Design, synthesis, and evaluation of a mitoxantrone probe (MXP) for biological studies

Mitoxantrone (MX) is a robust chemotherapeutic with well-characterized applications in treating certain leukemias and advanced breast and prostate cancers. The canonical mechanism of action associated with MX is its ability to intercalate DNA and inhibit topoisomerase II, giving it the designation of a topoisomerase II poison. Years after FDA approval, investigations have unveiled novel protein-binding partners, such as methyl-CpG-binding domain protein (MBD2), PIM1 serine/threonine kinase, RAD52, and others that may contribute to the therapeutic profile of MX. Moreover, recent proteomic studies have revealed MX's ability to modulate protein expression, illuminating the complex cellular interactions of MX. Although mechanistically relevant, the differential expression across the proteome does not address the direct interaction with potential binding partners. Identification and characterization of these MX-binding cellular partners will provide the molecular basis for the alternate mechanisms that influence MX's cytotoxicity. Here, we describe the design and synthesis of a MX-biotin probe (MXP) and negative control (MXP-NC) that can be used to define MX's cellular targets and expand our understanding of the proteome-wide profile for MX. In proof of concept studies, we used MXP to successfully isolate a recently identified protein-binding partner of MX, RAD52, in a cell lysate pulldown with streptavidin beads and western blotting.

An 8-step synthesis was used to generate a biotinylated-mitoxantrone probe (MXP).A pulldown of MXP demonstrated selectivity for RAD52, but not Replication Protein A.Western blot confirmed the identity of the isolated protein, RAD52.