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Modulators of HSP70/DnaK Function and Methods of Use Thereof

a technology of hsp70 and dnak, applied in the field of modulating hsp70/hsc70/dnak function, can solve the problems of affecting the prognosis of patients,

Inactive Publication Date: 2011-08-04
THE TRUSTEES OF THE UNIV OF PENNSYLVANIA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009]Thus, in accordance with one aspect of the present invention, there is provided a method for potentiating the effect of at least one chemotherapeutic ...

Problems solved by technology

Such enhanced HSP70 expression correlates with resistance of the tumor cells to caspase-dependent and -independent cell death and is associated with poor patient prognosis (Brodsky and Chiosis, 2006; Garrido et al., 2006; Guzhova and Margulis, 2006; Schmitt et al., 2006).
It is likely that the unfavorable conditions associated with the tumor microenvironment, such as hypoxia, nutrient deprivation, oxidative stress, oncogene activation, and exposure to chemotherapeutics lead to alterations in protein structure or processing, as well as upregulation of HSP70.
To date, however, only a limited number of compounds that specifically target HSP70 have been identified in chemical screens, and few are currently available to assess the physiologic impact of modulating HSP70 actions (Brodsky and Chiosis, 2006; Powers and Workman, 2007; Wisén and Gestwicki, 2008).

Method used

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  • Modulators of HSP70/DnaK Function and Methods of Use Thereof
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  • Modulators of HSP70/DnaK Function and Methods of Use Thereof

Examples

Experimental program
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Effect test

example i

PES Interacts with HSP70

[0120]PES was identified in a screen of the Chembridge DIVERSet library of drug-like small molecules for those exhibiting an ability to inhibit the direct mitochondrial pathway of p53-mediated apoptosis, and was referred to as PFTμ (Strom et al., 2006). Subsequently, we found that PES prevents the accumulation of p53 at mitochondria and inhibits caspase cleavage following cisplatin treatment of a human tumor cell line (Leu and George, 2007). In an attempt to better understand the molecular basis for these observations, we sought to identify intracellular targets of PES.

[0121]Based on the structure of PES (FIG. 2A), we used a thiol-cleavable amine-reactive reagent to synthesize a biotinylated form of the molecule (biotin-PES), as described (see Methods). Several mammalian cell lines were treated with biotin-PES, cell lysates were prepared, and biotin-PES complexes were captured using NeutrAvidin Resins. PES-interacting proteins were eluted using 100 mM DTT, wh...

example 2

[0151]Combined PES and Chemotherapy and / or Radiation Treatment Effectively Kills Cancer Cells

[0152]PES was originally identified as an inhibitor of mitochondrial p53. Hsp70s function to re-fold misfolded proteins, and to enable protein trafficking to mitochondria, and across membranes. Silencing HSP70 universally kills tumor cells, with little toxicity to normal cells. As discussed above in Example 1, PES inhibits autophagy which is known to protect cells from hypoxia mediated cell death. Hypoxia is well known to protect cancer cells from the impact of chemotherapeutic agents and radiation. Accordingly, we performed experiments to assess whether PES cooperated with either a chemotherapeutic agent or radiation to induce tumor cell death by inhibiting autophagy / survival. As shown in FIGS. 20A and 20B and described above, PES acts synergistically with cisplatin to kill FADU cells.

[0153]In further experiments we subjected FADU cells to a combination of PES (10 μM) and gamma irradiation ...

example 3

PES-Cl Interacts with HSP70

[0161]PES-Cl was shown to interact with HSP70. The free amide group of PBS-Cl was biotinylated and added to HA-tagged HSP70-transfected H1299 cells. The treatment of the cells occurred for 6 hours. The complexes were pulled down with NeutrAvidin beads, eluted with 100 mM DTT treatments, separated with SDS-PAGE and detected with anti-HA antibody (Millipore) by Western blot. Both PES and PES-Cl were determined to interact with HSP70 (FIG. 23).

PES-Cl Inhibits Autophagy in H1299 and FaDu Cells

[0162]The inhibition of autophagy by PES-Cl was observed in the H1299 and FaDu cell lines. The potential of PES-Cl induced apoptosis was also observed. Tumorogenic cells H1299 and FaDu cells were seeded in 10 cm plates and were treated with 10 μM PES or 10 μM PES-Cl. DMSO treated cells were used as a vehicle treated control. Twenty-four, 48 and 72 hours post-treatment the cells were scrapped and protein extracts were generated. Western blotting was performed as described ...

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Abstract

Compositions and methods for modulating HSP70 function, particularly for the targeted killing of cancer cells, are disclosed.

Description

[0001]This application is a 35 U.S.C. §365(c) filing of PCT / US09 / 57430 filed Sep. 18, 2009 which in turn claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 61 / 198,476 filed Sep. 19, 2008, the entire disclosure of each is being incorporated herein by reference as though set forth in full.[0002]Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S. Government has certain rights in the invention described, which was made in part with funds from the National Institutes of Health, Grant Numbers CA118761 and DK078025.FIELD OF THE INVENTION[0003]The present invention relates to the fields of medicine, oncology and protein folding and transport. More specifically, the invention provides compositions and methods for modulating HSP70 / HSC70 / DnaK function. Such compositions and methods can be used alone or in combination to effectively induce cell death in targeted cells, in antimicrobial applications and in drug screening assays.BACKGROUND OF THE INVENTION[0004...

Claims

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Application Information

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IPC IPC(8): A61K38/20C07C307/02A61K31/4355A61K31/675A61K31/18A61K31/196A61K31/175A61K31/519A61K31/513A61K31/7068A61K31/52A61K31/7076A61K31/4375A61K31/704A61K38/21A61K33/24A61K31/565A61K31/69A61K31/506A61K31/7042A61K38/04A61K31/7008A61K31/545A61K31/43A61K31/7048A61K31/65A61P35/00A61P31/04A61K33/243
CPCA61K31/18A61K45/06C07C311/13A61K31/436A61K33/24A61N5/1001A61K2300/00A61P31/04A61P35/00A61K33/243
Inventor GEORGE, DONNA L.LEU, JULIA I-JUMURPHY, MAUREEN
Owner THE TRUSTEES OF THE UNIV OF PENNSYLVANIA
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