SPECIFIC SUBSTRATE OF AN ALDH ISOENZYME

Abstract

The invention relates to a specific substrate on an ALDH isoenzyme, to a composition comprising at least one such substrate, to a diagnostic marker comprising such a substrate, and to the uses thereof and associated methods.

Claims

1. A specific substrate of an ALDH isoenzyme comprising a compound: (a) of formula (I): RCOOA (I) resulting from the esterification of a fluorescent tracer AOH with an acylating agent derived from the corresponding acid RCOOH, in which R is chosen in order to form a compound selected from the group consisting of retinoate, propionate, octanoate, benzoate, 4-aminobutyrate, hexanoate, 4-diethylaminobenzoate and 4-hydroxy-2-nonenoate; or (b) of formula (II): ##STR00040## in which: R and R, which are identical or different, are chosen in order to form a compound selected from the group consisting of retinoate, propionate, octanoate, benzoate, 4-aminobutyrate, hexanoate, 4-diethylaminobenzoate and 4-hydroxy-2-nonenoate.

2. The specific substrate according to claim 1, wherein AOH is chosen from the group consisting of 7-hydroxycoumarin, a fluorophore of the tokyo green family, resorufin and fluorescein.

3. The specific substrate according to claim 1, wherein the ALDH isoenzyme is ALDH1 and wherein R and R, which are identical or different, are chosen in order to form a compound selected from the group consisting of retinoate, hexanoate and propianoate.

4. The specific substrate according to claim 1, wherein the ALDH isoenzyme is ALDH3 and wherein R and R, which are identical or different, are chosen in order to form a compound selected from the group consisting of octanoate, the 4-hydroxy-2-nonenoate, the 4-diethylaminobenzoate and benzoate.

5. The specific substrate according to claim 1, wherein the ADLH isoenzyme is detected in a cell population.

6. A composition comprising at least one specific substrate according to claim 1.

7. A diagnostic marker comprising a specific substrate according to claim 1.

8. A method for quantifying at least one ALDH isoenzyme in a cell population, comprising the use of at least one specific substrate according to claim 1.

9. (canceled)

10. A method for diagnosing a disease involving deregulation of an ALDH isoenzyme comprising the use of a marker according to claim 7.

11. The method according to claim 10, wherein the disease is selected from the group consisting of cancers, sperm motility disorders, ischemia, head trauma and pancreatitis.

12. A method for determining whether a subject is capable of responding to a therapy that inhibits the activity of an ALDH isoenzyme and/or is directed against cancer stem cells comprising the use of a marker according to claim 7.

13. A method for distinguishing healthy stem cells from cancer stem cells comprising the use of at least one specific substrate according to claim 1.

14. The method according to claim 13 for distinguishing stem cells from solid cancers and/or hematological malignant tumors.

15. A method for characterizing the various stages of a cancer or the different stages of stem cell differentiation comprising the use of at least one specific substrate according to claim 1.

16. A method for distinguishing cells expressing at least one ALDH isoenzyme in a cell population, wherein the method comprises: (a) contacting the cell population with at least one specific substrate according to claim 1, (b) measuring the fluorescence of the cell population; and (c) identifying cells with increased fluorescence relative to the fluorescence of the cell population before this population is brought into contact with the at least one specific substrate.

17. A kit for quantifying an ALDH isoenzyme comprising at least one specific substrate according to claim 1.

18. The specific substrate according to claim 2, wherein the fluorophore of the tokyo green family is 2-methyl-4-methoxy-Tokyo Green.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0121] The present invention will be further illustrated by the following figures and examples.

[0122] FIG. 1: Michaelis-Menten plot to determine the K.sub.m and V.sub.max of resorufine propionate on ALDH1A1, ALDH2 and ALDH3A1.

[0123] FIG. 2: Test of ALDH1 activity by resorufine propionate after treatment with ALDH1A1 interfering the RNA.

[0124] FIG. 3: Test of ALDH1 activity by resorufin propionate after treatment with inhibitors of ALDH 1 and 3.

[0125] FIG. 4: ALDH1 activity detected by flow cytometry via resorufine propionate.

[0126] FIG. 5: Test of ALDH1 activity by resorufin retinoate and fluorescein di-retinoate and ALDH3 by resorufin benzoate and fluorescein dibenzoate after retinoic acid treatment.

[0127] FIG. 6: Test of ALDH1 activity by resorufin retinoate and fluorescein di-retinoate, and ALDH3 by resorufin benzoate and fluorescein dibenzoate after Disulfiram (DSF) treatment.

DETAILED DESCRIPTION

EXAMPLES

[0128] 1. Preparation of Specific Substrates According to the Invention

[0129] Operating Conditions

[0130] Commercial reagents and solvents (Fisher, Sigma, Fluorochem, etc.) were used without purification except for dichloromethane distilled under an inert atmosphere on CaH.sub.2. The reactions were monitored by thin layer chromatography on aluminum sheets coated with Macherey-Nagel silica gel ALUGRAM SIL G/UV.sub.254 (thickness 0.2 mm), the observation of the plates was carried out under a 254 and 312 nm ultraviolet lamp.

