Click chemistry promises a simple method to produce pyrrolo[2,1-c][1,4]benzodiazepine (PBD)-based DNA binders. DNA binding and covalent crosslinking capabilities have been validated for ‘clicked’ PBD heterodimers using thermal denaturation analysis, mass spectrometry and DNase I footprinting. One dimer induced strong DNA cross-linking and cellular cytotoxicity, indicating the future potential for dimerization in living cells.

Abstract

The covalent cross-linking of DNA duplex strands by small molecule drugs is a validated mechanism in anticancer therapy. The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) have been established as potent DNA binders that can achieve inter- and intrastrand DNA cross-links when dimerized, and have been used as cytotoxic payloads in multiple antibody-drug conjugates (ADCs). In this study, we explore the potential of click chemistry to obtain PBD heterodimers. The heterodimers D1–D4 were prepared by copper catalyzed or strain-promoted azide-alkyne cycloadditions (SPAACs) from the corresponding monomers M1 – M4 and MbA. The interactions of monomers and dimers with DNA were evaluated by DNA thermal denaturation analysis, a newly developed mass spectrometry-based method for the detection of single stand and double strand modifications, and by DNase I footprinting. All methods demonstrated the DNA binding and cross-linking capabilities of D1. Testing for cytotoxicity in three cell lines revealed that the monomers M1 and M4 and the dimers D1 and D4 were the most potent. The click chemistry approach opens an easy access to larger libraries of PBD-based DNA binders, including the option for dimerization in live cells.