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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 inhibits PrPSc-seeded misfolding during serial PMCA assay. The inhibition of PrPSc-seeded misfolding was evaluated by sPMCA assay by treating mNBH with either 1 or 4 μM DB772. Treated reaction mixtures were seeded with 10−4, 10−5, or 10−6 dilutions of 10% ScBH and processed for 4 rounds. Reaction mixtures were collected at each round and tested for PK-resistant PrPSc by immunoblotting. PMCA rounds were normalized to the initial round of PrPSc detection (i.e., the initial round of detection corresponds to round 1n). (A) A representative immunoblot from samples collected at round 1n is demonstrated. Band intensities were quantified by densitometry. (B and C) Densities of bands from DB772-treated reaction mixtures were normalized to those of vehicle control (0.4% DMSO) to obtain a percent reduction categorical score for DB772 at 1 μM (B) and 4 μM (C). Data columns represent the median score ±1 standard deviation. a, all three replicates scored 0. NT, reaction mixtures seeded with 10−5 or 10−6 ScBH and treated with 1 μM DB772 were not tested at round 3n.
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Figure 5: DB772 inhibits PrPSc-seeded misfolding during serial PMCA assay. The inhibition of PrPSc-seeded misfolding was evaluated by sPMCA assay by treating mNBH with either 1 or 4 μM DB772. Treated reaction mixtures were seeded with 10−4, 10−5, or 10−6 dilutions of 10% ScBH and processed for 4 rounds. Reaction mixtures were collected at each round and tested for PK-resistant PrPSc by immunoblotting. PMCA rounds were normalized to the initial round of PrPSc detection (i.e., the initial round of detection corresponds to round 1n). (A) A representative immunoblot from samples collected at round 1n is demonstrated. Band intensities were quantified by densitometry. (B and C) Densities of bands from DB772-treated reaction mixtures were normalized to those of vehicle control (0.4% DMSO) to obtain a percent reduction categorical score for DB772 at 1 μM (B) and 4 μM (C). Data columns represent the median score ±1 standard deviation. a, all three replicates scored 0. NT, reaction mixtures seeded with 10−5 or 10−6 ScBH and treated with 1 μM DB772 were not tested at round 3n.

Mentions: PrPSc-seeded misfolding was inhibited in the presence of DB772 at both 1 and 4 μM (Fig. 5A). While the inhibition was most pronounced for reaction mixtures using more dilute PrPSc seed (i.e., in reaction mixtures using 10−5 and 10−6 dilutions of ScBH) (Fig. 5B and C), the inhibitory effect of 1 μM DB772 was similar to, if not greater than, that at 4 μM. This suggests that micromolar DB772 is near its maximal effective concentration in this assay, which is consistent with its micromolar maximum antiprion activity in cell culture. It is interesting that the initial inhibitory action of DB772 was overcome with additional rounds of PMCA despite the fresh addition of DB772 at the beginning of each round. This indicates that micromolar DB772 may inhibit only a subpopulation of the seeds present in ScBH, thereby delaying rather than abolishing the accumulation of PK-resistant PrPSc in this assay. It is also possible that DB772 may select for a quasispecies more resistant to this inhibitory activity, similar to what has been observed with some antiprion compounds (e.g., swainsonine) (55). Nevertheless, 1 μM DB772 did reduce PrPSc-seeded misfolding by 1/2-log or greater (i.e., a score of 2) compared to the vehicle control, indicating that DB772 may directly inhibit PrP conversion.


Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System
DB772 inhibits PrPSc-seeded misfolding during serial PMCA assay. The inhibition of PrPSc-seeded misfolding was evaluated by sPMCA assay by treating mNBH with either 1 or 4 μM DB772. Treated reaction mixtures were seeded with 10−4, 10−5, or 10−6 dilutions of 10% ScBH and processed for 4 rounds. Reaction mixtures were collected at each round and tested for PK-resistant PrPSc by immunoblotting. PMCA rounds were normalized to the initial round of PrPSc detection (i.e., the initial round of detection corresponds to round 1n). (A) A representative immunoblot from samples collected at round 1n is demonstrated. Band intensities were quantified by densitometry. (B and C) Densities of bands from DB772-treated reaction mixtures were normalized to those of vehicle control (0.4% DMSO) to obtain a percent reduction categorical score for DB772 at 1 μM (B) and 4 μM (C). Data columns represent the median score ±1 standard deviation. a, all three replicates scored 0. NT, reaction mixtures seeded with 10−5 or 10−6 ScBH and treated with 1 μM DB772 were not tested at round 3n.
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Figure 5: DB772 inhibits PrPSc-seeded misfolding during serial PMCA assay. The inhibition of PrPSc-seeded misfolding was evaluated by sPMCA assay by treating mNBH with either 1 or 4 μM DB772. Treated reaction mixtures were seeded with 10−4, 10−5, or 10−6 dilutions of 10% ScBH and processed for 4 rounds. Reaction mixtures were collected at each round and tested for PK-resistant PrPSc by immunoblotting. PMCA rounds were normalized to the initial round of PrPSc detection (i.e., the initial round of detection corresponds to round 1n). (A) A representative immunoblot from samples collected at round 1n is demonstrated. Band intensities were quantified by densitometry. (B and C) Densities of bands from DB772-treated reaction mixtures were normalized to those of vehicle control (0.4% DMSO) to obtain a percent reduction categorical score for DB772 at 1 μM (B) and 4 μM (C). Data columns represent the median score ±1 standard deviation. a, all three replicates scored 0. NT, reaction mixtures seeded with 10−5 or 10−6 ScBH and treated with 1 μM DB772 were not tested at round 3n.
Mentions: PrPSc-seeded misfolding was inhibited in the presence of DB772 at both 1 and 4 μM (Fig. 5A). While the inhibition was most pronounced for reaction mixtures using more dilute PrPSc seed (i.e., in reaction mixtures using 10−5 and 10−6 dilutions of ScBH) (Fig. 5B and C), the inhibitory effect of 1 μM DB772 was similar to, if not greater than, that at 4 μM. This suggests that micromolar DB772 is near its maximal effective concentration in this assay, which is consistent with its micromolar maximum antiprion activity in cell culture. It is interesting that the initial inhibitory action of DB772 was overcome with additional rounds of PMCA despite the fresh addition of DB772 at the beginning of each round. This indicates that micromolar DB772 may inhibit only a subpopulation of the seeds present in ScBH, thereby delaying rather than abolishing the accumulation of PK-resistant PrPSc in this assay. It is also possible that DB772 may select for a quasispecies more resistant to this inhibitory activity, similar to what has been observed with some antiprion compounds (e.g., swainsonine) (55). Nevertheless, 1 μM DB772 did reduce PrPSc-seeded misfolding by 1/2-log or greater (i.e., a score of 2) compared to the vehicle control, indicating that DB772 may directly inhibit PrP conversion.

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.