EFFICIENT INHIBITION OF HSP27

Abstract

The present invention relates to novel HSP27 inhibitors, in particular purine derivatives according to general formula (I) or (II) and phenothiazine derivatives according to formula (V), and to their use as drugs for the selective inhibition of the heat shock protein HSP27 (HSPB1), in particular for use in the treatment of carcinomas or cystic fibrosis, said inhibitors having a particularly advantageous activity in the lower micromolar or sub-micromolar active ingredient concentration range with respect to HSP2.

Claims

1. Purine derivative according to formula (I) or (II) for use as a medicinal product in the treatment of carcinomas or mucoviscidosis: ##STR00033## wherein: R.sub.n is selected from hydrogen (H), ##STR00034## and wherein: U is O, a C.sub.1 to C.sub.4 alkyl residue or a vinyl residue, W is an optionally substituted methylene (—CH.sub.2—), ethylene (—CH.sub.2CH.sub.2—), ethoxy (—O—CH.sub.2CH.sub.2—) or ethene-1,2-diyl residue (—CH═CH—), W is a methylene (—CH.sub.2—), ethylene (—CH.sub.2CH.sub.2—), ethoxy (—O—CH.sub.2CH.sub.2—) or ethene-1,2-diyl radical (—CH═CH—) V is selected from —H, —OH, an AA, an optionally substituted heterocycle and a substituted and/or polycyclic aryl residue, A.sub.1 and A.sub.2 independently of one another are from —CH.sub.2—, —CHOR, —CHF— and —CHOC(═O)R, G is CH.sub.2, —CH.sub.2O— or O, Y is O, S, NR, carboxyl, carbonyl or amide, wherein R is an H or a C.sub.1 to C.sub.8 alkyl residue, the substituent R.sub.y is H, OH, an optionally substituted C.sub.1 to C.sub.7 alkyl residue, an optionally substituted cyclic or polycyclic aryl residue or an optionally substituted nitrogen heterocycle.

2. Purine derivative according to claim 1, characterized in that the substituent R.sub.y is selected from H, —CR.sub.aR.sub.bR.sub.c, general formula (III) or formula (IV) ##STR00035## wherein: is the covalent linkage to general formula (I) or (II), X is N or CH, Z is a single bond, CH.sub.2, O, C(═O), S or NR.sub.x, R.sub.x is an optionally substituted and/or branched C.sub.1 to C.sub.4 alkyl residue, R.sup.1, R.sup.2, R.sup.3 and R.sup.4, each independently of one another, are —H, -halogen, —NO.sub.2, —CN, —NR.sub.2, and —SR, —OR, —COOR, —COR, —R, a C.sub.1 to C.sub.4 vinyl residue or an aryl residue, wherein R is H or C.sub.1 to C.sub.8 alkyl residue, R.sub.a, R.sub.b and R.sub.c independently of one another are selected from H and cyclic residues.

3. Purine derivative according to claim 1, characterized in that R.sub.n is H, an optionally branched C1-C4 alkyl residue, wherein W is H or OH, an optionally substituted five-membered ring oxygen heterocycle or six-membered ring oxygen heterocycle.

4. Purine derivative according to claim 3, characterized in that Z is O, C(═O) or S and X is N or CH.

5. Purine derivative according to claim 1, characterized in that Y is O, NR or an amide group.

6. Purine derivative according to claim 2, characterized in that R.sup.1, R.sup.3 and optionally R.sup.4 are H.

7. Purine derivative according to claim 2, characterized in that for the substituent R.sub.y according to formula (IV), R.sup.1 is H, R.sup.2 and R.sup.3 are, independently of one another, R or an optionally substituted C.sub.1 to C.sub.4 vinyl residue, wherein R is H or an optionally OH-functionalized C.sub.1 to C.sub.5 alkyl residue.

8. Purine derivative according to claim 2, characterized in that for the substituent R.sub.y according to formula (III), R.sup.1, R.sup.3 and R.sup.4 are H and R.sup.2 is H, -halogen, —COR or an optionally substituted phenyl residue.

9. (canceled)

10. A pharmaceutical formulation comprising at least one purine derivative according to claim 1 in a physiologically effective concentration for the treatment of carcinomas or mucoviscidosis.

11. The pharmaceutical composition according to claim 10, further comprising one or more phenothiazine derivatives according to general formula (V): ##STR00036## wherein: X is N and Z is S, R.sub.x is an optionally substituted and/or branched C.sub.1 to C.sub.4 alkyl, alkenyl or alkynyl residue, the substituents R.sup.1 and R.sup.2 in ortho-, meta- or para-position are, independently of one another, —H, —OR, —C(═O)R, —CF.sub.3, -halogen, an aliphatic or aromatic heterocycle, Y is N or an amide group (—NR—C(═O)—), n and m independently of one another are an integer from 0 to 3, R.sup.5 and R.sup.6 independently of one another are H, a piperazine residue, a phenyl residue (—C.sub.6H.sub.5), a hydroxyphenyl residue (—OC.sub.6H.sub.5), or a phenylene residue (—C.sub.6H.sub.4—).

12. A method for treating cancer comprising administering a pharmaceutically effective amount of a purine derivative according to claim 1 to patient having cancer.

13. A method for treating cancer comprising administering an pharmaceutically effective amount of a pharmaceutical composition according to claim 10 to a patient having cancer who is undergoing chemotherapy, radiotherapy and/or immunotherapy.

14. A method for treating cancer comprising administering a pharmaceutically effective amount of the pharmaceutical formulation according to claim 11 to a patient having cancer who is undergoing chemotherapy, radiotherapy and/or cancer immunotherapy.

Description

[0142] Further features and advantages of the invention can be taken from the following schematic drawings and embodiment examples with the aid of which the invention is intended to be explained in more detail by way of example without limiting the invention thereto.

