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WO2022207813A1 - Sondes de ph mitochondrial et leurs utilisations - Google Patents

Sondes de ph mitochondrial et leurs utilisations Download PDF

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Publication number
WO2022207813A1
WO2022207813A1 PCT/EP2022/058616 EP2022058616W WO2022207813A1 WO 2022207813 A1 WO2022207813 A1 WO 2022207813A1 EP 2022058616 W EP2022058616 W EP 2022058616W WO 2022207813 A1 WO2022207813 A1 WO 2022207813A1
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Prior art keywords
fluorophore
mitochondria
compound
mitochondrial
alkyl
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Gennady NIKITIN
Karl Johan TRONSTAD
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Vestlandets Innovasjonsselskap AS
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Vestlandets Innovasjonsselskap AS
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/06—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups three >CH- groups, e.g. carbocyanines
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH

Definitions

  • the present invention relates to dual fluorophore probes and their use for measuring the pH and other metabolic parameters within mitochondria.
  • the probes comprise a pH- independent fluorophore comprising for example, a cyanine dye, linked to a pH- dependent fluorophore comprising a fluorescein.
  • Mitochondria are rod-shaped double membrane organelles in a cell that convert energy stored in nutrients into adenosine triphosphate (ATP), using oxygen as an oxidant, thereby providing energy for the cell to drive the cell’s metabolic activities. This process is known as aerobic respiration. As well as energy generation, mitochondria regulate cellular redox states, generate most of the cellular reactive oxygen species and initiate cellular apoptosis.
  • ATP adenosine triphosphate
  • Tumour cells often exhibit metabolic reprogramming involving a range of metabolic features, including aberrant mitochondrial metabolism, abnormal expression of metabolic enzymes, and increased dependence on glycolysis for ATP generation and biomolecule production. They also frequently exhibit dysregulation in other mitochondrial parameters, including deregulated mtDNA content, increased ROS production, and defects in oxidative phosphorylation, suggesting that these alterations can be indicative of carcinogenesis.
  • Such alterations to mitochondrial metabolism can provide tumour cells with survival advantages, contribute to the resistance to chemotherapy and can boost metastatic potential. At the same time, these alterations can render the cancer cells susceptible to metabolic therapeutics, that target specifically malignant and not the healthy cells, because healthy cells simply lack the metabolic alterations that are affected by these drugs.
  • the current invention relates to fluorescence-based probes which are optimized to have a quasilinear response in the pH range which is physiologically relevant for mitochondria. These probes can be used to measure the functional parameters of mitochondria of any cell type, based on the measurement of mitochondrial pH.
  • the probes of the invention are lipophilic and are delivered to cells in a non-active (proactive) form; these rapidly penetrate cellular membranes. This facilitates the use of the probes with any cell type and ensures that the invention may be used with actively drug-effluxing cells such as some cancer cell lines and hematopoietic stem cells.
  • mitochondrial esterases convert the pro-active forms of the probes into a more hydrophilic active form, thus ensuring the retention of the active probes within the mitochondria.
  • the probes of the invention comprise two fluorophores: a pH-independent fluorophore which provides an indication of the concentration of probe within the mitochondria; and a pH-dependent fluorophore, which provides an indication of the pH within the mitochondria.
  • the fluorescent readout is ratiometric (i.e. internally normalized), which makes the signal exclusively dependent on mitochondrial pH and not on any other factors, such as mitochondrial mass or total potential.
  • probes of the invention increase the compatibility of assays with additional fluorescent probes and, importantly, GFP-based fluorophores.
  • the crucial aspects of metabolic activity of mitochondria such as efficiency and mode of function of the ATP synthase and the degree of uncoupling activity in mitochondria, can be tested using the probes of the invention.
  • the basal level of mitochondrial pH is well known to correlate to oxidative stress in mitochondria. Since the measurement of mitochondrial pH is not time-dependent as opposed to the direct measurement of reactive oxygen species (ROS) production, it makes the mitochondrial pH a very convenient and reliable proxy for oxidative stress in the context of realistic clinical diagnostic method.
  • ROS reactive oxygen species
  • ATP-related activity and ROS generation in mitochondria present potential anti cancer therapeutic targets.
  • the degree of ROS generation and the mode of ATP synthase function can affect the efficacy of a particular anti-cancer drug. These influences can be independent of each other or synergistic, and the mitochondrial pH- based assays can provide information on which drugs target the cancer-specific mitochondrial features most potently.
  • the main methodology of measuring the vital functional mitochondrial parameters involves comparison of basic mitochondrial pH value with corresponding values when one of the key metabolic enzymes is inhibited.
  • oligomycin By inhibition with oligomycin, the positive or negative change in mitochondrial pH shows whether the ATP synthase produces energy (ATP) for the cell or consumes it, respectively.
  • genipin By inhibition with genipin, the amount of uncoupling activity and corresponding proton leak in mitochondria can be assessed.
  • a number of fluorescence-based mitochondrial pH probes have previously been developed to measure mitochondrial pH. Most of these probes are relatively hydrophilic and cannot be applied to actively drug-effluxing cells, however.
