| Abstract
| - We have investigated the activity of 60 bisphosphonates against the replication of Toxoplasmagondii in vitro and of three of the most active compounds, in vivo. The two most activecompounds found were n-alkyl bisphosphonates containing long (n = 9 or 10) hydrocarbonchains, not the nitrogen-containing species used in bone resorption therapy. The target of allof the most active bisphosphonates appears to be the isoprene biosynthesis pathway enzymefarnesyl pyrophosphate synthase (FPPS), as indicated by the correlations between T. gondiigrowth inhibition and FPPS (human and Leishmania major) enzyme inhibition and by thefact that a T. gondii strain engineered to overexpress FPPS required considerably higher levelsof bisphosphonates to achieve 50% growth inhibition, while the IC50 for atovaquone (whichdoes not inhibit FPPS) remained the same in the overexpressing strain. The phosphonateinhibitor of the non-mevalonate pathway, fosmidomycin, which inhibits the enzyme 1-deoxyxylulose-5-phosphate reductoisomerase, had no effect on T. gondii growth. To investigatestructure−activity relationships (SARs) in more detail, we used two three-dimensionalquantitative SAR methods: comparative molecular field analysis (CoMFA) and comparativemolecular similarity indices analysis (CoMSIA), to investigate all 60 bisphosphonates. Boththe CoMFA and CoMSIA models indicated a 60−70% contribution from steric interactions anda 30−40% contribution from electrostatic interactions and using four N = 55 training sets foreach method, we found on average between a factor of 2 and 3 error in IC50 prediction. Thethree most active compounds found in vitro were tested in vivo in a Smith-Webster mousemodel and the two most active bisphosphonates were found to provide up to an 80% protectionfrom death, a considerable improvement over that found previously with nitrogen-containingbisphosphonates. This effect may originate in the much higher therapeutic indices of thesealkyl bisphosphonates, as deduced from in vitro assays using LD50 values for growth inhibitionof a human cell line. Overall, these results indicate that alkyl bisphosphonates are promisingcompounds for further development as agents against Toxoplasma gondii growth, in vivo.
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