation signature in cancer. Nat Commun 12: 1602.
  4. Kurup, H.M., et al. (2022) Design, synthesis, and evaluation of a cross-linked oligonucleotide as the first nanomolar inhibitor of APOBEC3A. Biochemistry 61: 2568-2578.
  5. Harjes, S., …, Hale, T.K., Filichev, V.V., Harjes, E., Harris, R.S., Jameson, G.B. (2023). Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A. Nat Communications, 14: 6832.