EP3491143A1

EP3491143A1 - Specific substrate of an aldh isoenzyme

Info

Publication number: EP3491143A1
Application number: EP17749666.8A
Authority: EP
Inventors: Ismail Ceylan; Guillaume Martin; Gérard Quash; Milleidys PEREZ-ALEA; Guy Fournet
Original assignee: Advanced Biodesign; Centre National de la Recherche Scientifique CNRS; Universite Claude Bernard Lyon 1 UCBL
Current assignee: ADVANCED BIODESIGN
Priority date: 2016-07-28
Filing date: 2017-07-27
Publication date: 2019-06-05
Also published as: US11519014B2; JP2019525762A; CA3031587A1; WO2018019927A1; FR3054564A1; CN109715822A; JP7050748B2; IL264481B2; IL264481A; US20190233871A1; FR3054564B1; IL264481B1

Abstract

The invention relates to a specific substrate of 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.

Description

Specific substrate of an isoenzyme of ALDH

The present invention relates to a substrate specific for an isozyme I ^ALDH, a composition comprising at least one such substrate, a diagnostic marker comprising such a substrate, and their associated uses and methods.

Aldehydes dehydrogenases (ALDHs) are a group of enzymes that catalyze the oxidation (dehydrogenation) of aldehydes. To date, nineteen genes encoding ALDHs have been identified in the human genome. These genes participate in a wide variety of biological processes, including the detoxification of aldehydes generated exogenously and endogenously. The ALDHs are found in all subcellular regions including in the cytosol, in the mitochondria, in the endoplasmic reticulum and the core, many ^of them ending up in more than one compartment. Most ALDHs have a broad tissue distribution and display distinct substrate specificity.

Generally considered to be detoxifying enzymes, ALDHs have been shown to protect against aldehyde-induced cytotoxicity. ALDHs also have a central role in physiological functions and processes, such as embryogenesis and development.

In particular, the ALDH1 isoenzyme would play a central role in embryogenesis and development mediated by retinoic acid signaling. It would also be involved in the detoxification of methional while ^the ALDH3 be involved in the 4-hydroxynonal, these two compounds being apoptogenic endogenous aldehydes. ALDH1 and ALDH3 are also thought to be related to cellular defense mechanisms against UV radiation inducing damage in ocular tissue. ^The ALDH2 isozyme is itself a mitochondrial isozyme primarily related to the detoxifying acetaldehyde in the second step in the metabolism of alcohol.

More than 20 years ago, ALDHs were studied for their potential use as a universal marker of normal and cancer stem cells, since some of the ALH isoenzymes would have been identified as key elements of these cells. For example, ALDH 1 has been shown to be elevated in hematopoietic stem cells and could be used to isolate them.

A common method used to identify and isolate stem cells through their high ALDH activity is the use of the ALDEFLUOR ™ assay (Stemcell Technologies Inc.). This ALDEFLUOR ™ test uses a fluorescent substrate that can be metabolized by many isoenzymes of ALDH.

In this test, the substrate of ALDH: BODIPY-aminacétaldéhyde (BAAA) is converted to BODIPY aminoacetate in the presence ^of ALDH, and the latter ^accumulates in the cells and enhances their fluorescence by issuing a green color.

However, ^the ALDEFLUOR ™ does not differentiate the various isoenzymes ^of ALDH.

Despite active research, the role of the various isoenzymes ^of ALDH in areas such as stem cells or cancer remain enigmatic. Understanding the metabolism of various isoenzymes in the control of cell phenotype and during ^development, tissue homeostasis and repair, as well as in carcinogenesis may yet open up significant prospects in tissue biology.

There is therefore a need to identify new substrates that are specific for different isozymes ^of ALDH.

^It was in this context that the inventors of the present invention have discovered a tool for ^identifying the various isozymes of ALDH, by developing new specific substrates thereof.

The subject of the present invention is therefore a substrate specific ^for an isoenzyme of ALDH comprising a compound:

(a) of formula (I): R-COO-A (I) resulting from the esterification of a fluorescent tracer A-OH with an acylating agent derived from ^the corresponding acid RCOOH, wherein R is selected so as to form the retinoate, propionate, octanoate, benzoate, 4-aminobutyrate, ^the hexanoate, 4-diethylaminobenzoate or 4-hydroxy-2-nonenoate; or

(b) of formula (II):

form retinoate, propionate, octanoate, benzoate, 4- aminobutyrate, hexanoate, 4-diethylaminobenzoate or 4-hydroxy-2-nonenoate.

A substrate specific ^for an isoenzyme of ALDH is thus described comprising a compound:

(a) of formula (I): R-COO-A (I); or

(b) of formula (II):

(II)

in which :

R and R ^', identical or different, are chosen so as to form the retinoate, propionate, ^octanoate, benzoate, 4-aminobutyrate, hexanoate, 4-diethylaminobenzoate or 4-hydroxy-2-nonenoate; and A-OH is a fluorescent tracer.

A is thus the esterified form of A-OH which is a fluorescent tracer, when the latter is free.

The present invention also concerns the use ^of a specific substrate in an invention to quantify isozyme ^of ALDH in a cell population.

The present invention also relates to ^the use of a specific substrate of ^the invention to distinguish healthy stem cells from cancer stem cells.

The present invention also relates to ^the use ^of a specific substrate according to ^the invention to characterize the different stages of cancer or the various stages of differentiation of stem cells.

The present invention also relates to a composition comprising at least one specific substrate according to ^the invention.

The present invention also relates to a diagnostic marker comprising a specific substrate according to the invention.

The present invention also relates to the use ^of a marker according to ^the invention for ^the diagnosis of a disease involving dysregulation of a isozyme ^of ALDH. In particular, the marker is used to determine if a subject is likely to respond to a therapy that inhibits the activity of an isoenzyme of ALDH and / or directed against cancer stem cells.

