A Chemoproteomics Approach to Determine the Mechanism of Testicular Toxicity for the Bruton's Tyrosine Kinase Inhibitor CC-292

Abstract

During drug development, potential safety issues can occur at any time. Understanding the cause of a toxicity can help with deciding on how to advance the drug development program. Chemoproteomics provides a way to help understand the cause of a toxicity wherein the affected tissue is accessible and can be probed with a covalently binding compound that is analogous to the offending drug. In this case, N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292), a covalently binding Bruton's tyrosine kinase inhibitor, had produced testicular toxicity in rodents. Experiments were conducted using a CC-292 analog that could be chemically modified with biotin to probe rodent testes homogenates for potential binding sites that were subsequently recovered with avidin beads. These biotin-tagged proteins undergo trypsin digest on the avidin beads to yield peptides that are identified using mass spectrometry. Two proteins were identified from the testicular homogenates of both rats and mice, namely retinal dehydrogenase 1 (ALDH1A1) and retinal dehydrogenase 2 (ALDH1A2). Literature confirmed a link between inhibition of these enzymes and testicular toxicity. Subsequently, molecular modeling was used to demonstrate that CC-292 can be docked into both the nicotinamide adenine dinucleotide and retinal binding pockets of the analogous human ALDH1A2 enzyme. These data suggest that the off-target binding site for CC-292 on retinal dehydrogenase enzymes may provide a mechanistic explanation to the testicular toxicity observed in rodents and that there may be a potential concern for human male fertility. SIGNIFICANCE STATEMENT: Biotinylated covalently binding drug analogues are used to enrich bound proteins from tissue homogenates wherein toxicity was observed in rodents. Bound proteins were subsequently identified by mass spectroscopy. Competition of the analog binding with the parent inhibitor itself and three-dimensional molecular modeling were used to establish a likely link between the off-targets of CC-292, ALDH1A1, and ALDH1A2 with potential testicular toxicity.