[0131] The column chromatographies were carried out on Macherey-Nagel silica gel 60M (40-63 ?m) under air pressure.

[0132] Melting points were measured with a Tottoli B?chi SMP-20 instrument and were not corrected.

[0133] .sup.1H NMR spectra were recorded with Brucker ALS300 or DRX300 300 MHz devices. The .sup.13C NMR spectra were obtained on Brucker DRX300 75 MHz devices. The chemical shifts ? are expressed in parts per million (ppm), the residual peak of the solvent having been taken as the internal reference. The coupling constants J are expressed in Hz.

[0134] Mass spectra were recorded in positive mode on a hybrid time-of-flight mass spectrometer (MicroTOFQ-II, Bruker Daltonics, Bremen) with an electrospray source (ESI).

[0135] The spray gas flow was at 0.4 bar and the capillary voltage at 3500v. The solutions were perfused at 10 ?l/min in a solvent mixture (methanol/dichloromethane/water 45/40/15) with 1% formic acid. The mass range of the assay was 50-1000m-z and calibration was performed with sodium formate.

[0136] Structural Examples of Specific Substrates According to the Invention

TABLE-US-00003 TABLE 3 [00015] Resorufine propionate [00016] Resorufin hexanoate [00017] Resorufin octanoate [00018] Resorufine benzoate [00019] Resorufin 4- diethylaminobenzoate [00020] Resorufine retinoate [00021] 2-Methyl-4-methoxy-Tokyo Green hexanoate [00022] 2-Methyl-4-methoxy-Tokyo Green octanoate [00023] 4-hydroxycoumarin octanoate [00024] Fluorescein di-hexanoate [00025] Fluorescein dioctanoate [00026] Fluorescein di-retinoate

[0137] Resorufin Esters

[0138] Resorufin Propionate and Benzoate

[0139] The compounds were prepared from resorufin (sodium salt) (Sigma) with the corresponding acid chloride (2 equivalents) in dichloromethane (0.05M) in the presence of DIPEA (diisopropylethylamine, 2 equivalents).

[0140] For proprionate, a suspension of resorufin sodium salt (235.2 mg, 1.0 mmol) in 20 mL of anhydrous DCM was added DIPEA (2 equivalents) at room temperature and then propionyl chloride (2 equivalents) dropwise at 0? C. After 5 minutes at 0? C., the reaction medium was brought to room temperature and stirred overnight. 30 mL of water was then added and extraction with 3?30 mL of DCM was performed. The combined organic phases were washed with 30 ml of a saturated solution of NaHCO.sub.3 and then 30 ml of a saturated solution of NaCl. After drying over Na.sub.2SO.sub.4, filtration and evaporation of the solvent, the residue was taken up in EtOH. Sonication was performed and the solid obtained was sintered and washed with EtOH (twice). After drying under vacuum 218 mg (81%) of an orange solid were obtained.

Mp 176-177? C. (EtOH) (Guilbault et al Analytical Chem 1965, 37, 120-123:177? C.); .sup.1HRMN (300 MHz, DMSO) ? 7.89 (d, J=8.7 Hz, 1H), 7.57 (d, J=9.8 Hz, 1H), 7.40 (d, J=2.3 Hz, 1H), 7.25 (dd, J=8.7, 2.4 Hz, 1H), 6.84 (dd, J=9.8, 2.0 Hz, 1H), 6.31 (d, J=2.0 Hz, 1H), 2.66 (q, J=7.4 Hz, 2H), 1.15 (t, J=7.5 Hz, 3H); ESI-MS m/z 270.1 [M+H]+.

[0141] Weight: 269.2 g.mol.sup.?1.

[0142] Formula: C.sub.15H.sub.11NO.sub.4

##STR00027##

[0143] For benzoate, the procedure is identical to that of propionate. Benzoyl chloride was used (scale: 1.0 mmol). The isolated solid (166 mg) was purified by silica gel chromatography (MeOH/DCM: 1/99) to give 156 mg (49%) of pure benzoate.

[0144] Mp>210? C. (Guilbault et al Analytical Chem 1965, 37, 120-123: 203? C.); .sup.1HRMN (300 MHz, DMSO) ? 8.21-8.14 (m, 2H), 7.95 (d, J=8.7 Hz, 1H), 7.84-7.75 (m, 1H), 7.62 (ddd, J=11.7, 10.5, 8.1 Hz, 4H), 7.44 (dd, J=8.7, 2.4 Hz, 1H), 6.86 (dd, J=9.8, 2.1 Hz, 1H), 6.33 (d, J=2.1 Hz, 1H); ESI-MS m/z 318.1 [M+H]+.