The present invention also relates to a method for distinguishing cells expressing at least one isoenzyme ^of ALDH in a cell population, said method comprising:

(a) contacting the cell population with at least one specific substrate according to ^the invention,

(b) measuring the fluorescence of the cell population; and

(c) identifying cells with increased fluorescence relative to the fluorescence of the cell population before said population is contacted with at least one specific substrate according to the invention.

The present invention also relates to a kit for quantifying an isozyme ^of ALDH comprising at least one specific substrate according to ^the invention.

As noted above, ^the invention relates to a specific substrate ^of an isozyme ^of ALDH comprising a compound:

(a) of formula (I): R-COO-A (I) resulting from ^the ^{esterification} of a fluorescent tracer A-OH with an acylating agent derived from ^the corresponding acid RCOOH, wherein R is selected so as to form the retinoate, propionate, ^octanoate, benzoate, 4-aminobutyrate, ^the hexanoate, 4-diethylaminobenzoate or 4-hydroxy-2-nonenoate; or

(b) of formula (II):

wherein R and R ^', identical or different, are chosen so as to form the retinoate, propionate, ^octanoate, benzoate, 4-aminobutyrate, hexanoate, 4-diethylaminobenzoate or 4-hydroxy-2- nonenoate. The compound of formula (II) may also be described as having the formula: R-COO-A-OOC-R ', wherein the fluorescent tracer is of formula HO-A-OH and is fluorescein. By "specific substrate ^of an isoenzyme of ALDH" means a chemical molecule that will specifically interact with a particular isoenzyme of ALDH as ALDH1 I or I ALDH3 for example, to produce a chemical reaction which, in the scope of the present invention, will free a fluorescent molecule (a-OH) allowing to ^identify with certainty said particular isozyme ^of ALDH. Thus, in the context of the ^invention, the specific substrate is cleaved by an isozyme ^of ALDH that allow the release of the fluorescent tracer A-OH.

The specific substrate according to the invention results from the esterification of the fluorescent tracer A-OH with an acylating agent derived from the corresponding acid RCOOH or R'COOH.

For example, in the case where R and / or R ^{^'whose} acid RCOOH and / or ^R' is COOH propionic acid, the corresponding ester is propionate, in the case where R and / or R ^'whose RCOOH acid and / or R'COOH is hexanoic acid, the corresponding ester hexanoate, when R and / or R ^'whose acid RCOOH and / or R'COOH is a retinoic acid, ^the corresponding ester is the retinoate when R and / or R ^{^'whose} acid RCOOH and / or R'COOH is a benzoic acid, ^the corresponding ester is benzoate when R and R' of which ^the acid RCOOH and / or R ^'is a COOH 4-diethyl-aminobenzoic acid, ^the corresponding ester is diethyl-4-aminobenzoate, when R and R 'of which ^the acid RCOOH and / or R'COOH is 4-hydroxy-2-nonenoic acid, the corresponding ester is 4-hydroxy-2-nonenoate, when R and / or R ^' whose acid RCOOH and / or R'COOH is 4-aminobutanoic acid, the corresponding ester is 4-aminobutyrate.

Meanwhile, for example, in the case where R and / or R 'is heptyl, the corresponding ester is ^octanoate, when R and / or ^R' is ethyl, ^the corresponding ester is the propionate, and when R / or R 'is phenyl, ^the corresponding ester is benzoate, when R and / or ^R' is 3-aminopropyl, ^the corresponding ester is 4-aminobutyrate, etc.

"A" is defined such that the hydroxylated form of A which is A-OH), is a fluorescent tracer. The latter forms an ester with specific substrates by formulas (I) R-COO-A and (II) R-COO-A-OOC-R 'which after cleavage of the ester function leads to the release of ^the acid RCOOH and / or R'COOH and said fluorescent tracer A-OH. Table 1 below gives the structure of the substituents R and R ^' according to the invention, linked to the fluorescent tracer. The table also shows the specificity of each vis-à-vis substrate isoenzymes ^of ALDH.

Table 1

By "fluorescent tracer" is meant a chemical compound that can be identified by fluorescence. In particular, a fluorescent tracer of ^the invention is a fluorochrome or a fluorophore, ie a chemical substance capable of emitting fluorescence light after excitation.

In the context of this invention, the fluorophore is released under the action ^of an isozyme ^of ALDH.

The fluorophores are well known to ^those skilled in the art (see eg Manafi (2000) Int J Food Microbiol. 60:. 205-218).

Different fluorescent labels according to ^the invention appear in Table 2 below.

Table 2

In particular, in the context of the present invention, A-OH is chosen from 7-hydroxycoumarin, a fluorophore of the tokyo green family, in particular 2-methyl-4-methoxy-Tokyo Green, resorufin and fluorescein. . These tracers are known to the art and are either commercially available or can be synthesized by methods well known to ^the skilled person.

In particular, 2-methyl-4-methoxy-Tokyo Green is also named 6-Hydroxy-9- (4-methoxy-2-methylphenyl) -3H-xanthen-3-one, 2-Me-4-OMe TokyoGreen or 2-Me-4- OMe TG.

Thus, one can cite as a specific substrate of the present invention the following molecules: the retinoate resorufin, resorufin propionate, ^octanoate resorufin, benzoate resorufin, 4-aminobutyrate resorufin, ^the hexanoate resorufin, 4-diéthylaminobenzoate resorufin or 4-hydroxy-2-nonenoate resorufin, the retinoate 7-hydroxycoumarin propionate, 7-hydroxycoumarin, ^octanoate 7-hydroxycoumarin benzoate, 7-hydroxycoumarin, the 4- aminobutyrate 7-hydroxycoumarin, ^the hexanoate 7-hydroxycoumarin, 4-diéthylaminobenzoate of 7-hydroxycoumarin or 4-hydroxy-2-nonenoate 7- hydroxycoumarin, the retinoate 2-methyl-4-methoxy-Tokyo Green, propionate 2-methyl-4-methoxy-Tokyo Green, ^octanoate 2-methyl-4-methoxy-Tokyo Green benzoate, 2-methyl-4-methoxy-Tokyo Green, 4-aminobutyrate 2-methyl-4-methoxy-Tokyo Green, 2-methyl-4-metho hexanoate xy-Tokyo Green, 2-methyl-4-methoxy-Tokyo Green 4-diethylaminobenzoate or 2-methyl-4-methoxy-Tokyo Green 4-hydroxy-2-nonenoate, fluorescein di-retinoate, fluorescein propionate, fluorescein di-octanoate, fluorescein di-benzoate, fluorescein di-4-aminobutyrate, fluorescein di-hexanoate, fluorescein di-4-diethylaminobenzoate or di-4-hydroxy Fluorescein 2-nonenoate.

