INHIBITORS OF HSP70 PROTEIN

Abstract

This invention relates to compounds that are inhibitors of HSP70 protein, and applications thereof.

Claims

1. A method of treating tumors in a patient in need thereof by inhibiting HSP70 protein activity thereby inducing apoptosis of tumor cells and re-educating macrophages, comprising administering to the patient a therapeutically efficient amount of a compound of formula (I) or (II) ##STR00014## wherein R.sub.1 represents a hydrogen atom, an halogen atom, a C1-C8 alkyl group, R.sub.2 represents a hydrogen atom, an halogen atom, a C1-C8 alkyl group, R.sub.3 represents a hydrogen atom, an halogen atom, a C1-C8 alkyl group, R.sub.4 represents a C1-C8 alkyl group, and R.sub.5 represents a hydrogen atom, an halogen atom, or a C1-C8 alkyl group, ##STR00015## wherein R.sub.6 represents an heterocycle preferably a piperidinyl group, R.sub.7 represents a hydrogen atom or a halogen atom, R.sub.8 represents a hydrogen atom or a halogen atom, R.sub.9 represents a hydrogen atom or a halogen atom, R.sub.10 represents a hydrogen atom or a halogen atom, R.sub.11 represents a hydrogen atom or a halogen atom, and R.sub.12 represents a hydrogen atom or a halogen atom.

2. The method according to claim 1, wherein the compound of formula (I) is chosen from the group consisting of a compound of formula (B) ##STR00016## and a compound of formula (C) ##STR00017## and the compound of formula (II) is a compound of formula (A) ##STR00018##

3. Complex formed between a compound of formula (I) or (II) and a lipoprotein, ##STR00019## wherein R.sub.1 represents a hydrogen atom, an halogen atom, a C1-C8 alkyl group R.sup.2 represents a hydrogen atom, an halogen atom, a C1-C8 alkyl group, R.sub.3 represents a hydrogen atom, an halogen atom, a C1-C8 alkyl group, R.sub.4 represents a C1-C8 alkyl group, and R.sub.5 represents a hydrogen atom, an halogen atom, or a C1-C8 alkyl group; ##STR00020## wherein R.sub.6 represents an heterocycle preferably a piperidinyl group, R.sub.7 represents a hydrogen atom or a halogen atom, R.sub.8 represents a hydrogen atom or a halogen atom, R.sub.9 represents a hydrogen atom or a halogen atom, R.sub.10 represents a hydrogen atom or a halogen atom, R.sub.11 represents a hydrogen atom or a halogen atom, and R.sub.12 represents a hydrogen atom or a halogen atom.

4. The complex according to claim 3, wherein the compound of formula (I) is chosen from the group consisting of a compound of formula (B) ##STR00021## and a compound of formula (C) ##STR00022## and the compound of formula (II) is a compound of formula (A) ##STR00023##

5. Complex as defined in claim 3, further comprising a platinum compound selected from the group consisting of cisplatin, carboplatin, oxaliplatin, tetraplatin, iproplatin, satraplatin, nedaplatin, lobaplatin, picoplatin and ProLindac (polymere-platinate-DACH AP5346).

6. (canceled)

7. Pharmaceutical composition comprising, as active principle, the compound of formula (I) or (II) as defined in claim 1 or a complex formed between the compound and a lipoprotein, and a pharmaceutically acceptable excipient.

8. A method of treating tumors in a patient in need thereof by inhibiting the HSP70 protein activity, thereby inducing apoptosis of tumor cells and re-educating macrophages, comprising administering to the patient a therapeutically effective amount of comprising the complex.

9. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a combination of a compound of formula (I) or (II) as defined in claim 1, a complex formed between the compound of formula (I) or (II) and a lipoprotein or a pharmaceutical composition comprising the compound or the complex, and one or more anti-cancer agents, surgery, immunotherapy and/or radiotherapy.