  • W02019/180105 that relates to the production of mitochondrial pH probes and the use of such probes for drug screening.
  • W02019/180105 discloses dual fluorophore ratiometric mitochondrial pH probes comprising 5-aminofluoroscein molecules linked to the cyanine dyes Cy3 or Cy5.
  • the invention aims to overcome one or more of the above-mentioned problems by providing a new probes which are biologically stable, often for several hours after the initial staining.
  • the probes are also more hydrophobic and exhibit increased mitochondrial pH sensitivity.
  • the fluorescence from the pH- dependent fluorophore is not detected as a fluorescein channel (GFP-like green fluorescence), but exclusively as FRET from fluorescein to Cy3 channel.
  • FRET fluorescein to Cy3 channel.
  • This increases the brightness of pH-dependent channel by almost an order of magnitude.
  • the native fluorescein channel is decreased to almost zero.
  • the probe can now be used with a different green fluorophore (e.g. GFP, other fluoresceins or similar) for more complex measurements.
  • the present invention therefore provides dual fluorophore probes comprising a pH- independent fluorophore comprising a cyanine dye, linked to a pH-dependent fluorophore comprising a fluorescein.
  • the invention also provides methods of using the probes of the invention to measure mitochondrial pH and other metabolic parameters.
  • the invention provides a compound of Formula I:
  • A is a pH-independent fluorophore
  • L is a linker
  • B is a pH-dependent fluorophore of Formula II: wherein
  • X denotes the point of attachment to the linker, L
  • R1 is selected from a C1-5 alkyl, C2-5 alkenyl, C2-5-alkynyl or C1-5-haloalkyl group, or a 5- to 10-membered aryl or heteroaryl (e.g. phenyl or benzyl) group, optionally substituted, e.g. by one or more of halogen, C1 -6-alkyl (preferably C1 -3-alkyl), C2-4- alkenyl, C2-4-alkynyl, C1 -4-haloalkyl (e.g. CF 3 ), -CN or -N0 2 ; and R2 and R3 are independently protecting groups; or a stereoisomer or tautomer, or a pharmaceutically-acceptable salt thereof.
  • the compounds of the invention are dual fluorophores.
  • fluorophore means a fluorescent chemical compound that can re-emit light upon light excitation, emitted light being of a different wavelength than excitation light.
  • dual fluorophore means a molecule that contains two or more covalently-linked fluorophores that are tethered together by a linker. Upon light excitation, each fluorophore can re emit light.
  • FRET Formster resonance energy transfer
  • Moiety A is a pH-independent fluorophore.
  • pH-independent fluorophore means a fluorophore which emits light at a specific wavelength largely irrespective of the pH of the environment it is in. A pH-independent fluorophore therefore exhibits consistent fluorescence over variable pH conditions, for example both low and high pH.
  • the pH-independent fluorophore A is a cationic lipophilic fluorophore.
  • the probe can more easily pass through the plasma and mitochondrial membranes and the positive charge allows the probe to target the mitochondria.
  • the pH-independent fluorophore A is a mitochondrial-targeting moiety.
  • the pH-independent fluorophore A is selected from a fluorophore containing a positively-charged N ion; a fluorophore derived from fluorones, polymethines, pyrenes or acridines; or a fluorophore derived from a rosamine or cyanine.
  • pH-independent fluorophore A include: wherein X denotes the attachment site of L. ln further embodiments, the attachment site X may be positioned in a different part of the fluorophore provided the position does not adversely affect the ability of the fluorophore to fluoresce in a pH independent manner. More preferably, moiety A is a cyanine dye.
  • the cyanine dye may comprise one or two sulpho groups, thus rendering the dyes more water soluble. The cyanine due may also be PEGylated.
  • moiety A is Cy3 or Cy5: wherein R4 and R5 are methyl, ethyl or propyl (preferably methyl), and wherein X is the point of attachment to the linker, L.
  • R4 and R5 are methyl, ethyl or propyl (preferably methyl), and wherein X is the point of attachment to the linker, L.
  • Groups that do not affect the fluorescence properties of the fluorophore A can be modified and remain within the scope of the invention.
  • L is a chemical linker which links moieties A and B.
  • the probes of the present invention comprise two fluorophores covalently linked together. Covalently linking fluorophores leads to advantageous properties. If two non-covalently linked fluorophores were used, the ratiometric approach would rely on the same concentration of each probe being present in the mitochondria at the same time. In practice, this is difficult to achieve because different probes will influx and efflux at different rates. By covalently linking the two fluorophores together, the concentration ratio between them is kept constant, because it is defined by the stoichiometry of the molecule. In the present invention, the linker also provides efficient FRET between the two fluorophores.
  • linker refers to a sub-unit of the probe which serves to covalently link two other sub-units of said probe together.
  • the linker may be absent whereby the two units are directly linked to each other, or may be present and provide the link between the units.
  • a linker should not adversely affect the functions of the linked sub-units.
  • the linker should not prevent the individual fluorophores from fluorescing in a pH dependent or pH independent manner, as required, and the efficiency of FRET between pH-dependent and pH-independent fluorophore subunits should be preserved.