[0145] Weight: 317.3 g.mol.sup.?1

[0146] Formula: C.sub.19H.sub.11NO.sub.4

##STR00028##

[0147] Resorufin Hexanoate and Octanoate

[0148] The compounds were prepared and isolated in the same manner as for the two preceding esters using the corresponding acid chlorides. The respective yields obtained are 65% and 81%.

[0149] For hexanoate, the procedure is identical to that of propionate. Hexanoyl chloride (scale: 1.0 mmol) was used. The compound obtained was isolated by precipitation in EtOH, followed by 2 washes with EtOH, yield 65%.

[0150] .sup.Mp 130-132? C.; .sup.1HRMN (300 MHz, DMSO) ? 7.89 (d, J=8.7 Hz, 1H), 7.57 (d, J=9.8 Hz, 1H), 7.39 (d, J=2.3 Hz, 1H), 7.24 (dd, J=8.7, 2.4 Hz, 1H), 6.84 (dd, J=9.8, 2.0 Hz, 1H), 6.30 (d, J=2.0 Hz, 1H), 2.63 (t, J=7.4 Hz, 2H), 1.75-1.57 (m, 2H), 1.43-1.25 (m, 4H), 0.90 (t, J=7.0 Hz, 3H); ESI-MS m/z312.1 [M+H]+.

[0151] Weight: 311.3 g.mol.sup.?1

[0152] Formula: C.sub.18H.sub.17NO.sub.4

##STR00029##

[0153] For octanoate, the procedure is identical to that of propionate. Octanoyl chloride (scale: 1.0 mmol) was used. The compound obtained was isolated by precipitation in EtOH, followed by 2 washes with EtOH, yield 81%.

[0154] Mp 127-129? C.; .sup.1HRMN (300 MHz, DMSO) ? 7.89 (d, J=8.7 Hz, 1H), 7.57 (d, J=9.8 Hz, 1H), 7.39 (d, J=2.4 Hz, 1H), 7.23 (dd, J=8.7, 2.4 Hz, 1H), 6.84 (dd, J=9.8, 2.0 Hz, 1H), 6.30 (d, J=2.1 Hz, 1H), 2.63 (t, J=7.4 Hz, 2H), 1.72-1.57 (m, 2H), 1.44-1.19 (m, 8H), 0.87 (t, J=6.8 Hz, 3H); ESI-MS m/z 340.2 [M+H]+.

[0155] Weight: 339.4 g.mol.sup.?1

[0156] Formula: C.sub.20H.sub.21NO.sub.4

##STR00030##

[0157] Resorufin 4-diethylamino benzoate

[0158] The sodium salt of resorufin (235.2 mg, 1.0 mmol), 4-diethylaminobenzoic acid (1.1 equivalent), EDCI (1.1 equivalent) and 4-DMAP (0.1 equivalent) were placed under argon. 25 ml of DCM were added and the reaction medium was stirred overnight at room temperature. The solvent was evaporated and the residue purified by chromatography on silica gel (MeOH/DCM=1/99 to 2.5/87.5) to give 207 mg (53%) of product with a very slight impurity (visible UV) that the second column (MeOH/DCM=1/99) allows to eliminate. .sup.1HRMN (300 MHz, DMSO) ? 7.92 (d, J=9.0 Hz, 3H), 7.59 (d, J=9.8 Hz, 1H), 7.49 (s, 1H), 7.35 (d, J=8.5 Hz, 1H), 6.85 (d, J=9.6 Hz, 1H), 6.79 (d, J=9.2 Hz, 2H), 6.32 (d, J=2.0 Hz, 1H), 3.46 (q, J=7.0 Hz, 4H), 1.14 (t, J=7.0 Hz, 6H); ESI-MS m/z 389.1 [M+H]+.

[0159] Weight: 388.4 g.mol.sup.?1

[0160] Formula: C.sub.23H.sub.20N.sub.2O.sub.4

##STR00031##

[0161] (All) Trans Retinoate of Resorufin

[0162] The compound was prepared in the same manner as the preceding ester using commercial retinoic acid (40% yield). The crude (orange-red solid) obtained was washed with MeOH and purified by chromatography on silica gel (MeOH/DCM: 1/99). Further washing with EtOH then MeOH gave 100 mg (40%) of pure product. Mp 150-160 (decomposition); .sup.1HRMN(300 MHz, CDCl.sub.3) ? 7.82 (d, J=8.6 Hz, 1H), 7.46 (d, J=9.8 Hz, 1H), 7.25-7.10 (m, 3H), 6.89 (dd, J=9.8, 2.0 Hz, 1H), 6.46-6.31 (m, 3H), 6.26-6.14 (m, 2H), 6.00 (s, 1H), 2.45 (d, J=0.9 Hz, 3H), 2.10-2.00 (m, 5H), 1.75 (d, J=0.6 Hz, 3H), 1.70-1.58 (m, 2H), 1.54-1.46 (m, 2H), 1.06 (s, 6H); ESI-MS m/z 496.2 [M+H+].