[0143] There are shown in:

[0144] FIG. 1: a spectrophotometric protein aggregation assay at 43° C., a wavelength of 500 nm and a concentrations of the proteins: 1.44 μmol/L citrate synthase (CS), 481 nmol/L HSP27 (HSP) in a 40 mmol/L HEPES buffer (pH 7.4) during treatment with 14 or 28 μmol/L 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one (CS+HSP+test substance no. 2), with 750 μmol/L BVDU (CS+HSP+BVDU) and without (CS+HSP; reference);

[0145] FIG. 2: the cell counts over the number of treatment days of U937 cells treated with the cytostatic agent bortezomib (0.1 to 0.5 ng/ml) in each case combined with the HSP27 inhibitors acepromazine (+1 μM ACE) and BVDU (+30 μM BVDU) and without HSP27 inhibitor.

[0146] FIG. 3: the cell counts over the number of treatment days of U937 cells treated with the cytostatic agent bortezomib (0.2 to 0.4 ng/ml) in each case combined with the HSP27 inhibitors acepromazine (+1 μM ACE) and BVDU (+30 μM BVDU) and without HSP27 inhibitor.

[0147] FIG. 4 shows the inhibition of the development of resistance of the tumour cell line RPMI-8226 by acepromazine. FIG. 4a shows RPMI 8226 cells treated with cytostatic agent bortezomib 0.2-0.4 ng/ml (A), combination treatment with bortezomib plus 0.75 μM acepromazine (B) and combination treatment with bortezomib plus 30 μM BVDU (C). FIG. 4b shows the control without cytostatic agent: RPMI 8226 cells untreated (A), 0.75 μM acepromazine alone (B) and 30 μM BVDU alone (C).

[0148] FIG. 5 shows the inhibition of the development of resistance of the tumour cell line RPMI-8226 by chlorpromazine. FIG. 5a shows RPMI 8226 cells treated with cytostatic agent bortezomib 0.2-0.4 ng/ml (A), combination treatment with bortezomib plus 0.5 μM chlorpromazine (B) and combination treatment with bortezomib plus 30 μM BVDU (C). FIG. 5b shows the control without cytostatic agent: RPMI 8226 cells untreated (A), 0.5 μM chlorpromazine alone (B) and 30 μM BVDU alone (C).

[0149] FIG. 6 shows the inhibition of the development of resistance of the tumour cell line RPMI-8226 by 2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one. FIG. 6a shows RPMI 8226 cells treated with cytostatic agent bortezomib 0.1-0.3 ng/ml (A), combination treatment with bortezomib plus 1 μM 2-(4-butylphenylamino)-9-(2-hydroxy-ethoxymethyl)-1,9-dihydropurin-6-one (B) and combination treatment with bortezomib plus 30 μM BVDU (C). FIG. 6b shows the control without cytostatic agent: RPMI 8226 cells untreated (A), 1 μM 2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one alone (B) and 30 μM BVDU alone (C).

[0150] FIG. 7 shows the inhibition of the development of resistance of the tumour cell line RPMI-8226 by 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one. FIG. 7a shows RPMI 8226 cells treated with cytostatic agent bortezomib 0.1-0.3 ng/ml (A), combination treatment with bortezomib plus 1 μM 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one (B) and combination treatment with bortezomib plus 30 μM BVDU (C). FIG. 7b shows the control without cytostatic agent: RPMI 8226 cells untreated (A), 1 μM 2-(3-trichlorovinyl-phenylamino)-1,9-dihydropurin-6-one alone (B) and 30 μM BVDU alone (C).

[0151] FIG. 8 shows the inhibition of the development of resistance of the tumour cell line RPMI-8226 by 9H-xanthene-9-carboxylic acid [4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amide. FIG. 8a shows RPMI 8226 cells treated with cytostatic agent bortezomib 0.075-0.275 ng/ml (A), combination treatment with bortezomib plus 1 μM 9H-xanthene-9-carboxylic acid [4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amide (B) and combination treatment with bortezomib plus 30 μM BVDU (C). FIG. 8b shows the control without cytostatic agent: RPMI 8226 cells untreated (A), 1 μM 9H-xanthene-9-carboxylic acid [4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amide alone (B) and 30 μM BVDU alone (C).

[0152] FIG. 9 shows the inhibition of the development of resistance of the tumour cell line RPMI-8226 by 2-biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]-acetamide. FIG. 9a shows RPMI 8226 cells treated with cytostatic agent bortezomib 0.075-0.275 ng/ml (A), combination treatment with bortezomib plus 1 μM 2-biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]acetamide (B) and combination treatment with bortezomib plus 30 μM BVDU (C). FIG. 9b shows the control without cytostatic agent: RPMI 8226 cells untreated (A), 1 μM 2-biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]acetamide alone (B) and 30 μM BVDU alone (C).

[0153] FIG. 10 shows the inhibition of the development of resistance of the tumour cell line U-937 by acepromazine. FIG. 10a shows U-937 cells treated with cytostatic agent bortezomib 0.1-0.5 ng/ml (A), combination treatment with bortezomib plus 0.75 μM acepromazine (B) and combination treatment with bortezomib plus 30 μM BVDU (C). FIG. 11b shows the control without cytostatic agent: RPMI 8226 cells untreated (A), 0.75 μM acepromazine alone (B) and 30 μM BVDU alone (C).

[0154] FIG. 11 shows the inhibition of the development of resistance of the tumour cell line U-937 by 2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one. FIG. 11a shows U-937 cells treated with cytostatic agent bortezomib 0.1-0.5 ng/ml (A), combination treatment with bortezomib plus 0.75 μM 2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one (B) and combination treatment with bortezomib plus 30 μM BVDU (C). FIG. 11b shows the control without cytostatic agent: RPMI 8226 cells untreated (A), 0.75 μM 2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one alone (B) and 30 μM BVDU alone (C).

EXAMPLE 1—SPEEDSCREEN OF PURINE DERIVATIVES

[0155] For the experimental validation of low-molecular-weight compounds which act as potential HSP27 inhibitors according to the in silico prediction, a method based on gel filtration was employed.