  • a linker should not conjugate into the fluorescence-causing region of the fluorophores, or should not link in a way that adversely changes the properties of the fluorophores.
  • the linker L is selected from any suitable group that can covalently link the two fluorophores together without adversely affecting the fluorescence performance of either fluorophore.
  • the linker positioning and selection is designed to minimise any steric hindrance for the rotation of the two fluorophores around the linker relative to each other that may adversely affect the ability of the probe to travel through the cellular plasma membrane and mitochondrial membranes.
  • the linker is a covalent bond between the two fluorophores.
  • the linker L may be selected from one or more of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroalkyl, carbonyl, alkylcarbonyl; and/or combinations thereof optionally joined directly or by an alkyl, carbonyl, aminocarbonyl, thiocarbonyl group or by a heteroatom.
  • the moiety B i.e. the pH-dependent fluorophore of Formula II
  • the moiety B end of the linker does not end -CO-NH-.
  • linker L may be of Formula III:
  • LI-(L 3 )-L 2 (Formula III), wherein l_i is attached to moiety A and l_ 2 is attached to moiety B, wherein l_i and l_ 2 are independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroalkyl, carbonyl, and alkylcarbonyl; and l_ 3 is a bond covalently linking l_i and l_ 2 together; or l_ 3 is selected from heteroatom, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, heteroalkyl, carbonyl, and alkylcarbonyl.
  • l_i may be selected from alkyl, heteroaryl, heterocyclic, heteroalkyl and alkylcarbonyl. In a further preferred embodiment l_i may be selected from heteroaryl or alkylcarbonyl; more preferably 1 ,3,5-triazine or Ci- n alkyl-1 -carbonyl, wherein n is 2 to 12.
  • l_ 2 may be selected from alkyl, heteroaryl, heterocyclic, heteroalkyl and alkylcarbonyl. In a yet further preferred embodiment, l_ 2 may be selected from alkyl, heterocyclic or heteroalkyl; more preferably piperazine, 1-amino-n-thio-Ci- n - alkyl, or 1 ,n-diamino-Ci- n alkyl, wherein n is 2 to 12.
  • l_ 3 may be absent or may be selected from: NH or S. In a particularly preferred embodiment, l_ 3 is absent.
  • Moiety B is a pro-active form of a pH-dependent fluorophore.
  • pH-dependent fluorophore means a fluorophore whose fluorescence intensity changes depending on the pH of the environment it is in.
  • a pH-dependent fluorophore is sensitive to the pH environment and exhibits variable fluorescence depending on pH.
  • X denotes the point of attachment to the linker, L.
  • R1 is selected from C1-5 alkyl, C2-5 alkenyl, C2-5-alkynyl or C1-5-haloalkyl group, or a 5- to 10-membered aryl or heteroaryl (e.g. phenyl or benzyl) group, optionally substituted, preferably substituted by one or more of halogen, C1 -6-alkyl (preferably C1- 3-alkyl), C2-4-alkenyl, C2-4-alkynyl, C1-4-haloalkyl (e.g. CF 3 ), -CN or -N0 2.
  • R1 alkyl, alkenyl or alkynyl may be branched or unbranched, and may be substituted or unsubstituted.
  • R1 is preferably a C2-4 unbranched alkyl group, most preferably ethyl or propyl.
  • R2 and R3 are independently protecting groups.
  • Alkyl means groups which may be branched or unbranched, and preferably have from 1 to about 12 carbon atoms. One more preferred class of alkyl groups has from 1 to about 8 carbon atoms. Even more preferred are alkyl groups having 1 , 2, 3, 4, 5, 6, 7 or 8 carbon atoms. Propyl including isopropyl, butyl including sec-butyl and isobutyl, pentyl, including isopentyl, hexyl, heptyl and octyl are particularly preferred alkyl groups in the compounds of the present invention. Alkyl groups according to the present invention may be unsubstituted or substituted, e.g. with a halo group.
  • Alkenyl and alkynyl mean groups which may be branched or unbranched, have one or more unsaturated linkages and from 2 to about 12 carbon atoms. One more preferred class of alkenyl and alkynyl groups has from about 2 to about 8 carbon atoms. Even more preferred are alkenyl and alkynyl groups having 2, 3, 4, 5, 6, 7 or 8 carbon atoms.
  • Halo means fluoro (F), chloro (Cl), bromo (Br) or iodo (I).
  • the mitochondrial pH probe according to the present invention is in a protective (i.e. inactive) form; it is protected into an overall positive oxidation state. In this form, the probe is efficiently transported into the mitochondria of the cell.
  • the protection is removed inside mitochondrial matrix (e.g. via an esterase) to convert the probe into an active form with a variable oxidation state so that the probe is sensitive to changes to pH.
  • “Inactive form” or “proactive form” means the molecule has been modified such that it exists in a positive oxidation state and that pH-dependent fluorophore exhibits no FRET to pH-dependent fluorophore. In some embodiments, in inactive form, the molecule does not fluoresce in a pH dependent manner. The molecule may be in inactive form via protection of one or more functional groups. When in inactive form, the molecule is configured to facilitate transportation into the target location, for example the cell and/or mitochondria.