[0163] Weight: 495.6 g.mol.sup.?1

[0164] Formula: C.sub.32H.sub.33NO.sub.4

##STR00032##

[0165] Tokyo-Green Esters

[0166] 2-Me-4-MeO tokyo-green (CAS No. 643755-84-4: 6-hydroxy-9- (4-methoxy-2-methylphenyl) -3H-Xanthen-3-one) was synthesized in two steps: a bis-silylation of the commercial 3.6-dihydroxyxanth-9-one carried out according to the procedure described in the article J. Biol. Chem Vol. 264, No. 14. Issue of May 15, PP. 8171-8178, 1989 led to 3.6-bis (t-butyldimethylsilyloxy) xanthone. A second step carried out according to the procedure described in the article Chem. Eur. J. 2014, 20, 447-455 consisting of treatment with magnesium from 2-bromo-3-methoxytoluene followed by acid hydrolysis gave 2Me4MeO tokyo green.

##STR00033##

[0167] Hexanoate and Octanoate of 2Me4MeO Tokyo-Green

[0168] The compounds were prepared conventionally using the corresponding acid chlorides.

[0169] For hexanoate, from 0.15 mmol of 2Me4MeOTG, purification by chromatography on silica gel (MeOH/DCM: 1/99 to 10/90) was carried out, yield: 40% (non-crystallized resin).

[0170] .sup.1HRMN (300 MHz, MeOD) et 7.45 (d, J=2.0 Hz, 1H), 7.24 (d, J=8.8 Hz, 1H), 7.16 (d, J=9.6 Hz, 2H), 7.11 (dd, J=8.8, 2.1 Hz, 1H), 7.04 (d, J=2.1 Hz, 1H), 6.99 (dd, J=8.4, 2.3 Hz, 1H), 6.61 (dd, J=9.7, 1.9 Hz, 1H), 6.44 (d, J=1.9 Hz, 1H), 3.89 (s, 3H), 2.63 (t, J=7.4 Hz, 2H), 2.04 (s, 3H), 1.81-1.67 (m, 2H), 1.47-1.33 (m, 4H), 1.00-0.89 (m, 3H).

[0171] .sup.13CRMN(101 MHz, CDCl3) et 186.23, 171.64, 160.63, 158.85, 154.54, 153.29, 148.87, 138.07, 131.00, 130.88, 130.58, 129.38, 124.29, 120.84, 118.85, 118.56, 116.24, 111.80, 110.38, 106.18, 77.16, 55.51, 34.47, 31.32, 24.60, 22.42, 20.19, 14.05;

[0172] ESI-MS: [M+H]+: 431.1

[0173] Weight: 430.5 g.mol.sup.?1

[0174] Formula: C.sub.27H.sub.26O.sub.5

##STR00034##

[0175] For the octanoate, from 0.19 mmol of TG, purification by chromatography on silica gel (MeOH/DCM: 1/99 to 10/90), was carried out, yield: 20% (non-crystallized resin)

[0176] .sup.1H RMN (300 MHz, MeOD) et 7.46 (s, 1H), 7.25 (d, J=8.8 Hz, 1H), 7.17 (d, 1=8.9 Hz, 2H), 7.12 (dd, J=8.8, 1.6 Hz, 1H), 7.05 (d, J=2.1 Hz, 1H), 7.00 (cid, 1=8.4, 2.2 Hz, 1H), 6.62 (dd, J=9.7, 1.5 Hz, 1H), 6.45 (s, 1H), 3.90 (s, 3H), 2.64 (t, J=7.4 Hz, 2H), 2.05 (s, 3H), 1.81-1.67 (m, 2H), 1.49-1.26 (m, 9H), 0.96-0.87 (m.3H).

[0177] .sup.13CRMN(101 MHz, CDCl3) et 186.20, 171.66, 160.61, 158.78, 154.49, 130.92, 130.59, 129.34, 124.33, 120.89, 118.86, 106.23, 77.16, 55.51, 34.51, 31.76, 29.14, 29.02, 24.92, 22.73, 20.19, 14.21.

[0178] ESI-MS: [M+H]+: 459.2

[0179] Weight: 485 g.mol.sup.?1

[0180] Formula: C.sub.29H.sub.30O.sub.5

##STR00035##

[0181] Fluorescein Diesters

[0182] The identical preparation is as described above using 3.0 eq. corresponding acid chlorides and 3 eq. basic. Hexanoate and octanoate have already been described [litt. Ge, Feng-Yan; Dyes and Pigments 2007, 72 (3), 322-326].