[0156] The method for detecting molecular bonds using gel filtration was described in 2004 as “SpeedScreen” [Muckenschnabel et al., 2004] and was adapted to meet the requirements of the invention. The method of separation consists in the fact that larger molecules, such as e.g. proteins, can pass through a matrix of Sephadex G-25, while low-molecular-weight compounds (small molecules) penetrate into the matrix and do not pass through it. For the separation, PD SpinTraps G-25 from GE Healthcare were used. During the 2-minute centrifugation at 800×g (according to operating instructions), these columns allow molecules with a molecular weight of greater than or equal to 5000 g/mol to pass through, while smaller molecules are retained in the matrix.

[0157] The target protein HSP27 has a molecular weight of 27000 g/mol and therefore passes through the matrix. The HSP27 inhibitors of general formula (I) described according to the invention are low-molecular-weight compounds, which can only pass through the Sephadex G-25 matrix if they have previously bound to the target protein HSP27 and are transported thereby through the matrix. Non-bound low-molecular-weight compounds are retained by the matrix. Molecules that have passed through the matrix can then be detected by mass spectroscopy. This enables “binders” to be differentiated from “non-binders”.

Result

[0158] Representatives of the purine derivatives according to general formula (I) tested by SpeedScreen are: [0159] 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one [0160] 2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one [0161] 2-(3-hydroxymethylphenylamino)-3,7-dihydropurin-6-one

[0162] Furthermore, acepromazine (as acepromazine maleate) was tested positively as a representative of the phenothiazine derivatives by means of SpeedScreen.

[0163] The three purine derivatives pass through the Sephadex G-25 matrix and are detected in the medium after the passage. The binding of the phenothiazine derivatives acepromazine (as acepromazine maleate) and chlorpromazine to HSP27 could also be positively detected by means of bio-layer interferometry with a K.sub.D=72 μmol/L and 770 μmol/L respectively.

EXAMPLE 2—SPEEDSCREEN OF THYMINE DERIVATIVES

[0164] For the experimental validation of low-molecular-weight compounds which act as potential HSP27 inhibitors according to the in silico prediction, a method based on gel filtration was employed.

[0165] The method for detecting molecular bonds using gel filtration was described in 2004 as “SpeedScreen” [Muckenschnabel et al., 2004] and was adapted to meet the requirements of the invention. The method of separation consists in the fact that larger molecules, such as e.g. proteins, can pass through a matrix of Sephadex G-25, while low-molecular-weight compounds (small molecules) penetrate into the matrix and do not pass through it. For the separation, PD SpinTraps G-25 from GE Healthcare were used. During the 2-minute centrifugation at 800×g (according to operating instructions), these columns allow molecules with a molecular weight of greater than or equal to 5000 g/mol to pass through, while smaller molecules are retained in the matrix.

[0166] The target protein HSP27 has a molecular weight of 27000 g/mol and therefore passes through the matrix. The HSP27 inhibitors of general formula (I) described according to the invention are low-molecular-weight compounds, which can only pass through the Sephadex G-25 matrix if they have previously bound to the target protein HSP27 and are transported thereby through the matrix. Non-bound low-molecular-weight compounds are retained by the matrix. Molecules that have passed through the matrix can then be detected by mass spectroscopy. This enables “binders” to be differentiated from “non-binders”.

Result

[0167] Representatives of the thymine derivatives according to general formula (VI) tested by SpeedScreen are: [0168] 9H-xanthene-9-carboxylic acid [4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amide [0169] 2-biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]-acetamide

[0170] Both thymine derivatives pass through the Sephadex G-25 matrix and are detected in the medium after the passage.

[0171] The binding of the phenothiazine derivatives acepromazine (as acepromazine maleate) and chlorpromazine to HSP27 could also be positively detected by means of bio-layer interferometry with a K.sub.D=72 μmol/L and 770 μmol/L respectively.

EXAMPLE 3—DETECTION OF BINDING BY BIO-LAYER INTERFEROMETRY

[0172] Bio-layer interferometry is a technique for detecting biomolecular interactions. It is an analytical technique that measures the optical interference of white light which is reflected by two surfaces: in each case, one surface consists of a layer of the immobilized target protein on the tip of the biosensor and one is an internal reference surface. The binding of ligands to the target protein alters the interference pattern and can be measured in real time.

[0173] For the measurements, an instrument of the ForteBio Octet Red 384 brand was used. The target protein HSP27 was biotinylated and bound to the appropriate sensors (SSA). One great advantage of this analytical method consists in the fact that the analytes do not have to be either immobilized or labelled. The purine derivatives of general formula (I) or (II) pre-identified by means of in silico screening are employed for these measurements in a concentration range (K.sub.D) of between 333 μmol/L and 10 nmol/L.

[0174] Using the bio-layer interferometry method, the purine derivative 2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one could be positively detected as an HSP27-binding low-molecular-weight compound with a K.sub.D=15 μmol/L.

[0175] The binding of the phenothiazine derivatives acepromazine (as acepromazine maleate) and chlorpromazine to HSP27 could also be positively detected by means of bio-layer interferometry with a K.sub.D=72 μmol/L or 770 μmol/L respectively.

EXAMPLE 4—SPECTROPHOTOMETRIC PROTEIN AGGREGATION ASSAY

[0176] To measure the efficacy of the purine derivatives according to the invention, robust experiments for the chaperone function of the HSP27 protein were performed:

[0177] A known client protein (=substrate on which HSP27 acts as a chaperone) of HSP27 is citrate synthase. Citrate synthase is denatured when the temperature increases to 43° C. This heat denaturation is prevented or delayed by the presence of HSP27 [Jakob U, 1993]. By adding the novel active ingredients, their efficacy can thus be measured through the inhibition of the HSP27 chaperone function.