  • Active form means the molecule is configured to produce its desired effect, e.g. measure mitochondrial pH. In active form, the molecule will fluoresce in a pH-dependent manner to enable mitochondrial pH measurement. In addition, the FRET between pH- dependent and pH-independent fluorophores is activated only in active form. The molecule may be converted into an active form via removal of the protecting group(s).
  • Oxidation state means the overall charge of an atom, part of a molecule or a molecule. A single positive charge in a molecule corresponds to an oxidation state of +1 and a single negative charge corresponds to an oxidation state of -1. The oxidation state of a molecule or part of a molecule can be condition dependent or independent.
  • a nitrogen with four alkyl bonds exists in a +1 oxidation state irrespective of the surrounding conditions (e.g. pH).
  • an alcohol molecule ROH exemplifies a variable oxidation state as it can exist at multiple oxidation states depending on the pH of its surroundings. At low pH, the oxygen will bond to H+, leaving the alcohol ROH2+ in a +1 oxidation state. Alternatively, at a high pH, H+ may be lost, leading to a molecule in a -1 oxidation state as RO-.
  • a molecule with the fixed oxidation state nitrogen and the variable oxidation state alcohol both present would display a variable oxidation state, moving from +1 in inactive form of the molecule to 0 for the active form of the molecule at low pH to -1 for the active form of the molecule at high pH.
  • the mitochondrial pH probes of the present invention are protected such that they exist in a positive oxidation state.
  • the probe is in a fixed oxidation state of +1.
  • the probe By fixing the probe into a positive oxidation state, the probe will preferentially accumulate within the mitochondria. Additionally, in the inactive form the probe is much more lipophilic, thus permeating the cellular membranes much more rapidly.
  • Mitochondria are distinguished from other cellular organelles by their inner mitochondrial membranes having a high electrochemical potential. This electrostatic potential pulls the positively-charged probe into the mitochondria leading to a concentration of probe in the mitochondria several orders of magnitude higher than the rest of the cell. The positive charge on the probe also helps with the initial delivery of the probe into the cell since the cell plasma membrane also has an electrochemical potential of the same polarity of the mitochondria, albeit at a much lower magnitude.
  • the probe is attracted into the mitochondria, again due to its positive oxidation state.
  • the probe then accumulates inside the mitochondria in the mitochondrial matrix.
  • the probe is converted via cellular enzymes (for example esterases) into active form. While this conversion will take place within the whole cell, the preferential accumulation into the mitochondria due to the positive probe charge leads to an overall accumulation and entrapment inside the mitochondria.
  • the probe is protected by one or more protecting groups (PGs).
  • PGs protecting groups
  • protecting group means a group which has been introduced into a molecule by modification of a functional group which prevents said functional group from undergoing further changes (e.g. reactions) until the protecting group has been removed. Suitable protecting groups can be deprotected intracellularly.
  • the pH dependent fluorophore B comprises oxygen groups which can be protected to lock or cage the molecule into a positive oxidation state, and to remove FRET between fluorophores A and B.
  • the pH dependent fluorophore B in inactive form comprises one or more protected oxygen(s).
  • Any suitable protecting group can be used that is able to both achieve a positive oxidation state and also be removed intracellularly.
  • Suitable protecting groups include alcohol protecting groups, amine protecting groups, carboxylic acid protecting groups.
  • alcohol protecting groups include esters, formed with saturated and aromatic carboxylic acids, organic carbonate esters.
  • amine protecting groups include amides, formed with saturated and aromatic carboxylic acids, carbamates, thiocarbamates.
  • carboxylic acid protecting groups include acetoxymethyl ester, anhydrides, formed with saturated and aromatic carboxylic acids.
  • Other examples of possible protecting groups include acyl, propionyl, butyryl, isobutyryl, pivaloyl or benzoyl.
  • An especially preferred protecting group is acyl.
  • Particularly preferred mitochondrial pH probes of the invention include:
  • the compounds herein described may contain one or more chiral centres and may therefore exist in different stereoisomeric forms.
  • stereoisomer refers to compounds which have identical chemical constitution but which differ in respect of the spatial arrangement of the atoms or groups. Examples of stereoisomers are enantiomers and diastereomers.
  • enantiomers refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.
  • diastereoisomers refers to stereoisomers with two or more chiral centres which are not mirror images of one another.
  • the invention is considered to extend to the use of diastereomers and enantiomers, as well as racemic mixtures.
  • the compounds herein described may be resolved into their enantiomers and/or diastereomers.
  • these may be provided in the form of a racemate or racemic mixture (a 50:50 mixture of enantiomers) or may be provided as pure enantiomers, i.e. in the R- or S-form.
  • Any of the compounds which occur as racemates may be separated into their enantiomers by methods known in the art, such as column separation on chiral phases or by recrystallization from an optically active solvent.
