title: Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function.
abstract: We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone - chaperone and chaperone - client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 -dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 -induced activation of ER stress signaling and maintained mTOR activity; AR-12 -mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types.
authors.affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Laurence
authors.initials: L
authors.lastname: Booth
authors.affiliation: Urologic and Diagnostic Radiology, Yale School of Medicine, New Haven, CT 06520-8058, USA.|Urologic Oncology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Shuch
authors.affiliation: Department of Chemistry and Physics, Augusta University, Augusta, GA 30912, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Albers
authors.affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Jane L
authors.initials: JL
authors.lastname: Roberts
authors.affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Mehrad
authors.initials: M
authors.lastname: Tavallai
authors.affiliation: Arno Therapeutics, Flemington, NJ 08822, USA.
authors.forename: Stefan
authors.initials: S
authors.lastname: Proniuk
authors.affiliation: Arno Therapeutics, Flemington, NJ 08822, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Zukiwski
authors.affiliation: Molecular and Translational Science, United States Medicinal Chemistry and Pharmacognosy, School of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Dasheng
authors.initials: D
authors.lastname: Wang
authors.affiliation: Molecular and Translational Science, United States Medicinal Chemistry and Pharmacognosy, School of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Ching-Shih
authors.initials: CS
authors.lastname: Chen
authors.affiliation: Urologic Oncology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Don
authors.initials: D
authors.lastname: Bottaro
authors.affiliation: School of Biological Sciences and Illawarra Health and Medical Research Institute, University of Wollongong, NSW 2522, Australia.
authors.forename: Heath
authors.initials: H
authors.lastname: Ecroyd
authors.affiliation: Department of Chemistry and Physics, Augusta University, Augusta, GA 30912, USA.
authors.forename: Iryna O
authors.initials: IO
authors.lastname: Lebedyeva
authors.affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Dent
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D007191:Indazoles; D062105:Molecular Docking Simulation; D009536:Niacinamide / Q000031:analogs & derivatives / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011720:Pyrazoles / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology; D000077157:Sorafenib; D013449:Sulfonamides / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D007191:Indazoles; C500894:OSU 03012; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D013449:Sulfonamides; D009536:Niacinamide; C516667:pazopanib; D000077157:Sorafenib
keywords: ATPase; OSU-03012; chaperones; pazopanib; sorafenib