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.

Multi-kinase inhibitors interact with sildenafil and ERBB1/2/4 inhibitors to kill tumor cellsin vitroandin vivo

“…The BT474 line expresses a mutant p53 protein and over-expresses the plasma membrane receptor ERBB2 (HER2). In agreement with our prior in vitro data combining [sorafenib + sildenafil + afatinib], and its effect on chaperone activity and total ERBB2 expression, in vivo in athymic mice the three drug combination of [regorafenib + sildenafil + lapatinib] profoundly suppressed BT474 mammary tumor growth to a significantly greater extent than the two drug [regorafenib + sildenafil] combination (Figure 14A, p < 0.05) [9]. We next grew afatinib resistant H1975 tumors in the rear flanks of beige SCID mice and determined the response of these cells in vivo to [sorafenib + sildenafil + afatinib] and to [pazopanib + BYL719].…”

“…We transformed bacteria with a plasmid to make a GST fusion protein of the NH 2 -terminal portion of HSP90; the domain that contains the ATP binding site and ATPase activity of the chaperone. Sorafenib and pazopanib, but not regorafenib, reduced chaperone ATPase activity, as measured on the isolated purified NH 2 -terminal HSP90-GST protein fragment in vitro (Figure 1A) see additional data in Booth et al, 2016: reference [9]. Based on chemical structure alone, the only difference between sorafenib and regorafenib is the inclusion of a single fluorine atom in regorafenib.…”

“…Based on chemical structure alone, the only difference between sorafenib and regorafenib is the inclusion of a single fluorine atom in regorafenib. It should be noted, however, that in our recent biochemical studies using mammalian cell synthesized HSP90 and HSP70; the PKG-dependent phosphorylation of these chaperones facilitated the ATPase inhibitory activity of regorafenib [9]. In silico docking of pazopanib into the amino-terminal ATP binding domain of HSP90 resulted in the identification of two predominant poses.…”

“…Asn51 interacts with the indazole nitrogen, there is a possible π-cation interaction between Lys58 and the pyrimidine ring, and Lys112 and the amide hydrogen are creating hydrogen bonds with the sulfonamide group (Figure 1C). When combined with sildenafil both sorafenib and pazopanib modestly decreased HSP90 expression in 6 h as measured using a COOH terminal antibody but strongly reduced expression of its essential co-chaperone CDC37 that also correlated with reduced co-localization as judged by the pure-yellow merged signal becoming more orange in hue (Figure 1D) [9]. …”

“…The protein kinase ULK-1 is considered to be an essential gate-keeper kinase for the regulation of autophagosome formation, and whose activity is negatively regulated by Serine 757 phosphorylation, catalyzed by mTOR [9, 11, 12]. The reduced mTOR S2448 phosphorylation caused by drug exposure and shown in Figure 3 correlated with the drug-induced dephosphorylation of ULK-1 at Serine 757, which collectively in turn correlated with increased phosphorylation of the autophagosome formation regulatory protein ATG13 at Serine 318 seen in Figure 3 (Figure 4A) [20, 21].…”