Albumin Conjugates of Thiosemicarbazone and Imidazole-2-thione Prochelators: Iron Coordination and Antiproliferative Activity

Abstract

The central role of iron in tumor progression and metastasis motivates the development of iron-binding approaches in cancer chemotherapy. Disulfide-based prochelators are reductively activated upon cellular uptake to liberate thiol chelators responsible for iron sequestration. Herein, a trimethyl thiosemicarbazone moiety and the imidazole-2-thione heterocycle are incorporated in this prochelator design. Iron binding of the corresponding tridentate chelators leads to the stabilization of a low-spin ferric center in 2 : 1 ligand-to-metal complexes. Native mass spectrometry experiments show that the prochelators form stable disulfide conjugates with bovine serum albumin, thus affording novel bioconjugate prochelator systems. Antiproliferative activities at sub-micromolar levels are recorded in a panel of breast, ovarian and colorectal cancer cells, along with significantly lower activity in normal fibroblasts.

Keywords: albumin; cancer; imidazole-2-thione; iron chelation; thiosemicarbazone.

Figure 1.

Structures of reported iron chelators and disulfide-based prochelators.

Figure 2.

Disulfide-based prochelators characterized in this study.

Figure 3.

Crystal structures of [Fe(244mTC−H)₂]⁺ (left) and [Fe(IT1−H)₂]⁺ (right) showing a partial atom labeling scheme. Thermal ellipsoids are scaled to the 50% probability level. Carbon-bound hydrogen atoms in calculated positions and [BF₄]^– counter ions are not shown (CCDC, left: 2060902, right: 2060901)

Figure 4.

EPR spectra of [Fe(244mTC−H)₂]⁺ (top) and [Fe(IT1−H)₂]⁺ (bottom) in frozen DMSO/water solution (70/30%, v/v). The pH of the solution was estimated at 7.0 using colorimetric indicator strips with a sensitivity of 0.5 pH units. Experimental conditions: microwave frequency, 9.4 GHz; microwave power, 2 mW; magnetic field modulation amplitude, 0.2 mT; temperature, 77 K.

Figure 5.

Covalent modifications of BSA by disulfide prochelators as monitored by native mass spectrometry. Spectra are shown on the left with annotated charge states. Corresponding deconvolved mass distributions are shown on the right. Solutions of BSA (1.0 μM) in aqueous ammonium acetate (0.2 M, pH 6.8) were incubated in the presence of prochelators (244mTC-S)₂ (panels a, b), (IT1-S)₂ (c, d), (IT2-S)₂ (e, f), or control thioether TE1 (g, h). The concentration of all tested compounds was 10 μM, and the incubations were conducted at room temperature for 24 h.