  • Those compounds with at least two asymmetric carbon atoms may be resolved into their diastereomers on the basis of their physical-chemical differences using methods known perse, e.g. by chromatography and/or fractional crystallization, and where these compounds are obtained in racemic form, they may subsequently be resolved into their enantiomers.
  • tautomer refers to structural isomers which readily interconvert by way of a chemical reaction which may involve the migration of a proton accompanied by a switch of a single bond and adjacent double bond. It includes, in particular, keto-enol tautomers. Dependent on the conditions, the compounds may predominantly exist either in the keto or enol form and the invention is not intended to be limited to the particular form shown in any of the structural formulae given herein.
  • the compounds of the invention must be cationic for targeting to mitochondria; therefore they may be provided as salts.
  • pharmaceutically-acceptable salt refers to any pharmaceutically-acceptable organic or inorganic salt of any of the compounds herein described.
  • a pharmaceutically acceptable salt may include one or more additional molecules such as counter-ions.
  • the counter-ions may be any organic or inorganic group which stabilizes the charge on the parent compound. If the compound for use in the invention is a base, a suitable pharmaceutically acceptable salt may be prepared by reaction of the free base with an organic or inorganic acid. If the compound for use in the invention is an acid, a suitable pharmaceutically acceptable salt may be prepared by reaction of the free acid with an organic or inorganic base. Non-limiting examples of suitable salts are described herein.
  • the term “pharmaceutically-acceptable” means that the compound or composition is chemically and/or toxicologically compatible with other components of the formulation or with the patient’s mitochondria.
  • the salt is a chloride, tetrafluoroborate or perchlorate, or any other strong acid’s anion.
  • a pharmaceutical composition is meant a composition in any form suitable to be used for a medical purpose.
  • the probes of the present invention are ratiometric. By covalently linking a pH-dependent fluorophore with a pH-independent fluorophore, it is possible to take concentration-independent pH measurements. This allows for results to be normalised and compared between biological samples with variable nature.
  • “Ratiometric” means a method of measuring the fluorescence of the fluorophores and determining the fluorescence ratio. Applying a ratiometric approach avoids fluorophore concentration effects from influencing the pH measurement. If a probe comprises a single pH dependent fluorophore, the intensity of the emission will be dependent not only on pH but also on the fluorophore concentration. This makes it difficult to compare results between different biological samples. By applying a ratiometric approach, internal normalisation of the fluorescence readings is possible. While the absolute fluorescence intensities between different samples may vary depending on the fluorophore concentrations, the fluorescence ratio will remain independent of concentration and will only vary depending on pH.
  • the ratiometric probes of the present invention while the absolute fluorescent intensities of different samples may vary dependent on the corresponding fluorophore concentrations, the fluorescence ratio remains independent of the concentration and only responds to the change in pH.
  • the two fluorophores have spectrally separated fluorescence bands and/or absorption bands. This improves the sensitivity of the readings and allows for more accurate measurements to be taken.
  • the probes of the present invention may be made by any suitable means. According to one embodiment, there is provided a method of manufacturing a dual fluorophore ratiometric mitochondrial pH probe of Formula I:
  • A is a pH independent fluorophore
  • B is a pH dependent fluorophore
  • L is a chemical linker, conjugating the pH independent fluorophore A to the pH dependent fluorophore B, or L is a covalent bond; as defined herein, comprising protecting the pH dependent fluorophore B such that it is put into a positive oxidation state.
  • the mitochondrial pH probes of the present invention may be used to measure mitochondrial pH.
  • a method of measuring mitochondrial pH comprising the steps of:
  • the probes of the present invention When in inactive form, the probes of the present invention are configured to more easily pass through lipid membranes and to target the mitochondria. This leads to an accumulation of the probe in the mitochondria. As already discussed above, this accumulation is defined by the rate of influx into the cell and mitochondria and the rate of efflux by the cell’s defence mechanisms to remove the probe. By ensuring rapid influx and effective mitochondrial targeting, the probe will accumulate in the mitochondria and influx will override efflux.
  • the advantageous properties of the probes of the present invention are such that, in some embodiments, sufficient accumulation of active probe in the mitochondria can take place within 15-20 minutes. This enables rapid mitochondrial pH measurements to take place.
  • the sensitivity and update speed of the probe gives the ability to monitor real time changes to mitochondrial pH in response to any stimulus or condition being applied to the cell.
  • the cells are washed with the medium, not containing the probe. All the accumulated probe inside the mitochondria of the cells is converted into active form almost instantaneously. It is retained inside the mitochondria due to its ability to be trapped because of the membrane impermeability of the active form.
  • the cells are then ready to be analyzed by fluorescent light microscopy or flow cytometry. The ratiometric fluorescent signal is acquired and quantified.
  • the probe Prior to taking a pH measurement, it may be necessary to calibrate the probe to align the fluorescence ratiometric readings to a specific pH value. This may be undertaken by analysing the fluorescence ratio in the set of control experiments, including staining the control cell samples in the set of buffers with defined pH. These buffers must contain the protonophore or ionophore or cation exchanging agents, allowing the equilibration of the mitochondrial matrix pH with the pH of the surrounding buffer.
