TRFRET-based cell-based primary high throughput screening assay to identify inhibitors of cell surface Prion Protein (PRPC)
Name: TRFRET-based cell-based primary high throughput screening assay to identify inhibitors of cell surface Prion Protein (PRPC) ..more
BioActive Compounds: 1596
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Corinne Lasmezas, The Scripps Research Institute Molecular Screening Center
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA035192-01
Grant Proposal PI: Corinne Lasmezas, The Scripps Research Institute Molecular Screening Center
External Assay ID: PRPC_INH_TRFRET_1536_1X%INH PRUN
Name: TRFRET-based cell-based primary high throughput screening assay to identify inhibitors of cell surface Prion Protein (PRPC)
Prion diseases are lethal infectious neurodegenerative diseases affecting animals and humans . In humans, Creutzfeldt-Jakob Disease (CJD) is either sporadic, affecting mainly people over 60 years, iatrogenic, or genetic with a high penetrance. Prion diseases are characterized by the accumulation, in brain and lymphoid tissue of PrPsc , a misfolded, aggregated form of the host prion protein PrP [3, 4]. PrPsc is thought to be the main or only constituent of the prion, the infectious agent [5-8]. PrP is a glycosylphosphatidylinositol (GPI)-anchored protein of 254 amino acids attached to the cell surface in microdomains known as lipid rafts [9-11], and tends to aggregate into rod-like structures. The encoded protein contains a highly flexible region of five tandem octapeptide repeats. Mutations in the repeat region as well as elsewhere in this gene have been associated with Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler disease.
Prion replication is based on the structural conversion of the host prion protein into its harmful misfolded counterpart. In the sporadic form of CJD, the initial misfolding of PrP may be a rare, stochastic event. In familial CJD, this event is triggered by certain mutations in the protein. When the disease is transmitted, PrPsc acts as a template and "seeds" the misfolding of the host PrP. Prion diseases are not curable and the survival time is typically 6 to 12 months after the onset of symptoms. Bovine spongiform encephalopathy (BSE), or mad cow disease, is transmissible to humans [12, 13]. While about 200 individuals are known to have succumbed to "human BSE" (variant CJD), the number of infected but as yet asymptomatic people is unknown because there is no preclinical diagnostic test for prion diseases and the incubation time may extend to decades; this is of particular concern because the disease is transmissible by blood transfusion. As a result, the identification of compounds that suppress cell surface PrP and thereby suppress prion replication may provide useful tools for the therapy of prion diseases, and for study of PrP biosynthetic and cellular trafficking pathways. [14-18].
1. Prusiner, S.B., Novel proteinaceous infectious particles cause scrapie. Science, 1982. 216(4542): p. 136-44.
2. Doi, S., et al., Western blot detection of scrapie-associated fibril protein in tissues outside the central nervous system from preclinical scrapie-infected mice. J Gen Virol, 1988. 69 ( Pt 4): p. 955-60
3. Pan, K.M., et al., Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci U S A, 1993. 90(23): p. 10962-6.
4. Gasset, M., et al., Perturbation of the secondary structure of the scrapie prion protein under conditions that alter infectivity. Proc Natl Acad Sci U S A, 1993. 90(1): p. 1-5.
5. Legname, G., et al., Synthetic mammalian prions. Science, 2004. 305(5684): p. 673-6
6. Couzin, J., Biomedicine. An end to the prion debate? Don't count on it. Science, 2004. 305(5684): p. 589
7. Weissmann, C., A 'unified theory' of prion propagation. Nature, 1991. 352(6337): p. 679-83.
8. Lasmezas, C.I., Putative functions of PrP(C). Br Med Bull, 2003. 66: p. 61-70.
9. Naslavsky, N., et al., Characterization of detergent-insoluble complexes containing the cellular prion protein and its scrapie isoform. J Biol Chem, 1997. 272(10): p. 6324-31.
10. Sarnataro, D., et al., PrP(C) association with lipid rafts in the early secretory pathway stabilizes its cellular conformation. Mol Biol Cell, 2004. 15(9): p. 4031-42.
11. Vey, M., et al., Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains. Proc Natl Acad Sci U S A, 1996. 93(25): p. 14945-9.
12. Will, R.G., et al., A new variant of Creutzfeldt-Jakob disease in the UK. Lancet, 1996. 347(9006): p. 921-5.
13. Lasmezas, C.I., et al., BSE transmission to macaques. Nature, 1996. 381(6585): p. 743-4.
14. Trevitt, C.R. and J. Collinge, A systematic review of prion therapeutics in experimental models. Brain, 2006. 129(Pt 9): p. 2241-65.
15. Charveriat, M., et al., New inhibitors of prion replication that target the amyloid precursor. J Gen Virol, 2009. 90(Pt 5): p. 1294-301.
