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

// Laurence Booth 1, * , Brian Shuch 2, 3, * , Thomas Albers 7, * , Jane L. Roberts 1 , Mehrad Tavallai 1 , Stefan Proniuk 5 , Alexander Zukiwski 5 , Dasheng Wang 4 , Ching-Shih Chen 4 , Don Bottaro 3 , Heath Ecroyd 6 , Iryna O. Lebedyeva 7 , Paul Dent 1 1 Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA 2 Urologic and Diagnostic Radiology, Yale School of Medicine, New Haven, CT 06520-8058, USA 3 Urologic Oncology Branch, National Cancer Institute, Bethesda, MD 20892, USA 4 Molecular and Translational Science, United States Medicinal Chemistry and Pharmacognosy, School of Pharmacy, The Ohio State University, Columbus, OH 43210, USA 5 Arno Therapeutics, Flemington, NJ 08822, USA 6 School of Biological Sciences and Illawarra Health and Medical Research Institute, University of Wollongong, NSW 2522, Australia 7 Department of Chemistry and Physics, Augusta University, Augusta, GA 30912, USA * These authors contributed equally to this work Correspondence to: Paul Dent, e-mail: pdent@vcu.edu Keywords: OSU-03012, sorafenib, pazopanib, chaperones, ATPase Received: January 09, 2016      Accepted: January 16, 2016      Published: February 12, 2016 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 NH 2 -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.