10. (canceled)

11. Kit comprising, the complex of claim 3 and an anti-cancer agent and/or a platinum compound.

12. The method of claim 1, wherein the tumors are solid tumors.

13. The method of claim 1, wherein one or more of R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is a C1-C3 alkyl group.

14. The method of claim 1, wherein R.sub.6 is a piperidinyl group.

15. The complex of claim 3, wherein the lipoprotein is a High Density Lipoprotein (HDL).

16. The method of claim 3, wherein one or more of R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is a C1-C3 alkyl group.

17. The method of claim 3, wherein R.sub.6 is a piperidinyl group.

18. The method of claim 8, wherein the tumors are solid tumors.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0117] Table 1. High throughput screening of chemical agonists of the A18 peptide aptamer. The screening was based on the ability of the molecules to inhibit the association A18/HSP70.

[0118] Table 2. Selected “Hits” with high inhibitory activity against A18 binding to HSP70. Their chemical structure is shown as well as their ability to block HSP70/A18 interaction (Biacore).

[0119] FIG. 1: Mouse embryonic fibroblasts (MEF) knock out for heat shock factor-1 (HSF1−/−) do not express HSP70. A western blot of inducible HSP70 and constitutive HSC70 is shown.

[0120] FIG. 2: Small molecules hits A, B and C inhibit HSP70 chaperone activity. A) Protein aggregation was determined in the presence or absence of recombinant HSP70 (10 ng). When indicated A18 or molecules A, B and C were added (1 μM). B) Protein aggregation was determined in the presence or absence of recombinant HSP70, HSC70 or HSP90 (10 ng).

[0121] When indicated A18 or molecules A, B and C were added (1 μM). A0, a non-relevant peptide aptamer was used here as a negative control. Values are normalized for each HSP. 100% chaperone effect being the inhibition of protein aggregation induced by addition of the recombinant chaperones.

[0122] FIG. 3. The chemical molecule hits sensitize cancer cells to cisplatin. A) Human cervix HeLa or B) mouse colorectal cancer CT-26 cells were incubated during 48 h with cisplatin (25 μM) in the presence of absence of molecules A, B and C (2 μM). Cell survival was measured by a colorimetric assay staining.

[0123] FIG. 4. The chemical molecules lose their chemo-sensitizing properties in cells not expressing HSP70. MEF HSF1−/− cells were incubated during 48 h with cisplatin (25 μM) in the presence of absence of molecules A, B and C (1 μM). Cell death was measured by a colorimetric assay staining.

[0124] FIG. 5: Chemical HSP70 inhibitors increase the number of apoptotic cells. Hela cells were treated with cisplatin (CDDP, 25 μM, 24 h) in the presence or absence of the chemical molecules A, B and C (2 μM). Apoptosis mas measured by flow cytometry (AnnexinV+/IP−).

[0125] FIG. 6: Molecule B-vectorized with HDL induces ROS from macrophages. A: LDL and HDL were purified by density gradient ultracentrifugation. Total cholesterol in lipoproteins was adjusted to 1 mM. Lipoproteins were then incubated with the HSP70 inhibitor Molecule B (100 μM) for 3 hours at 37° C., then submitted to dialysis (two times against 1L PBS, cutoff 8000 Da). Total molecule B concentration in lipoproteins was then measured by mass spectrometry. B: For macrophages activation, human M2 macrophages were treated for 2 hours with Molecule B (10 μM in DMSO) or vectorized in LDL or HDL (10 μM final Molecule B concentration). Percentage of ROS positive macrophages are represented as mean value +/− SEM. n=4, **: p<0.01, NS=Not Significant.