As previously indicated "ALDH" is used for "aldehyde dehydrogenases" and represents a group of dehydrogenase-like enzymes that exist in constitutive and inducible forms.

In humans, 19 ALDHs have been identified, including as many genes. They are divided into subgroups: ALDHI comprising ALDH1 A1, ALDH1 A2, ALDH1 A3, ALDH1 B1, ALDH1 L1 and ALDH1 L2, ALDH2, I ALDH3 comprising ALDH3A1, ALDH3A2, ALDH3B1 and ALDH3B2, I ALDH4, ALDHS, I ALDH6, TALDH7 the ALDH8, I ALDH9 the ALDHI 6, ^and ALDH18.

In particular, a specific substrate of the invention is a substrate specific for ^the ALDHI or ^of ALDH3. According to one embodiment of the present invention, when the isozyme ^of ALDH is ^the ALDHI, R and R 'identical or different, are chosen so as to form the retinoate, hexanoate or propionate.

In particular, when the isoenzyme ALDH is ALDHI, a specific substrate according to ^the invention is selected from retinoate resorufin, ^the hexanoate resorufin, resorufin propionate, retinoate 7-hydroxycoumarin, the ^hexanoate 7- hydroxycoumarin propionate, 7-hydroxycoumarin, the retinoate 2-methyl-4- methoxy-Tokyo Green, hexanoate, 2-methyl-4-methoxy-Tokyo Green, propionate 2-methyl- 4-methoxy-Tokyo Green, fluorescein di-retinoate, fluorescein dipropionate, fluorescein di-hexanoate.

According to another embodiment of the present invention, when ^isozyme ^of ALDH is ALDH3 I, R and R ^'identical or different, are chosen so as to obtain octanoate, 4-hydroxy-2-nonenoate, the 4-diethylaminobenzoate or benzoate.

In particular, when ^isozyme ^of ALDH is I ALDH3, a specific substrate according to ^the invention is selected from ^octanoate resorufin, 4-hydroxy-2-nonenoate resorufin, benzoate resorufin, 4- diéthylaminobenzoate resorufin octanoate 7-hydroxycoumarin, 4-hydroxy-2-nonenoate 7-hydroxycoumarin benzoate, 7-hydroxycoumarin, 4-diéthylaminobenzoate of 7-hydroxycoumarin, ^octanoate 2-methyl-4- methoxy-Tokyo Green, 2-methyl-4-methoxy-Tokyo Green 4-hydroxy-2-nonenoate, 2-methyl-4-methoxy-Tokyo Green benzoate, 2-methyl-4-methoxy hexanoate -Tokyo Green, fluorescein di-octanoate, fluorescein di-4-hydroxy-2-nonenoate, fluorescein di-benzoate and fluorescein di-4-diethylaminobenzoate.

According to another embodiment of the present invention, when ^the isoenzyme ALDH is ^the ALDH9, R and R 'identical or different, are chosen so as to obtain 4-aminobutyrate.

In particular, when ^the isoenzyme ALDH is ALDH9 I, a specific substrate of the invention is selected from 4-aminobutyrate resorufin, 4-aminobutyrate 7-hydroxycoumarin, 4-aminobutyrate, 2-methyl- 4-methoxy-Tokyo Green, fluorescein di-4-aminobutyrate.

According to one of these aspects, the present invention also relates to a composition comprising at least one specific substrate according to the invention.

The composition according to the invention thus comprises 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 17, 18, 19 or more specific substrates according to the ^invention.

Said composition makes it possible to directly detect and identify several isozymes ^of ALDH. For example, the composition comprises a substrate specific for ^the ADLHI and a specific substrate of the ALDH3 or a specific substrate of the ALDHI and I ALDH9, or ALDH3 and ^ALDH 9 or ^the ALDHI to TALDH3 and ALDH9.

Thus, the composition according to the invention may for example comprise one or more of the following specific substrates: the retinoate resorufin, resorufin propionate, ^octanoate resorufin, benzoate resorufin, 4-aminobutyrate resorufin, ^the hexanoate resorufin or 4-hydroxy-2-nonenoate resorufin, 4- diéthylaminobenzoate resorufin, the retinoate 7-hydroxycoumarin propionate, 7-hydroxycoumarin, ^octanoate 7-hydroxycoumarin, 7-hydroxycoumarin benzoate, 7-hydroxycoumarin 4-aminobutyrate, 7-hydroxycoumarin hexanoate or 7-hydroxycoumarine 4-hydroxy-2-nonenoate, 7-hydroxycoumarine 4-diethylaminobenzoate, 2-methyl-4-methoxy retinoate Tokyo Green, propionate 2-methyl-4-methoxy-Tokyo Green, ^octanoate 2-methyl-4- methoxy-Tokyo Green benzoate, 2-methyl-4-methoxy-Tokyo Green, 4- aminobutyrate of 2-methyl-4-methoxy-Tokyo Green, 2-methyl hexanoate 1-4-methoxy-Tokyo Green or 2-methyl-4-methoxy-Tokyo Green 4-hydroxy-2-nonenoate, 2-methyl-4-methoxy-Tokyo Green 4-diethylaminobenzoate, di-retinoate fluorescein, fluorescein dipropionate, fluorescein di-octanoate, fluorescein dibenzoate, fluorescein di-4-aminobutyrate, fluorescein di-hexanoate or di-4-hydroxy-2-nonenoate. fluorescein, fluorescein di-4-diethylaminobenzoate.

