Abstract
The transmissible spongiform encephalopathies are fatal neurodegenerative disorders characterized by the misfolding of the native cellular prion protein (PrPC) into the accumulating, disease-associated isoform (PrPSc). Despite extensive research into the inhibition of prion accumulation, no effective treatment exists. Previously, we demonstrated the inhibitory activity of DB772, a monocationic phenyl-furan-benzimidazole, against PrPSc accumulation in sheep microglial cells. In an effort to determine the effect of structural substitutions on the antiprion activity of DB772, we employed an in vitro strategy to survey a library of structurally related, monothiophene- and furan-based compounds for improved inhibitory activity. Eighty-nine compounds were screened at 1 μM for effects on cell viability and prion accumulation in a persistently infected ovine microglia culture system. Eleven compounds with activity equivalent to or higher than that of DB772 were identified as preliminary hit compounds. For the preliminary hits, cytotoxicities and antiprion activities were compared to calculate the tissue culture selectivity index. A structure-activity relationship (SAR) analysis was performed to determine molecular components contributing to antiprion activity. To investigate potential mechanisms of inhibition, effects on PrPC and PrPSc were examined. While inhibition of total PrPC was not observed, the results suggest that a potential target for inhibition at biologically relevant concentrations is through PrPC misfolding to PrPSc. Further, SAR analysis suggests that two structural elements were associated with micromolar antiprion activity. Taken together, the described data provide a foundation for deeper investigation into untested DB compounds and in the design of effective therapeutics.
Highlights
The transmissible spongiform encephalopathies are fatal neurodegenerative disorders characterized by the misfolding of the native cellular prion protein (PrPC) into the accumulating, disease-associated isoform (PrPSc)
Infectious agents that cause fatal neurodegenerative disorders collectively known as the transmissible spongiform encephalopathies (TSEs)
Structural derivatives of furamidine are of broad interest because of potent activity demonstrated against parasitic microorganisms (e.g., Trypanosoma brucei) [29] and for bovine viral diarrhea virus (BVDV) [25, 26, 30, 31]
Summary
A summary flow chart of the experimental in vitro strategy used to screen the DB compound library is provided as Fig. S1 in the supplemental material. The average percent reduction following DB compound treatment was determined from at least three independent experiments and statistically compared using a one-way ANOVA and Dunnett’s multiple-comparison test for post hoc comparison of each compound to the vehicle control or the cutoff value of 68% reduction PrPSc-positive cells were passaged, plated, and treated as previously described for the WST-1 assay, except that mitotic activity was measured on each day of the 4-day treatment using a colorimetric BrdU cell proliferation enzyme-linked immunosorbent assay (ELISA) kit (Abcam, Cambridge, MA). The fold changes in PRNP were mean centered and autoscaled as previously described [45], and the average fold change for each sample, from at least three independent experiments, was statistically compared to the vehicle control using a two-tailed t test The average relative percent PrPSc, from at least 5 independent experiments, was statistically compared to the vehicle control using a two-tailed t test (P Ͻ 0.05; SigmaPlot ver. 13)
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