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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.


DB772 and DB948 stabilize PrPSc to reduce PK digestion. The stabilization of PrPSc and subsequent reduction in PK digestion by selected compounds were evaluated by treating scrapie-positive sheep brain homogenate with the indicated concentration and DB compound. Aliquots of brain homogenate were treated with the indicated dilutions of DB772 (A) or selected DB compounds (B) and tested for PK-resistant PrPSc by immunoblotting. Band intensities were quantified as described above and normalized to the vehicle control. Columns and error bars represent the mean relative percent PrPSc and 95% confidence intervals, respectively, from at least 5 independent experiments. Mean values were statistically compared to the vehicle control using individual t tests (*, P < 0.05).
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Figure 7: DB772 and DB948 stabilize PrPSc to reduce PK digestion. The stabilization of PrPSc and subsequent reduction in PK digestion by selected compounds were evaluated by treating scrapie-positive sheep brain homogenate with the indicated concentration and DB compound. Aliquots of brain homogenate were treated with the indicated dilutions of DB772 (A) or selected DB compounds (B) and tested for PK-resistant PrPSc by immunoblotting. Band intensities were quantified as described above and normalized to the vehicle control. Columns and error bars represent the mean relative percent PrPSc and 95% confidence intervals, respectively, from at least 5 independent experiments. Mean values were statistically compared to the vehicle control using individual t tests (*, P < 0.05).

Mentions: As has been associated with LCPs (39), candidate antiprion compounds may also inhibit PrPSc accumulation through the stabilization of PrPSc amyloids. To determine the effect of preliminary hit compounds on the stability of PrPSc against PK digestion, a scrapie-positive sheep brain homogenate was treated with a dilution series of DB772 (range, 38.1 to 1.2 μM) similar to previous work done using polythiophenes (39). Relative to vehicle-treated controls, preincubation of ScBH with DB772 significantly increased PK-resistant PrPSc bands in samples treated with 38.1 μM (107% increase, P = 0.0018) (Fig. 7A) but not at the lower concentrations that were effective in inhibiting PrPSc accumulation in cell culture and sPMCA assays; it is possible that this disparity between effective concentrations may just reflect differences in assay matrices (e.g., abundance of lipids in brain homogenate, etc.), but this was not further investigated.


Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System
DB772 and DB948 stabilize PrPSc to reduce PK digestion. The stabilization of PrPSc and subsequent reduction in PK digestion by selected compounds were evaluated by treating scrapie-positive sheep brain homogenate with the indicated concentration and DB compound. Aliquots of brain homogenate were treated with the indicated dilutions of DB772 (A) or selected DB compounds (B) and tested for PK-resistant PrPSc by immunoblotting. Band intensities were quantified as described above and normalized to the vehicle control. Columns and error bars represent the mean relative percent PrPSc and 95% confidence intervals, respectively, from at least 5 independent experiments. Mean values were statistically compared to the vehicle control using individual t tests (*, P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4997874&req=5

Figure 7: DB772 and DB948 stabilize PrPSc to reduce PK digestion. The stabilization of PrPSc and subsequent reduction in PK digestion by selected compounds were evaluated by treating scrapie-positive sheep brain homogenate with the indicated concentration and DB compound. Aliquots of brain homogenate were treated with the indicated dilutions of DB772 (A) or selected DB compounds (B) and tested for PK-resistant PrPSc by immunoblotting. Band intensities were quantified as described above and normalized to the vehicle control. Columns and error bars represent the mean relative percent PrPSc and 95% confidence intervals, respectively, from at least 5 independent experiments. Mean values were statistically compared to the vehicle control using individual t tests (*, P < 0.05).
Mentions: As has been associated with LCPs (39), candidate antiprion compounds may also inhibit PrPSc accumulation through the stabilization of PrPSc amyloids. To determine the effect of preliminary hit compounds on the stability of PrPSc against PK digestion, a scrapie-positive sheep brain homogenate was treated with a dilution series of DB772 (range, 38.1 to 1.2 μM) similar to previous work done using polythiophenes (39). Relative to vehicle-treated controls, preincubation of ScBH with DB772 significantly increased PK-resistant PrPSc bands in samples treated with 38.1 μM (107% increase, P = 0.0018) (Fig. 7A) but not at the lower concentrations that were effective in inhibiting PrPSc accumulation in cell culture and sPMCA assays; it is possible that this disparity between effective concentrations may just reflect differences in assay matrices (e.g., abundance of lipids in brain homogenate, etc.), but this was not further investigated.

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.