The formation of a novel oxime adduct with abasic (AP) sites in duplex DNA with the alkoxyamine O-benzylhydroxylamine
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[EMBARGOED UNTIL 12/01/2026] Apurinic/apyrimidinic (AP) sites are among the most abundant DNA lesions, arising from spontaneous base loss or enzymatic activity during base excision repair (BER). These sites exist in equilibrium between a ring-closed hemiacetal and a reactive ring-open aldehyde, the latter serving as a potential target for covalent modification. Methoxyamine (MX), an aminooxy compound, has been investigated as a pharmacological agent capable of forming oxime adducts with AP sites to inhibit AP endonuclease 1 (APE1), a critical enzyme in BER. However, MX-AP adducts are incompletely resistant to APE1 processing, limiting their therapeutic potential. In this work, we report the formation of a novel oxime adduct between O-benzylhydroxylamine (OBHA) and AP sites in duplex DNA. Using gel electrophoresis and MALDI-TOF mass spectrometry, we confirmed covalent adduct formation under physiological conditions. Enzymatic assays demonstrated that, unlike MX, OBHA-AP adducts are completely resistant to APE1 cleavage, even at elevated enzyme concentrations and extended incubation times. Structural characterization revealed altered electrophoretic mobility consistent with a bulky, stable modification at the lesion site. These findings establish OBHA as a potent inhibitor of APE1-mediated repair of AP sites, highlighting its potential utility in synthetic lethality strategies for cancer therapy. By irreversibly blocking BER at abasic lesions, OBHA may enhance the cytotoxicity of DNAdamaging agents and overcome resistance mechanisms associated with elevated APE1 activity. This study provides mechanistic insight into oxime adduct formation at AP sites and identifies OBHA as a promising candidate for further evaluation in DNA repairtargeted therapeutics.
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M.S.