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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.


Selected DB compounds inhibit PrPSc-seeded misfolding during serial PMCA assay. The inhibition of PrPSc-seeded misfolding was evaluated by sPMCA assay by treating mNBH with 1 μM indicated compound. Reaction mixtures were collected at each round and tested for PK-resistant PrPSc by immunoblotting, and band intensities were analyzed as previously described (Fig. 5). (A) A representative immunoblot from samples collected at round 2n is demonstrated. (B and C) Percent reduction categorical scores were evaluated from reaction mixtures seeded with 10−5 (B) or 10−6 (C) dilutions of 10% ScBH. Data columns represent the median score ±1 standard deviation. a, all three replicates scored 0.
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Figure 6: Selected DB compounds inhibit PrPSc-seeded misfolding during serial PMCA assay. The inhibition of PrPSc-seeded misfolding was evaluated by sPMCA assay by treating mNBH with 1 μM indicated compound. Reaction mixtures were collected at each round and tested for PK-resistant PrPSc by immunoblotting, and band intensities were analyzed as previously described (Fig. 5). (A) A representative immunoblot from samples collected at round 2n is demonstrated. (B and C) Percent reduction categorical scores were evaluated from reaction mixtures seeded with 10−5 (B) or 10−6 (C) dilutions of 10% ScBH. Data columns represent the median score ±1 standard deviation. a, all three replicates scored 0.

Mentions: In the next experiment (Fig. 6), reaction mixtures were treated with 1 μM three preliminary hit compounds (DB818A, DB932A, and DB948), two structurally similar compounds that had demonstrated weak inhibitory activity in cell culture (DB771 and DB302), and DB772 as a control. DB771, which has only one substitution compared to DB772 and is a weak cell culture inhibitor, lacked any measurable inhibitory activity against PrPSc-seeded misfolding. Interestingly, DB948, the compound with the strongest cell-based inhibitory activity, also lacked detectable inhibition in the sPMCA reaction mixtures. DB932A, DB818A, and the remaining weak cell culture inhibitor, DB302, demonstrated strong inhibitory activity against PrPSc-seeded misfolding (Fig. 6A). The peak inhibitory activity of DB772 was observed at round 1n, particularly in reaction mixtures seeded with 10−6 dilutions of ScBH. DB932A and DB302 exhibited strong inhibition following round 1n, regardless of the seed concentration. Inhibition declined slightly following round 2n but remained in reaction mixtures seeded with a 10−6 dilution of ScBH. Finally, DB818A inhibited PrPSc-seeded misfolding in all reaction mixtures regardless of the seed concentration or round (Fig. 6). Taken together, the data suggest that DB772 and some of the structurally similar analogs may act by reducing prion-associated conversion of PrPC to PrPSc. As demonstrated by DB948 and DB302, the cell-based inhibitory activity does not always follow that of seeded misfolding inhibition, which may reflect alternative mechanisms of PrPSc inhibition (e.g., for DB948) and alternative cellular distribution and/or metabolism (e.g., for DB302).


Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System
Selected DB compounds inhibit PrPSc-seeded misfolding during serial PMCA assay. The inhibition of PrPSc-seeded misfolding was evaluated by sPMCA assay by treating mNBH with 1 μM indicated compound. Reaction mixtures were collected at each round and tested for PK-resistant PrPSc by immunoblotting, and band intensities were analyzed as previously described (Fig. 5). (A) A representative immunoblot from samples collected at round 2n is demonstrated. (B and C) Percent reduction categorical scores were evaluated from reaction mixtures seeded with 10−5 (B) or 10−6 (C) dilutions of 10% ScBH. Data columns represent the median score ±1 standard deviation. a, all three replicates scored 0.
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Related In: Results  -  Collection

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Figure 6: Selected DB compounds inhibit PrPSc-seeded misfolding during serial PMCA assay. The inhibition of PrPSc-seeded misfolding was evaluated by sPMCA assay by treating mNBH with 1 μM indicated compound. Reaction mixtures were collected at each round and tested for PK-resistant PrPSc by immunoblotting, and band intensities were analyzed as previously described (Fig. 5). (A) A representative immunoblot from samples collected at round 2n is demonstrated. (B and C) Percent reduction categorical scores were evaluated from reaction mixtures seeded with 10−5 (B) or 10−6 (C) dilutions of 10% ScBH. Data columns represent the median score ±1 standard deviation. a, all three replicates scored 0.
Mentions: In the next experiment (Fig. 6), reaction mixtures were treated with 1 μM three preliminary hit compounds (DB818A, DB932A, and DB948), two structurally similar compounds that had demonstrated weak inhibitory activity in cell culture (DB771 and DB302), and DB772 as a control. DB771, which has only one substitution compared to DB772 and is a weak cell culture inhibitor, lacked any measurable inhibitory activity against PrPSc-seeded misfolding. Interestingly, DB948, the compound with the strongest cell-based inhibitory activity, also lacked detectable inhibition in the sPMCA reaction mixtures. DB932A, DB818A, and the remaining weak cell culture inhibitor, DB302, demonstrated strong inhibitory activity against PrPSc-seeded misfolding (Fig. 6A). The peak inhibitory activity of DB772 was observed at round 1n, particularly in reaction mixtures seeded with 10−6 dilutions of ScBH. DB932A and DB302 exhibited strong inhibition following round 1n, regardless of the seed concentration. Inhibition declined slightly following round 2n but remained in reaction mixtures seeded with a 10−6 dilution of ScBH. Finally, DB818A inhibited PrPSc-seeded misfolding in all reaction mixtures regardless of the seed concentration or round (Fig. 6). Taken together, the data suggest that DB772 and some of the structurally similar analogs may act by reducing prion-associated conversion of PrPC to PrPSc. As demonstrated by DB948 and DB302, the cell-based inhibitory activity does not always follow that of seeded misfolding inhibition, which may reflect alternative mechanisms of PrPSc inhibition (e.g., for DB948) and alternative cellular distribution and/or metabolism (e.g., for DB302).

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.