[0126] FIG. 7: HSP70 inhibitor vectorization in HDL prevents tumor growth by targeting macrophages. A: Balb-c mice were injected with CT-26 colorectal cancer cells (10.sup.6 cells/mice, s.c.). At the indicated times mice were treated either with PBS, or HDL-Molecule B (100 μM cholesterol, 10 μM Molecule B, 100 μl/mouse). n=4. B: Tumor volume was measured every 3 days, and represented as mean value +/− SEM, ***: p<0.001. C: Apoptosis and macrophage infiltration were t-determined in histological slides labeled with a cleaved caspase-3 antibody (Green) and a F4/80 antibody (Red), and with DAPI. Pictures were chosen in random fields and are representative of five pictures taken for each condition. n=4, scale bar=50 μm. D: ROS production in tumors was measured in histological slides by DAPI/DHE staining. Pictures, taken in random chosen fields, are representative of five pictures taken for each condition. n=4, scale bar=E, F & G: Quantifications of the immunofluorescence intensity of Cleaved Caspase-3 (E), F4/80 (F) and DHE (G). Data are represented as mean increase vs. CTL +/− SEM. n=4, *: p<0.05, ***: p<0.001, ****: p<0.0001.

[0127] FIG. 8: In vivo effects of cisplatin vectorization in LDL and Molecule B vectorization in HDL. A: Balb-c mice were injected with CT-26 colorectal cancer cells (10.sup.6 cells/mice, s.c.). At the indicated times mice were treated either with PBS, HDL-Molecule B (100 μM cholesterol, 10 μM Molecule B, 100 μ/mouse) or LDL-Cisplatin (100 μM cholesterol, 1.5 mg/kg cisplatin)+HDL-Molecule B (100 μM cholesterol, 10 μM Molecule B, 100 l/mouse). n=4 mice per groups. B: Tumor volume was measured every 3 days and represented as mean value+/−SEM. *: p<0.05. C: Apoptosis and macrophage infiltration were t-determined in histological slides labeled with a cleaved caspase-3 antibody (Green) and a F4/80 antibody (Red), and with DAPI. Pictures were chosen in random fields and are representative of five pictures taken for each condition. n=4, scale bar=50 μm. D: ROS production in tumors was measured in histological slides by DAPI/DHE staining. Pictures, taken in random chosen fields, are representative of five pictures taken for each condition. n=4, scale bar=50 μm. E, F & G: Quantifications of the immune-fluorescence intensity of Cleaved Caspase-3 (E), F4/80 (F) and DHE (G). Data are represented as mean increase vs. CTL+/−SEM. n=4, *: p<0.05, ****: p<0.0001.

EXAMPLES

Material and Methods

[0128] High Throughput Screening of Chemical Agonists of A18 Peptide Aptamer.

[0129] A high-throughput screening assay, AptaScreen™ (developed by Aptanomics SA; (Baines I C et al. Drug Discov Today. 2006;11:334-341; Rerole A L, et al. Cancer Res 20119;71:484-495)) was performed by the Company Imaxio (Lyon, France) on a library of almost 60,000 small molecules. The AptaScreen™ assay is based on an automated dual luminescence (luc and ruc reporter genes) yeast two-hybrid assay, with HSP70 expressed as a ‘bait’ and A18 aptamer expressed as a ‘prey’. The HSP70/A18 interaction directs the transcription of the luc reporter gene, while in the same assay a control protein/aptamer couple directs the transcription of the ruc reporter. A small molecule can be considered as ‘hit candidate’ when it inhibits the interaction between HSP70 and A18 (i.e. decreases the luciferase signal), but not the control interaction (i.e. no or little change in the ruc signal). Molecules were screened at a concentration of 10 μM and then hit candidates were confirmed by dose-response assays, following the standard protocol (Rerole A L, et al. Cancer Res 20119;71:484-495).

[0130] Cells, Plasmids and Transfections. HeLa cells, HSF1−/− MEFs, and CT-26 cells were grown as monolayers in a controlled atmosphere (37° C., 5% CO2) using RPMI 1640 medium or DMEM glucose 4.5 g. L-1 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (all mediums and FBS come from Lonza, Switzerland). HSF1−/− MEFs were transiently transfected with HSP70 cloned into HA-tagged pcDNA3.1 vector or co-transfected with HSP70 and with A18 peptide aptamer, or A14 as control aptamer, cloned into Myc/6His-tagged pcDNA3.1, or with the empty vector as control.

