Identification of a Binding Site on Hsc70 for the Immunosuppressant 15-Deoxyspergualin

Nadler, Steven G.; Dischino, Douglas D.; Malacko, Alison R.; Cleaveland, Jeffrey S.; Fujihara, Sheri M.; Marquardt, Hans

doi:10.1006/bbrc.1998.9775

Cited by 60 publications

(49 citation statements)

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“…The natural product analog, 15-deoxyspergualin, is an agonist of Hsp70 that binds outside the nucleotide-binding site (63)(64)(65). Recent work by Brodsky and co-workers (66,67) has shown that certain synthetically accessible dihydropyrimidines, which structurally mimic 15-deoxyspergualin, can likewise enhance ATPase activity and regulate protein translocation.…”

Section: Resultsmentioning

confidence: 99%

Heat Shock Proteins 70 and 90 Inhibit Early Stages of Amyloid β-(1–42) Aggregation in Vitro

Evans

Wisén

Gestwicki

2006

Journal of Biological Chemistry

340

304

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Alzheimer disease is a neurological disorder that is characterized by the presence of fibrils and oligomers composed of the amyloid ␤ (A␤) peptide. In models of Alzheimer disease, overexpression of molecular chaperones, specifically heat shock protein 70 (Hsp70), suppresses phenotypes related to A␤ aggregation. These observations led to the hypothesis that chaperones might interact with A␤ and block self-association. However, although biochemical evidence to support this model has been collected in other neurodegenerative systems, the interaction between chaperones and A␤ has not been similarly explored. Here, we examine the effects of Hsp70/40 and Hsp90 on A␤ aggregation in vitro. We found that recombinant Hsp70/40 and Hsp90 block A␤ self-assembly and that these chaperones are effective at substoichiometric concentrations (ϳ1:50). The antiaggregation activity of Hsp70 can be inhibited by a nonhydrolyzable nucleotide analog and encouraged by pharmacological stimulation of its ATPase activity. Finally, we were interested in discerning what type of amyloid structures can be acted upon by these chaperones. To address this question, we added Hsp70/40 and Hsp90 to pre-formed oligomers and fibrils. Based on thioflavin T reactivity, the combination of Hsp70/40 and Hsp90 caused structural changes in oligomers but had little effect on fibrils. These results suggest that if these chaperones are present in the same cellular compartment in which A␤ is produced, Hsp70/40 and Hsp90 may suppress the early stages of self-assembly. Thus, these results are consistent with a model in which pharmacological activation of chaperones might have a favorable therapeutic effect on Alzheimer disease.The amyloid diseases are a collection of protein misfolding disorders associated with the formation of distinctive fibrils (reviewed in Refs. 1-5). Alzheimer disease is one of the most common amyloid diseases, and it is characterized by fibrils composed of A␤, 2 a 39 -43-amino acid proteolytic fragment of the amyloid precursor protein (reviewed in Ref. 6). Upon release from amyloid precursor protein, A␤ becomes enriched in ␤-sheet structure and acquires the propensity to self-assemble (7). Initial theories to explain the pathology of AD focused on the involvement of the visually striking fibrils, but more recent evidence has strongly supported a role for soluble oligomers (reviewed in Refs. 3 and 8 -10). A␤ oligomers can be prepared in vitro (11,12), biosynthesized by cultured cells (13,14), or collected from AD tissues (15), and in all cases, these structures are highly neurotoxic. Despite these important observations, numerous questions about the basis of disease are unanswered. For example, although there is active research in this area (8,11,16), the number of A␤ monomers present in an oligomer has not been fully described. Moreover, the subcellular site(s) of oligomer production and the conditions that lead to their assembly are still debated; fibrils are found in extracellular space, but there is growing evidence that oligomers may be pr...

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Section: Resultsmentioning

confidence: 99%

Heat Shock Proteins 70 and 90 Inhibit Early Stages of Amyloid β-(1–42) Aggregation in Vitro

Evans

Wisén

Gestwicki

2006

Journal of Biological Chemistry

340

304

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“…While many have suggested Hsp70 as an excellent target for therapeutic intervention in the treatment of cancer, no drug-like inhibitors have yet emerged [32]. Previously described inhibitors have centred on 15-deoxyspergulin [33,34], dihydropyrimidines [35,36] and 3 0 -sulfogalactolipids [37,38]. While these compounds have demonstrated effects on Hsc70/Hsp70 chaperone activity in vitro and possess modest anti-tumor cellular activity (GI 50 of 2.4-50 lM), no evidence exists for their cellular activity being via Hsc70/Hsp70 inhibition.…”