  • Step (d) may comprise using this ratio to determine the mitochondrial pH by comparing the obtained ratio to ratios obtained from mitochondria of intact cells or isolated mitochondria under control conditions.
  • Such agents might include: carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP) as described in To, M. S. et al. Mitochondrial uncoupler FCCP activates proton conductance but does not block store-operated Ca 2+ current in liver cells. Arch.
  • FCCP carbonylcyanide-4-trifluoromethoxyphenylhydrazone
  • the cells initially are stained with the mitochondrial pH probe in the same conditions, as used in the main experiments. Then the medium, containing the probe, is washed away and substituted with the corresponding series of defined pH buffers, containing the means to equilibrate the mitochondrial pH and the buffer pH, preferably FCCP or CCCP. The series of control cells then analysed after 15 minutes with the same method, as used for the main experiments, to quantify the ratiometric fluorescent signal, corresponding to the exact mitochondrial pH value, defined by the buffer pH in the case of the control experiment. The dependence of ratiometric signal over the pH value is then analytically approximated with the mathematical equation, preferably linear equation. This equation is later used to convert the value of ratiometric fluorescent signal, obtained in the main experiments, to the mitochondrial pH value, observed experimentally.
  • the basal level of mitochondrial pH is well known to correlate to oxidative stress in mitochondria. Since the measurement of mitochondrial pH is not time-dependent (as opposed to the direct measurement of reactive oxygen species (ROS) production), it makes the mitochondrial pH a very convenient and reliable proxy for oxidative stress in the context of a clinical diagnostic method.
  • ROS reactive oxygen species
  • the main methodology of measuring the vital functional mitochondrial parameters involves comparison of basic mitochondrial pH value with corresponding values when one of the key metabolic enzymes is inhibited.
  • oligomycin By inhibition with oligomycin, the positive or negative change in mitochondrial pH shows whether the ATP synthase produces energy for the cell or consumes it, respectively.
  • genipin By inhibition with genipin, the amount of uncoupling activity and corresponding proton leak in mitochondria can be assessed.
  • These two assays test the most general adaptations of cancer cell mitochondria: the exit from reliance on mitochondrial energy production (also known as the “Warburg effect”) and the increased resistance to oxidative stress. Importantly, the assays are performed in a quantitative manner, so both the presence and the severity of metabolic alterations can be tested.
  • ATP synthase acts in its forward mode, wherein protons flow into mitochondria to produce ATP from ADP (as illustrated in Figure 2).
  • ATP synthase acts in reverse mode, wherein ATP is converted to ADP and protons are pumped out of the mitochondria.
  • the ATP synthase inhibitor oligomycin inhibits proton conductance by ATP synthase, removing its contribution to the proton gradient. If mitochondrial ATP synthase acts in the normal mode, inhibition with oligomycin would increase mitochondrial pH, while in the reverse mode it would decrease mitochondrial pH. In the case of pre-existing natural inhibition of ATP synthase, inhibition with oligomycin would not noticeably change mitochondrial pH.
  • the invention provides a method of obtaining an indication of whether a subject’s mitochondria may be usable as a tumour-specific target in a subject suffering from cancer, the method comprising the steps:
  • Step (d) comparing the first and second ratios obtained in Step (c), wherein a first ratio which is higher than the second ratio is indicative of the subject’s mitochondria being usable as a tumour-specific target.
  • Determining the first and second ratios in Step (c) may be carried out in either order, or simultaneously, or consecutively, when the first ratio is measure before the second.
  • ATP synthase inhibitors include oligomycin, Gboxin, BTB06584, hydroxyglutaric acid, and Apoptolidin.
  • an inhibitor of the regulator of ATP synthase e.g. ANT
  • the invention provides a method of obtaining an indication of whether a subject’s mitochondria may be usable as a tumour-specific target in a subject suffering from cancer, the method comprising the steps:
  • Step (d) comparing the first and second ratios obtained in Step (c), wherein a first ratio which is higher than the second ratio is indicative of the subject’s mitochondria being usable as a tumour-specific target.
  • Determining the first and second ratios in Step (c) may be carried out in either order, or simultaneously, or consecutively, when the first ratio is measure before the second.
  • Regulators of ATP synthase include ANT1 , ANT2, ANT3, IF1 proteins.
  • Inhibitors of regulators of ATP synthase include ANT proteins: bongkrekic acid, carboxyatractyloside, isobongkrekic, acidatractyloside, GSAO (4-(N-(S- glutathionylacetyl)amino) phenylarsonous acid) and PENAO (4-(N-(S- penicillaminylacetyl)amino) phenylarsonous acid).
  • IF1 can be inhibited by IF1 -targeting shRNA
  • the probes of the invention may also be used to assess the level of proton leakage within mitochondria.
  • An uncoupling protein is a mitochondrial inner membrane protein that is a regulated proton channel or transporter. An uncoupling protein is thus capable of dissipating the proton gradient generated various metabolic activities of mitochondria. The energy lost in dissipating the proton gradient via UCPs is not used to do biochemical work; instead, heat is generated. UCPs belong to the mitochondrial carrier (SLC25) family. This is illustrated in Figure 3.