16. Achyuthan, K.E. and D.G. Whitten, Design considerations for high-throughput screening and in vitro diagnostic assays. Comb Chem High Throughput Screen, 2007. 10(6): p. 399-412.
17. Kocisko, D.A., et al., Evaluation of new cell culture inhibitors of protease-resistant prion protein against scrapie infection in mice. J Gen Virol, 2004. 85(Pt 8): p. 2479-83.
18. Kocisko, D.A., et al., New inhibitors of scrapie-associated prion protein formation in a library of 2000 drugs and natural products. J Virol, 2003. 77(19): p. 10288-94.
PRPC, PrPC, PRNP, ASCR, CD230, CJD, GSS, KURU, PRIP, PrP, PrP27-30, PrP33-35C, PrPc, p27-30, LD9, LD9 cells, fibroblasts, L929, Brefeldin A, brefeldine A, BFA, primary, singlicate, biochemical, inhibit, inhibitor, inhibition, inh, TRFRET, TR-FRET, HTRF, fluorescence, fluor, cell, surface, antibodies, antibody, donor, acceptor, prion, CJD, Creutzfeldt-Jakob Disease, AD, Alzheimer, amyloid, plaque, mad cow, BSE, HTS, high throughput screen, 1536, Scripps, Scripps Florida, MLSMR, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that act as inhibitors of PRPc cell surface expression. This assay detects PrPc on the surface of living LD9 fibroblast cells using two PRPC-specific antibodies which carry donor and acceptor fluorophores, respectively.
In this assay, the antibodies are directed against distinct, non-overlapping PrPc epitopes. Antibody occupancy of both epitopes of a PrPc molecule results in FRET signal emission. Free antibody or single antibody occupancy is not detected. The assay employs long half-life lanthanide donors (Europium or Terbium cryptate) and acceptor fluorophores XL665 or d2: SAF32 (aa53-93) and D18 (aa133-157) labeled with the donor and acceptor fluorophores, respectively. Ratios of 665 nm to 620 nm measurements are calculated, to take into account non-specific absorption of 620 nm light by the assay matrix. Compounds are tested in singlicate at a final nominal concentration of 13.8 microM.
The LD9 cell line (subclone of L929, which is a mouse fibroblast cell line) was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Opti-MEM I Reduced Serum Medium (no phenol red) supplemented with 5% v/v Bovine Growth Serum Supplemented Calf and 1X Penicillin-Streptomycin.
The day before the assay, 3 uL of cell growth media was dispensed into the first two columns of 1536 well microtiter plates and 1500 cells in 3 uL of cell growth media were seeded into the remaining wells. Next, 42 nL of test compound in DMSO, Brefeldin A (58 uM final concentration) in DMSO, or DMSO alone (1.38% final concentration) were dispensed to the appropriate wells. The plates were then incubated for 21 hours at 37 C, 5% CO2, and 95 % RH.
The assay was started by adding 1 uL of SAF32-Tb at a final concentration of 0.072ug/mL and 1ul of D18-d2 at a final concentration of 0.66ug/mL to all wells, respectively. Labeled Antibodies were prepared in 1X PBS. Plates were centrifuged and after 3 hours of incubation at room temperature, well TRFRET was read on a ViewLux microplate reader (Perkin Elmer, Turku, Finland). Measurements were performed by exciting the plates at 340 nM, and monitoring well fluorescence at 618 nm (Tb) and 671 nm (d2) with the ViewLux microplate reader.
To normalize data, values measured from both fluorescence emission wavelengths were used to calculate a ratio for each well, according to the following mathematical expression:
Ratio = I671nm / I618nm
I represents the measured fluorescence emission intensity at the enumerated wavelength in nm.
The percent inhibition was calculated from the median ratio as follows:
%_Inhibition = 100 * ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) )
Test_Compound is defined as wells containing cells, test compounds and DMSO.
High_Control is defined as wells containing cells, Brefeldin A and DMSO.
Low_Control is defined as the median of the wells containing test compounds.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-18, and for inactive compounds 18-0.
List of Reagents:
LD9 cells (supplied by Assay Provider)
Opti-MEM Reduced Serum Medium (Life Technologies, part 11058-021)
Bovine Growth Serum Supplemented Calf (Hyclone, part SH30541.03)
Penicillin-Streptomycin (100X) (Life Technologies, part 15140-122)
Brefeldin A (Control, Sigma, part B7651)
SAF32-Tb (Cisbio, custom labeled antibody)
D18-d2 (Cisbio, custom labeled antibody)
PBS (Life Technologies, part 10010-031)
Detachin Cell Detachment Reagent (Genlantis, part T100100)
T-175 Flasks (Nunc, part 159910)
1536-well plates (Corning, part 7298)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)