In particular, the specific substrate according to ^the invention is characterized in that ^isozyme ^of ALDH is detected in a cell population.

By "cell population" is meant a set of cells of the same origin or of different origin and whose characteristics (genetic sequences, levels of expression, state of differentiation) are identical or different. In particular, the cell population comprises at least 2 cells, for example 10, 100, 1000 or 1 000 cells.

Thus, according to one embodiment of the present invention, the specific substrate is detected in vitro or ex vivo by ^the use of the fluorescence technique plate of flow cytometry technique and / ^or immunofluorescence.

The specific substrates according to the invention are useful for identifying the different isoenzymes of ALDH. They allow in particular to ^identify cells expressing different ALDH isoenzymes (eg, certain types of stem cells) and distinguish in a mixed population of those who ^do not isoenzyme expression ^of ALDH or not the same isozyme ^of ALDH. The substrates of ^the invention can also be used to distinguish cells that express isozyme of ALDH to a high degree of the cells that ^{express it} to a lesser degree.

Thus, the present invention relates to the use ^of at least one specific substrate according to ^the invention to quantify at least one isoenzyme of ALDH in a cell population.

Thus, the present invention also relates to the use of at least one specific substrate according to the invention to isolate and / or select a portion ^of a cell population of overexpressing an isoenzyme ^ALDH.

Thus, the present invention also relates to the use ^of at least one specific substrate according to ^the invention to sort all or part of a cell population according to their expression ^of at least one isoenzyme of ALDH.

"At least one" isoenzyme of ALDH means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 17, 18 or 19 isoenzymes of ALDH. The 19 isoenzymes have been described previously.

"At least one" specific substrate according to the invention has the same meaning as previously described.

The present invention also relates to a method for quantifying at least one isoenzyme of ALDH in a cell population, comprising ^the ^use of at least one specific substrate according to ^the invention.

Quantification can be performed using a plate fluorescence technique. The ^{identification} and / or quantification of the various isozymes ^of ALDH thus enables to distinguish and / or identify different cell types.

Thus, the present invention also relates to the use ^of at least one specific substrate of the invention to distinguish healthy stem cells from cancer stem cells.

It also relates to a method for distinguishing healthy stem cells from cancer stem cells comprising ^the use ^of at least one specific substrate of the invention.

Said method and said use also make it possible ^to isolate these cells, including for example a step ^of isolation of cells exhibiting fluorescence.

By "cancerous stem cells" is meant, for example, stem cells of cancer of the bladder, breast, cervix, colon, head and neck, liver, lung, pancreas, prostate, ovarian, leukemia.

In particular, the specific substrate according to ^the invention is used to distinguish the stem cells of solid cancers and / or hematologic malignancies. By "solid cancer" is meant, for example, breast, lung or prostate cancer.

For example, "hematological malignancies" means leukemia, lymphomas or myeloma.

In particular, the stem cells of interest are cancerous or healthy hematopoietic cells. The ^{identification} and / or quantification of different isoenzymes of ALDH also allows to characterize different stages ^of a disease or differentiation of cells.

Thus, the present invention also relates to the use ^of at least one specific substrate according to ^the invention to characterize the different stages of cancer or the various stages of differentiation of stem cells.

It also relates to a method for characterizing the various stages of cancer or the various stages of stem cell differentiation comprising ^the use of at least one specific substrate according to the invention.

By "different steps of the differentiation of stem cells" are meant the steps which are well known to those skilled in the art, in particular the following steps: undifferentiated cells, poorly differentiated cells, moderately differentiated cells and well differentiated cells.

By "different stages ^of cancer" refers to the steps that are well known to those skilled in the art, in particular the following steps: undifferentiated tumor or cancer, tumor or cancer poorly differentiated, moderately differentiated tumor or cancer, or tumor well differentiated cancer.

Specific substrates according to the invention, allowing the ^{identification} and / or quantification of the various isozymes of ALDH, are useful as a diagnostic marker.

The present invention therefore also relates to a diagnostic marker comprising a specific substrate according to the invention.

By "diagnostic marker" is meant the meaning commonly attributed to these terms by a person skilled in the art, that is to say a characteristic element making it possible to confirm or invalidate a diagnosis.

According to one embodiment of the present invention, the diagnostic marker according to the invention is a specific substrate according to the invention.

Thus, the present invention also relates to the use ^of a marker according to the invention for the diagnosis ^of a disease involving deregulation of an isoenzyme of ALDH. It also relates to a method for diagnosing a disease involving dysregulation ^of an isoenzyme of ALDH comprising ^the use of a marker according to the invention.

A disease involving deregulation ^of an isoenzyme of ALDH is a disease involving said isoenzyme will be overexpressed or under expressed in the subject patient relative to the so-called normal expression, ^that is to say the expression observed in a holy subject.

By "overexpressed" or "overexpression" means a level of ^expression in the patient about higher than the healthy subject.

By "under expressed" or "sub-expression" means a level of ^expression in the subject sick less than that of healthy subjects.

Such a disease can be selected from cancers, disorders of sperm motility, ischemia, head trauma or pancreatitis.

By "cancer" is meant for example leukemia, breast cancer or lung cancer.

"Sperm motility disorders" means disorders affecting the rate at which sperm can move and pass through the woman's cervix, uterus and fallopian tubes.

The present invention also relates to ^the use ^of a marker according to the invention for determining whether a subject is likely to respond to therapy inhibiting ^the activity ^of an isoenzyme ^of ALDH and / or directed against cancer stem cells.

