Bisphosphonate Inhibitors ofToxoplasmagondiGrowth:  In Vitro,QSAR, and In Vivo Investigations

Abstract

We have investigated the activity of 60 bisphosphonates against the replication of Toxoplasma gondii in vitro and of three of the most active compounds, in vivo. The two most active compounds found were n-alkyl bisphosphonates containing long (n = 9 or 10) hydrocarbon chains, not the nitrogen-containing species used in bone resorption therapy. The target of all of the most active bisphosphonates appears to be the isoprene biosynthesis pathway enzyme farnesyl pyrophosphate synthase (FPPS), as indicated by the correlations between T. gondii growth inhibition and FPPS (human and Leishmania major) enzyme inhibition and by the fact that a T. gondii strain engineered to overexpress FPPS required considerably higher levels of bisphosphonates to achieve 50% growth inhibition, while the IC(50) for atovaquone (which does not inhibit FPPS) remained the same in the overexpressing strain. The phosphonate inhibitor of the non-mevalonate pathway, fosmidomycin, which inhibits the enzyme 1-deoxyxylulose-5-phosphate reductoisomerase, had no effect on T. gondii growth. To investigate structure-activity relationships (SARs) in more detail, we used two three-dimensional quantitative SAR methods: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), to investigate all 60 bisphosphonates. Both the CoMFA and CoMSIA models indicated a 60-70% contribution from steric interactions and a 30-40% contribution from electrostatic interactions and using four N = 55 training sets for each method, we found on average between a factor of 2 and 3 error in IC(50) prediction. The three most active compounds found in vitro were tested in vivo in a Smith-Webster mouse model and the two most active bisphosphonates were found to provide up to an 80% protection from death, a considerable improvement over that found previously with nitrogen-containing bisphosphonates. This effect may originate in the much higher therapeutic indices of these alkyl bisphosphonates, as deduced from in vitro assays using LD(50) values for growth inhibition of a human cell line. Overall, these results indicate that alkyl bisphosphonates are promising compounds for further development as agents against Toxoplasma gondii growth, in vivo.

The Farnesyl-diphosphate/Geranylgeranyl-diphosphate Synthase of Toxoplasma gondii Is a Bifunctional Enzyme and a Molecular Target of Bisphosphonates

“…T. gondii tachyzoites of the 2F1 clone expressing ␤-galactosidase were a gift from Dr. L. David Sibley (21) and were routinely maintained in vitro in the same media as used for the RH strain. In vitro growth inhibition was done using confluent human fibroblast cells in 96-well plates as described previously (22). Tachyzoites of the 2F1 clone at 10 4 per ml were used for infection, and the plates were incubated at 37°C.…”

“…Tachyzoites of the 2F1 clone at 10 4 per ml were used for infection, and the plates were incubated at 37°C. At 48 h post-infection, the plates were processed for ␤-galactosidase activity as described previously (22).…”

“…IC 50 values were calculated using the same methods as described previously for in vitro testing (22).…”

“…TgFPPS Is a Molecular Target for Bisphosphonates-The inhibition test with the purified enzyme showed that 3 of the 10 best T. gondii growth inhibitors (22) were also potent inhibitors of TgFPPS activity ( Table 3). The best T. gondii inhibitor, compound 1 (an alkyl bisphosphonate), has an IC 50 of 0.022 Ϯ 0.003 M, for TgFPPS.…”

Lipidomics of host–pathogen interactions

“…Lysobisphosphatidic acid and phosphoinositides regulate release of VSV nucleocapsid into cytoplasm [13] SigD/SopB is a type III bacterial derived phosphoinositide phosphatase [29,112] Molecular species of phosphatidylcholine and phosphatidylethanolamine are altered in host plasmamembrane after Plasmodium infection [116] GP Ethanolamine phospholipids required for Sindbis virus production [111] Intracellular mycobacteria release a heterogeneous mixture of lipids [27,113] Growth arrest of Plasmodium [117] and Toxoplasma [85] by disruption of phosphatidylcholine synthesis Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity [14] Phosphatidylinositol mannosides stimulate fusion of early endosomes with mycobacterial phagosomes [30] Glycosylated phosphatidylinositol causes phagosome maturation arrest [114] SapM, a mycobacterial derived phosphatase hydrolyses PI3P contributing to inhibition of phagolysosome maturation [115] Sphingosine 1-kinase is recruited to nascent phagosomes [118] Inhibition of sphingolipid biosynthesis in T. gondii blocks replication [119] SP Sphingomyelin metabolism important for P. falciparum development [120] Inhibition of cholesterol biosynthesis inhibits Hepatitis C virus RNA replication [15] Inhibition of cholesterol acquisition by the host lowers T. gondii replication [40] ST Cholesterol esterification essential for optimal T. gondii proliferation [121,122] Isopreonoid synthesis inhibitors with anti-malarial [123] and anti-T. gondii activity [124] PR Block of protein farnesylation as antiapicomplexan therapies [125] 5…”