A propidium iodide-based in vitro screen of the "Bug Box" against Babesia duncani reveals potent inhibitors.

Abstract

The incidence and endemic range of human babesiosis are expanding. Standard therapy for human babesiosis consists of antimicrobials developed for other indications. While these treatments are adequate in immunocompetent hosts, infections in the immunocompromised can be severe, relapsing, and drug-resistant despite the use of multi-drug regimens. Existing drugs are ineffective in the immunocompromised because they cannot achieve and maintain adequate serum concentrations to inhibit Babesia. Discovery of improved agents against Babesia spp. is of growing importance, and efficient techniques for high-throughput compound screening can assist in this effort. We developed a high-throughput in vitro drug screening assay for Babesia duncani that is conducted in 384-well plates and makes use of the fluorescent DNA stain propidium iodide (PI) with relative fluorescence measured by a microplate reader. A Z' factor of 0.82 was calculated, which suggests an excellent ability to detect inhibitory compounds. A screen of the 41-compound library Structural Genomics Consortium Bug Box was conducted, yielding five hits: trimethoprim, atovaquone, SDDC M7, diphenyleneiodonium chloride, and panobinostat. Panobinostat, a histone deacetylase complex (HDAC) inhibitor, was selected for further evaluation given that its target had not been previously explored in B. duncani. Dose-response testing of structurally related compounds revealed multiple potential leads, including nanatinostat and quisinostat, both of which were potent at the nanomolar level and showed favorable selectivity index in cytotoxicity studies. High-throughput screening using PI and 384-well plates is an advance in drug discovery for babesiosis, and HDAC inhibitors show promise as lead compounds worthy of further investigation.