It also relates to a method for determining whether a subject is likely to respond to therapy inhibiting the activity ^of an isoenzyme of ALDH and / or directed against cancer stem cells comprising ^the use of a marker according to the invention .

By "subject" is meant in the context of the present invention, a warm-blooded animal such as a mammal, animal or human, particularly a human being. The subject may be a healthy subject or a subject who is suffering from, or has the potential to be afflicted with, one or more diseases and / or conditions described within the scope of the present invention.

By "therapy inhibit ^the activity ^of an isozyme ^of ALDH" refers to a therapy that directly or indirectly target would be an isozyme ^of ALDH as such I'ALDHI, ALDH3, I ALDH9 or more isoenzymes ALDH. By "therapy directed against cancer stem cells" refers to a therapy which would target cancer stem cells, one of the features is the high level ^of ALDH.

According to one of these aspects, the present invention also relates to a method for distinguishing cells expressing at least one isoenzyme from ALDH in a cell population, said method comprising:

(b) measuring the fluorescence of the cell population; and

(c) identifying cells with increased fluorescence relative to the fluorescence of the cell population before said population is contacted with said at least one specific substrate.

By "bringing into contact" is meant in particular the incubation with at least one specific substrate according to the invention for a defined time ranging from a few minutes, for example 30 minutes to several hours, for example 4 hours or more, with the cell population.

A "cell population" is as defined above. "At least one isozyme ^of ALDH" and "at least one specific substrate of the invention" are as defined above.

The fluorescence measurement can be performed by any method known to ^the skilled person. One can cite as an ^example the use of the fluorometer, the flow cytometer or fluorescence microscopy.

By "an increased fluorescence with respect to the fluorescence of the cell population before said population is brought into contact with the specific substrate", it is meant a florescence of the studied cell population that is greater than the fluorescence of this same cell population before that population. it has been brought into contact with the specific substrate according to the invention.

If at least two substrates specific for at least two different ALDH isoenzymes are contacted with the cell population, said method may also include an additional step d) comprising distinguishing between cells expressing the at least two isoenzymes of the ^ALDH.

This distinction can be achieved for example by observing different fluorescent colors depending on ^the detected isoenzyme.

By way of example, if the method comprises bringing the cell population into contact with resorufin retinoate and 7-hydroxycoumarin octanoate, the cells with increased red fluorescence will be identified as expressing ALDH1 whereas cells with increased blue fluorescence will be identified as expressing ALDH3.

According to one aspect, the present invention also relates to a kit for the uses mentioned in the context of the present invention, particularly for quantifying an isozyme ^of ALDH, particularly in a cell population, comprising at least one specific substrate according to the invention.

The kit may be a kit for diagnosing ^a disease involving dysregulation of an isozyme of ALDH, wherein said disease is selected for example from: cancers, disorders of sperm motility ischemia, head trauma or pancreatitis; for determining whether a subject is likely to respond to therapy inhibit ^the activity ^of an isoenzyme of ALDH and / or directed against cancer stem cells; to distinguish healthy stem cells from cancer stem cells, for example to distinguish between stem cells from solid cancers and / or hematological malignancies; or to characterize the different stages of cancer or the different stages of stem cell differentiation.

The kits of the invention may for example further comprise instructions for use of said kit for determining the amount of isoenzyme ALDH, particularly in a cell population, for the diagnosis ^of a disease involving deregulation an ALDH isoenzyme, for determining whether a subject is likely to respond to therapy inhibit ^the activity ^of an isoenzyme of ALDH and / or directed against cancer stem cells, to distinguish healthy stem cells cancer stem cells, or to characterize the different stages ^of cancer or the various stages of differentiation of stem cells.

The various compounds included in a kit according to the invention may be provided in the form of a solid (for example freeze-dried) or in liquid form.

Kits of the present invention may optionally include different containers (eg, ampule, test tube, vial or bottle) for each compound. Each compound will usually be aliquoted in its container or provided in a concentrated form. Other suitable containers for carrying out certain steps of the methods described in the context of the present invention may also be provided.

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

Figure 1: Plot of ichaelis- enten to determine the K _m and V _max of resorufine propionate on ALDH1A1, ALDH2 and ALDH3A1.

Figure 2: Assay of ALDH activity 1 by resorufin propionate after treatment with interfering RNA ALDH1A1.

Figure 3: Assay of ALDH1 activity by resorufin propionate after treatment with inhibitors of ALDH 1 and 3.

Figure 4: Activity ALDH1 detected by flow cytometry via resorufin propionate. Figure 5: Determination ^of activity by the ALDH1 retinoate resorufin and fluorescein di-retinoate and ALDH3 by benzoate resorufin and fluorescein di- benzoate after treatment with retinoic acid.

Figure 6: Assay of ALDH1 activity by resorufin retinoate and fluorescein di-retinoate and ALDH3 by resorufin benzoate and fluorescein dibenzoate after Disulfiram (DSF) treatment.

Examples

1. Preparation of Specific Substrates According to the Invention

Operating conditions

Commercial reagents and solvents (Fisher, Sigma, Fluorochem, etc.) were used without purification except dichloromethane distilled under an inert atmosphere on CaH ₂ . The reactions were monitored by thin layer chromatography on ^aluminum sheets coated with silica gel Macherey-Nagel SIL G ALUGRAM / UV _{April 25} (thickness 0.2 mm), observation of the plates having been carried out under ultraviolet light at 254 and 312 nm.

The column chromatographies were carried out on Macherey-Nagel 60M silica gel (40-63 μηι) under air pressure.

Melting points were measured with a Tottoli Buchi SMP-20 and were not corrected.

¹ H NMR spectra were recorded with Brucker ALS300 or DRX300 300 MHz devices. ¹³ C NMR spectra were obtained on Brucker DRX300

75 MHz. The chemical shifts δ are expressed in parts per million (ppm), the residual peak of the solvent having been taken as internal reference. The coupling constants J are expressed in Hz. Mass spectra were recorded in positive mode on a hybrid time-of-flight mass spectrometer (MicroTOFQ-11, Bruker Daltonics, Bremen) with an electrospray source (ESI).

