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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.

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Identification of DB compounds inhibitory at 1 μM in PrPSc-positive hTERT-ovine microglia. Following 4 days of incubation with 1 μM (each) DB compound, cell viability and PrPSc inhibition were evaluated by WST-1 assay and SSCA, respectively. Values were expressed as the percent change in viability or PrPSc spot number versus the vehicle control (0.1% DMSO). (A) Distribution of tested compounds with regard to effects on cell viability. Values along the x axis represent the mean percent viability from a minimum of three independent experiments. Values along the y axis represent a number of the total compound population that falls within a value of percent change. Compounds were compared to the vehicle control, and those lacking significant effects on cell viability or of distinct structural interest were selected for inhibition screening. (B) Distribution of selected compounds with regard to effects on PrPSc accumulation. Values along the x axis represent the mean percent change in spot number from a minimum of three independent experiments. Values along the y axis represent the number of the total compound population that falls within a value of percent change. Compounds with a percent reduction of greater than or equal to 68% (+ standard deviation) were identified as preliminary hits and selected for further testing (white bars). (C) Correlation between average percent reduction in PrPSc and average percent viability following 1 μM treatment with DB compounds with (open circles, PDun < 0.05) or without (closed circles) significant effects on cell viability. The black line represents the best fit line following linear regression analysis. (D) Average percent reduction in PrPSc by the 12 preliminary hits selected from panel B. Values were statistically compared to that of the reference molecule, DB772, to determine enhanced inhibitory activity (*, PDun < 0.05). Error bars represent ±1 standard deviation of the mean from at least 3 independent experiments.
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Figure 1: Identification of DB compounds inhibitory at 1 μM in PrPSc-positive hTERT-ovine microglia. Following 4 days of incubation with 1 μM (each) DB compound, cell viability and PrPSc inhibition were evaluated by WST-1 assay and SSCA, respectively. Values were expressed as the percent change in viability or PrPSc spot number versus the vehicle control (0.1% DMSO). (A) Distribution of tested compounds with regard to effects on cell viability. Values along the x axis represent the mean percent viability from a minimum of three independent experiments. Values along the y axis represent a number of the total compound population that falls within a value of percent change. Compounds were compared to the vehicle control, and those lacking significant effects on cell viability or of distinct structural interest were selected for inhibition screening. (B) Distribution of selected compounds with regard to effects on PrPSc accumulation. Values along the x axis represent the mean percent change in spot number from a minimum of three independent experiments. Values along the y axis represent the number of the total compound population that falls within a value of percent change. Compounds with a percent reduction of greater than or equal to 68% (+ standard deviation) were identified as preliminary hits and selected for further testing (white bars). (C) Correlation between average percent reduction in PrPSc and average percent viability following 1 μM treatment with DB compounds with (open circles, PDun < 0.05) or without (closed circles) significant effects on cell viability. The black line represents the best fit line following linear regression analysis. (D) Average percent reduction in PrPSc by the 12 preliminary hits selected from panel B. Values were statistically compared to that of the reference molecule, DB772, to determine enhanced inhibitory activity (*, PDun < 0.05). Error bars represent ±1 standard deviation of the mean from at least 3 independent experiments.

Mentions: When screening candidate antiprion compounds, it is critical to recognize those that significantly alter cell viability, as such an effect may influence the measurement of PrPSc accumulation. For example, increasing the rate of cell division decreases the rate of PrPSc accumulation in murine neuroblastoma cells (53). Conversely, cytotoxic effects would reduce the number of cell targets and may decrease PrPSc replication and accumulation. Thus, DB compounds were initially screened for effects on the viability of hTERT-ovine microglia under the same culture conditions subsequently used to screen for antiprion activity (i.e., viability was measured by WST-1 assay 4 days post-exposure to 1 μM DB compound). Consistent with our previous findings (24), 1 μM DB772 did not significantly affect cell viability (96.4% ± 8.7% of vehicle control). Cell viability was significantly decreased by one DB compound (DB948; 60%) and increased by 24 DB compounds (range, 127 to 266%); however, the majority (64 total) demonstrated no significant effect when tested at 1 μM (Fig. 1A). Of the DB compounds selected for further testing, 59 had no significant micromolar effect on cell viability (range, 76 to 156%). Seventeen additional DB compounds had significant effects on cell viability but were also selected for further testing because of distinct structural interests: one significant inhibitor (60%) and 16 significant enhancers (range, 127 to 239%).


Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System
Identification of DB compounds inhibitory at 1 μM in PrPSc-positive hTERT-ovine microglia. Following 4 days of incubation with 1 μM (each) DB compound, cell viability and PrPSc inhibition were evaluated by WST-1 assay and SSCA, respectively. Values were expressed as the percent change in viability or PrPSc spot number versus the vehicle control (0.1% DMSO). (A) Distribution of tested compounds with regard to effects on cell viability. Values along the x axis represent the mean percent viability from a minimum of three independent experiments. Values along the y axis represent a number of the total compound population that falls within a value of percent change. Compounds were compared to the vehicle control, and those lacking significant effects on cell viability or of distinct structural interest were selected for inhibition screening. (B) Distribution of selected compounds with regard to effects on PrPSc accumulation. Values along the x axis represent the mean percent change in spot number from a minimum of three independent experiments. Values along the y axis represent the number of the total compound population that falls within a value of percent change. Compounds with a percent reduction of greater than or equal to 68% (+ standard deviation) were identified as preliminary hits and selected for further testing (white bars). (C) Correlation between average percent reduction in PrPSc and average percent viability following 1 μM treatment with DB compounds with (open circles, PDun < 0.05) or without (closed circles) significant effects on cell viability. The black line represents the best fit line following linear regression analysis. (D) Average percent reduction in PrPSc by the 12 preliminary hits selected from panel B. Values were statistically compared to that of the reference molecule, DB772, to determine enhanced inhibitory activity (*, PDun < 0.05). Error bars represent ±1 standard deviation of the mean from at least 3 independent experiments.
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Related In: Results  -  Collection

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Figure 1: Identification of DB compounds inhibitory at 1 μM in PrPSc-positive hTERT-ovine microglia. Following 4 days of incubation with 1 μM (each) DB compound, cell viability and PrPSc inhibition were evaluated by WST-1 assay and SSCA, respectively. Values were expressed as the percent change in viability or PrPSc spot number versus the vehicle control (0.1% DMSO). (A) Distribution of tested compounds with regard to effects on cell viability. Values along the x axis represent the mean percent viability from a minimum of three independent experiments. Values along the y axis represent a number of the total compound population that falls within a value of percent change. Compounds were compared to the vehicle control, and those lacking significant effects on cell viability or of distinct structural interest were selected for inhibition screening. (B) Distribution of selected compounds with regard to effects on PrPSc accumulation. Values along the x axis represent the mean percent change in spot number from a minimum of three independent experiments. Values along the y axis represent the number of the total compound population that falls within a value of percent change. Compounds with a percent reduction of greater than or equal to 68% (+ standard deviation) were identified as preliminary hits and selected for further testing (white bars). (C) Correlation between average percent reduction in PrPSc and average percent viability following 1 μM treatment with DB compounds with (open circles, PDun < 0.05) or without (closed circles) significant effects on cell viability. The black line represents the best fit line following linear regression analysis. (D) Average percent reduction in PrPSc by the 12 preliminary hits selected from panel B. Values were statistically compared to that of the reference molecule, DB772, to determine enhanced inhibitory activity (*, PDun < 0.05). Error bars represent ±1 standard deviation of the mean from at least 3 independent experiments.
Mentions: When screening candidate antiprion compounds, it is critical to recognize those that significantly alter cell viability, as such an effect may influence the measurement of PrPSc accumulation. For example, increasing the rate of cell division decreases the rate of PrPSc accumulation in murine neuroblastoma cells (53). Conversely, cytotoxic effects would reduce the number of cell targets and may decrease PrPSc replication and accumulation. Thus, DB compounds were initially screened for effects on the viability of hTERT-ovine microglia under the same culture conditions subsequently used to screen for antiprion activity (i.e., viability was measured by WST-1 assay 4 days post-exposure to 1 μM DB compound). Consistent with our previous findings (24), 1 μM DB772 did not significantly affect cell viability (96.4% ± 8.7% of vehicle control). Cell viability was significantly decreased by one DB compound (DB948; 60%) and increased by 24 DB compounds (range, 127 to 266%); however, the majority (64 total) demonstrated no significant effect when tested at 1 μM (Fig. 1A). Of the DB compounds selected for further testing, 59 had no significant micromolar effect on cell viability (range, 76 to 156%). Seventeen additional DB compounds had significant effects on cell viability but were also selected for further testing because of distinct structural interests: one significant inhibitor (60%) and 16 significant enhancers (range, 127 to 239%).

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 &mu;M 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.


Related in: MedlinePlus