[0131] HSP70 Chaperone Activity Study In Vitro.

[0132] HSP70 chaperone activity was evaluated with a protein thermolability assay. HSF1−/− MEFs extracts were incubated 20 minutes on ice in lysis buffer (50 mM HEPES, 150 mM NaCl, 5 mM EDTA, 0.1% NP40) supplemented with protease inhibitors (Roche, France). Supernatants were recovered after g×14,000 centrifugation at 4° C., during 15 minutes, and a protein concentration assay was performed (Dc Assay Kits, Bio-Rad, France) against bovine serum albumin (BSA) standard range.

[0133] Cellular extracts, to which recombinant HSP70, HSP90 or HSC70 were added with or without the small molecules to test, were diluted to a final concentration of 2 mg. mL-1 in pH 7 Tris-HCl buffer and heated at 55° C. during 1 hour. After g−16,000 centrifugation at 4° C. during 10 minutes, supernatant native protein quantity was determined by Lowry method (Dc Assay Kits, Bio-Rad). This final protein concentration was then compared to the initial protein concentration in supernatants to quantify denatured proteins.

[0134] Cell Death Analysis.

[0135] 3.5×10.sup.4 adherent cells were plated onto 24-well culture plates in complete medium. Cells were treated with small molecules at various concentrations (1 μM to 30 μM) for 4 hours to determine cellular IC50. Then, cells of half of each 24-well culture plates were treated with cisplatin (CDDP, 25 μM) for 48 hours. Cell death was measured by the crystal violet colorimetric assay staining.

[0136] Lipoprotein Purification

[0137] HDL were purified from non-therapeutic healthy plasma (EFS Besançon, France) by density gradient ultracentrifugation as described previously by Redgrave et al. 1975. Anal Biochem.; 65(1-2):42-9). Briefly, plasma density was adjusted to 1.21 by the addition of KBr salt (Sigma Aldrich, #746444), 5 ml of plasma was then transferred to a 13.2 ml Ultra-Clear centrifuge tube (Beckman Coulter, 344059). 3m1 of 1.063 density KBr solution (ddw, 0.1 g/l EDTA, 0.02 g/l Sodium Azide), then 2 ml 1.019 density KBr solution (ddw, 0.1 g/1 EDTA, 0.02 g/1 Sodium Azide) and finally 2 ml of 1.000 density NaCl solution (ddw, 0.1 g/l EDTA, 0.02 g/l Sodium Azide) were gently layered on top of the plasma. Samples were then centrifuged for 24 hours at 40.000 rpm on a Beckmann ultracentrifuge (XXL-80) equipped with a swinging rotor SW 41 TI, with low acceleration and no brake. After centrifugation, the HDL fraction was collected at the interface of the 1.063 and 1.21 solutions. Total cholesterol was dosed using the Cholesterol Quantitation kit (CliniSciences, JM-K603-100) as indicated by the manufacturer's instructions, and the final cholesterol concentration was adjusted to 1mM in each sample by the addition of PBS.

[0138] Molecule B Incorporation in Lipoproteins.

[0139] Molecule B was first diluted in pure DMSO to a final concentration of 100 μM, then diluted 10 times in 1 mM HDL fractions and incubated 4 hours at 37° C. Unbound molecule B and DMSO were then removed by two successive dialyses using Spectrum™ Spectra/Por™ 1 RC Dialysis Membrane Tubing (6000 to 8000 Dalton Cut Off, Fisher Scientific, 08-670C), against 1000 times the volume of PBS. Molecule B incorporation HDL was then assessed by mass spectrometry.