  • UCPs are positioned in the same membrane as the ATP synthase, which is also a proton channel.
  • the two proteins thus work in parallel with one generating heat and the other generating ATP from ADP and inorganic phosphate, the last step in oxidative phosphorylation.
  • Mitochondrial respiration is coupled to ATP synthesis (ADP phosphorylation) but is regulated by UCPs.
  • UCPs decrease mitochondrial energetic efficiency, but reduce the mitochondrial oxidative stress. UCP action is associated with decrease in mitochondrial matrix pH.
  • the invention provides a method of obtaining an indication of whether a subject’s mitochondria may be usable as a tumour-specific target in a subject suffering from cancer, the method comprising the steps:
  • Step (d) comparing the first and second ratios obtained in Step (c), wherein a significant positive difference between the second ratio and the first ratio is indicative of the subject’s mitochondria being usable as a tumour-specific target.
  • UCP inhibitors include genipin.
  • Significance may be measured by any suitable technique, e.g. Student’s t-test (p ⁇ 0.05). In some embodiments, a pH difference of more than 0.1 , 0.2 or 0.3 pH units is considered to be significant.
  • the probes of the invention may also be used for screening of the therapeutic efficiency of libraries of candidate drugs.
  • a method of measuring the impact of drugs or other conditions on the mitochondrial pH comprising applying the drug or other condition to a cell and measuring the change in mitochondrial pH using a dual fluorophore ratiometric mitochondrial pH probe as described herein. “Measuring the impact of drugs or other conditions” means using the probes of the present invention to determine the impact of drug(s) or other situations on mitochondrial pH.
  • the probes of the present invention may be used to determine how mitochondrial pH changes in response to the application of a drug into a cellular system and/or the removal of a drug from a cellular system.
  • the probe of the present invention may also be used to determine how mitochondrial pH changes in response to other changes to cellular conditions, for example changes to temperature or the like.
  • the probes of the present invention may be used to monitor real time changes to mitochondrial pH in response to any stimulus or condition being applied to the cell.
  • the biological sample comprising mitochondria may be obtained from any suitable source.
  • the biological sample is a sample of cells comprising mitochondria or of purified or substantially purified mitochondria.
  • the cells or mitochondria may be obtained from any suitable biological sample, including a tissue sample, biopsy or blood sample.
  • the biological sample is a blood sample.
  • Certain population of cells within the sample may be purified or isolated from the biological sample prior to use.
  • the method of the invention will be carried out ex vivo, or in vitro.
  • the biological samples are samples which have previously been obtained from a subject.
  • the cells or mitochondria are obtained from a subject who is suffering from a cancer, who is suspected to be suffering from a cancer or who is at risk of suffering from a cancer.
  • the subject is preferably a mammalian subject.
  • the mammal may be human or non-human.
  • the subject may be a farm mammal (e.g. sheep, horse, pig, cow or goat), a companion mammal (e.g. cat, dog or rabbit) or a laboratory test mammal (e.g. mouse, rat or monkey).
  • the subject is a human.
  • the subject may be male or female.
  • the human may, for example, be 0-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90- 100 or above 100 years old.
  • Cancers are classified by the type of cell that the tumour cells resemble and is therefore presumed to be the origin of the tumour. These types include:
  • Carcinoma Cancers derived from epithelial cells. This group includes many of the most common cancers and include nearly all those in the breast, prostate, lung, pancreas and colon.
  • Sarcoma Cancers arising from connective tissue (i.e. bone, cartilage, fat, nerve), each of which develops from cells originating in mesenchymal cells outside the bone marrow.
  • Germ cell tumour Cancers derived from pluripotent cells, most often presenting in the testicle or the ovary (seminoma and dysgerminoma, respectively).
  • Blastoma Cancers derived from immature "precursor" cells or embryonic tissue.
  • the cancer may therefore be a carcinoma, sarcoma, germ cell tumour or a blastoma.
  • the cancer is a blood cancer, e.g. a leukaemia, lymphoma or myeloma.
  • a blood cancer e.g. a leukaemia, lymphoma or myeloma.
  • leukaemia There are four main types of leukaemia: Acute myeloid leukaemia (AML); Acute lymphoblastic leukaemia (ALL); Chronic myeloid leukaemia (CML); and Chronic lymphocytic leukaemia (CLL).
  • AML Acute myeloid leukaemia
  • ALL Acute lymphoblastic leukaemia
  • CML Chronic myeloid leukaemia
  • CLL Chronic lymphocytic leukaemia
  • leukaemia include: acute promyelocytic leukaemia (APL); hairy cell leukaemia (HCL); large granular lymphocytic leukaemia (LGL); T-cell acute lymphoblastic leukaemia (T-ALL); and chronic myelomonocytic leukaemia (CMML)
  • APL acute promyelocytic leukaemia
  • HCL hairy cell leukaemia
  • LGL large granular lymphocytic leukaemia
  • T-ALL T-cell acute lymphoblastic leukaemia
  • CMML chronic myelomonocytic leukaemia
  • the leukaemia is AML.