[0183] Fluorescein Di-Hexanoate:

[0184] [7364-90-1] Scale 0.8 mmol, purification by chromatography on silica gel (PE/EtOAc: 90/10). White solid, yield: 98%. Mp 103-105? C. (pentane washings) (100? C., Ge, Feng-Yan, Dyes and Pigments 2007, 72 (3), 322-326);

[0185] .sup.1H NMR (300 MHz, DMSO) ? 8.09-8.03 (m, 1H), 7.83 (td, J=7.4, 1.4 Hz, 1H), 7.77 (td, J=7.4, 1.2 Hz, 1H), 7.44-7.38 (m, 1H), 7.28 (d, J=2.0 Hz, 2H), 6.94 (dd, J=8.7, 2.2 Hz, 2H), 6.88 (d, J=8.6 Hz, 2H), 2.60 (t, J=7.4 Hz, 4H), 1.71-1.58 (m, 4H), 1.40-1.25 (m, 8H), 0.95-0.81 (m, 6H);

[0186] ESI-MS: [M+H]+: 529.2

[0187] Weight: 528.9 g.mol.sup.?1

[0188] Formula: C.sub.32H.sub.32O.sub.7

##STR00036##

[0189] Fluorescein Dioctanoate:

[0190] [19722-86-2] Scale 0.8 mmol, purification by chromatography on silica gel (PE/EtOAc: 90/10 to 80/20). White solid, 92%. Mp 50-52?. (lit 49? C., Ge, Feng-Yan, Dyes and Pigments 2007, 72 (3), 322-326).

[0191] .sup.1H NMR (300 MHz, DMSO) ? 8.09-8.04 (m, 1H), 7.83 (td, J=7.4, 1.3 Hz, 1H), 7.77 (td, J=7.3, 1.0 Hz, 1H), 7.41 (d, J=7.3 Hz, 1H), 7.27 (d, J=2.1 Hz, 2H), 6.94 (dd, J=8.7, 2.2 Hz, 2H), 6.88 (d, J=8.6 Hz, 2H), 2.59 (t, J=7.4 Hz, 4H), 1.70-1.57 (m, 4H), 1.41-1.20 (m, 16H), 0.92-0.81 (m, 6H);

[0192] ESI-MS: [M+H]+: 585.3.

[0193] Weight: 584.7 g.mol.sup.?1

[0194] Formula: C.sub.36H.sub.40O.sub.7

##STR00037##

[0195] Fluorescein Di-Retinoate:

[0196] The compound was prepared from 2.1 equivalents of retinoic acid, 2.1 equivalents of EDCI, 0.1 equivalents of 4-DMAP. Scale: 0.5 mmol, purification by chromatography on silica gel (PE/EtOAc: 80/20). Yellow solid, 45% yield. M.p. 145-150? C. (dec);

[0197] .sup.1H NMR (300 MHz, DMSO) ? 8.07 (d, J=7.3 Hz, 1H), 7.89-7.73 (m, 2H), 7.42 (d, J=7.3 Hz, 1H), 7.31 (d, J=2.3 Hz, 1H), 7.20 (dd, J=15.0, 11.5 Hz, 2H), 6.98 (dd, J=8.7, 2.2 Hz, 2H), 6.88 (d, J=8.7 Hz, 2H), 6.54 (d, J=15.0 Hz, 2H), 6.39-6.15 (m, 6H), 6.11 (s, 2H), 2.38 (s, 6H), 2.07-1.95 (m, 10H), 1.70 (s, 6H), 1.63-1.51 (m, 4H), 1.50-1.40 (m, 4H), 1.03 (s, 12H).

[0198] ESI-MS: [M+H]+: 897.3

[0199] Weight: 879.1 g.mol.sup.?1

[0200] Formula: C.sub.60H.sub.64O.sub.7

##STR00038##

[0201] Esters of 7-hydroxycoumarin

[0202] These esters were prepared in the same manner as previously from 7-hydroxycoumarin and acid chloride in the presence of DIPEA.

[0203] 7-hydroxycoumarin octanoate:

[0204] Scale 2 mmol, purification by chromatography on silica gel (PE/EtOAc: 70/30) yield: 92%. Mp 58-59? C.;

[0205] .sup.1H NMR (300 MHz, DMSO) ? 8.08 (d, J=9.3 Hz, 1H), 7.77 (d, J=8.5 Hz, 1H), 7.26 (d, J=2.2 Hz, 1H), 7.15 (dd, J=8.4, 2.2 Hz, 1H), 6.48 (d, J=9.6 Hz, 1H), 2.61 (t, J=7.4 Hz, 2H), 1.65 (s, 2H), 1.42-1.21 (m, 8H), 0.93-0.81 (m, 3H);

[0206] ESI-MS: [M+H]+: 289.1

[0207] Weight: 288.3 g.mol.sup.?1

[0208] Formula: C.sub.17H.sub.20O.sub.4

##STR00039##

[0209] 2. Activity of Specific Substrates According to the Invention

[0210] Material and Methods

[0211] Cell Lines

[0212] NCI-H522 and A549 lung cancer cells as well as leukemia lines were used. The cells were obtained from American Type Culture Collection (ATCC), the European Collection of Cell Cultures (ECACC) and Deutsche Sammlung von Mikroorganismen and Zellkultruren (DSMZ).

[0213] Determination of the Km and Vmax of Resorufine Propionate with the Different Isoenzymes ALDH1A1, ALDH2 and ALDH3A1 Purified.

