Characterization of the Activities of Dinuclear Thiolato-Bridged Arene Ruthenium Complexes against Toxoplasma gondii

Abstract

The in vitro effects of 18 dinuclear thiolato-bridged arene ruthenium complexes (1 monohiolato compound, 4 dithiolato compounds, and 13 trithiolato compounds), originally designed as anticancer agents, on the apicomplexan parasite Toxoplasma gondii grown in human foreskin fibroblast (HFF) host cells were studied. Some trithiolato compounds exhibited antiparasitic efficacy at concentrations of 250 nM and below. Among those, complex 1 and complex 2 inhibited T. gondii proliferation with 50% inhibitory concentrations (IC₅₀s) of 34 and 62 nM, respectively, and they did not affect HFFs at dosages of 200 μM or above, resulting in selectivity indices of >23,000. The IC₅₀s of complex 9 were 1.2 nM for T. gondii and above 5 μM for HFFs. Transmission electron microscopy detected ultrastructural alterations in the matrix of the parasite mitochondria at the early stages of treatment, followed by a more pronounced destruction of tachyzoites. However, none of the three compounds applied at 250 nM for 15 days was parasiticidal. By affinity chromatography using complex 9 coupled to epoxy-activated Sepharose followed by mass spectrometry, T. gondii translation elongation factor 1α and two ribosomal proteins, RPS18 and RPL27, were identified to be potential binding proteins. In conclusion, organometallic ruthenium complexes exhibit promising activities against Toxoplasma, and the potential mechanisms of action of these compounds as well as their prospective applications for the treatment of toxoplasmosis are discussed.

Keywords: Toxoplasma gondii; affinity chromatography; electron microscopy; in vitro culture; mitochondrion; ruthenium complex; toxoplasmosis; translation elongation factor 1α.

FIG 1

Structures of complexes 1 to 18 used in this study. Note that compounds 1, 2, and 9 were further characterized.

FIG 2

Ultrastructure of T. gondii tachyzoites grown in HFFs. (A) Low-magnification view of infected HFFs. The boxed area is shown at a higher magnification in panel B. Tachyzoites proliferate within a parasitophorous vacuole surrounded by a parasitophorous vacuole membrane. nuc, nucleus; dg, dense granules; mic, micronemes; rop, rhoptries; mito, mitochondrion; con, conoid. The boxed area in panel B shows the mitochondrial matrix and is enlarged in panel C. Bars = 1.8 μm (A), 0.3 μm (B), and 0.1 μm (C).

FIG 3

Ultrastructure of T. gondii tachyzoites grown in HFFs and treated with ruthenium complexes 1 and 9. Treatments were carried out using 200 nM complex 1 (A to D) or complex 9 (E, F). (A) Low-magnification view of parasites treated with complex 1 for 6 h. The boxed areas are enlarged in panels B and C. (D) Parasites exposed to complex 1 for 48 h. (E, F) Parasites exposed to complex 9 for 24 h. Note the distinct alterations in the mitochondria (mito) in panels B, C, and E and the still intact host cell mitochondria (h-mito) in panel D. The boxed area in panel F is enlarged in panel E. Bars = 1 μm (A, F), 0.4 μm (B, C, E), and 0.8 μm (D).

FIG 4

Compound 9 inhibits T. gondii tachyzoite proliferation only when it is applied early during infection. HFF monolayers grown in 96-well plates were treated with complex 9 (100 nM) either 10 min prior to infection or 1 h, 5 h, or 24 h after infection with T. gondii tachyzoites. The proliferation of tachyzoites was measured after 2 days of culture by beta-galactosidase assay, as described in Materials and Methods. OD570, optical density at 570 nm.

FIG 5

Identification of complex 9-binding proteins. (A) SDS-PAGE and silver staining of tandem (the column with mock epoxy-Sepharose and the column with compound 9-Sepharose) affinity chromatography of a protein extract prepared from T. gondii-infected HFFs. The soluble extract and the nonbinding fraction (flowthrough) are shown on the left, followed by wash and eluate fractions of the column with mock epoxy-Sepharose and the column with complex 9-Sepharose. The two arrows point to the two bands of 50 kDa and 20 kDa, which were cut out and analyzed by LC-MS/MS. (B) Amino acid sequence of the 50-kDa band identified as TgEF1α. The peptide sequences identified by LC-MS/MS are underlined.