[0178] To determine the activity (i.e. the effect on the aggregation behaviour of the citrate synthase (CS) after thermal incubation) of 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one in comparison to BVDU, four different batches containing 1.44 μmol/L CS, 481 nmol/L HSP27 (HSP) in a 40 mmol/L HEPES buffer (pH 7.4) were prepared and BVDU (750 μmol/L) or various concentrations of 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one (14 μmol/L; 28 μmol/L) were added. The samples were then incubated at 43° C. and the aggregation behaviour of the CS was monitored in a spectrometer (PerkinElmer LS55, PerkinElmer Inc.) at a wavelength of 500 nm.

[0179] It can be seen from FIG. 1 that the protein citrate synthase (graph CS alone) is denatured by increasing the temperature to 43° C. and forms aggregates. The aggregation is measured in the spectrometer at 500 nm (light scattering by protein aggregates). The presence of HSP27 (HSP) counteracts the aggregation (cf. graph, CS+HSP). Since the addition of the test substances (BVDU and 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one) inhibits the chaperone function of HSP27, the activity of the respective test substance can be determined by measuring the aggregation behaviour of the CS.

[0180] It is clear that CS alone forms aggregates very rapidly; these CS aggregates then precipitate in the measuring cuvette, so that the red graph falls again after reaching the peak. Through the presence of HSP27, CS aggregation is prevented over the period of one hour shown here. BVDU partially cancels the chaperone function of HSP27 at a concentration of 750 μmol/L BVDU. The test substance 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one shows a comparable effect at a concentration of 14 μmol/L and a significantly more pronounced effect at 28 μmol/L.

[0181] The inhibition of HSP27 correlates to the binding strength of a low-molecular-weight organic compound to the active ingredient binding pocket of HSP27. The active concentration of a low-molecular-weight organic compound interacting with HSP27 can be converted to BVDU equivalents via the activity of BVDU, i.e. equal to the HSP27 inhibiting dose in mg BVDU. Based on 100 mg BVDU, the test substance 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one has a BVDU equivalent dose of less than 1.9 mg.

EXAMPLE 5—CAPILLARY ELECTROPHORESIS OF THE CS PROTEIN PRECIPITATES WITH PURINE DERIVATIVES

[0182] Since the heat-denatured protein aggregates of the citrate synthase from Example 1 are not water-soluble, these can be separated completely from the supernatant by means of centrifugation at room temperature at 16,000×g, 10 min. By using capillary electrophoresis, the compositions of the separated precipitates can be shown and quantified. The relative quantity of protein of precipitated citrate synthase serves as a measure of the efficacy of the test substances.

[0183] In each case, 481 nmol/L HSP27 and 1.44 μmol/L citrate synthase in 40 mmol/L HEPES buffer, pH 7.4, were employed. The relative quantity of protein of the precipitated citrate synthase in each case was normalized using a defined quantity of BSA as internal standard. The relative quantity of protein that precipitated when using 750 μmol/L BVDU was set at 1. The other test substances (2 representatives of phenothiazine derivatives of general formula (V), 2 representatives of purine derivatives of formula (I) or (II)) were each employed at a concentration of 10 μmol/L.

[0184] Table 1 below shows the separation by capillary electrophoresis of heat-denatured protein aggregates of citrate synthase after thermal treatment of the samples at 43° C. for 120 minutes.

Overview of Test Results:

[0185]

TABLE-US-00002 Rel. quantity of Efficacy protein compared Dose Substance employed prec. CS with BVDU 750 μmol/L  BVDU 1 10 μmol/L Chlorpromazine 1.06 79.5 x  10 μmol/L Acepromazine 0.99 74.25 x 10 μmol/L 2-(4-Butylphenylamino)-9- 0.92 69.0 x  (2-hydroxyethoxymethyl)- 1,9-dihydropurin-6-one 10 μmol/L 2-(3-Trichloro- 0.71 53.25 x vinylphenylamino)- 1,9-dihydropurin-6-one

[0186] The four test substances proved significantly more effective than the reference substance BVDU when used at a concentration of 10 μmol/L in each case, and can therefore be given in a dose in the range of between 53 and 79 times lower to achieve an effect comparable with that of the conventional BVDU. In particular, the purine derivative 2-(4-butyl-phenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one shows a 69 times higher activity than BVDU for the inhibition of the HSP27 protein (BVDU equivalent dose is 1.4 mg). However, the purine derivative 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one also shows a 53 times higher activity for the inhibition of the HSP27 protein compared with BVDU, which corresponds to a BVDU equivalent dose of less than 1.9 mg.

EXAMPLE 6—TESTING THE HSP27 INHIBITORS IN CANCER CELLS

[0187] Cancer cells develop resistances extremely rapidly when they are treated with cytostatic agents. This is the reason for numerous failures in chemotherapy. HSP27 favours the development of chemoresistances, e.g. by interacting with apoptosis proteins and thus preventing the sought cell death of the cancer cells. For bortezomib (Velcade) in particular, a modern cytostatic agent (proteasome inhibitor), the HSP27-driven development of resistance is well documented [Chauhan D, 2004].

[0188] In the cell experiments described here, it is shown that, by administering the novel HSP27 inhibitors, the development of resistance to the cytostatic agent bortezomib is prevented in U937 cells. In this case, acepromazine was tested in comparison with BVDU.

[0189] For this purpose, U937 cells (histiocytic lymphoma) were cultured in DMEM culture medium (+10% FCS) at 37° C., 5% CO.sub.2 in an H.sub.2O-saturated atmosphere. 100,000 cells/ml were seeded and incubated with the cytostatic agent bortezomib (0.1 ng/ml) and the respective test substance (dosage in the current experiment: 1 μM/L acepromazine). The reference substance, BVDU, was employed at a concentration of 30 μM/L. The cells were in each case passaged before the cell count reached 1,000,000 cells/ml. At each passage, 100,000 cells/ml were again seeded and the dose of the cytostatic agent bortezomib was increased stepwise (0.1 ng/mL or 0.05 ng/mL). The concentration of the test substances remained constant. As a result of the stepwise increase in the dosage of the cytostatic agent, resistance developed in the U937 cells. The development of resistance is prevented or inhibited in the case of a positively tested HSP27 inhibitor.