[0214] In 50 ?l of 0.1 M phosphate buffer pH6.00; 0.2 M KCl; 2 mM NADP+; 2 mM EDTA, a range of resorufine propionate was made: 250, 200, 150, 125, 100, 90, 80 and 70 ?M and 0 ?M. 50 ?L of recombinant enzyme solution at 2.5 mU/well ALDH1A1 (R&D Sytems, 5869-DH), ALDH2 (abcam, ab87415) and ALDH3A1 (R&D Sytems, 6705-DH) was added. The incubation was carried out for 30 minutes at +37? C. and then the fluorescence reading was made (Em: 590 nm, Ex: 530 nm). The data were then converted to resorufin released (nM.min-1.?g.sup.?1) and Km and Vmax were calculated using the Michaelis-Menten equation using GraphPad Prism 5.0.

[0215] Treatment of Cells with Specific Inhibitors of ADLH1 and 3

[0216] In a 96-well plate, the HL-60 cells were inoculated at a concentration of 50.000 cells/well in RPMI-1640 medium without phenol red supplemented with L-Glutamine, Penicillin, Streptomycin and 10% Fetal Calf Serum (FCS) supplemented with dimethyl ampalthiolester (DIMATE) a specific inhibitor ALDH 1 and 3, morpholino ampal thiolester (MATE) a specific inhibitor of ALDH3, at concentrations of 8 ?M respectively. After incubation for 6 hours, the substrates of the different ALDHs respectively resorufine propionate for ALDH1 and resorufin 4-diethylaminobenzoate for ALDH3, were added to a final concentration of 10 ?M and then incubated for one hour at +37? C. The plate was then read using an Appliskan fluorescent plate reader (ex=560 nm, Em=600). The data were expressed in relative fluorescence units produced by an equal number of cells.

[0217] Treatment of Cells with Disulfiram (DSF)

[0218] In a 96-well plate, the HL-60 cells were inoculated at a concentration of 50.000 cells/well in RPMI-1640 medium without phenol red supplemented with L-Glutamine, Penicillin, Streptomycin and 10% Fetal Calf Serum (FCS) supplemented with disulfiram (DSF), an inhibitor of ALDH activity, at concentrations of 250 nM and 1000 nM. The cells were then incubated for 1 hour. After incubation, substrates of the different ALDHs, resorufin retinoate and fluorescein di-retinoate for ALDH1, and fluorescein benzoate and fluorescein di-benzoate for ALDH 3, were added to a final concentration of 5 ?M then incubated for 30 minutes at +37? C. The plate was then read using a SpectraMax? fluorescent plate reader, Molecular Devices (Ex=560 nm, Em=600 nm for resorufin and ex=485 nm, em=535 nm for fluorescein). The data were expressed in relative fluorescence units.

[0219] Treatment of Cells with Retinoic Acid

[0220] In a 96-well plate, the HL-60 cells were inoculated at a concentration of 50.000 cells/well in RPMI-1640 medium without phenol red supplemented with L-Glutamine, Penicillin, Streptomycin and 10% Fetal Calf Serum (FCS) supplemented with retinoic acid, known to be an inhibitor of ALDH activity at 1 ?M and 10 ?M concentrations. The cells were then incubated for 72 hours. After incubation, the substrates of the different ALDHs, resorufine retinoate and fluorescein di-retetinoate for ALDH1, and fluorescein benzene and fluorescein di-benzote for ALDH3, were added to a final concentration of 5 ?M then incubated for 30 minutes at +37? C. The plate was then read using a SpectraMax? fluorescent plate reader, Molecular Devices (Ex=560 nm, Em=600 nm for resorufin and Ex=485 nm, Em=535 nm for fluorescein). The data were expressed in relative fluorescence units.

[0221] Treatment of Cells with Interfering RNA ALDH1A1

[0222] In a 60 mm Petri dish, the NCI-H522 cells were seeded at a concentration of 250.000 cells/plate, corresponding to a confluence of 40-50%, in RPMI-1640 medium supplemented with L-Glutamine, Penicillin, Streptomycin and 10% Fetal Calf Serum (FCS) overnight at 37? C. The next day, the medium was replaced by the same medium without FCS. The transfection solution was then prepared by diluting 100 nM in 500 ?L of culture medium without FCS, to which 500 ?L of culture medium without FCS supplemented with 3 ?L of lipofectamine 2000 (Invitrogen) was added. The solution was then incubated for 30 minutes at room temperature and then drops added to the cell solution. The mixture was incubated at +37? C. in a 5% CO.sub.2 incubator for 8 hours. After the incubation, the medium was changed by medium supplemented with FCS and the cells were left in culture for 48 hours. The inhibition of the protein was validated by Western Blot. The specific activity of ALDH1A1 was then assayed by fluorescence.