[0140] Cell Culture

[0141] Human macrophages were differentiated from peripheral blood monocytes obtained from Buffy Coats of healthy donors (EFS Besancon, France). Briefly, to extract monocytes, 15 ml of blood (diluted 2-times in PBS) was gently layered on a 46-65% Percoll gradient solution (Sigma Aldrich P1644-1L), and centrifuged for 30 min at 550 g, RT. After centrifugation, the upper ring containing monocytes was recovered and seeded on 12-well plates (5.10.sup.5 monocytes per well) in RPMI 10%FBS, 100 UI/ml PSA, 37° C. 5% CO.sub.2 and differentiated into macrophages by stimulating cells 7 days with M-CSF (100 ng/ml, Miltenyi Biotechnology #130-095-372).

[0142] Flow Cytometry Analysis

[0143] For macrophage ROS production analysis by flow cytometry, cells were cultured for 30 min at 37° C. and 5% CO.sub.2, in DHE (10 μM in PBS), scraped out and centrifuged (10 min, 1500 rpm, 4° C.). Cells were fixed for 5 min in a PBS 4% PFA solution and analyzed using an LSRII flow cytometer (Becton Dickinson). Primary Size-Granularity dot plot allowed us to discriminate cells from debris, and DHE positive cells were obtained by comparing red fluorescence vs. unstained samples.

[0144] Mouse Procedures

[0145] 6-8 week-old female Balb/c mice were purchased from Charles River. The Balb/c-derived mouse colon carcinoma cell line CT26 (CRL2638™) was purchased from American Type Culture Collection (ATCC), and cultivated according to the manufacturer's instruction. CT26 cells (10.sup.6 cells/mice) were injected subcutaneously in the left flank. Time 0 was considered when the size of the tumor-reached 6 mm.sup.3. For tumor growth experiments, mice were treated i.p. at day 7, 14 and 21, with PBS, HDL-Molecule B (100 μM cholesterol, 10 μM Molecule B, 100 μl mouse) and euthanized at on day 25 (n=5 mice per groups). Tumors were measured every three days with a digital caliper (tumor volume was determined using the ½×Length×Width.sup.2 formula). Experiments were approved by the ethical comity of the Université de Bourgogne (protocol N3613).

Results

Screening of a Chemical Library against the A18 Aptamer—HSP70 interaction.

[0146] A high-throughput screening assay, AptaScreen™ (developed by Aptanomics SA; .sup.22,23), was performed by the Company Imaxio (Lyon, France) on a library of almost 60,000 small molecules including most of the marketed drugs. The AptaScreen™ assay is based on an automated dual luminescence (luc and ruc reporter genes) yeast two-hybrid assay, with HSP70 expressed as a ‘bait’ and a peptide aptamer (A18), the inventors previously isolated that bound to the ATP domain of HSP70 expressed as a ‘prey’ (Rerole A L, et al. Cancer Res 20119;71:484-495). A small molecule can be considered as ‘hit candidate’ when it inhibits the interaction between HSP70 and A18 (i.e. decreases the luciferase signal). Molecules were screened at a concentration of 10 μM. Eight molecules (hit candidates) were initially identified. From the dose-response studies, three of them were retained on the basis of their specificity and high inhibitory activity against A18 binding to HSP70 (Table 1 and 2). Interestingly, molecules 2 and 3 are analogues.

TABLE-US-00001 TABLE 1 Screening Confirmation IC50 HSP70 Molecule inhibition inhibition (μM) specificity A 105 34 19.6 YES B 105 59 0.2 YES C 103 61 0.46 YES

TABLE-US-00002 TABLE 2 Activity profil/50% Name, formula and structure effective concentration A [00011] EC.sub.50 = 19.6 μM B [00012]  EC.sub.50 = 0.2 μM C [00013] EC.sub.50 = 0.46 μM

[0147] The Small Molecule Hit-Candidates Inhibits HSP70 Chaperone Activity In Vitro.

