Inhibitors of Molecular Chaperones as Therapeutic Agents

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Expert Opin Ther Targets. Heat shock proteins as emerging therapeutic targets. Br J Pharmacol. Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol. Calderwood, Jianlin Gong.

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Molecular chaperones in mammary cancer growth and breast tumor therapy. Heat shock protein the cancer chaperone. J Biosci. Heat shock protein Curr Opin Oncol. Differential effects of Hsp90 inhibition on protein kinases regulating signal transduction pathways required for myoblast differentiation. Exp Cell Res. Geldanamycin and its allylamino- demethoxy analogue antagonize the action of cisplatin in human colon adenocarcinoma cells: differential caspase activation as a basis for interaction.

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Cancer Res. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Targeting of multiple signalling pathways by heat shock protein 90 molecular chaperone inhibitors. Endocr Relat Cancer. The data are shown as cell index graphs. All experiments were made in triplicates. To test the effect of AEAC in combination with an anticancer drug, we used doxorubicin, which is currently used in clinical oncology.

The data from measuring LDH activity in medium CytoTox96 assay showed that the addition of AEAC to cell culture increased the toxicity of doxorubicin in both concentrations.

The Chaperoning System

Similarly, 1. We confirmed these data using an MTT test and demonstrated that in the presence of 2.

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  4. The parameter is based on impedance measurements and reflects the number of cells attached to the surface electrode of a multi-well chamber. Because doxorubicin is a potent inducer of apoptosis 14 , we analyzed whether AEAC can increase the pro-apoptotic effects of the drug. The percentage of cells that demonstrated apoptotic nuclear morphology visualized by Acridine orange staining increased from Importantly, AEAC alone in concentration 2. To determine whether AEAC is able to elevate antitumor capacity of doxorubicin, we used two animal models: mouse melanoma B16 growing subcutaneously and the intracranial rat C6 glioblastoma 15 , 16 , After tumor cell inoculation and treatment with doxorubicin alone or in combination with AEAC see Materials and Methods for details , we measured tumor size in Bbearing mice on the day 20 after tumor grafting.

    Treatment with doxorubicin alone inhibited tumor growth by approximately Combined action of doxorubicin with AEAC slowed tumor growth even more; the average tumor size in this group was 0. Interestingly, treatment with AEAC alone also suppressed tumor growth by Seven days later, doxorubicin or AEAC alone or in combination in DMSO was applied every two days to the injection area; c C6 glioma cells were grafted intracranially, and seven days later rats were injected intraperitoneally with doxorubicin or AEAC alone or in combination.

    The therapy continued to day 27 after grafting, and application was repeated each second day. The cumulative proportion surviving Kaplan—Meier curve is presented. We also evaluated the lifespan of tumor-bearing mice after various treatment modes.

    Treatment with doxorubicin alone increased the lifespan by 10—12 days, compared to the control group The therapy with AEAC alone also was effective, and it resulted in a 5-day delay in animal death compared to control groups Fig. A similar animal survival profile was demonstrated in rats with intracranial C6 glioblastoma. Interestingly, the therapeutic efficacy of doxorubicin alone and AEAC alone was much lower; the death was postponed for 3.

    The fact that Hsp70 is found in a high quantity in numerous tumors is well documented and prompts researchers to search for a way to inhibit the chaperone. To date, over 20 small-molecule inhibitors of Hsp70 have been identified and most of them are shown to possess anti-cancer activity in a variety of cell and animal models.

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    HSP-Molecular Chaperones in Cancer Biogenesis and Tumor Therapy: An Overview

    Hsp70 inhibitors are created using rational drug designs to identify a specific part of the chaperone molecule that can serve as the target for the careful selection of the most affine one It is clear that scaffold compounds that are generated in these experiments do not necessarily inhibit chaperone activity or protective function. To specifically target the chaperone capacity of Hsp70, we modified two assays that have been previously used for the same purpose 19 , 20 , and adapted them for high-throughput analysis of chemical compounds.

    The substrate-binding assay revealed approximately 30 modulators of Hsp70 activity from the selection of more than representatives of maximally diverse chemicals collected in InterBioScreen library Fig. The hits were investigated further using a refolding assay that resulted in 10 to 12 of the 30 previously chosen Hsp70 inhibitor compounds retaining both the substrate-binding and refolding activities. The absence of correlation between the efficacy of denatured protein recognition and refolding activity found for remaining 20 compounds led us to suggest that the recognition of the substrate and the conversion of its molecule to a native state may be differently regulated.

    An example of similar discoordination between the client protein binding and ATP-ase was demonstrated for a bacterial member of the Hsp70 family Notably, we have not employed ATP in substrate-binding assay because its addition to reaction mixture completely inhibited the Hspsubstrate interaction by passing the chaperone to intermediate state.

    This was true irrespective of whether the interaction occurred with a substrate CMLA and pure Hsp70 or the chaperone existing in cell extract as shown earlier Indeed, both compounds inhibited substrate-binding activity of Hsp To expand the application of both assays to the simultaneous analysis of penetrative activity of the compounds and their ability to regulate Hsp70 chaperone function we employed tests in vivo imitating both above assays. The system comprising cells expressing luciferase is similar to that of Nollen with coauthors and allows to study the recovery of luciferase under the action of HspHsp40 chaperones in cultured cells We employed this system and found that AEAC not only penetrated cells and proved its relevance for the screening of Hsp70 inhibitors.

