Vaughn Ewing posted an update 3 months, 1 week ago
The mule deer-derived brain fibroblast cell line has been used to demonstrate the anti-prion LEE011 activity of pentosan polysulfate and a porphyrin compound. Besides the varying effectiveness of anti-prion compounds in different systems, another consideration for model development is the potential for significant effects of co-infecting agents. It has been shown that small ruminant lentivirus infection is associated with enhanced distribution of PrPSc in naturally co-infected sheep. This effect in sheep may be related to virus-enhanced, intracellular accumulation of PrPSc, as has been demonstrated in vitro using primary sheep microglial cells. It is unknown if other virus families have similar effects. Flaviviruses are a group of enveloped, positive-sense stranded RNA viruses that can infect monocyte-lineage cells, establish persistent infections in vivo, and establish noncytopathic infections in vitro. Rov9 cells, as derivatives of RK13 cells, and sheep microglial cells are susceptible to bovine viral diarrhea virus infection. To cure cells of a potentially confounding, co-infecting virus, 2- -5- furan dihydrochloride, a known BVDV inhibitor, was used. In addition to inhibiting BVDV, this treatment also inhibited PrPSc accumulation. Here we describe the anti-prion activity of DB772, a monocationic phenyl-furanbenzimidazole, which belongs to a chemical category previously untested for anti-PrPSc activity. In summary, only one study has investigated in vitro chemical inhibition of prions in a cell system derived from a natural host and no studies have tested for anti-prion activity in a sheep cell culture system or in microglial cells from any species, despite the relevance of sheep scrapie and monocyte-derived cells to prion diseases. Reported herein is the discovery of anti-prion activity of a compound belonging to a previously untested chemical category using sheep-origin PrPSc and sheep microglial cells. Despite previous research investigating compounds with anti- PrPSc activity, no effective chemotherapeutics exist for the treatment or prevention of prion diseases. Identification of new classes of anti-prion compounds is therefore vital, not only for the practical application of in vivo chemotherapeutics, but also for investigations studying the mechanisms of PrPSc conversion and accumulation. The data herein describe the discovery of in vitro anti-prion activity of a novel aromatic monocation. The anti-prion activity was demonstrated in two different cell culture models, including a cell type that is relevant to natural prion disease. While the anti-prion effects described herein were discovered while using DB772 to eliminate BVDV from primary sheep microglial cells and Rov9 cells, to the authors’ knowledge there are no published reports of phenyl-furan-benzimidazole cations with anti-prion activity. In addition to the PrPSc inhibition, DB772 treatment also inhibited BVDV in both cell lines; however, it did not cure most of the cell replicates as BVDV antigen and BVDV RNA returned to detectable levels in one microglial replicate and in all of the Rov9 cell replicates. This incomplete pestivirus inhibition is different from what was demonstrated in primary bovine fibroblasts. The differing results may be due to differences in the strains of BVDV that were tested, as well as the different cell types. The cytotoxicity of DB772 was evaluated in sheep microglial cells and Rov9 cells. The 50% cytotoxicity point was similar between sheep microglial cells and Rov9 cells and is also similar to the previously demonstrated CC50 of 8.6 mM in B16 melanoma cells. These values are in contrast to previous cytotoxicity studies using DB772 in Madin-Darby bovine kidney cells, in which the CC50 was substantially higher at 215 mM. The discrepancy between these CC50 values is possibly a reflection of the different cell types, but may also be a result of the different culture conditions used. Initial investigations into the mechanism of action were conducted and while no mechanism was identified, some potential mechanisms have been ruled out. Expression of PRNP is required for PrPSc permissiveness and the level of expression correlates with PrPSc permissiveness ; thus, one obvious mechanism of PrPSc inhibition would be the partial to complete inhibition of PRNP expression. There was no evidence that DB772 inhibited PRNP expression, as PRNP transcript levels and total PrP protein levels were not decreased. In fact at passage four, microgliaSc/DB772 and microgliaC/DB772 cells have significant increases in PRNP transcript and total PrP protein levels as compared to the untreated controls. This confirms that DB772 does not inhibit PrPC expression in microglial cells and suggests that PrPC expression may increase in response to DB772 exposure.