[0190] It can be seen from FIG. 2 that, by treating U937 cells with acepromazine (+1 μM ACE), no development of resistance occurred and therefore no living cells were detectable after the end of the experiment. In contrast, for U937 cells treated with BVDU (+30 μM BVDU), a final cell count of 430,000 cells/ml was detected whereas the final cell count in the culture without the addition of an HSP27 inhibitor amounted to 790,000 cells/ml. As a control, U937 cells were treated with the respective test substances alone. At the same dosage, the test substances alone have no effect on cell growth, since HSP27 is not needed in a large quantity by the cancer cells without the pressure by the cytostatic agent. The harmful effect of HSP27 for the patient on the development of resistance only becomes clear when the cytostatic agent is administered simultaneously.

EXAMPLE 7—TESTING THE HSP27 INHIBITORS IN CANCER CELLS

[0191] The experiments from Example 6 were repeated in the same way, wherein U937 cells (histiocytic lymphoma) are cultured in DMEM culture medium (+10% FCS) at 37° C., 5% CO.sub.2 in an H.sub.2O— saturated atmosphere. 100,000 cells/ml were seeded and incubated with an initial concentration of 0.2 ng/ml of the cytostatic agent bortezomib and both without an inhibitor and with acepromazine as an HSP27 inhibitor at a concentration of 1 μM/L. The reference substance, BVDU, was employed at a concentration of 30 μM/L. The cells were passaged in each case before the cell count reached 1,000,000 cells/ml. At each passage, 100,000 cells/ml were again seeded and the dose of the cytostatic agent bortezomib was increased stepwise by 0.1 ng/mL. The concentration of the test substance and the reference substance remained constant. As a result of the stepwise increase in the dosage of the cytostatic agent, resistance developed in the U937 cells. The development of resistance is prevented or inhibited in the case of a positively tested HSP27 inhibitor.

[0192] It can be seen from FIG. 3 that, by treating U937 cells with acepromazine (+1 μM ACE), no development of resistance occurred and therefore no living cells were detectable after the end of the experiment. In contrast, for U937 cells treated with BVDU (+30 μM BVDU), a final cell count of 60,000 cells/ml was detected whereas the final cell count in the culture without the addition of an HSP27 inhibitor amounted to 230,000 cells/ml.

[0193] As a control, U937 cells were treated with the respective test substances alone. At the same dosage, the test substances alone have no effect on cell growth, since HSP27 is not needed in a large quantity by the cancer cells without the pressure by the cytostatic agent. The harmful effect of HSP27 for the patient on the development of resistance only becomes clear when the cytostatic agent is administered simultaneously.

EXAMPLE 8—CAPILLARY ELECTROPHORESIS OF THE CS PROTEIN PRECIPITATES WITH THYMINE DERIVATIVES

[0194] Since the heat-denatured protein aggregates of the citrate synthase from Example 2 are not water-soluble, these can be separated completely from the supernatant by means of centrifugation at room temperature at 16,000×g, 10 min. By using capillary electrophoresis, the compositions of the separated precipitates can be shown and quantified. The relative quantity of protein of precipitated citrate synthase serves as a measure of the efficacy of the test substances.

[0195] In each case, 481 nM HSP27 and 1.44 μmol/L citrate synthase in 40 mmol/L HEPES buffer, pH 7.4, were employed. The relative quantity of protein of the precipitated citrate synthase in each case was normalized using a defined quantity of BSA as internal standard. The relative quantity of protein that precipitated when using 750 μmol/L BVDU was set at 1. The other test substances (2 representatives of phenothiazine derivatives of general formula (V), 2 representatives of thymine derivatives of formula (I) or (II)) were employed at a concentration of 10 μmol/L in each case.

[0196] Table 2 below shows the separation by capillary electrophoresis of heat-denatured protein aggregates of citrate synthase after thermal treatment of the samples at 43° C. for 120 minutes.

Overview of the Test Results:

[0197]

TABLE-US-00003 Rel. quantity Efficacy of protein compared Dose Substance employed prec. CS with BVDU 750 μmol/L  BVDU 1 10 μmol/L Chlorpromazine 1.06 79.5 x  10 μmol/L Acepromazine 0.99 74.25 x 10 μmol/L 9H-Xanthene-9-carboxylic 0.65 48.75 x acid[4-(5-methyl-2,4-dioxo- 3,4-dihydro-2H-pyrimidin-1- yl)-but-2-enyl]amide 10 μmol/L 2-Biphenyl-4-yl-N- 0.81 60.75 x [4-(5-methyl-2,4-dioxo- 3,4-dihydro-2H-pyrimidin-1- yl)-but-2-enyl]acetamide

[0198] The four test substances proved significantly more effective than the reference substance BVDU when used at a concentration of 10 μmol/L in each case, and can therefore be given in a dose in the range of between 49 and 79 times lower to achieve an effect comparable with that of the conventional BVDU. In particular, the thymine derivative 2-biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]acetamide shows a 61 times higher activity than BVDU for the inhibition of the HSP27 protein (BVDU equivalent dose is 1.6 mg). However, the thymine derivative 9H-xanthene-9-carboxylic acid[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amide also shows a 49 times higher activity for the inhibition of the HSP27 protein compared with BVDU, which corresponds to a BVDU equivalent dose of less than 2 mg.

