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Oncotarget. 2016 Mar 15;7(11):12975-96. doi: 10.18632/oncotarget.7349.

Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function.

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Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA.
Urologic and Diagnostic Radiology, Yale School of Medicine, New Haven, CT 06520-8058, USA.
Urologic Oncology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
Department of Chemistry and Physics, Augusta University, Augusta, GA 30912, USA.
Arno Therapeutics, Flemington, NJ 08822, USA.
Molecular and Translational Science, United States Medicinal Chemistry and Pharmacognosy, School of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
School of Biological Sciences and Illawarra Health and Medical Research Institute, University of Wollongong, NSW 2522, Australia.


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.


ATPase; OSU-03012; chaperones; pazopanib; sorafenib

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