[0223] Determination of ALDH1 Activity by Resorufine Propionate

[0224] The cells were inoculated at a concentration of 1?10.sup.4 cells/well in a 96-well plate in 100 ?l in RPMI-1640 medium without phenol red supplemented with L-Glutamine, Penicillin, Streptomycin and 10% Fetal Calf Serum (FCS) supplemented with 10 ?M resorufin propionate. The cells were then incubated for 1 hour and then the plate was read using an Appliskan fluorescent plate reader (ex=560 nm, Em=600). The data were expressed in relative fluorescence units produced by an equal number of cells.

[0225] Double Localization of Resorufin Propionate and ALDH1A1 by Fluorescent Microscopy

[0226] In a 24-well plate with microscope glasses previously washed with ethanol and then placed in the wells, the NCI-H522 cells were added at a cell concentration of 50.000 cells/well and then incubated in RPMI-1640 medium supplemented with L-Glutamine, Penicillin, Streptomycin and 10% Fetal Calf Serum overnight at +37? C. with a 5% CO.sub.2 atmosphere. The next day, the culture medium was changed with either DIMATE (5 ?M) or the treatment vehicle and incubated for 6 hours. The cells were incubated for 30 minutes with culture medium supplemented with 10 ?M of resorufin propionate, were then washed with cold PBS and then fixed with paraformaldehyde for 15 minutes at 37? C. The cells were permeabilized and saturated with PBS; 3% bovine albumin; 0.3% triton for 1 hour. The incubation was carried out with the anti-ALDH1A1 antibody (R&D System, MAB5869) for 1 hour at +37? C., then with an anti-mouse antibody coupled to fluorescein for 1 hour at room temperature in the dark, with PBS washes between the two steps. 3 washes with PBS were carried out then the microscopy glasses were mounted with an anti-decoloration support supplemented with DAPI. The cells were then observed under a microscope.

[0227] Identification of Positive ALDH1 Cells by Flow Cytometry

[0228] In a 60mm Petri dish, 500.000 cells were incubated with RPMI-1640 medium supplemented with L-Glutamine, Penicillin, Streptomycin and 10% Fetal Calf Serum overnight at +37? C. with 5% atmosphere. CO2. For the negative control, the cells were taken up in medium supplemented with 15 ?M of dimethyl ampal thiolester (Dimate), a specific inhibitor of ALDH1 and 3, then incubated for 5 hours at 37? C., 5% CO.sub.2. After trypsination, the cells were taken up in supplemented RPMI medium and centrifuged at 800 g for 5 minutes. The cell pellet was then taken up in fresh supplemented medium containing 10 ?M of resorufin propionate, and then incubated for 1 hour in a polycarbonate tube at 37? C., 5% CO.sub.2. After incubation, the cells were then centrifuged and then washed in cold PBSx1 and finally taken up in 200 mL of cold PBSx1. The solution was then analyzed by flow cytometry (Ex 590 nm/Em560 nm or red laser).

[0229] Results

[0230] The results are shown in FIGS. 1 to 5.

[0231] In particular, FIG. 1 shows the data obtained to determine the K.sub.m and V.sub.max of resorufine propionate. The results are also shown in Table 4 below.

TABLE-US-00004 TABLE 4 ALDH1 ALDH2 ALDH3 Vmax 12132 6693 8141 Km 115.5 116.0 445.1 Km/Vmax 105.04 57.69 18.29 Increase 1 0.54 0.17

[0232] The results of the assay of ALDH1 activity by resorufine propionate after treatment with RNA interfering ALDH1A1 are illustrated in FIG. 2 which shows that a complete inhibition of ALDH1A1 is observed at 100nM of siRNA which induces a decrease significant (P<0.05) signal resorufine propionate.

[0233] The results of the test of ALDH1 activity by resorufin propionate after treatment with inhibitors of ALDH 1 and 3 are illustrated in FIG. 3 and shows a significant inhibition of fluorescence (P<0.001) after treatment with DIMATE (inhibitor of ALDH 1 and 3) unlike MATE which is a specific inhibitor of ALDH3.

[0234] In addition, the conversion of resorufin propionate to resorufin, a molecule that fluoresces in the red, was detected by fluorescent microscopy. The reaction is inhibited by DIMATE, an inhibitor of ALDH 1, showing the specificity of the substrate (results not shown). In addition, the fluorescent signal of resorufine propionate can be colocalized with ALDH1A1 suggesting specific activity with the enzyme (results not shown).

[0235] FIG. 4 illustrates ALDH1 activity detected by flow cytometry via resorufin propionate. The untreated condition demonstrates the presence of live positive ALDH1 cells (Calcein-AM).

[0236] In the presence of an inhibitor of ALDH1, DIMATE, positive ALDH1 cell inhibition is observed for living cells as well as a decrease in viability, due to treatment.

[0237] Results of the test of ALDH1 activity by resorufin retinoate and fluorescein di-retinoate and ALDH3 activity by resorufin benzoate and fluorescein di-benzoate after retinoic acid treatment are illustrated by FIG. 5 which shows that a significant (P<0.001) or (P<0.01) level of activity inhibition of ALDH1 for resorufin retinoate and fluorescein di-retinoate and ALDH3 for benzoate of resorufin and fluorescein di-benzoate.