The spray gas flow was at 0.4 bar and the capillary voltage at 3500v. The solutions were perfused at 10 μl / 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 the calibration was performed with sodium formate.

Examples of specific substrates structure according to ^the invention

Table 3

Resorufine propionate

Resorufin Hexanoate

Resorufin Octanoate

Resorufine benzoate

Resorufin 4-diethylaminobenzoate

Resorufine retinoate

2-methyl-4-methoxy-Tokyo Green Hexanoate

2-methyl-4-methoxy-Tokyo Green Octanoate 4-hydroxycoumarin octanoate

Fluorescein di-hexanoate

Fluorescein dioctanoate

Fluorescein di-retinoate

Esters of resorufin

Propionate and resorufine benzoate

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

For the proprionate, to a suspension of resorufin sodium salt (235.2 mg, 1.0 mmol) in 20 mL of anhydrous DCM was added DI PEA (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 were then added and extraction with 3x30 ml of DCM was carried out. The combined organic phases were washed with 30 ml of a saturated solution of NaHCO ₃ and then 30 ml of a saturated solution of NaCl. After drying over Na ₂ SO 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 (2 times). 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); ¹ H MN (300 MHz, DMSO) δ 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] + Weight: 269.2 g.mol ^"1

Formula: Ci ₅ H N0 ₄

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.

Mp> 210 ° C (Guilbault et al. Analytical Chem 1965, 37, 120-123: 203 ° C); HRMN (300 MHz, DMSO) δ 8.21 - 8.14 (m, 2H), 7.95 (d, J = 8.7 Hz, 1H), 7.84 - 7.75 (m, 1H), 7.62 (ddd, J = 1.7), 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] +.

Weight: 317.3 g. mol ^"1

Formula: C ₁₉ HiiN0 ₄

Hexanoate and resorufine octanoate

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%.

For ^the 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%.

^Mp 130-132 C; ¹ HRMN (300 MHz, DMSO) δ 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] +.

Weight: 31 1.3 g. mol ^"1

Formula: C ₁₈ H ₁₇ N0 ₄

For ^the 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%.

Mp 127-129 ° C; HRMN (300 MHz, DMSO) δ 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, 2.0Hz, 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] +.

Weight: 339.4 g. mol ^"1

Formula: C ₂ oH2i 0 ₄

Resorufin 4-diethylamino benzoate

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 second column (MeOH / DCM = 1/99) allows to eliminate. Mp 206-208 ° C; HRMN (300 MHz, DMSO) δ 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] +.

Weight: 388.4 g. mol ^"1

Formula: C ₂ 3H2oN ₂ 0 ₄

(All) trans retinoate of resorufin

The compound was prepared in the same manner as ^the previous ester using commercial retinoic acid (yield. 40%). 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 followed by MeOH gave 100 mg (40%) of pure product. Mp 150-160 (decomposition); H NMR (300 MHz, CDCl ₃ ) δ 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 +].

Weight: 495.6 g. mol ^"1

Formula: C ₃ 2H ₃₃ 04

Tokvo-qreen Esters

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 3.6- commercial dihydroxyxanth-9-one produced according to the procedure described in ^the article "J. Biol. ChemVol. 264, No. 14. Issue of May 15, PP. 8171-8178, 1989 "led to the 3,6-bis (tbutyldiméthylsilyloxy) xanthone. A second step carried out according to the procedure described in ^the article" Chem. Eur. J. 2014, 20, 447. 455 "consisting of a treatment using magnesium from 2-Bromo-3-methoxytoluene followed by acid hydrolysis gave 2-Me-4-MeO tokyo green.

Hexanoate and octanoate of 2-Me-4-MeO tokyo-green

The compounds were prepared conventionally using the corresponding acid chlorides. For hexanoate, from 0.15 mmol of 2-Me-4-MeO-TG, purification by chromatography on silica gel (MeOH / DCM: 1/99 to 10/90) was carried out, yield: 40% (non-crystallized resin).

HRMN (300 MHz, MeOD) and 7.45 (d = J = 2.0 Hz, 1H), 7.24 (d, J = 8.8 Hz, 1H), 7.16 (d, J = 9.6 Hz, 2H), 7.1 ( 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 (M, 2H), 1.47-1.33 (m, 4H), 1.00-0.89 (m, 3H).

¹³ CRMN (101 MHz, CDCl3) and 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, 18.85, 18.56, 16.24, 11.1. 1.80, 1 10.38, 106.18, 77.16, 55.51, 34.47, 31.32, 24.60, 22.42, 20.19, 14.05.

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

Weight: 430.5 g. mol ^"1

Formula: C ₂ H ₂₆ 0 _S

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)

¹ H NMR (300 MHz, MeOD) and 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 = 2.1Hz, 1H), 7.00 (cid, 1 = 8.4, 2.2Hz, 1H), 6.62 (dd, J = 9.7, 1.5Hz, 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).

¹³ CRMN (101 MHz, CDCl3) and 186.20, 171.66, 160.61, 158.78, 154.49, 130.92, 130.59, 129.34, 124.33, 120.89, 18.86, 106.23, 77.16, 55.51, 34.51, 31.76, 29.14, 29.02, 24.92, 22.73, 20.19, 14.21.

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

Weight: 485 g. mol ^"1

Formula: C2 ₉ H ₃ o0 ₅

Fluorescein diesters

The identical preparation is as described above using 3.0 eq. ^chlorides and 3 eq corresponding acids. basic. ^The hexanoate and ^octanoate were already described [litt. Ge, Feng-Yan; Dyes and Pigments 2007, 72 (3), 322-326].