Section: Discussionmentioning

confidence: 99%

A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells

Massey¹,

Williamson²,

Browne³

et al. 2009

Cancer Chemother Pharmacol

269

254

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Purpose The anti-apoptotic function of the 70 kDa family of heat shock proteins and their role in cancer is well documented. Dual targeting of Hsc70 and Hsp70 with siRNA induces proteasome-dependent degradation of Hsp90 client proteins and extensive tumor specific apoptosis as well as the potentiation of tumor cell apoptosis following pharmacological Hsp90 inhibition. Methods We have previously described the discovery and synthesis of novel adenosine-derived inhibitors of the 70 kDa family of heat shock proteins; the first inhibitors described to target the ATPase binding domain. The in vitro activity of VER-155008 was evaluated in HCT116, HT29, BT474 and MDA-MB-468 carcinoma cell lines. Cell proliferation, cell apoptosis and caspase 3/7 activity was determined for VER-155008 in the absence or presence of small molecule Hsp90 inhibitors. Results VER-155008 inhibited the proliferation of human breast and colon cancer cell lines with GI 50 s in the range 5.3-14.4 lM, and induced Hsp90 client protein degradation in both HCT116 and BT474 cells. As a single agent, VER-155008 induced caspase-3/7 dependent apoptosis in BT474 cells and non-caspase dependent cell death in HCT116 cells. VER-155008 potentiated the apoptotic potential of a small molecule Hsp90 inhibitor in HCT116 but not HT29 or MDA-MB-468 cells. In vivo, VER-155008 demonstrated rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level. Conclusion These data suggest that small molecule inhibitors of Hsc70/Hsp70 phenotypically mimic the cellular mode of action of a small molecule Hsp90 inhibitor and can potentiate the apoptotic potential of a small molecule Hsp90 inhibitor in certain cell lines. The factors determining whether or not cells apoptose in response to Hsp90 inhibition or the combination of Hsp90 plus Hsc70/ Hsp70 inhibition remain to be determined.

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“…Despite the uncertainty in the actual mechanism of action of DSG, DSG is known to bind cellular chaperones, Hsp70 and Hsp90, potently, via their regulatory C-terminal motif EEVD (3,4). DSG does not inhibit their chaperone activity (3)(4)(5).…”

mentioning

confidence: 99%

“…DSG does not inhibit their chaperone activity (3)(4)(5). However, it selectively enhances the ATPase activity of heat shock proteins that have the C-terminal EEVD motif, and this could be the basis of a specific modulation of the function of proteins that bind the EEVD motif of heat shock proteins (5).…”

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confidence: 99%

15-Deoxyspergualin Primarily Targets the Trafficking of Apicoplast Proteins in Plasmodium falciparum

Ramya

Karmodiya

Surolia

et al. 2007

Journal of Biological Chemistry

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15-Deoxyspergualin, an immunosuppressant with tumoricidal and antimalarial properties, has been implicated in the inhibition of a diverse array of cellular processes including polyamine synthesis and protein synthesis. Endeavoring to identify the mechanism of antimalarial action of this molecule, we examined its effect on Plasmodium falciparum protein synthesis, polyamine biosynthesis, and transport. 15-Deoxyspergualin stalled protein synthesis in P. falciparum through Hsp70 sequestration and subsequent phosphorylation of the eukaryotic initiation factor eIF2␣. However, protein synthesis inhibition as well as polyamine depletion were invoked only by high micromolar concentrations of 15-deoxyspergualin, in contrast to the submicromolar concentrations sufficient to inhibit parasite growth. Further investigations demonstrated that 15-deoxyspergualin in the malaria parasite primarily targets the hitherto underexplored process of trafficking of nucleus-encoded proteins to the apicoplast. Our finding that 15-deoxyspergualin kills the malaria parasite by interfering with targeting of nucleus-encoded proteins to the apicoplast not only exposes a chink in the armor of the malaria parasite, but also reveals new realms in our endeavors to study this intriguing biological process. 15-Deoxyspergualin (DSG)4 ( Fig. 1), an analog of spergualin isolated from the culture broth of Bacillus laterosporus, inhibits the growth of the malaria parasite, Plasmodium, in vitro and in vivo, presumably by depleting its polyamine reserves (1, 2). However, other known inhibitors of polyamine biosynthesis, methylglyoxal-bis-(guanylhydrazone) analog (MGBCP), irinotecan hydrochloride (CPT11), and ␣-difluoromethyl ornithine (DFMO) are known to be incapable of suppressing malaria in infected mice (2). This led us to question the speculated mechanism of antimalarial action of DSG.Despite the uncertainty in the actual mechanism of action of DSG, DSG is known to bind cellular chaperones, Hsp70 and Hsp90, potently, via their regulatory C-terminal motif EEVD (3, 4). DSG does not inhibit their chaperone activity (3-5). However, it selectively enhances the ATPase activity of heat shock proteins that have the C-terminal EEVD motif, and this could be the basis of a specific modulation of the function of proteins that bind the EEVD motif of heat shock proteins (5).One of the cellular milieus in which heat shock proteins are speculated to be involved is translational regulation. Translation of an mRNA requires the formation of a ternary complex of the methionyl-tRNA, the mRNA and the 40 S subunit of the ribosome, a feat achieved through the intervention of the GTPbound form of the initiation factor eIF2 (6). Phosphorylation of eIF2␣ at Ser 51 blocks protein synthesis by preventing eIF2 recycling and depleting the cell of its pool of eIF2-GTP required for translation initiation (7). Several conditions of cellular stress trigger the phosphorylation of eIF2␣ via the activation of four different eIF2␣ kinases: PERK (PKR-like endoplasmic reticulum-associated kina...

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Identification of a Binding Site on Hsc70 for the Immunosuppressant 15-Deoxyspergualin

Cited by 60 publications

References 0 publications

Heat Shock Proteins 70 and 90 Inhibit Early Stages of Amyloid β-(1–42) Aggregation in Vitro

Heat Shock Proteins 70 and 90 Inhibit Early Stages of Amyloid β-(1–42) Aggregation in Vitro

A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells

15-Deoxyspergualin Primarily Targets the Trafficking of Apicoplast Proteins in Plasmodium falciparum

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