[0238] In the presence of DSF at both the 250 nM and 1000 nM concentrations, a significant (P<0.001) inhibition of ALDH1 activity for resorufin retinoate and fluorescein di-retinoate and ALDH3 for resorufin benzoate and fluorescein di-benzoate, are illustrated in FIG. 6.

[0239] 3. Application of the Use of Substrates

[0240] Materials and Methods

[0241] Patients

[0242] Bone marrow (Mo) and blood (Sg) were obtained from 33 patients with their enlightened chords. The evaluations were performed on whole blood after lysis of red blood cells or bone marrow.

[0243] Isolation of Blast Cells and Evaluation of ALDH1 and ALDH3 Activity

[0244] The isolation of the blast cells was performed by a Navios flow cytometer (Beckman Coulter?) according to the phenotype of the latter indicated in Table 5 (CD34 +, CD117+ or CD45 weak).

[0245] The ALDH1 and ALDH3 activity was evaluated by incubating the reagents: resorufin retinoate or resorufin octanoate at 5 ?mol.sup.?1 and fluorescein di-retinoate or fluorescein dioctanoate at 0.8 ?mol.sup.?1 in the blood total after lysis of red blood cells or in bone marrow extract for 30 minutes at 37? C. The fluorescence observed is analyzed by cytometry making it possible to give a value of the median fluorescence intensity (MFI) corresponding to the relative activity of each ALDH isoform of the blast cells.

[0246] Results

[0247] The results are shown in Table 4 below.

TABLE-US-00005 TABLE 4 Patient parameters included in the study of the different activities of Aldehydes Dehydrogenases. Resorufin Resorufin Fluorescein Fluorescein Type of Relapse Phenotypes Retinoate Octanoate di-retinoate octanoate Pt No Age Pathology sampling (Y/N) of blasts (IFM) (IFM) (IFM) (IFM) 1 74 LAM Sg Y CD34 pos. 59.47 11.05 147.18 65.23 2 50 LAM Sg N CD34 pos. 34.98 10.26 56.44 34.07 3 50 LAM Mo N CD34 pos. 129.14 20.1 104.36 214.5 4 44 LAM Sg Y CD34 pos. 121.23 12.72 230.7 103.44 5 91 LAM Mo Y CD45 weak 2.01 3.7 16.23 6.11 6 91 LAM Sg Y CD117 pos. 69 61.13 299.48 37.47 7 82 LAM Sg N CD45 weak 3.03 10.66 8 84 LAM Sg N CD45 weak 2.08 25 151 9.05 9 93 LAM Sg N CD45 weak 8.99 25.83 169 8.74 10 78 LAM Sg N CD45 weak 1.5 1.93 27.5 3.55 11 67 AREB Mo N CD34 pos. 40.46 20.32 72.4 9.5 12 74 LAM Sg Y CD34 pos. 548 12.45 349 64 13 74 LAM Sg Y CD34 pos. 83.29 6.5 91.03 245.88 14 80 AREB Sg Y CD34 pos. 14.8 13.71 114.2 1.07 15 80 AREB Sg Y CD34 pos. 86.29 5.94 144.98 87.99 16 71 LAM Sg Y CD34 pos. 15.18 670 773 14.3 17 71 LAM Sg Y CD34 pos. 74.79 8.82 139.26 94.44 18 39 LAM Mo N CD117 pos. 160.58 11.53 119.1 176.9 19 39 LAM Mo N CD117 pos. 59.03 8.01 54.01 25.93 20 39 LAM Sg N CD117 pos. 252.73 4.35 28.27 422.55 21 53 LAM Sg N/D CD117 pos. 66 78.82 246 35.26 22 53 LAM Mo N/D CD34 pos. 64.7 8.02 33.11 2.87 23 53 LAM Mo N CD34 pos. 26.71 568 2.41 17.67 24 53 LAM Mo N CD34 pos. 83.04 14.64 131.04 78.47 25 78 LAM Sg Y CD34 pos. 71.71 46.24 176.61 64.29 26 71 LAM Mo Y CD34 pos. 101.54 11.6 144.5 64.3 27 93 LAM Sg Y CD34 pos. 73.52 6.57 217.5 123.02 28 70 AREB Sg Y CD34 pos. 97.79 9.79 112 48.19 29 60 LAM Mo N CD34 pos. 171.6 13.23 74.26 133.5 30 70 LAM Mo Y CD34 pos. 39.26 91.95 139.19 29.68 31 70 LAM Sg Y CD34 pos. 34.26 15.22 101.1 26.3 32 72 LAM Sg N CD34 pos. 25.06 4.08 27.7 19.04 33 72 LAM Mo N CD34 pos. 70 3.58 25.66 33.82 AML, Acute Leukemia Myeloide; AREB, Refractory Anemia with Excess Blasts; Sg, blood; Mo, bone marrow; IFM, value of the Median Fluorescence Intensity.