Fluorescein di-hexanoate:

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

¹ H NMR (300 MHz, DMSO) δ 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 = 2.0Hz, 2H), 6.94 (dd, J = 8.7, 2.2Hz, 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);

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

Weight: 528.9 g. mol ^"1

Formula: C ₃₂ H ₃₂ 0 _/

Fluorescein dioctanoate:

[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 C () (49 C, Ge, Feng-Yan, Dyes and Pigments 2007, 72 (3), 322-326).

¹ H NMR (300 MHz, DMSO) δ 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);

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

Weight: 584.7 g. mol ^"1

Fluorescein di-retinoate:

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

¹ H NMR (300 MHz, DMSO) δ 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, 1.15 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).

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

Weight: 879.1 g. mol ^"1

Formula: C6oH ₆₄ 0 ₇

Esters of 7-hydroxycoumarin

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

7-hydroxylamine octanoate:

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

H NMR (300 MHz, DMSO) δ 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);

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

Weight: 288.3 g. mol ^"1

Formula: C ₁₇ H ₂ O0 ₄

2. Activity of specific substrates according to the invention

Material and methods

Cell lines

NCI-H522 and A 549 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 und Zellkultruren (DSMZ).

Determination of the Km and Vmax of resorufine propionate with the different purified ALDH1A 1. ALDH2 and ALDH3A 1 isoenzymes.

In 50 μl of 0.1 M phosphate buffer pH6.00; 0.2 M KCl; 2mM NADP +; 2 mM EDTA, a range of resorufine propionate was made: 250, 200, 150, 125, 100, 90, 80 and 70 μΜ and 0 μΜ. 50 μί solution ^of recombinant enzyme at 2.5 mU / well ALDH1 A1 (R & D Sytems, 5869-DH), ALDH2 (Abeam, 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 was then converted to resorufin salted (nM.min ^{".pg ^1"} ¹⁾ and the Km and Vmax were calculated via ^the Michaelis-Menten equation using GraphPad Prism 5.0.

Treatment of cells with specific inhibitors of IADLH1 and 3

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 ( SVF) supplemented with dimethyl ampalthiolester (DIMATE) a specific inhibitor ALDH1 and 3, morpholino ampal thiolester (MATE) a specific inhibitor of ALDH3, at concentrations of 8 μΜ respectively. After incubation for 6 hours, the substrates of the different ALDHs respectively resorufine propionate for ALDH1 and resorufin 4-diethylaminobenzoate for ALDH 3 were added at a final concentration of 10 μΜ 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.

Treatment of cells with Disulfiram (DSF) In a 96-well plate, the HL-60 cells were inoculated at a concentration of 50,000 cells / well in PMI-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 250 nM and 1000N. The cells were then incubated for 1 hour. After incubation, the substrates of different ALDHs the retinoate respectively resorufin and fluorescein di- retinoate for I ALDH1 and benzoate and fluorescein di-benzoate fluorescein ^for ALDH 3 were added at a final concentration of 5 μΜ 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.

Treatment of cells with retinoic ^acid

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 ( SVF) supplemented with retinoic acid, known to be an inhibitor of ALDH activity at concentrations of 1 μΜ and 10 μΜ. The cells were then incubated for 72 hours. After incubation, the substrates of different ALDHs the retinoate resorufin respectively and di-réetinoate fluorescein for I ALDH 1 and benzote fluorescein and di-benzote fluorescein ^for ALDH 3 were added at a final concentration of 5 μΜ 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 = 535nm for fluorescein). The data were expressed in relative fluorescence units.

Treatment of cells with interfering RNA ALDH1A 1

In a 60mm Petri dish, the NCI-H522 cells were seeded at a concentration of 250,000 cells / plates, corresponding to a confluence of 40-50%, in RPMI-1640 medium supplemented with L-Glutamine, Penicillin, Streptomycin and 10% Fetal Calf Serum (FCS) on the night at + 37 C. The next day, the medium was replaced with the same medium without FBS. The transfection solution was then prepared by diluting 100 nM in 500 μl of culture medium without FCS, to which 500 μl of culture medium without FCS supplemented with 3 μl 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 2 incubator for 8 hours. After 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.

Determination ^of activity by ALDH1 propionate resorufin

The cells were inoculated at a concentration of 1 × 10 ⁴ cells / well in a 96-well plate in 100 μl in RPMI-1640 medium without phenol red supplemented with L-Glutamine, Penicillin, Streptomycin and 10% Fetal Calf Serum (FBS). supplemented with 10 μΜ resorufine 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.

Double Localization of Resorufin Propionate and ALDHIA 1 by Fluorescent Microscopy

In a 24-well plate with microscopy 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 + 37C with 5% CO2. The next day, the culture medium was changed with either DIMATE (5μΜ) or the treatment vehicle and incubated for 6 hours. The cells were incubated for 30 minutes with culture medium supplemented with 10 μl 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 a PBS solution. ; 3% Bovine albumin; 0.3% Triton for 1 hour. ^The incubation was carried out with ^anti-ALDM antibody A1 (R & D System, MAB5869) 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 PBS washes were performed and microscopy glasses were mounted with support ^of antifading supplemented with DAPI. The cells were then observed under a microscope.

Identification of positive ALDH1 cells by flow cytometry

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 + 37C with 5% C02 atmosphere. . For the negative control, the cells were taken up in medium supplemented with 15 μl of dimethyl ampalthiolester (DIMATE) a specific inhibitor of ALDH1 and 3, then incubated for 5 hours at 37 C, 5% CO2. After trypsination, the cells were taken up in supplemented RPMI medium and centrifuged at 800g for 5 minutes. The cell pellet was then taken up in fresh supplemented medium containing 10 μl of resorufin propionate and then incubated for 1 hour in a tube of polycarbonate at 37 ° C., 5% CO 2. After incubation, the cells were then centrifuged and then washed in cold PBSxl and finally taken up in 200 ml of cold PBSxl. The solution was then analyzed by flow cytometry (Ex 590nm / Em 560 nm or red laser).

Results

The results are shown in Figures 1 to 5.

