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Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Dose-response curves show variable effect on cell viability by preliminary hit DB compounds. PrPSc-positive hTERT-ovine microglia were treated for 4 days with a 1/4-log dilution series of each compound. Cell viability was subsequently determined by WST-1 assay. (A) DB compounds that exhibited a concentration-dependent cytotoxic effect. (B) DB compounds that did not induce a significant effect on cell viability up to a concentration of 3.16 μM. (C) DB compounds that led to a concentration-dependent increase in cell viability. Data points represent the mean percent viability ±1 standard deviation from at least 3 independent experiments.
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Figure 3: Dose-response curves show variable effect on cell viability by preliminary hit DB compounds. PrPSc-positive hTERT-ovine microglia were treated for 4 days with a 1/4-log dilution series of each compound. Cell viability was subsequently determined by WST-1 assay. (A) DB compounds that exhibited a concentration-dependent cytotoxic effect. (B) DB compounds that did not induce a significant effect on cell viability up to a concentration of 3.16 μM. (C) DB compounds that led to a concentration-dependent increase in cell viability. Data points represent the mean percent viability ±1 standard deviation from at least 3 independent experiments.

Mentions: Of the 12 DB compounds identified as preliminary hits, concentration-dependent cytotoxicity was observed for only three (Fig. 3A). The cytotoxicity of DB772 at a concentration greater than 1 μM is consistent with our previous finding, which used primary ovine microglia (24). The structures of DB948 and DB932A represent minimal chemical modifications of DB772; thus, it is not surprising that the CC50 values associated with these compounds were similar (Table 1). Significant cytotoxicity was not observed for the other 9 preliminary hit compounds, three of which (DB2228, DB191, and DB1033) failed to demonstrate any effect on cell viability (Fig. 3B) and six of which were associated with a concentration-dependent increase in cell viability (Fig. 3C). The CC50 for these 9 preliminary hit compounds was thus nonestimable and is instead reported as >3.16 μM—the highest concentration tested.


Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System
Dose-response curves show variable effect on cell viability by preliminary hit DB compounds. PrPSc-positive hTERT-ovine microglia were treated for 4 days with a 1/4-log dilution series of each compound. Cell viability was subsequently determined by WST-1 assay. (A) DB compounds that exhibited a concentration-dependent cytotoxic effect. (B) DB compounds that did not induce a significant effect on cell viability up to a concentration of 3.16 μM. (C) DB compounds that led to a concentration-dependent increase in cell viability. Data points represent the mean percent viability ±1 standard deviation from at least 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4997874&req=5

Figure 3: Dose-response curves show variable effect on cell viability by preliminary hit DB compounds. PrPSc-positive hTERT-ovine microglia were treated for 4 days with a 1/4-log dilution series of each compound. Cell viability was subsequently determined by WST-1 assay. (A) DB compounds that exhibited a concentration-dependent cytotoxic effect. (B) DB compounds that did not induce a significant effect on cell viability up to a concentration of 3.16 μM. (C) DB compounds that led to a concentration-dependent increase in cell viability. Data points represent the mean percent viability ±1 standard deviation from at least 3 independent experiments.
Mentions: Of the 12 DB compounds identified as preliminary hits, concentration-dependent cytotoxicity was observed for only three (Fig. 3A). The cytotoxicity of DB772 at a concentration greater than 1 μM is consistent with our previous finding, which used primary ovine microglia (24). The structures of DB948 and DB932A represent minimal chemical modifications of DB772; thus, it is not surprising that the CC50 values associated with these compounds were similar (Table 1). Significant cytotoxicity was not observed for the other 9 preliminary hit compounds, three of which (DB2228, DB191, and DB1033) failed to demonstrate any effect on cell viability (Fig. 3B) and six of which were associated with a concentration-dependent increase in cell viability (Fig. 3C). The CC50 for these 9 preliminary hit compounds was thus nonestimable and is instead reported as >3.16 μM—the highest concentration tested.

View Article: PubMed Central - PubMed

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.

No MeSH data available.