A Latent DNA–Protein Cross-Link Agent as a Chemical Adjuvant for Photodynamic Therapy

Photodynamic therapy (PDT) induces cell death in part through singlet oxygen (1O2)-mediated oxidation of DNA and other biomolecules. However, PDT effectiveness can be compromised in cancer cells by increased capacity for base excision repair (BER) of DNA lesions and/or proteostasis-associated tolerance of oxidative protein damage. We envisioned that using 1O2 to create DNA–protein cross-links (DPCs) would reduce the protective effect of BER overexpression and help overcome cellular PDT-resistance. Here, we describe a furan-containing adjuvant (MCF) that yields DPCs under PDT conditions. As a half-nitrogen mustard, MCF alkylates DNA and subsequently undergoes 1O2-mediated furan oxidation to generate a DNA-tethered 1,4-dicarbonyl electrophile that reacts with protein nucleophiles to form DPCs. MCF-dependent DPC formation was observed in nucleosome core particles and human cells under PDT conditions. DPC formation in NCPs and human cells increased in D2O, consistent with 1O2-mediated oxidation of MCF DNA adducts. DPC levels were also elevated in cells lacking functional SPRTN, a protease involved in DPC repair. Photochemical activation of MCF increased cellular toxicity. This effect was amplified by SPRTN truncation or proteasome inhibition, implicating proteolytic DPC processing in protection from MCF-induced phototoxicity. Importantly, MCF retained its cytotoxic enhancement in cells that overexpress the BER protein APE1. MCF also substantially reduced the detectable survival advantage of a HeLa cell population selected for resistance to PDT conditions. These results support MCF as a latent DPC precursor that potentiates PDT in cells with reduced sensitivity to oxidative photodamage.