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

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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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Prion protein expression is not inhibited by preliminary hit DB compounds. The effects of preliminary hit DB compounds on PRNP transcript levels (A) and total PrP (B) were evaluated. (A) RNA was collected from treated cells and tested by qRT-PCR for PRNP transcript levels. PRNP transcripts were normalized to GAPDH transcript and compared to the vehicle control. (B) Total protein was collected from treated cells and tested for total PrPC using a commercial ELISA. Total PrP was normalized to the vehicle control. Columns represent the fold change in PRNP transcripts or total PrP. Increasing bars indicate compound enhancement, and decreasing bars indicate compound inhibition. Fold change values were statistically compared to the vehicle control using individual t tests (*, P < 0.05). The y axis reference lines indicate the established 2-fold cutoff level for biologically significant inhibition or enhancement. Error bars represent the 95% confidence intervals from the mean fold change from at least 3 independent experiments.
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Figure 4: Prion protein expression is not inhibited by preliminary hit DB compounds. The effects of preliminary hit DB compounds on PRNP transcript levels (A) and total PrP (B) were evaluated. (A) RNA was collected from treated cells and tested by qRT-PCR for PRNP transcript levels. PRNP transcripts were normalized to GAPDH transcript and compared to the vehicle control. (B) Total protein was collected from treated cells and tested for total PrPC using a commercial ELISA. Total PrP was normalized to the vehicle control. Columns represent the fold change in PRNP transcripts or total PrP. Increasing bars indicate compound enhancement, and decreasing bars indicate compound inhibition. Fold change values were statistically compared to the vehicle control using individual t tests (*, P < 0.05). The y axis reference lines indicate the established 2-fold cutoff level for biologically significant inhibition or enhancement. Error bars represent the 95% confidence intervals from the mean fold change from at least 3 independent experiments.

Mentions: To elucidate a possible mechanism of inhibition, the preliminary hit compounds were examined for effects on PrPC expression. Evidence has supported the reliance of prion replication on multiple PrPC-specific factors, including PRNP expression and total PrPC (46, 54). Therefore, a reduction in PrPC expression represents a possible target for antiprion compounds. To determine if PrPC expression is inhibited, the effects on total cellular content of PrP transcripts (qRT-PCR) (Fig. 4A) and protein (ELISA) (Fig. 4B) were determined in PrPSc-negative cells after 4 days of incubation with preliminary hit compounds at 1 μM. Results were normalized to vehicle-treated cells, and fold changes were statistically compared to the vehicle control. Consistent with our previous finding (24), a significant effect on PrPC expression was not detected for DB772. None of the remaining 11 preliminary hit compounds caused a 2-fold change in either PRNP transcript or total PrP levels. The only statistically significant changes were in PRNP transcripts after incubation with DB948 (P = 0.0262) and DB932A (P = 0.0467), although both were less than 2-fold. Significant correlations of antiprion activity at 1 μM with PRNP transcript (P = 0.1084) or total PrPC (P = 0.8333) levels were not detected, strongly suggesting that inhibition of PrPC expression is not the general mechanism by which these selected DB compounds inhibit PrPSc.


Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System
Prion protein expression is not inhibited by preliminary hit DB compounds. The effects of preliminary hit DB compounds on PRNP transcript levels (A) and total PrP (B) were evaluated. (A) RNA was collected from treated cells and tested by qRT-PCR for PRNP transcript levels. PRNP transcripts were normalized to GAPDH transcript and compared to the vehicle control. (B) Total protein was collected from treated cells and tested for total PrPC using a commercial ELISA. Total PrP was normalized to the vehicle control. Columns represent the fold change in PRNP transcripts or total PrP. Increasing bars indicate compound enhancement, and decreasing bars indicate compound inhibition. Fold change values were statistically compared to the vehicle control using individual t tests (*, P < 0.05). The y axis reference lines indicate the established 2-fold cutoff level for biologically significant inhibition or enhancement. Error bars represent the 95% confidence intervals from the mean fold change from at least 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Prion protein expression is not inhibited by preliminary hit DB compounds. The effects of preliminary hit DB compounds on PRNP transcript levels (A) and total PrP (B) were evaluated. (A) RNA was collected from treated cells and tested by qRT-PCR for PRNP transcript levels. PRNP transcripts were normalized to GAPDH transcript and compared to the vehicle control. (B) Total protein was collected from treated cells and tested for total PrPC using a commercial ELISA. Total PrP was normalized to the vehicle control. Columns represent the fold change in PRNP transcripts or total PrP. Increasing bars indicate compound enhancement, and decreasing bars indicate compound inhibition. Fold change values were statistically compared to the vehicle control using individual t tests (*, P < 0.05). The y axis reference lines indicate the established 2-fold cutoff level for biologically significant inhibition or enhancement. Error bars represent the 95% confidence intervals from the mean fold change from at least 3 independent experiments.
Mentions: To elucidate a possible mechanism of inhibition, the preliminary hit compounds were examined for effects on PrPC expression. Evidence has supported the reliance of prion replication on multiple PrPC-specific factors, including PRNP expression and total PrPC (46, 54). Therefore, a reduction in PrPC expression represents a possible target for antiprion compounds. To determine if PrPC expression is inhibited, the effects on total cellular content of PrP transcripts (qRT-PCR) (Fig. 4A) and protein (ELISA) (Fig. 4B) were determined in PrPSc-negative cells after 4 days of incubation with preliminary hit compounds at 1 μM. Results were normalized to vehicle-treated cells, and fold changes were statistically compared to the vehicle control. Consistent with our previous finding (24), a significant effect on PrPC expression was not detected for DB772. None of the remaining 11 preliminary hit compounds caused a 2-fold change in either PRNP transcript or total PrP levels. The only statistically significant changes were in PRNP transcripts after incubation with DB948 (P = 0.0262) and DB932A (P = 0.0467), although both were less than 2-fold. Significant correlations of antiprion activity at 1 μM with PRNP transcript (P = 0.1084) or total PrPC (P = 0.8333) levels were not detected, strongly suggesting that inhibition of PrPC expression is not the general mechanism by which these selected DB compounds inhibit PrPSc.

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