Further Test Results Table 3 below shows the results of the capillary electrophoretic separation of heat-denatured protein aggregates of citrate synthase after thermal treatment of the samples at 43° C. for 70 minutes for further thymine derivatives. Otherwise, the experiments were performed as described above:

TABLE-US-00004 Rel. quantity of Efficacy protein prec. compared with Dose Substance employed CS BVDU 750 μmol/L  BVDU 1 10 μmol/L N-[(2Z)-4-(2,4-Dioxo-3H-pyrimidin-1-yl)but-2-en-1-yl]-2- 1.15 86.25 x (naphthalen-2-yl)acetamide 10 μmol/L N-[(2Z)-4-(2,4-Dioxo-3H-pyrimidin-1-yl)but-2-en-1-yl]-2-(4- 0.88 66.15 x phenylphenyl)acetamide 10 μmol/L N-[(2Z)-4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)but-2-en- 0.86 64.31 x 1-yl]-9H-fluorene-9-carboxamide 10 μmol/L 3′-Deoxy-3′-[4-(3,4-dichlorophenyl)-1H-1,2,3-triazol-1-yl]- 0.86 64.21 x thymidine 10 μmol/L N-[(2Z)-4-(2,4-Dioxo-3H-pyrimidin-1-yl)but-2-en-1-yl]-9,9a- 0.85 63.38 x dihydro-4aH-xanthene-9-carboxamide 10 μmol/L 3′-Deoxy-3′-[4-(2-pyridinyl)-1H-1,2,3-triazol-1]-yl]thymidine 0.84 62.86 x 10 μmol/L N-[(2Z)-4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)but-2-en- 0.83 62.51 x 1-yl]-2-(naphthalen-2-yl)acetamide 10 μmol/L N-[(2Z)-4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)but-2-en- 0.82 61.19 x 1-yl]-2,2-diphenylacetamide 10 μmol/L 1-[(4S,5S)-4-[4-(3-Bromophenyl)-1H-1,2,3-triazol-1-yl]-5- 0.81 60.88 x (hydroxymethyl)oxolan-2-yl]-5-methyl-1,2,3,4- tetrahydropyrimidine-2,4-dione 10 μmol/L N-[(2Z)-4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)but-2-en- 0.81 60.44 x 1-yl]-2-(naphthalen-1-yl)acetamide 10 μmol/L 3′-Deoxy-3′-(4-propyl-1H-1,2,3-triazol-1-yl)thymidine 0.79 59.59 x 10 μmol/L 3′-Deoxy-3′-(4-phenyl-1H-1,2,3-triazol-1-yl)thymidine 0.79 59.07 x 10 μmol/L N-[4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)butyl]-2,2- 0.79 58.97 x diphenylacetamide 10 μmol/L N-[(2Z)-4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)but-2-en- 0.78 58.73 x 1-yl]naphthalene-1-carboxamide 10 μmol/L 3′-(4-Butyl-1H-1,2,3-triazol-1-yl)-3′-deoxythymidine 0.76 57.08 x 10 μmol/L N-[4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)butyl]-2- 0.75 56.36 x (naphthalen-2-yl)acetamide 10 μmol/L 1-[(4S,5S)-5-(Hydroxymethyl)-4-[4-(4-methoxyphenyl)-1, 0.75 55.89 x 2,3-triazol-1-yl]oxolan-2-yl]-5-methyl-3H-pyrimidine-2,4- dione 10 μmol/L 3′-Deoxy-3′-[4-(2-phenylethyl)-1H-1,2,3-triazol-1-yl]- 0.74 55.67 x thymidine 10 μmol/L 3′-Deoxy-3′-(4-pentyl-1H-1,2,3-triazol-1-yl)thymidine 0.70 52.57 x 10 μmol/L N-[4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)butyl]-9H- 0.68 51.16 x xanthene-9-carboxamide 10 μmol/L 3′-[4-(4-Chlorophenyl)-1H-1,2,3-triazol-1-yl]-3′- 0.67 50.53 x deoxythymidine 10 μmol/L N-[4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)butyl]-2-(4- 0.67 50.52 x phenylphenyl)acetamide 10 μmol/L 3′-Deoxy-3′-[4-(4-fluorophenyl)-1H-1,2,3-triazol-1-yl]- 0.66 49.69 x thymidine 10 μmol/L 3′-[4-(Cyclopentylmethyl)-1H-1,2,3-triazol-1-yl]-3′- 0.66 49.63 x deoxythymidine 10 μmol/L 1-[(4S,5S)-4-[4-(4-Bromophenyl)-1,2,3-triazol-1-yl]-5- 0.64 48.14 x (hydroxymethyl)oxolan-2-yl]-5-methyl-3H-pyrimidine-2,4- dione 10 μmol/L 3′-Deoxy-3′-[4-(2-methyl-2-propanyl)-1H-1,2,3-triazol-1- 0.61 46.01 x yl]thymidine 10 μmol/L N-[4-(5-Methyl-2,4-dioxo-3H-pyrimidin-1-yl)butyl]-9H- 0.58 43.30 x fluorene-9-carboxamide

EXAMPLE 9—TREATMENT OF MUCOVISCIDOSIS

[0199] To determine the effects on the concentration of deletion mutant ΔF508 CFTR functionally integrated into the membrane, the mucoviscidosis cell line CCD-186Sk (ATCC® CRL-1563™) was used.

[0200] The mucoviscidosis cell line CCD-186Sk is a homozygous carrier of the ΔF508 mutation, to which the CFTR gene relates. The deletion of the phenylalanine at position 508 of the CFTR gene is typical of this disease and affects over 70% of patients. Because of this deletion, the CFTR protein that forms is not folded entirely correctly and is therefore supplied to the cellular degradation processes and not—like the healthy protein—transported to the cell membrane and incorporated there as a transmembrane protein. HSP27 plays a crucial role in isolating the delta F508 CFTR protein for degradation. The experiments serve to prove the hypothesis that delta F508 CFTR protein which is not isolated and degraded immediately upon its formation is transported to the cell membrane where, as a functioning transmembrane protein, it regulates the transport of water and salt through the plasma membrane of the cells.

