Abstract
Catalytic disulfide cleavage is an essential mechanism in cells for protein folding and synthesis; however, the detailed enzymatic mechanism of disulfide bond cleavage in xenobiotics is not well understood. This report describes an enzymatic mechanism of disulfide bond cleavage in xenobiotic small molecules and antibody drug conjugate (ADC) linkers. The chemically stable disulfide bonds in substituted disulfide-containing pyrrolobenzodiazepine (PBD) monomer prodrugs in presence of glutathione or cysteine were found to be unstable in incubations in whole blood of humans and rats. Thioredoxin (TRX) and glutaredoxin (GRX) were the enzymes determined to be involved in this reaction. For a diverse set of drug-linker conjugates, TRX generated cleaved products in the presence of TRX-reductase and NADPH that are consistent with catalytic disulfide cleavage and linker immolation. GRX was less rigorously studied mainly due to availability and lower activity than TRX but its role in the catalytic cleavage was also confirmed for this set of compounds. Collectively, these in vitro experiments demonstrate that TRX, as well as GRX, can catalyze the cleavage of disulfide bonds in both small molecules and ADC linkers.
SIGNIFICANCE STATEMENT N/A
- drug design
- enzyme mechanism
- glutathione
- HPLC
- mass spectrometry/MS
- metabolite identification
- prodrugs
- structure-activity relationships
- The American Society for Pharmacology and Experimental Therapeutics