[0148] A method to study the chaperone activity of HSPs was set up. Proteins were extracted from mouse embryonic HSF1−/− cells and they were heat shocked. HSF1 is the main transcription factor responsible of HSP expression after a stress. Therefore, this genetic background allowed us to work with reduced contamination with endogenous inducible HSPs like HSP70 (FIG. 1). An equal amount of proteins was submitted to a heat shock (55° C., 1 h) and the percentage of protein aggregation (which is directly related to the amount of denatured proteins) was determined in the presence or absence of recombinant HSP70, with or without the chemical molecules to be tested. As shown in FIG. 2A, HSP70 was able to reduce the amount of aggregated proteins in a very significant manner, demonstrating its chaperone function. A similar effect was observed when adding HSC70 or HSP90. As expected, when the inventors added the A18 aptamer, a strong inhibition of HSP70 chaperone activity was observed that was not observed with a control aptamer (A0). This inhibitory effect was specific because A18 was unable to block recombinant HSC70 or HSP90 chaperone activity. All four small molecule hits inhibited HSP70 chaperone activity, albeit to a different extent. Molecule A only partially blocked HSP70 chaperone function but this effect seemed specific. Molecules B and C provoked a much more important inhibition (FIG. 2B).

[0149] Chemo-Sensitizing Properties of the Chemical Molecules Targeting HSP70

[0150] To study the effect of the molecules in cancer cells, the inventors used two different cancer cells lines: Human cervix cancer Hela cells and mouse colorectal cancer CT-26. Cells were treated with the small molecule hit (2 μM) either alone or together with cisplatin for 48 hours and cell survival was determined. As shown in FIGS. 3A and 3B, the molecules synergistically increased cell death induced by cisplatin, the most important effect been found for the molecule B. This cell death was associated with the inhibition of HSP70 because the molecules had hardly any effect on the MEF HSF1−/− cells (FIG. 4) which, as shown in FIG. 1, do not express inducible HSP70. Cells seem to die mainly by apoptosis, as detected by the amount of Annexin V positive/propidium iodide negative cells obtained after treatment with the molecule (FIG. 5). The inventors conclude that the hit candidates block HSP70 chaperone activity and sensitize cancer cells to apoptosis induced by cisplatin. Since throughout these results the molecule B seemed, in terms of specificity (IC50) and effect, the most promising, the inventors selected this molecule for the studies in vivo.

[0151] Molecule B Displays an Anti-Tumor effect in Mice bearing a Colorectal Cancer, which Involve Cytotoxic Macrophages

[0152] To study the molecule B in vivo, the inventors decided to vectorize the molecule with lipoproteins of high density (HDL) because these natural nano-vectors have been reported to solve solubility problems in hydrophobic and hardly soluble molecules (like all four molecules selected here including the molecule B) and to favor cellular uptake.

[0153] A syngeneic model in which mouse colon cancer CT-26 cells were injected into Balb/c mice was used. When tumor size reached about 0.9 mm.sup.3, mice were treated with the molecule B complexed to HDL (FIG. 6A) at 5 mg/kg (average concentration described in the literature for similar small chemical compounds), which was given i.p. every three days until the end of the experiment (determined for ethical reasons by the size of the tumor in the control group) (FIG. 7A). Treatment by compound B induced a decrease in tumor growth of 60% (FIG. 7B). In accord with the previous results, this tumor regression effect was associated with an increase in tumor cells apoptosis (caspase-3 activation. FIG. 7C, E). But the most remarkable effect induced by the treatment with the vectorized molecule B was a strong accumulation within the regressing tumor of macrophages with a cytotoxic (anti-tumor) phenotype (FIG. 7C-F), as shown by their ability to produce reactive oxygen species (ROS) (FIG. 7C-G).

[0154] This effect of the-molecule B favoring cytotoxic macrophages was confirmed in vitro, in macrophages isolated from buffy coats. HDL-molecule B was able to induced ROS production (FIG. 6B) whereas the same amount of molecule complexed to LDL did not have any effect, indicating the importance of the lipoprotein used as a nano-vector.

[0155] This molecule, the first described experimental therapeutic HSP70 inhibitor targeting macrophages in vitro and in vivo, may pave the way to a new type of immunotherapeutic molecules against chemo resistant cancers.

