In Vitro and In Vivo Activities of Sulfur-Containing Linear Bisphosphonates against Apicomplexan Parasites

Abstract

We tested a series of sulfur-containing linear bisphosphonates against Toxoplasma gondii, the etiologic agent of toxoplasmosis. The most potent compound (compound 22; 1-[(n-decylsulfonyl)ethyl]-1,1-bisphosphonic acid) is a sulfone-containing compound, which had a 50% effective concentration (EC₅₀) of 0.11 ± 0.02 μM against intracellular tachyzoites. The compound showed low toxicity when tested in tissue culture with a selectivity index of >2,000. Compound 22 also showed high activity in vivo in a toxoplasmosis mouse model. The compound inhibited the Toxoplasma farnesyl diphosphate synthase (TgFPPS), but the concentration needed to inhibit 50% of the enzymatic activity (IC₅₀) was higher than the concentration that inhibited 50% of growth. We tested compound 22 against two other apicomplexan parasites, Plasmodium falciparum (EC₅₀ of 0.6 ± 0.01 μM), the agent of malaria, and Cryptosporidium parvum (EC₅₀ of ∼65 μM), the agent of cryptosporidiosis. Our results suggest that compound 22 is an excellent novel compound that could lead to the development of potent agents against apicomplexan parasites.

Keywords: Cryptosporidium parvum; Plasmodium falciparum; Toxoplasma gondii; bisphosphonates; farnesyl diphosphate synthase; isoprenoids.

FIG 1

Basic structure of the bisphosphonates and aminobisphosphonates used clinically.

FIG 2

(A) Chemical structures of representative lineal bisphosphonic acids as putative antiparasitic agents. (B and C) Synthetic approaches used to produce the designed linear sulfur-containing 1,1-bisphosphonates. rt, room temperature.

FIG 3

(A and B) Inhibition curves for compound 22 against the growth of the T. gondii RH strain and Prugniard strains. (C) Fluorescence values at days 3, 4, and 5 of red parasites of the Prugniard strain growing in fibroblasts in 96-well plates. Notice that these cells have slow growth and that they are still replicating at day 5. (D) Growth curve of T. gondii RH-RFP cells and inhibition of the RH strain by compound 22 at 0.9 μM, which is the concentration calculated from the data shown in panel A that inhibits 90% of growth. (E) FPPS activity with different concentrations of compound 22. The protocol to measure the activity is explained in Materials and Methods. Values shown in panels A to D are means ± standard deviations of three independent experiments (n = 3).

FIG 4

Dose-response curve showing the inhibition of growth of Plasmodium falciparum by compound 22. R² was 0.9935. Details of the experiment are explained in Materials and Methods. Values are means ± standard deviations of two independent experiments, each one in triplicate.

FIG 5

Dose-response graph for compound 22 against C. parvum growth. BK, background counts without parasites; Parom, paromomycin control. The protocol for measuring inhibition of growth and growth of C. parvum is explained in Materials and Methods. Results are from one representative experiment of three. LU, light units; DMSO, dimethyl sulfoxide.

FIG 6

In vivo results for the T. gondii RH mouse model of infection. (A) Mouse survival after treatment with 0.05, 0.1, 0.5, and 1 mg/kg of compound 22 i.p. for 10 days. Surviving mice were challenged with 5,000 tachyzoites at day 30. (B) Mouse survival after treatment with 0.05 mg/kg of compound 22 i.p. twice a day for 10 days compared to treatment with 0.1 mg/kg daily for 10 days. Surviving mice were challenge with 5,000 tachyzoites at day 30. All of these experiments were repeated twice with 5 mice for each dose and the results were combined. p.i., postinfection.