Babesiosis is a malaria-like illness found worldwide and endemic in the United States. The disease is caused by intraerythrocytic parasites of the genus Babesia. Babesia microti and Babesia duncani, which are responsible for most cases reported in the United States, are transmitted to humans by Ixodes scapularis and Dermacentor albipictus ticks, respectively, and can also be introduced by blood transfusion. The current treatment for human babesiosis consists of combination therapies with atovaquone+azithromycin or clindamycin+quinine. However, these drugs are associated with high rate of recrudescence, treatment failures and adverse events. Furthermore, recent studies in mice infected with B. microti and in vitro with B. duncani showed that these parasites are naturally tolerant to these drugs. These limitations emphasize the need for more effective and safer therapies for treatment of human babesiosis. We have recently reported the development of a continuous in vitro culture system for B. duncani in human red blood cells. Using this assay, we screened a library of FDA-approved drugs and identified fosinopril (the prodrug of the dipeptidyl carboxypeptidase ACE inhibitor fosinoprilat) as a potent antibabesial inhibitor. The compound was also effective against both B. duncani and B. microti in mouse models of babesiosis at 10 mg/kg. Interestingly, neither fosinoprilat nor other ACE inhibitors affected the growth of B. duncani in vitro. Equally interesting, analysis of the structure of fosinopril and bortezomib (another potent inhibitor of B. duncani identified in the chemical screen and a known proteasome inhibitor) revealed similarities between the two compounds. Together these data suggest that the antibabesial activity of fosinopril could be due to inhibition of either an ACE-like peptidase and/or a proteasome activity of Babesia parasites. The primary goals of this application are to identify analogs of fosinopril with more potent antibabesial but no ACE activity, and to elucidate the mechanism of action of these compounds in Babesia parasites. Building upon our preliminary data, we propose the following three specific aims. In Aim 1, we will determine whether or not the prodrug form of fosinopril is the active compound, and characterize the efficacy of fosinopril and newly synthesized analogs against B. duncani and B. microti clinical and field isolates in vitro and examine the structure activity relationship specific to these parasites. In Aim 2, we will characterize the in vivo efficacy of the most active compounds alone or in combination with other drugs as a strategy for elimination of Babesia infections. In Aim 3, we will implement biochemical, mass spectrometry and genetic assays to elucidate the mode of action of and possible mechanisms of Babesia resistance to fosinopril and its analogs. The success of the proposed three aims will guide future clinical trials to create an ideal regimen for the treatment of human babesiosis with no rec...