    Another test we developed was substrate-binding assay transmitted to in vitro format. Using the test, we proved that AEAC not only penetrated cells of completely different origin and also dose-dependently reduced the substrate-recognition activity of Hsp We conclude that the assays developed in this study can be applied to the screening of a wide spectrum of Hsp70 modulators, and that they are less time-consuming and labor-intensive than the current methods.

    Since chaperone-inhibitory activity of AEAC may mean that its molecule binds Hsp70, we investigated this suggestion using three different methods and found that according to data of microscale thermophoresis the K D value is approximately 0. Similar values of the constant have been reported for other small-molecule binders of Hsp70, which suggests that the efficiency of their anti-Hsp70 effects is similar Using molecular docking and dynamics simulation we found that there are three binding sites for AEAC on Hsp70 molecule Figs.

    It is meaningful that site two locates near the helical part of the SBD that is involved in the conversion from open to closed conformations of Hsp70 Fig. Recently, this site was found to be potentially druggable on DnaK 24 , One of the goals of the study was to explore the possibility of using AEAC in therapeutic protocols in combination with doxorubicin, a well-established drug. In a more recent study, Zeng et al. We conclude that AEAC alone does not affect cell viability and growth as strongly as other Hsp70 inhibitors do, and tested it in combination with doxorubicin for anti-tumor activity in rat glioblastoma C6 and mouse melanoma B16 cells.

    The results show that a concentration of 2. The same phenomenon of AEAC-mediated sensitization of tumors was observed in experiments in vivo with tumors of quite diverse origin; in both cases we observed the prolongation of survival of animals with melanoma or glioma by We found only a few reports concerning application of anti-chaperone compounds in combination with antitumor drugs. First, the combinations of triptolide a known inhibitor of the heat shock response with several anticancer drugs were shown to be extremely effective against human breast cancer cells grafted onto nude mice Despite the promising effects of the combination, this study does not include detailed information about the toxicity of triptolide alone, which is a limiting factor in the clinical application of the drug.

    Secondly, in a study that is similar to our present work, McKeon et al It is clear that for the development of more effective therapeutic tools based on well-established anticancer drugs in combinations with Hsp70 chaperone inhibitors the search for concrete antitumor partners is needed.

    In conclusion, our work has demonstrated a few novel test systems that support the search of possible modulators of Hsp70 chaperone proteins. One such molecule was identified as a potent co-factor in combinational anticancer therapy. The compounds chosen for the screen pcs had chemical structures belonging to maximally diverse chemical groups.

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    Recombinant human Hsp70 was purified from bacteria that was transformed with a pMSHsp70 plasmid using a two-step chromatography procedure previously described in The substrate-binding activity of Hsp70 was measured using a modified enzyme immunoassay 19 , After the addition of tetramethylbenzene in a citrate buffer pH 4.

    The luciferase refolding assay was designed according to the protocol reported by Cassel et al. Luciferase activity was measured by luminometric analysis, which was completed using the Charity Fluorofot multipurpose reader Probanauchpribor, Russia and expressed as percentage of the activity before inactivation. BF10 mouse melanoma cells were obtained from Dr. The effects of AEAC on intracellular substrate-binding Hsp70 activity were explored, as described by For the in vitro refolding analysis, which provided additional support to the suggestion of AEAC permeability, C6 or B16 cells were transfected with a pcDNA3 plasmid that contained luciferase gene kindly provided by Dr.

    C6 or B16 cells were placed in well plates and after overnight cultivation AEAC was added in the concentration indicated. Twenty-four hours later, cytotoxicity and the viability value were measured. Acridine orange staining was used to measure the number of apoptotic cells.

    Molecular chaperone therapy- the future in cancer

    Petersburg, Russia. Mice were subcutaneously grafted with 10 6 B16 cells and divided into five groups, with seven mice in each group. AEAC was applied to a shaven area of skin surrounding the tumor, as described earlier 16 , and the application was repeated every second day until day 27 of the experiment. Tumor length L and width W were measured using a sliding caliper. Measurements were taken for all groups on day 20 following inoculation with B16 cells.

    Multi-pronged approach

    Wistar rats were anesthetized before mounting in a stereotactic frame David Kopf Instruments, Tujunda, CA , as described earlier Rats were divided into the same groups as described above, but 10 rats were included in each group. Treatment was started on day 7 after surgery.

    AEAC and doxorubicine were administered intraperitoneally and treatment continued to day 27 one application every two days.

    Inhibitors of Molecular Chaperones as Therapeutic Agents Inhibitors of Molecular Chaperones as Therapeutic Agents
    Inhibitors of Molecular Chaperones as Therapeutic Agents Inhibitors of Molecular Chaperones as Therapeutic Agents
    Inhibitors of Molecular Chaperones as Therapeutic Agents Inhibitors of Molecular Chaperones as Therapeutic Agents
    Inhibitors of Molecular Chaperones as Therapeutic Agents Inhibitors of Molecular Chaperones as Therapeutic Agents
    Inhibitors of Molecular Chaperones as Therapeutic Agents Inhibitors of Molecular Chaperones as Therapeutic Agents
    Inhibitors of Molecular Chaperones as Therapeutic Agents Inhibitors of Molecular Chaperones as Therapeutic Agents
    Inhibitors of Molecular Chaperones as Therapeutic Agents Inhibitors of Molecular Chaperones as Therapeutic Agents

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