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.

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 high potential of such metabolic anti-cancer therapy is recognized in the scientific literature. The practical clinical situation is, however, very complicated. There are several types of metabolic adaptations in mitochondria of cancer cells, and an effective therapy must rely on the precise diagnosis of which type is present in a particular patient. Currently, there is no reliable method to test the function of mitochondria in cancer cells derived from a clinical patient.

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.

Importantly, these functions can be measured only in live and intact cells; the reporting fluorophore is therefore non-toxic and stable in the living cell environment.

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.

Once in the mitochondrial matrix, 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. Thus 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.

The ability of probes of the invention to comprise different pH-independent fluorophores and the FRET nature of pH-dependent fluorophore increases 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. In addition, 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.

Both 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. 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. 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 Warburg effect) and the increased resistance to oxidative stress. Importantly, the assays are done in a quantitative manner, so both the presence and the severity of metabolic alterations can be tested. The ability to accurately and sensitively measure these parameters within living cells can therefore provide an indication of the presence of vulnerabilities in AML mitochondria in a subject. This ability also provides the basis for an assay for the assessment of the efficacy of drugs for the treatment of AML.

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.

One exception is 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.

There remains a need, however, for probes which are more stable, which can provide a more sensitive pH measurement and increased fluorescence.

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.

In particular, in some embodiments of the invention, 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. This increases the brightness of pH-dependent channel by almost an order of magnitude. One consequence of this is that the native fluorescein channel’s fluorescence is decreased to almost zero. This means that 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.

In one embodiment, the invention provides a compound of Formula I:

A - L - B (Formula I) wherein

A is a pH-independent fluorophore;

L is a linker; and

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. As used herein, the term “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 (Forster resonance energy transfer) is a process, when the energy of light is used to excite one fluorophore (donor), while the emission light of different wavelength is produced by another fluorophore (acceptor), and that is not a typical emission wavelength of the donor fluorophore.

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.

Preferably, the pH-independent fluorophore A is a cationic lipophilic fluorophore. By using a cationic and/or 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. In a preferred embodiment, the pH-independent fluorophore A is a mitochondrial-targeting moiety.

In a preferred embodiment, 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.

Particular non-limiting examples of 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.

Most preferably, 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. 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. As used herein, the term “linker” refers to a sub-unit of the probe which serves to covalently link two other sub-units of said probe together. In the present invention, 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. In particular, 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. For example, 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. In a preferred embodiment, 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.

In one embodiment, the linker is a covalent bond between the two fluorophores.

In another embodiment, 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.

In some embodiments, the moiety B (i.e. the pH-dependent fluorophore of Formula II) is not directly attached to the NH of an NH-CO amide group (as part of the linker, L, i.e. the moiety B end of the linker does not end -CO-NH-.

In a further embodiment, the 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.

In a preferred embodiment, 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.

In a further preferred embodiment, 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.

In a yet further preferred embodiment, l_ ₃ may be absent or may be selected from: NH or S. In a particularly preferred embodiment, l_ ₃ is absent.

Especially preferred linkers include: Most preferably, the linker is -(CH ₂ )n-(C=0)-NH-CH ₂ CH ₂ -S-(CH ₂ )n- wherein n is 2-8, preferably 4-6, and more preferably n is 5.

Moiety B is a pro-active form of a pH-dependent fluorophore. As used herein, the term “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.

B is a compound of Formula II:

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. The 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. For example, a nitrogen with four alkyl bonds exists in a +1 oxidation state irrespective of the surrounding conditions (e.g. pH). By comparison, 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-. As a further example, 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.

In an inactive form, the mitochondrial pH probes of the present invention are protected such that they exist in a positive oxidation state. In a preferred embodiment, the probe is in a fixed oxidation state of +1.

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.

Once in the cytoplasm, 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.

In an inactive form, the probe is protected by one or more protecting groups (PGs).

As used herein, the term “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.

In particular, 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. In such an embodiment, 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. Examples of alcohol protecting groups include esters, formed with saturated and aromatic carboxylic acids, organic carbonate esters. Examples of amine protecting groups include amides, formed with saturated and aromatic carboxylic acids, carbamates, thiocarbamates. Examples of 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. The term “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. The term “enantiomers” refers to two stereoisomers of a compound which are non-superimposable mirror images of one another. The term “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. For example, where these contain only one chiral centre, 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.

The term “tautomer” as used herein 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.

The term “pharmaceutically-acceptable salt” as used herein 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. Preferably, the salt is a chloride, tetrafluoroborate or perchlorate, or any other strong acid’s anion.

By “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.

With a single fluorophore approach, there is just a single wavelength region of a pH- dependent fluorophore, where fluorescence intensity is measured. Different biological samples can only be compared between each other if the concentrations of the fluorophore in these samples are equal. With 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. According to a preferred embodiment, 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.

When two emission or excitation spectral bands used for the ratiometric approach correspond to the same single fluorophore, these bands are usually highly overlapping spectrally. This makes the pH-dependent fluorescence ratio change relatively small and thus the probe sensitivity low. While it is possible to use specially designed optical filters to increase probe sensitivity, this requires modification of the microscopes and thus causes impracticality. In contrast, if the two emission/excitation bands are completely spectrally separated, and are within the standard optical filter wavelength range (in this case green (GFP/FITC) and red (Cy3) or deep red (Cy5) ranges), then the sensitivity of the probe is greatly increased and can be used with any standard imaging system.

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-L-B (Formula I); wherein:

A is a pH independent fluorophore;

B is a pH dependent fluorophore; and

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.

Further details of the production of such probes may be found in the appended Examples and in W02019/180105. The mitochondrial pH probes of the present invention may be used to measure mitochondrial pH. In one embodiment, there is provided a method of measuring mitochondrial pH, the method comprising the steps of:

(a) contacting a cell with a mitochondrial pH probe of the invention;

(b) allowing the probe to accumulate in the mitochondria of the cell, and to convert into an active form;

(c) determining the ratio of fluorescence between the pH-independent fluorophore A and the pH dependent fluorophore B; and

(d) using this ratio to determine the mitochondrial pH.

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.

Once sufficient accumulation has taken place, it is possible to begin measuring the fluorescence of the mitochondrial pH probe. After initial incubation time with the medium, containing probe in the inactive form, 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.

Other methods, including but not limited plate-reading bulk analysis, can be used to analyze the stained cells. Overall, the staining and washing procedures can be performed within 30 minutes, after which the cells are ready for analysis.

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.

Hence 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.

Biochem. Biophys. 495, 152-158 (2010); and/or carbonylcyanide-3- chlorophenylhydrazone (CCCP) as described in Ruas, J. S. et al. Underestimation of the maximal capacity of the mitochondrial electron transport system in oligomycin- treated cells. PLoS One 11 , 1-20 (2016); and/or other mitochondrial protonophore and/or ionophore and/or ion-exchanging agent, such as valinomycin, nigericin antimycin and others, for example Lou, P.-H. et al. Mitochondrial uncouplers with an extraordinary dynamic range. Biochem. J. 407, 129-140 (2007); Kadenbach, B. Intrinsic and extrinsic uncoupling of oxidative phosphorylation. Biochim. Biophys. Acta - Bioenerg. 1604, 77- 94 (2003). 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.

Other crucial aspects of the metabolic activity of mitochondria, such as efficiency and mode of function of the ATP synthase and the degree of uncoupling activity in mitochondria, can also be tested using the probes of the invention. Such information can be particularly useful in the context of cancer diagnosis and treatment.

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. 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. 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.

Normal cells primarily produce energy through glycolysis followed by mitochondrial citric acid cycle and oxidative phosphorylation. However, most cancer cells predominantly produce their energy through a high rate of glycolysis followed by lactic acid fermentation even in the presence of abundant oxygen. "Aerobic glycolysis" is less efficient than oxidative phosphorylation in terms of adenosine triphosphate production, but leads to the increased generation of additional metabolites that may particularly benefit proliferating cells such as cancer cells.

In normal healthy cells, ATP synthase acts in its forward mode, wherein protons flow into mitochondria to produce ATP from ADP (as illustrated in Figure 2). In cancer cells, 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.

Consequently, the measurement of mitochondrial pH in the presence and absence of an ATP synthase inhibitor may be used to determine the extent to which the ATP synthase molecules are acting in forward or reverse mode, and hence to give an indication of the cancerous nature of the cells. In yet a further embodiment, therefore, 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:

(a) contacting a biological sample comprising mitochondria from the subject with a probe of the invention;

(b) allowing the probe to accumulate in the mitochondria, and to convert into an active form;

(c) determining a first ratio of fluorescence of the pH-dependent fluorophore B to the pH-independent fluorophore A in the absence of an ATP synthase inhibitor, and determining a second ratio of fluorescence of the pH-dependent fluorophore B to the pH-independent fluorophore A in the presence of the ATP synthase inhibitor;

(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.

Instead of using an ATP synthase inhibitor, an inhibitor of the regulator of ATP synthase, e.g. ANT, may be used. In yet a further embodiment, therefore, 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:

(a) contacting a biological sample comprising mitochondria from the subject with a probe of the invention;

(b) allowing the probe to accumulate in the mitochondria, and to convert into an active form; (c) determining a first ratio of fluorescence of the pH-dependent fluorophore B to the pH-independent fluorophore A in the absence of an inhibitor of the regulator of ATP synthase, and determining a second ratio of fluorescence of the pH- dependent fluorophore B to the pH-independent fluorophore A in the presence of the inhibitor of the regulator of ATP synthase;

(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

It is also known that cancer cells have an increased resistance to oxidative stress; in many cases, this is due to the upregulation of proton leakage. The probes of the invention may also be used to assess the level of proton leakage within mitochondria.

An uncoupling protein (UCP) 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.

In yet another embodiment, therefore, 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:

(a) contacting a biological sample comprising mitochondria from the subject with a probe of the invention;

(b) allowing the probe to accumulate in the mitochondria, and to convert into an active form;

(c) determining a first ratio of fluorescence of the pH-dependent fluorophore B to the pH-independent fluorophore A in the absence of a UCP inhibitor, and determining a second ratio of fluorescence of the pH-dependent fluorophore B to the pH-independent fluorophore A in the presence of the UCP inhibitor;

(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. In a yet further aspect there is provided 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. For example, 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. Preferably, 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. Preferably, the biological sample is a blood sample.

Certain population of cells within the sample (e.g. cancer cells, such as AML cells) may be purified or isolated from the biological sample prior to use.

Generally, the method of the invention will be carried out ex vivo, or in vitro. Generally, the biological samples are samples which have previously been obtained from a subject.

Preferably, 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. For example, 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). Preferably, 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:

(i) 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.

(ii) 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.

(iii) Germ cell tumour: Cancers derived from pluripotent cells, most often presenting in the testicle or the ovary (seminoma and dysgerminoma, respectively).

(iv) Blastoma: Cancers derived from immature "precursor" cells or embryonic tissue.

The cancer may therefore be a carcinoma, sarcoma, germ cell tumour or a blastoma.

Preferably, the cancer is a blood cancer, e.g. a leukaemia, lymphoma or myeloma. There are four main types of leukaemia: Acute myeloid leukaemia (AML); Acute lymphoblastic leukaemia (ALL); Chronic myeloid leukaemia (CML); and Chronic lymphocytic leukaemia (CLL).

Other types of 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)

In some preferred embodiments, the leukaemia is AML.

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.

Preferably, the method steps are carried out in the order specified.

The disclosure of each reference set forth herein is specifically incorporated herein by reference in its entirety.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Mitochondrial pH responsiveness and calibration of a probe of the invention in the physiologically-relevant pH range.

Figure 2. Warburg Effect: cancer cell ATP synthase reversal upon aerobic glycolysis. pH gradient might be created not from oxidative phosphorylation, but by reversal of ATP-synthase (cancer cells); this is crucial for tumour invasiveness and aggressive development. These two scenarios can be distinguished by changes in mitochondrial pH upon ATP-synthase inhibition. This provides a quick and reliable test for one feature of mitochondrial metabolism which is a characteristic of cancer cells.

Figure 3. Proton leakage in mitochondria.

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).

Figure 5. Know-how readouts may be used to find novel drug combinations for variety of AML phenotypes (left); and ultimately lead to personalized medicine package, when the metabolically-assisted therapy will be optimized for every AML patient, based on the responsiveness of his/her own cancer cells to a library of drug candidates (right).

Figure 6. Proof-of-concept demonstration of high-throughput readout of metabolic parameters of cancer cell mitochondria in the plate-reader format.

Figures 7A-7D. Photomicrographs of mitochondria stained with probes of the invention. The images were taken on a confocal microscope at 60x magnification. EXAMPLES

The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1 : Production of probes

In the following Examples, the compound having the following structure is referred to as 6-FA-NDS-Cy3-DA. 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:

Synthesis of 6-NDS-CA

To a solution of 1 eq. of 6-aminofluorescein in DMAC a solution of 1.1 eq. of 5- chloropentanal in DMAC was added. The mixture was stirred for 5 min at room temperature and then 2eq. of sodium cyanoborohydride was added. The mixture was stirred for 1 h. NMR spectra showed that the reaction was completed. After that, to the reaction mixture 15eq. of acetaldehyde was added, followed by 2.5eq. of sodium triacetoxyborohydride. The conversion of the reaction was controlled by NMR. Next, to the resulting reaction mixture 10eq. of cysteamine was added in MeOH-water solution. 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.

Synthesis of 6FA-NDS-Cv3

1eq. of Cy3-COOH chloride was dissolved in DMAC in a 2 ml Eppendorf tube. To the resulting solution 2eq. of carboxydiimidazole was added in 0.5 ml of DMAC. The mixture was stirred for 1 h. The completion of conversion of Cy3-acid to imidazolide was controlled by NMR. Next, to the reaction mixture a solution of 1eq. of 6-FA-NDS-CA in 0.5 ml of DMAC was added. The reaction mixture was stirred overnight at room temperature. After completion of the reaction (confirmed by NMR) the reaction mixture was precipitate by diethyl ether. This precipitate was separated by centrifugation and the liquid layer was discarded. Next, to the remaining precipitate 0.3 ml of THF was added and the mixture sonicated for 2 minutes. The precipitate was separated by centrifugation again and the sonication-centrifugation was repeated 7 times. The precipitate was dried in the open Eppendorf tube in the darkness. The purity of the product was controlled by NMR. Synthesis of 6-FA-NDS-Cv3-DA

To the solution of 1eq. 6-FA-NDS-Cy3-DA in DMAC, a solution of 6eq. EDCI hydrochloride, 20eq. of AcOH and 6eq. of DIPEA in DMAC was added. The mixture was stirred overnight at room temperature and the degree of acylation was controlled by NMR. Upon completion of acylation, the reaction mixture was transferred to 15 ml falcon tube and diluted with 2 ml of chloroform. The resulting mixture was extracted with 2 ml of 0.05 M HCI 5 times. The water layers were discarded and the remaining chloroform layer was transferred to a 2 ml Eppendorf tube and precipitated with 1 ml of diethyl ether to separate the product. 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.

Similar probes may also be produced by such methods, mutatis mutandis.

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.

Figure 1 illustrates the mitochondrial pH responsiveness and calibration of a probe of the invention in the physiologically-relevant pH range.

Example 3: Responsiveness of probe using cancer cells

In order to ensure that the diagnostic methods could be applied to any patient, 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

The most cost-effective and commonly available solution for high-throughput screening is a plate-reader format. To demonstrate the principle possibility of platereader-based readout, we performed a proof-of-concept screening, demonstrating the influence of different pH of the extracellular medium on metabolic parameters of tumor cells (Figure 6). The whole experiment took less than 5min. Importantly, the latest generation of platereaders can provide much better sensitivity and more detailed data with the same screening speed. In addition, they often come in the format of high-throughput screening (HTS) imagers, which allow to work with image arrays and extract much more useful data from the measurements, such as of mitochondrial functions on a single-cell level; and increase the sensitivity of the signal by subtracting the background fluorescence values. This makes the implementation of plate-reader-based approach for the analysis of patient-derived AML cells and simultaneous drug screening very realistic.

Myalgic encephalomyelitis (chronic fatigue syndrome) patients are suspected to have abnormalities in mitochondrial metabolism that manifest themselves in the symptoms of the disease. We used 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. We have found that ME-CFS patients exhibit decreased level of uncoupling activity in comparison to healthy controls, which is indicative of cellular energetic stress (Figure 4).

Example 5: Drug screening

Potential drug candidates are screened as shown in Figure 5.

In the first step, 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.

Example 6: Staining of mitochondria using probes of the invention

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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