Abstract
Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks.
Highlights
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a family of sequence-selective DNA minor-groove binding agents [1,2,3,4,5] which are growing in importance due to their use as payloads in Antibody-Drug Conjugates (ADCs) [6,7,8]
The naturally occurring PBDs produced by Streptomyces and Micrococcus species are monomeric and form singly-alkylated DNA-adducts, whereas the synthetic PBD Dimers consist of two PBD units joined through a C8/C80-linker and can form interstrand or intrastrand DNA cross-links in addition to mono-adducts [9,10,11,12]
The DNA-binding affinity and cytotoxicity of PBD Monomers has been enhanced by attaching heterocyclic units to the C8-position (e.g., GWL-78, 3, Fig 1), and molecules of this type have been shown to inhibit the binding of certain transcription factors to their consensus
Summary
The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a family of sequence-selective DNA minor-groove binding agents [1,2,3,4,5] which are growing in importance due to their use as payloads in Antibody-Drug Conjugates (ADCs) [6,7,8]. One PBD Dimer, SJG-136 (2, Fig 1), successfully completed Phase I clinical trials [13,14,15], and reached Phase II evaluation in ovarian and hematological cancers. The DNA-binding affinity and cytotoxicity of PBD Monomers has been enhanced by attaching heterocyclic units to the C8-position (e.g., GWL-78, 3, Fig 1), and molecules of this type have been shown to inhibit the binding of certain transcription factors to their consensus. Paul Jackson (PJ) received the balance of his salary from King’s College London, and Julia Mantaj was a self-funded PhD student during most of the period of the study
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