  • Non-Hodgkin lymphoma There are two main types of lymphoma: Non-Hodgkin lymphoma and Hodgkin lymphoma.
  • the information provided by the probes of the invention may be used to produce a metabolic profile of the subject’s mitochondria and/or of the disease in question.
  • One or more additional mitochondrial parameters such as total mitochondrial potential, mitochondrial DNA sequencing, mitochondrial mRNA and mitochondrial protein expression may also be included in such a profile.
  • Such profiles may be correlated with particular diseases or disorders.
  • the method steps are carried out in the order specified.
  • Figure 4 Difference between the level of uncoupling activity in cultured cell mitochondria, induced by the ME-CFS patient-derived serum and healthy control.
  • HC healthy control samples, 1 to 4 (from four human donors).
  • ME myalgic encephalomyelitis (samples from patients 1 to 4).
  • FIGS. 7A-7D Photomicrographs of mitochondria stained with probes of the invention. The images were taken on a confocal microscope at 60x magnification. EXAMPLES
  • 6-FA-NDS-Cy3-DA comprises the fluorophore 6-FA (fluorescein) linked to the fluorophore Cy3, joined via a double alkyl-N-substituted linker.
  • the 6-FA unit has been diacetylated, leading to the designation DA.
  • 6-FA-NDS-Cy3-DA was synthesised from commercially available 6-aminofluorescein according to the synthetic scheme below:
  • the resulting mixture then was transferred to a falcon tube and distilled water was added to precipitate the crude product, which was separated by centrifugation. Next, 7 ml of water was added to the separated precipitate and the resulting suspension sonicated for 2 minutes. The procedure was repeated 5 times and the purity of the product was controlled by NMR.
  • the formed precipitate was separated by centrifugation. Next, to the precipitate 1 ml of distilled water was added and the mixture was sonicated for 2 minutes, the resulting precipitate was separated by centrifugation again. This procedure was repeated 5 times. The purity of the product was controlled by NMR.
  • Example 2 Mitochondrial pH responsiveness and calibration of a probe
  • Calibration of the probe was performed by subjecting the cells stained with 6-FA-NDS- Cy3-DA to series of PBS-HEPES buffers of various pH, containing protonophore (preferably FCCP or CCCP) to equilibrate pH of the buffer and mitochondrial pH.
  • protonophore preferably FCCP or CCCP
  • Figure 1 illustrates the mitochondrial pH responsiveness and calibration of a probe of the invention in the physiologically-relevant pH range.
  • the mitochondrial pH-based assays described above were tested in wide range of adherent and non-adherent cancer cells, including a leukemic cell line, as well as in healthy blood cell types, such as hematopoietic stem cell and progenitors and T-cell populations.
  • the stability of the probe provided reliable measurement of the signal independent on the cell type. No toxicity related to the staining or mitochondrial pH measurements were observed.
  • the pH sensitivity was quasi-linear in the pH range (Figure 1), that is relevant to physiological mitochondrial levels.
  • Example 4 Influence of different pH of the extracellular medium on metabolic parameters of tumour cells
  • Myalgic encephalomyelitis (chronic fatigue syndrome) patients are suspected to have abnormalities in mitochondrial metabolism that manifest themselves in the symptoms of the disease.
  • 6FA-Cy3-NDS-DA and described above UCP inhibition assay to compare the level of uncoupling activity in ME-CFS patients, as compared to healthy donor controls.
  • ME-CFS patients exhibit decreased level of uncoupling activity in comparison to healthy controls, which is indicative of cellular energetic stress (Figure 4).
  • drug candidates are tested on cells from different tumour types (e.g. A- D) and compared against the effect of an oligomycin control or genipin control (to show whether the ATP synthase produces energy for the cell or consumes it; and the degree of uncoupling activity, respectively).
  • Mitochondria which consume energy are potential therapy targets.
  • Mitochondria that exhibit excessive uncoupling activity are another potential class of therapy targets.
  • the degree to which the potential drug successfully targets mitochondrial metabolism can be determined by comparing the results in the presence and absence of the drug for a particular tumour type.
  • Mitochondria of HeLa cells were stained with a 2mM solution of 6-FA-NDS-Cy3-DA for 30 minutes and then washed with imaging medium. Images were subsequently acquired with a spinning disk confocal microscope at 60x magnification ( Figures 7A- 7D).
  • the images show the pH dependent and pH independent channels. At first glance, the pairs of images look the same; this is because the probe is a molecule which is labelled with a coloured label, so it stains exactly the same mitochondria (as it is supposed to). The intensity of the signal between the channels is different, however, and this is what is quantified as a pH readout.

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Abstract

La présente invention se rapporte à des sondes à double fluorophore et à leur utilisation pour mesurer le pH et d'autres paramètres métaboliques dans les mitochondries. Les sondes comprennent un fluorophore indépendant du pH comprenant par exemple un colorant cyanine, lié à un fluorophore dépendant du pH comprenant une fluorescéine.
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