A rapid cellular FRET assay of polyglutamine aggregation identifies a novel inhibitor.
Many neurodegenerative diseases, including tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and the polyglutamine diseases, are characterized by intracellular aggregation of pathogenic proteins. It is difficult to study modifiers of this process in intact cells in a high-throughput and quantitative manner, although this could facilitate molecular insights into disease pathogenesis. Here we introduce a high-throughput assay to measure intracellular polyglutamine protein aggregation using fluorescence resonance energy transfer (FRET). We screened over 2800 biologically active small molecules for inhibitory activity and have characterized one lead compound in detail. Y-27632, an inhibitor of the Rho-associated kinase p160ROCK, diminished polyglutamine protein aggregation (EC(50) congruent with 5 microM) and reduced neurodegeneration in a Drosophila model of polyglutamine disease. This establishes a novel high-throughput approach to study protein misfolding and aggregation associated with neurodegenerative diseases and implicates a signaling pathway of previously unrecognized importance in polyglutamine protein processing.
Pollitt
Sonia K
SK
Genentech, Inc., South San Francisco, CA 94080, USA.
Pallos
Judit
J
Shao
Jieya
J
Desai
Urvee A
UA
Ma
Aye Aye K
AA
Thompson
Leslie Michels
LM
Marsh
J Lawrence
JL
Diamond
Marc I
MI
D000577:Amides / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D030801:Animals, Genetically Modified; D001681:Biological Assay / Q000379:methods*; D019556:COS Cells; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D004195:Disease Models, Animal; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004331:Drosophila melanogaster; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D000071058:Huntingtin Protein; D002479:Inclusion Bodies / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins / Q000172:deficiency / Q000235:genetics; D019636:Neurodegenerative Diseases / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000172:deficiency / Q000235:genetics; D010455:Peptides / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017956:Photoreceptor Cells, Invertebrate / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D017510:Protein Folding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology* / Q000627:therapeutic use; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D019680:Trinucleotide Repeat Expansion / Q000187:drug effects / Q000235:genetics; D054460:rho-Associated Kinases
D000577:Amides; D004791:Enzyme Inhibitors; C086055:HTT protein, human; D000071058:Huntingtin Protein; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011725:Pyridines; C108830:Y 27632; C097188:polyglutamine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases