Novel anti-Cryptosporidium activity of known drugs identified by high-throughput screening against parasite fatty acyl-CoA binding protein (ACBP)

Background: Cryptosporidium parvum causes an opportunistic Infection in AIDS patients, and no effective treatments are yet available. This Parasite possesses a single fatty acyl-CoA binding protein (CpACBP1) that is localized to the unique parasitophorous vacuole membrane (PVM). The major goal of this study was to identify inhibitors from known drugs against CpACBP1 as potential new anti-Cryptosporidium agents.

Methods: A fluorescence assay was developed to detect CpACBP1 activity and to identify inhibitors by screening known drugs. Efficacies of top CpACBP1 inhibitors against Cryptosporidium growth in vitro were evaluated using a quantitative RT-PCR assay.

Results: Nitrobenzoxadiazole-labelled palmitoyl-CoA significantly increased the fluorescent emission upon binding to CpACBP1 (excitation/emission 460/538 nm), which was quantified to determine the CpACBP1 activity and binding kinetics. The fluorescence assay was used to screen a collection of 1040 compounds containing mostly known drugs, and identified the 28 most active compounds that could inhibit CpACBP1 activity with sub-micromolar IC(50) values. Among them, four compounds displayed efficacies against Parasite growth in vitro with low micromolar IC(50) values. The effective compounds were broxyquinoline (IC(50) 64.9 μM), cloxyquin (IC(50) 25.1 μM), cloxacillin sodium (IC(50) 36.2 μM) and sodium dehydrocholate (IC(50) 53.2 μM).

Conclusions: The fluorescence ACBP assay can be effectively used to screen known drugs or Other compound libraries. Novel anti-Cryptosporidium activity was observed in four top CpACBP1 inhibitors, which may be further investigated for their potential to be repurposed to treat cryptosporidiosis and to serve as leads for drug development.