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

Abstract

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.

Keywords: Biotin; DNA repair; Mitoxantrone; Probe; RAD52.

Figure 1.

Examples of biotinylated or clickable small molecule probes used for mechanistic studies.

Figure 2.

Isolating RAD52 via MXP pulldown in RAD52-overexpressing E. coli cell lysate. A. SDS-PAGE gel stained with SYPRO^™ Ruby dye showing supernatant (S) and elution € samples from a pulldown using MXP. A protein standard of purified RAD52 (white arrow at approximately 48 kDa) and purified RPA (white arrowheads representing the heterotrimer at approximately 70 kD, 32 kDa, 14 kDa) were included for reference. B. Automated western blot results of the supernatant and elution using RAD52 antibody and RPA antibody for protein detection. Purified RPA was spiked into the E. coli lysate to determine if the MXP would interact with RPA or RAD52. RPA was only observed in the supernatant and not the elution, while RAD52, an identified protein-binding partner of MX, was detected in both the supernatant and the elution.

Scheme 1.

Reagents and conditions: (i) NaH, propargyl alcohol, DMF, 0°C to rt, 15 h; (ii) (BOC)₂O, THF:EtOH (1:1), rt, 16 h; (iii) NaH, 2, DMF, 0°C to rt, 15 h; (iv) 4M HCl in Dioxane, DCM, rt, 12 h; (v) AlCl₃, NaCl, 200°C, 2 h; (vi) 6, DIPEA, DMF, 50°C, 6 h; (vii) 3, DMF, 50°C, 6 h; (viii) Biotin-PEG3-azide, Sodium abcorbate, TBTA, CuSO₄.5H₂O, DMSO, H₂O, ^t-BuOH, rt, 8 h; (ix) NaH, propargyl alcohol, DMF, 0°C to rt, 15 h; (x) Biotin-PEG3-azide, Sodium abcorbate, TBTA, CuSO₄.5H₂O, DMSO, H₂O, ^t-BuOH, rt, 8 h.