In particular, Figure 1 shows the data obtained to determine the K _m and Vmax of resorufine propionate.

The results are also shown in Table 3 below.

Table 4

The results of the assay of ^the activity ALDH1 by resorufin propionate after treatment with ^the interfering RNA aldh1a1 are illustrated by Figure 2 which shows ^that complete inhibition of I aldh1a1 is measured at 100nM siRNA which induces a significant decrease (P <0.05) of the resorufin propionate signal.

The results of the activity assay ALDH1 by resorufin propionate after treatment with inhibitors ^of ALDH 1 and 3 are illustrated in Figure 3 and shows a significant inhibition of the fluorescence (P <0.001) after treatment with DIMATE (inhibitor ^ALDH 1 and 3) in contrast to the MATE which is a specific inhibitor TALDH3.

In addition, the conversion of resorufine 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 may be colocalized with ALDH 1A1 suggesting specific activity with the enzyme (results not shown). Figure 4 illustrates ALDH1 activity detected by flow cytometry via resorufin propionate. The untreated condition demonstrates the presence of living positive ALDH1 cells (Calcein-AM).

In the presence ^of an inhibitor ^of ALDHI the DIMATE, inhibition of ALDH1 positive cells was observed to living cells as well as ^a decrease in viability due to treatment.

The results of the assay 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 Figure 5 which shows that a significant inhibition (P <0.001) or (P <0.01) level of ^activity of the ALDHI for retinoate resorufin and fluorescein di-retinoate and ^the ALDH3 for benzoate of resorufin and fluorescein di-benzoate.

In the presence of DSF at both the 250nM and 1000nM 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 Figure 6.

3. Application of the use of substrates

Materials and methods

patients

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.

Isolation of Masonic Cells and Evaluation of ALDH1 and ALDH3 Activity

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 +, CD1 17+ or CD45 weak).

ALDH1 and ALDH3 activity was evaluated by incubating reagents: resorufin retinoate or resorufine octanoate at 5 pmol.L ^"1 and fluorescein di-retinoate or fluorescein dioctanoate at 0.8 pmol.L ^{" 1} in blood after total lysis of the erythrocytes or in ^the extract bone marrow for 30 minutes at 37 C. the observed fluorescence is analyzed by cytometry to give a median fluorescence intensity value (MFI) corresponding to the relative activity of each isoform ^of ALDH blast cells.

Results The results are shown in Table 4 below.

Table 4: Patient parameters included in the study of the different activities of Aldehydes Dehydrogenases. AML, Acute Leukemia Myeloide; AREB, Refractory Anemia with Excess Blasts; Sg, blood; Mo, bone marrow; I FM, value of the Median Fluorescence Intensity.

Claims

1. Specific substrate of an ALDH isoenzyme comprising a compound:

(a) of formula (I): R-COO-A (I) resulting from the esterification of an A-OH fluorescent tracer with an acylating agent derived from the corresponding acid

RCOOH wherein R is selected so as to form the retinoate, propionate, ^octanoate, benzoate, 4-aminobutyrate, ^the hexanoate, 4-diethylaminobenzoate or 4-hydroxy-2-nonenoate; or

(b) of formula (II):

(II)

in which :

R and R ', identical or different, are chosen so as to form the retinoate, propionate, octanoate, benzoate, 4-aminobutyrate, ^the hexanoate, 4-diethylaminobenzoate or 4-hydroxy-2-nonenoate.

2. Specific substrate according to claim 1, characterized in that A-OH is chosen from 7-hydroxycoumarin, a fluorophore of the tokyo green family, in particular 2-methyl-4-methoxy-Tokyo Green, resorufin and fluorescein.

3. Specific substrate according to claim 1 or 2, characterized in that ^isozyme ^of ALDH is I ^'ALDHI and in that R and ^R' identical or different, are chosen so as to form the retinoate, ^the hexanoate or propianoate.

4. Specific substrate according to claim 1 or 2, characterized in that ^the ALDH isoenzyme is ALDH3 I and R and R 'identical or different, are chosen so as to form octanoate, 4-hydroxy -2- nonenoate, 4-diethylaminobenzoate or benzoate.

5. Specific Substrate according to ^one of the preceding claims characterized in that ^the isoenzyme of ADLH is detected in a cell population.

6. A composition comprising at least one specific substrate according to ^one of claims 1 to 5.

A diagnostic marker comprising a specific substrate according to any one of claims 1 to 5.

8. Use ^of at least one specific substrate according ^to any one of claims 1 to 5 for quantizing at least one isoenzyme of ALDH in a cell population.

9. Use of at least one specific substrate according ^to any one of claims 1 to 5 for sorting all or part of a cell population according to their expression ^of at least one isoenzyme ^of ALDH.

10. Use ^of a marker according to claim 7 for ^the diagnosis of a disease involving dysregulation of a isozyme ^of ALDH.

The use of claim 10, wherein said disease is selected from: cancers, sperm motility disorders, ischemia, head trauma or pancreatitis.

12. Use ^of a marker according to claim 7 for determining whether a subject is likely to respond to therapy inhibiting the activity of an isoenzyme ^of ALDH and / or directed against cancer stem cells.

13. Use of at least one specific substrate according ^to any one of claims 1 to 5 for distinguishing healthy stem cells of cancer stem cells.

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

15. Use of at least one specific substrate according to any one of claims 1 to 5 to characterize the different stages of a cancer or the different stages of differentiation of stem cells.

16. A method for distinguishing cells expressing at least one isoenzyme from ALDH in a cell population, said method comprising:

(a) contacting the cell population with at least one specific substrate according to ^one of claims 1 to 5,

(b) measuring the fluorescence of the cell population; and

(c) the ^{identification} of cells having an increased fluorescence compared to the fluorescence of the cell population before said population is contacted with said at least one specific substrate.

17. Kit for quantifying an isoenzyme of ALDH comprising at least one specific substrate according to any one of claims 1 to 5.