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Novel compounds lowering the cellular isoform of the human prion protein in cultured human cells.

ABSTRACT: Previous studies showed that lowering PrP(C) concomitantly reduced PrP(Sc) in the brains of mice inoculated with prions. We aimed to develop assays that measure PrP(C) on the surface of human T98G glioblastoma and IMR32 neuroblastoma cells. Using these assays, we sought to identify chemical hits, confirmed hits, and scaffolds that potently lowered PrP(C) levels in human brains cells, without lethality, and that could achieve drug concentrations in the brain after oral or intraperitoneal dosing in mice.We utilized HTS ELISA assays to identify small molecules that lower PrP(C) levels by ?30% on the cell surface of human glioblastoma (T98G) and neuroblastoma (IMR32) cells.From 44,578 diverse chemical compounds tested, 138 hits were identified by single point confirmation (SPC) representing 7 chemical scaffolds in T98G cells, and 114 SPC hits representing 6 scaffolds found in IMR32 cells. When the confirmed SPC hits were combined with structurally related analogs, >300 compounds (representing 6 distinct chemical scaffolds) were tested for dose-response (EC??) in both cell lines, only studies in T98G cells identified compounds that reduced PrP(C) without killing the cells. EC?? values from 32 hits ranged from 65 nM to 4.1 ?M. Twenty-eight were evaluated in vivo in pharmacokinetic studies after a single 10 mg/kg oral or intraperitoneal dose in mice. Our results showed brain concentrations as high as 16.2 ?M, but only after intraperitoneal dosing.Our studies identified leads for future studies to determine which compounds might lower PrP(C) levels in rodent brain, and provide the basis of a therapeutic for fatal disorders caused by PrP prions.

SUBMITTER: Silber BM

PROVIDER: S-EPMC3984052 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Novel compounds lowering the cellular isoform of the human prion protein in cultured human cells.

Silber B Michael BM Gever Joel R JR Rao Satish S Li Zhe Z Renslo Adam R AR Widjaja Kartika K Wong Casper C Giles Kurt K Freyman Yevgeniy Y Elepano Manuel M Irwin John J JJ Jacobson Matthew P MP Prusiner Stanley B SB

Bioorganic & medicinal chemistry 20140109 6

<h4>Purpose</h4>Previous studies showed that lowering PrP(C) concomitantly reduced PrP(Sc) in the brains of mice inoculated with prions. We aimed to develop assays that measure PrP(C) on the surface of human T98G glioblastoma and IMR32 neuroblastoma cells. Using these assays, we sought to identify chemical hits, confirmed hits, and scaffolds that potently lowered PrP(C) levels in human brains cells, without lethality, and that could achieve drug concentrations in the brain after oral or intraper ...[more]

PMID: 24530226

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Project description:Anti-apoptotic properties of physiological and elevated levels of the cellular prion protein (PrPc) under stress conditions are well documented. Yet, detrimental effects of elevated PrPc levels under stress conditions, such as exposure to staurosporine (STS) have also been described. In the present study, we focused on discerning early apoptotic STS-induced proteome and phospho-proteome changes in SH-SY5Y human neuroblastoma cells stably transfected either with an empty or PRNP-containing vector, expressing physiological or supraphysiological levels of PrPc, respectively. PrPc-overexpression per se appears to stress the cells under STS-free conditions as indicated by diminished cell viability of PrPc-overexpressing versus control cells. However, PrPc-overexpression becomes advantageous following exposure to STS. Thus, only a short exposure (2 h) to 1 μM STS results in lower survival rates and significantly higher caspase-3 activity in control versus PrPc-overexpressing cells. Hence, by exposing both experimental groups to the same apoptotic conditions we were able to induce apoptosis in control, but not in PrPc-overexpressing cells (as assessed by caspase-3 activity), which allowed for filtering out proteins possibly contributing to protection against STS-induced apoptosis in PrPc-overexpressing cells. Among other proteins regulated by different PrPc levels following exposure to STS, those involved in maintenance of cytoskeleton integrity caught our attention. In particular, the finding that elevated PrPc levels significantly reduce profilin-1 (PFN-1) expression. PFN-1 is known to facilitate STS-induced apoptosis. Silencing of PFN-1 expression by siRNA significantly increased viability of PrPc-overexpressing versus control cells, under STS treatment. In addition, PrPc-overexpressing cells depleted of PFN-1 exhibited increased viability versus PrPc-overexpressing cells with preserved PFN-1 expression, both subjected to STS. Concomitant increase in caspase-3 activity was observed in control versus PrPc-overexpressing cells after treatment with siRNA- PFN-1 and STS. We suggest that reduction of PFN-1 expression by elevated levels of PrPc may contribute to protective effects PrPc-overexpressing SH-SY5Y cells confer against STS-induced apoptosis.

Dataset Information

Novel compounds lowering the cellular isoform of the human prion protein in cultured human cells.

Publications

Novel compounds lowering the cellular isoform of the human prion protein in cultured human cells.

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