title: Proteasome activation is a mechanism for pyrazolone small molecules displaying therapeutic potential in amyotrophic lateral sclerosis.
abstract: Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative disease. Pyrazolone containing small molecules have shown significant disease attenuating efficacy in cellular and murine models of ALS. Pyrazolone based affinity probes were synthesized to identify high affinity binding partners and ascertain a potential biological mode of action. Probes were confirmed to be neuroprotective in PC12-SOD1(G93A) cells. PC12-SOD1(G93A) cell lysates were used for protein pull-down, affinity purification, and subsequent proteomic analysis using LC-MS/MS. Proteomics identified the 26S proteasome regulatory subunit 4 (PSMC1), 26S proteasome regulatory subunit 6B (PSMC4), and T-complex protein 1 (TCP-1) as putative protein targets. Coincubation with appropriate competitors confirmed the authenticity of the proteomics results. Activation of the proteasome by pyrazolones was demonstrated in the absence of exogenous proteasome inhibitor and by restoration of cellular protein degradation of a fluorogenic proteasome substrate in PC12-SOD1(G93A) cells. Importantly, supplementary studies indicated that these molecules do not induce a heat shock response. We propose that pyrazolones represent a rare class of molecules that enhance proteasomal activation in the absence of a heat shock response and may have therapeutic potential in ALS.
authors.affiliation: Department of Chemistry, ‡Department of Molecular Biosciences, Rice Institute for Biomedical Research, and §Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Trippier
authors.forename: Kevin Tianmeng
authors.initials: KT
authors.lastname: Zhao
authors.forename: Susan G
authors.initials: SG
authors.lastname: Fox
authors.forename: Isaac T
authors.initials: IT
authors.lastname: Schiefer
authors.forename: Radhia
authors.initials: R
authors.lastname: Benmohamed
authors.forename: Jason
authors.initials: J
authors.lastname: Moran
authors.forename: Donald R
authors.initials: DR
authors.lastname: Kirsch
authors.forename: Richard I
authors.initials: RI
authors.lastname: Morimoto
authors.forename: Richard B
authors.initials: RB
authors.lastname: Silverman
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000690:Amyotrophic Lateral Sclerosis / Q000378:metabolism*; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D000071183:Autophagy-Related Proteins; D019921:Biotinylation; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004195:Disease Models, Animal; D004789:Enzyme Activation / Q000187:drug effects; D006358:Hot Temperature; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016716:PC12 Cells; D040901:Proteomics; D047069:Pyrazolones / Q000737:chemistry* / Q000494:pharmacology*; D051381:Rats; D013482:Superoxide Dismutase / Q000235:genetics; D053719:Tandem Mass Spectrometry; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000894:Anti-Inflammatory Agents, Non-Steroidal; D000071183:Autophagy-Related Proteins; D018797:Cell Cycle Proteins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008164:Luminescent Proteins; D047069:Pyrazolones; C405662:UBQLN2 protein, human; D014452:Ubiquitins; C038362:pyrazolone; C109415:SOD1 G93A protein; D013482:Superoxide Dismutase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: Amyotrophic lateral sclerosis; Neurodegeneration; Drug Discovery; Proteasome activator; Pyrazolone; Target identification