[0201] For this purpose, the CCD-186Sk cells are seeded at a density of 100,000 cells/ml and incubated with: [0202] 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one [0203] 2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one [0204] 2-(3-hydroxymethyl-phenylamino)-3,7-dihydropurin-6-one

[0205] as representatives of the purine derivatives in the respective concentration. Every 24 hours, cells are harvested and tests are performed to see whether the treated cells present more CFTR at the cell surface than untreated cells. To detect the CFTR protein at the cell surface, an antibody is used which specifically labels the extracellular “loop” (amino acids 103-117) of the CFTR protein. This antibody is labelled with the fluorescent dye FITC and detected by flow cytometry.

EXAMPLE 10—BIO-LAYER INTERFEROMETRY

[0206] For measurements by bio-layer interferometry, biotinylated HSP27 protein was first bound to Super-Streptavidin sensors (SSA, Fortebio). The loaded sensors were then incubated (“quenched”) with biocytin. As reference, unloaded SSA sensors were incubated with biocytin. In the case of a specific binding to HSP27 of the analyte to be measured, the loaded HSP27 sensor generates a stronger signal (response) than the concurrent reference sensor. After the “baseline” has been established by incubation with the reaction buffer (PBST 0.1% Tween 20 plus 3% DMSO), first the association and then the dissociation of the respective analyte is measured in real time. The analytes were measured in increasing concentrations in each case: 1.23-3.7-11.11-33.33-100-300 μM/l.

Results:

[0207]

TABLE-US-00005 Dissociation constant Substance K.sub.D [μM] Chlorpromazine 770 Acepromazine 72 5-Phenyl-2′-deoxyuridine 60 2-(3-Hydroxymethylphenylamino)-3,7-dihydropurin-6-one 710 2-(4-Butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9- 15 dihydropurin-6-one 9H-Xanthene-9-carboxylic acid 267 [4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)- but-2-enyl]amide 2-Biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H- 424 pyrimidin-1-yl)-but-2-enyl]acetamide

EXAMPLE 11—INHIBITION OF THE DEVELOPMENT OF RESISTANCE OF THE TUMOUR CELL LINE RPMI-8226 BY ACEPROMAZINE

[0208] The cell line RPMI-8226 (DMSZ 402) is a “multiple myeloma” cell line and was chosen because bortezomib is employed as a standard therapy for multiple myeloma. RPMI-8226 cells respond to bortezomib, express HSP27 and develop resistance to Velcade.

[0209] RPMI-8226 cells were seeded at a cell count of 100,000 cells/ml in each case and regularly passaged. FIG. 4a shows that, as a result of the treatment with 0.75 μmol/L acepromazine, the development of resistance to the cytostatic agent bortezomib was significantly inhibited and only 30,000 living cells per ml medium could be detected at the end of the experiment. In contrast, for RPMI 8226 cells that were treated with 30 μmol/L BVDU, a final cell count of 160,000 cells/ml was detected whereas the final cell count for the culture without the addition of an HSP27 inhibitor amounted to 350,000 cells/ml. As a control, RPMI 8226 cells were treated with the respective test substances alone, FIG. 4b. At the same dosage, the test substances alone do not affect cell growth, since HSP27 is not needed in a large quantity by the cancer cells without the pressure from the cytostatic agent.

EXAMPLE 12—INHIBITION OF THE DEVELOPMENT OF RESISTANCE OF THE TUMOUR CELL LINE RPMI-8226 BY CHLORPROMAZINE

[0210] RPMI-8226 cells were seeded with a cell count of 100,000 cells/ml in each case and regularly passaged. FIG. 5a shows that, as a result of the treatment with 0.5 μmol/L chlorpromazine, the development of resistance to the cytostatic agent bortezomib was significantly inhibited and only 70,000 living cells per ml medium could be detected at the end of the experiment. In contrast, for RPMI 8226 cells that were treated with 30 μmol/L BVDU, a final cell count of 160,000 cells/ml was detected whereas the final cell count in the culture without the addition of an HSP27 inhibitor amounted to 350,000 cells/ml. As a control, RPMI 8226 cells were treated with the respective test substances alone, FIG. 5b. At the same dosage, the test substances alone do not affect cell growth.

EXAMPLE 13—INHIBITION OF THE DEVELOPMENT OF RESISTANCE OF THE TUMOUR CELL LINE RPMI-8226 BY 2-(4-BUTYLPHENYLAMINO)-9-(2-HYDROXYETHOXYMETHYL)-1,9-DIHYDROPURIN-6-ONE

[0211] RPMI-8226 cells were seeded with a cell count of 100,000 cells/ml in each case and regularly passaged. FIG. 6a shows that, as a result of the treatment with 1 μM/L 2-(4-butylphenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one, the development of resistance to the cytostatic agent bortezomib was significantly inhibited. At the end of the experiment, 240,000 living cells per ml medium could be detected. For the treatment with 30 μM/L BVDU, a final cell count of 290,000 cells/ml was detected whereas the final cell count in the culture without the addition of an HSP27 inhibitor amounted to 950,000 cells/ml. As a control, RPMI 8226 cells were treated with the respective test substances alone, FIG. 6b. At the same dosage, the test substances alone do not affect cell growth.

EXAMPLE 14—INHIBITION OF THE DEVELOPMENT OF RESISTANCE OF THE TUMOUR CELL LINE RPMI-8226 BY 2-(3-TRICHLOROVINYLPHENYLAMINO)-1,9-DIHYDROPURIN-6-ONE

[0212] RPMI-8226 cells were seeded with a cell count of 100,000 cells/ml in each case and regularly passaged. FIG. 7a shows that, as a result of the treatment with 1 μmol/L 2-(3-trichlorovinylphenylamino)-1,9-dihydropurin-6-one, the development of resistance to the cytostatic agent bortezomib was significantly inhibited. At the end of the experiment, 270,000 living cells per ml medium could be detected. For the treatment with 30 μmol/L BVDU, a final cell count of 290,000 cells/ml was detected whereas the final cell count in the culture without the addition of an HSP27 inhibitor amounted to 950,000 cells/ml. As a control, RPMI 8226 cells were treated with the respective test substances alone, FIG. 7b. At the same dosage, the test substances alone do not affect cell growth.

EXAMPLE 15—INHIBITION OF THE DEVELOPMENT OF RESISTANCE OF THE TUMOUR CELL LINE RPMI-8226 BY 9H-XANTHENE-9-CARBOXYLIC ACID [4-(5-METHYL-2,4-DIOXO-3,4-DIHYDRO-2H-PYRIMIDIN-1-YL)-BUT-2-ENYL]AMIDE

[0213] RPMI-8226 cells were seeded with a cell count of 100,000 cells/ml in each case and regularly passaged. FIG. 8a shows that, as a result of the treatment with 1 μmol/L 9H-xanthene-9-carboxylic acid [4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]amide, the development of resistance to the cytostatic agent bortezomib was significantly inhibited. At the end of the experiment, 290,000 living cells per ml medium could be detected. For the treatment with 30 μmol/L BVDU, a final cell count of 340,000 cells/ml was detected whereas the final cell count in the culture without the addition of an HSP27 inhibitor amounted to 510,000 cells/ml. As a control, RPMI 8226 cells were treated with the respective test substances alone, FIG. 8b. At the same dosage, the test substances alone do not affect cell growth.

EXAMPLE 16—INHIBITION OF THE DEVELOPMENT OF RESISTANCE OF THE TUMOUR CELL LINE RPMI-8226 BY 2-BIPHENYL-4-YL-N-[4-(5-METHYL-2,4-DIOXO-3,4-DIHYDRO-2H-PYRIMIDIN-1-YL)-BUT-2-ENYL]-ACETAMIDE

[0214] RPMI-8226 cells were seeded with a cell count of 100,000 cells/ml in each case and regularly passaged. FIG. 9a shows that, as a result of the treatment with 1 μmol/L 2-biphenyl-4-yl-N-[4-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-but-2-enyl]-acetamide, the development of resistance to the cytostatic agent bortezomib was significantly inhibited. At the end of the experiment, 380,000 living cells per ml medium could be detected. For the treatment with 30 μmol/L BVDU, a final cell count of 340,000 cells/ml was detected whereas the final cell count in the culture without the addition of an HSP27 inhibitor amounted to 510,000 cells/ml. As a control, RPMI 8226 cells were treated with the respective test substances alone, FIG. 9b. At the same dosage, the test substances alone do not affect cell growth.

EXAMPLE 17: INHIBITION OF THE DEVELOPMENT OF RESISTANCE OF THE TUMOUR CELL LINE U-937 BY ACEPROMAZINE

[0215] The cell line U-937 is a histiocytic lymphoma (DSMZ ACC 5). This cell line also responds to treatment with bortezomib, expresses HSP27 and develops resistance to bortezomib. Bortezomib is not the standard therapy for this lymphoma, but is being discussed as a possible therapeutic agent.

[0216] U-937 cells were seeded with a cell count of 100,000 cells/ml in each case and regularly passaged. FIG. 10a shows that, as a result of the treatment with 0.75 μmol/L acepromazine, the development of resistance to the cytostatic agent bortezomib was significantly inhibited. At the end of the experiment, 70,000 living cells per ml medium could be detected. For the treatment with 30 μmol/L BVDU, a final cell count of 250,000 cells/ml was detected whereas the final cell count in the culture without the addition of an HSP27 inhibitor amounted to 450,000 cells/ml. As a control, U-937 cells were treated with the respective test substances alone, FIG. 10b. At the same dosage, the test substances alone do not affect cell growth.

EXAMPLE 18: INHIBITION OF THE DEVELOPMENT OF RESISTANCE OF THE TUMOUR CELL LINE U-937 BY 2-(4-BUTYLPHENYLAMINO)-9-(2-HYDROXYETHOXYMETHYL)-1,9-DIHYDROPURIN-6-ONE

[0217] U-937 cells were seeded with a cell count of 100,000 cells/ml in each case and regularly passaged. FIG. 11a shows that, as a result of the treatment with 0.75 μmol/L 2-(4-butyl-phenylamino)-9-(2-hydroxyethoxymethyl)-1,9-dihydropurin-6-one, the development of resistance to the cytostatic agent bortezomib was significantly inhibited. At the end of the experiment, 30,000 living cells per ml medium could be detected. For the treatment with 30 μmol/L BVDU, a final cell count of 250,000 cells/ml was detected whereas the final cell count in the culture without the addition of an HSP27 inhibitor amounted to 450,000 cells/ml. As a control, U-937 cells were treated with the respective test substances alone, FIG. 11b. At the same dosage, the test substances alone do not affect cell growth.

REFERENCES

[0218] Straume O, Shimamura T, Lampa M J, Carretero J, Øyan A M, Jia D, Borgman C L, Soucheray M, Downing S R, Short S M, Kang S Y, Wang S, Chen L, Collett K, Bachmann I, Wong K K, Shapiro G I, Kalland K H, Folkman J, Watnick R S, Akslen L A, Naumov G N. (2012) Suppression of heat shock protein 27 induces long-term dormancy in human breast cancer. Proc Natl Acad Sci USA. 2012 May 29; 109(22):8699-704. [0219] Jakob U, Gaestel M, Engel K, and Buchner J. (1993) Small Heat Shock Proteins Are Molecular Chaperones. J Biol Chem 268(3): 1517-1520. [0220] Muckenschnabel I, Falchetto R, Mayr L M, Filipuzzi I. (2004) SpeedScreen: label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands. Anal Biochem. 2004 Jan. 15; 324(2):241-9. [0221] Chauhan D, Li G, Auclair D, Hideshima T, Podar K, Mitsiades N, Mitsiades C, Chen L B, Munshi N, Saxena S, Anderson K C. (2004) 2-Methoxyestardiol and bortezomib/proteasome-inhibitor overcome dexamethasone-resistance in multiple myeloma cells by modulating Heat Shock Protein-27. Apoptosis. 2004 March; 9(2): 149-55.