Examination of a Chimeric Bis-Electrophile for Selective DNA-Protein Cross-Linking and Mechlorethamine Reveals an Unknown Source of Nitrogen Mustard Cytotoxicity.

DNA-protein cross-links (DPCs) are cytotoxic lesions whose study in cells is complicated by the lack of exogenous agents that produce them selectively over DNA-DNA interstrand cross-links (ICLs). The synthesis and reactivity of a chimeric bis-electrophile (MEBAC) that is comprised of a highly reactive alkylating agent and a lysine selective -ethynyl benzaldehyde is described. DPC formation in nucleosome core particles (NCPs) by MEBAC is >40-times greater than that of ICLs. Cell viability experiments and the single cell Comet assay are consistent with NCP reactivity. Compared to a nitrogen mustard (mechlorethamine, MCE) MEBAC produces higher DPC yields and lower ICL yields in NCPs and in cells at comparable cytotoxicity. Cell viability experiments show that while DPCs from MEBAC are repaired by the metalloprotease SPRTN and the proteasome, only the former repairs such lesions produced by MCE. The inability of the proteasome to repair DPCs in MCE-treated cells likely contributes to the cytotoxicity of the nitrogen mustard. Proteomic analysis identifies several cysteine-rich E3 ligases involved in ubiquitination that are cross-linked to DNA in MCE-treated but not MEBAC-treated cells and suggests a chemical basis for why DPCs produced by the nitrogen mustard are not repaired by the proteasome. This investigation reveals a previously unknown source of nitrogen mustard cytotoxicity and indicates that MEBAC and molecules like it will be useful tools for studying DPCs in cells.