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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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Effect of preliminary hit DB compounds on cell proliferation. PrPSc-positive hTERT-ovine microglia were treated for 4 days with 1 μM DB compound, during which cell proliferation was quantified daily by 24-h BrdU incorporation. Bars represent the average percent BrdU incorporation in DB compound-treated cells relative to that in day-matched, vehicle control (0.1% DMSO)-treated cells. Error bars represent the 95% confidence intervals from the averages from 3 independent experiments. Values were statistically compared to the day-matched vehicle controls to determine enhanced or reduced cell proliferation (*, PDun < 0.05).
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Figure 2: Effect of preliminary hit DB compounds on cell proliferation. PrPSc-positive hTERT-ovine microglia were treated for 4 days with 1 μM DB compound, during which cell proliferation was quantified daily by 24-h BrdU incorporation. Bars represent the average percent BrdU incorporation in DB compound-treated cells relative to that in day-matched, vehicle control (0.1% DMSO)-treated cells. Error bars represent the 95% confidence intervals from the averages from 3 independent experiments. Values were statistically compared to the day-matched vehicle controls to determine enhanced or reduced cell proliferation (*, PDun < 0.05).

Mentions: The effects of DB compounds on antiprion activity were not solely attributed to cell viability; however, it remained possible that additional effects on cell proliferation had impacted PrPSc accumulation (53). Therefore, the preliminary hit DB compounds were examined for effects at 1 μM on the proliferation of hTERT-ovine microglia throughout the 4-day course of treatment. Given its large cytotoxic effect, the effect of 1 μM DB948 on cell proliferation was not tested. A significant effect on cell proliferation was not detected over the 4-day treatment period for DB772, DB1310, DB1264, DB2228, DB192, and DB1504 (range, 76 to 106% of vehicle control) (Fig. 2). Conversely, significant decreases in cell proliferation were detected on days 2 through 4 following treatment with DB932A (range, 56 to 70%; PDun < 0.05) and DB2214 (range, 66 to 70%; PDun < 0.01). Significant decreases were detected only on day 4 following treatment with DB1033 (68%; PDun = 0.003), DB818A (76%; PDun = 0.048), and DB191 (74%; PDun = 0.027). Thus, we cannot exclude the possibility that a reduced rate of cell division might contribute to the antiprion activity of some of these compounds. However, significant correlations were not detected between effects on cell proliferation and cell viability (P = 0.9893), cell number (P = 0.1930), and antiprion activity (P = 0.0731) after 4 days of treatment. This further suggests that while a subset of DB compounds may target cell division or viability, these effects are likely not the sole cause of PrPSc inhibition. A summary of the cell-based effects (i.e., PrPSc inhibition, cell counts, cell viability, and BrdU incorporation) of the preliminary hit DB compounds is provided in Table S2 in the supplemental material and highlights that compounds such as DB1310, which significantly inhibits PrPSc but has no detectable effect on cell count, viability, or BrdU incorporation, are compounds of future interest.


Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System
Effect of preliminary hit DB compounds on cell proliferation. PrPSc-positive hTERT-ovine microglia were treated for 4 days with 1 μM DB compound, during which cell proliferation was quantified daily by 24-h BrdU incorporation. Bars represent the average percent BrdU incorporation in DB compound-treated cells relative to that in day-matched, vehicle control (0.1% DMSO)-treated cells. Error bars represent the 95% confidence intervals from the averages from 3 independent experiments. Values were statistically compared to the day-matched vehicle controls to determine enhanced or reduced cell proliferation (*, PDun < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effect of preliminary hit DB compounds on cell proliferation. PrPSc-positive hTERT-ovine microglia were treated for 4 days with 1 μM DB compound, during which cell proliferation was quantified daily by 24-h BrdU incorporation. Bars represent the average percent BrdU incorporation in DB compound-treated cells relative to that in day-matched, vehicle control (0.1% DMSO)-treated cells. Error bars represent the 95% confidence intervals from the averages from 3 independent experiments. Values were statistically compared to the day-matched vehicle controls to determine enhanced or reduced cell proliferation (*, PDun < 0.05).
Mentions: The effects of DB compounds on antiprion activity were not solely attributed to cell viability; however, it remained possible that additional effects on cell proliferation had impacted PrPSc accumulation (53). Therefore, the preliminary hit DB compounds were examined for effects at 1 μM on the proliferation of hTERT-ovine microglia throughout the 4-day course of treatment. Given its large cytotoxic effect, the effect of 1 μM DB948 on cell proliferation was not tested. A significant effect on cell proliferation was not detected over the 4-day treatment period for DB772, DB1310, DB1264, DB2228, DB192, and DB1504 (range, 76 to 106% of vehicle control) (Fig. 2). Conversely, significant decreases in cell proliferation were detected on days 2 through 4 following treatment with DB932A (range, 56 to 70%; PDun < 0.05) and DB2214 (range, 66 to 70%; PDun < 0.01). Significant decreases were detected only on day 4 following treatment with DB1033 (68%; PDun = 0.003), DB818A (76%; PDun = 0.048), and DB191 (74%; PDun = 0.027). Thus, we cannot exclude the possibility that a reduced rate of cell division might contribute to the antiprion activity of some of these compounds. However, significant correlations were not detected between effects on cell proliferation and cell viability (P = 0.9893), cell number (P = 0.1930), and antiprion activity (P = 0.0731) after 4 days of treatment. This further suggests that while a subset of DB compounds may target cell division or viability, these effects are likely not the sole cause of PrPSc inhibition. A summary of the cell-based effects (i.e., PrPSc inhibition, cell counts, cell viability, and BrdU incorporation) of the preliminary hit DB compounds is provided in Table S2 in the supplemental material and highlights that compounds such as DB1310, which significantly inhibits PrPSc but has no detectable effect on cell count, viability, or BrdU incorporation, are compounds of future interest.

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