[0156] Combinational Effect of Molecule B-HDL and Cisplatin-LDL Complexes

[0157] Finally, the inventors tested the impact of the association of cisplatin-LDL complexes together with molecule B-HDL complexes. LDL were purified by density gradient ultracentrifugation from buffy coats and incubated with cisplatin (to a final concentration of 1 mg/ml) for 4 hours at 37° C. Mice bearing CT-26 tumors were treated with LDL-Cisplatin alone, HDL-molecule B alone or the combination of both. The inventors observed a stronger decrease in tumor growth when using the combinational therapy (FIGS. 8A and B) Immunofluorescent staining revealed i) a strong induction of cancer cell apoptosis (FIGS. 8C and E), comparable to that observed in the animals treated with LDL-Cisplatin alone ii) a strong burst in macrophage infiltration comparable to that observed with the HDL-molecule B alone (FIGS. 8C and F). of note, a milder induction of ROS production was observed in this context (FIGS. 8D and G), probably due to a more advanced tumor regression in response to the combined treatment. This suggest that this combined strategy aiming to simultaneous target cancer cells with one drug (cisplatin-LDL) and tumor-infiltrating macrophages with the other (molecule B-HDL) allows a complementary additive effect, with no apparent increase in the drugs side-effects toxicity -as determined by the unchanged weight of the animals and the absence of apoptosis in the animals' epithelial cells in the intestinal crypts (data not shown).

[0158] Discussion

[0159] In this work, the inventors have identified small chemical molecules, agonists of the peptide aptamer A18. A18 is a thioredoxin-based aptamer with a 13 aminoacid variable region that binds to the ATP domain of HSP70 (Rerole A L, et al. Cancer Res 20119;71:484-495). The hits interfere with the chaperone activity of HSP70 in vitro, thus strongly suggesting that, as A18, they bind to the ATP domain of HSP70.

[0160] The four “drug candidates” described here sensitize cancer cells to death induced by cisplatin. Macrophages are essential component of the anti-cancer immune response and the inventors and others have reported a role for HSP70 in macrophages differentiation/maturation (Vega V L, et al. J Immnuol 2008;180:4299-307). Confirming these results, in the present application, the inventors have demonstrated that the i.p. injection of the molecule B in syngeneic mice bearing a tumor induced tumor regression that was associated with an impressive accumulation within the tumor of inflammatory cytotoxic macrophages (M1-like). Interestingly, to avoid problems of solubility, in these in vivo experiments the inventors vectorized the molecule by complexing it to natural HDL. The fact that the vectorization with HDL, but not with LDL, favors the effect of the molecule B in cultured macrophages suggests the importance of the nano-vector used and how the macrophages uptake of the HDL-molecule B complexes may involve specific receptors.

[0161] Cancer cells must extensively rewire their metabolic and signal transduction pathways, thereby becoming dependent on proteins that are dispensable for the survival of normal cells.

[0162] This HSPs-addition is the basis for the use of inhibitors of HSPs in cancer therapy. Today, with the exception of an inhibitor of HSP27 (an oligonucleotide antisense) all inhibitors in advanced clinical trials target HSP90 with somehow deceiving results and often unaccepted toxicity, which may be why they induce HSP70 expression that by its strong cell survival properties may counteract the efficacy of the HSP90 inhibitors. HSP70 can be considered as a protein that although it is not an oncogene, its presence is indispensable for the survival of cancer cells. HSP70 has a well-demonstrated role in apoptosis inhibition and autophagic cell death. Unfortunately, to date only a limited number of compounds that specifically target HSP70 have been identified. Leu et al described that the 2-phenylethynesulfonamide (PES), also known as pifithrin-α and originally identified as a molecule that interferes with the P53-induced apoptosis, specifically associated to the peptide binding domain of HSP70, induced autophagic cell death in cancer cells (but not apoptosis) and, in intraperitoneal administration, was able to inhibit the development of lymphomas in mice (Leu J I, et al. Mol Cell. 2009;36:15-27).

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