GLIOMA THERAPY

Abstract

The invention is directed to a method for identifying a candidate anti-glioma drug that is a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener, the method comprising: a. providing an admixture comprising: i. a candidate drug; ii. a submitochondrial vesicle (SMV) preparation; and iii. an H.sup.+ probe, wherein the H.sup.+ probe remains outside of the SMV(s); b. contacting the admixture with ATP; c. measuring a level of H.sup.+ outside of the SMV(s) via the H.sup.+ probe; d. comparing the level of H.sup.+ at step c) with a level of H.sup.+ in a control admixture lacking the candidate drug; and identifying the drug as a candidate anti-glioma drug that is a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener, when the level of H.sup.+ is higher compared to the level of H.sup.+ in the control admixture; or identifying that the drug is not a candidate anti-glioma drug that is a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener, when the level of H.sup.+ is the same or lower compared to the level of H.sup.+ in the control admixture.

Claims

1. A method for identifying a candidate anti-glioma drug that promotes proton (H.sup.+) leakage through a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel, the method comprising: a. providing an admixture comprising: i. a candidate drug; ii. a submitochondrial vesicle (SMV) preparation; and iii. an H.sup.+ probe; b. contacting the admixture with adenosine triphosphate (ATP), wherein the presence of ATP promotes translocation of protons across the SMV membrane and into the internal space of the SMV(s); c. detecting H.sup.+ external to the SMV(s) with the H.sup.+ probe, and determining a value of H.sup.+ external to the SMV(s); d. comparing the H.sup.+ level determined at step c) with that of a corresponding reference H.sup.+ level for a control admixture that lacks the candidate drug; and i. identifying the drug as a candidate anti-glioma drug that promotes H.sup.+ leakage, when the H.sup.+ level is higher compared to the reference H.sup.+ level for the control admixture; or ii. identifying that the drug is not a candidate anti-glioma drug that promotes H.sup.+ leakage, when the H.sup.+ level is the same or lower compared to the reference H.sup.+ level for the control admixture.

2. Use of an assay that measures proton (H+) leakage through the mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel, for identifying a candidate anti-glioma drug, the assay comprising: a. providing an admixture comprising: i. a candidate drug; ii. a submitochondrial vesicle (SMV) preparation; and iii. an H.sup.+ probe; b. contacting the admixture with ATP, wherein the presence of ATP promotes translocation of protons across the SMV membrane and into the internal space of the SMV(s); c. detecting H.sup.+ external to the SMV(s) with the H.sup.+ probe, and determining a value of H.sup.+ external to the SMV(s); d. comparing the H.sup.+ level determined at step c) with that of a corresponding reference H.sup.+ level for a control admixture that lacks the candidate drug; and i. identifying the drug as a candidate anti-glioma drug that promotes H.sup.+ leakage, when the H.sup.+ level is higher compared to the reference H.sup.+ level for the control admixture; or ii. identifying that the drug is not a candidate anti-glioma drug that promotes H.sup.+ leakage, when the H.sup.+ level is the same or lower compared to the reference H.sup.+ level for the control admixture.

3. A method for identifying a glioma patient's suitability for treatment with a medicament comprising a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener (preferably wherein said medicament was identified by a method or use according to claims 1-2), the method comprising: a. contacting an isolated glioma sample obtained from the patient with the medicament comprising F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener, and incubating the sample; b. detecting the presence or absence of glioma suppression when compared to a control glioma sample incubated in the absence of the medicament comprising leak channel opener; and i. identifying the patient as being suitable for treatment with the medicament comprising leak channel opener when suppression is detected; or ii. identifying the patient as being unsuitable for treatment with the medicament comprising leak channel opener when suppression is not detected.

4. A method for identifying the suitability of medicament comprising a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener for treating a glioma patient (preferably wherein said medicament was identified by a method or use according to claims 1-2), the method comprising: a. contacting an isolated glioma sample with the medicament comprising a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener, and incubating the sample; b. detecting the presence or absence of glioma suppression when compared to a control glioma sample incubated in the absence of the medicament comprising leak channel opener; and i. identifying the leak channel opener as being suitable for treating glioma when suppression is detected; or ii. identifying the leak channel opener as being unsuitable for treating glioma when suppression is not detected.

5. A screening method for identifying an anti-glioma drug, the method comprising: a. obtaining a candidate drug that has been confirmed to be a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener (preferably by a method or use according to any of claims 1-2); b. contacting an isolated glioma sample with the candidate drug, and incubating the sample; c. detecting the presence or absence of glioma suppression when compared to a control glioma sample incubated in the absence of the candidate drug; and i. identifying the candidate drug as suitable for use as an anti-glioma drug when suppression is detected; or ii. identifying the candidate drug as unsuitable for use as an anti-glioma drug when suppression is not detected.

6. The method or use according to claim 1 or 2, wherein the H.sup.+ probe is 9-Amino-6-chloro-2-methoxyacridine (ACMA).

7. The method or use according to any one of claims 1-6, wherein the glioma is temozolomide (TMZ) resistant.

8. The method or use according to any one of claims 1-7, wherein the glioma is glioblastoma multiforme (GBM).

9. The method according to any one of claims 3-8, wherein the leak channel opener or the candidate drug has been confirmed to be a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener (e.g. has been confirmed to induce proton leakage through the F.sub.1F.sub.o ATP Synthase c-subunit leak channel) by an assay.

10. The method according to claim 9, wherein the leak channel opener or the candidate drug has been confirmed to be a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener (e.g. has been confirmed to promote proton leakage through the F.sub.1F.sub.o ATP Synthase c-subunit leak channel) by an assay comprising: a. admixing an isolated submitochondrial vesicle (SMV) preparation with the leak channel opener or candidate drug and 9-Amino-6-chloro-2-methoxyacridine (ACMA), to provide an admixture comprising: i. leak channel opener or candidate drug; ii. ACMA; and iii. SMV preparation; b. contacting the admixture with ATP, wherein the presence of ATP promotes translocation of protons across the SMV membrane and into the internal space of the SMV; c. detecting H.sup.+ external to the SMV(s) by measuring a level of fluorescence (using excitation and emission wavelengths of 410 nm and 483 nm, respectively) from ACMA; d. comparing the level of fluorescence measured at step c) with a reference level of fluorescence for a corresponding control admixture lacking the leak channel opener or candidate drug; and e. confirming that the leak channel opener or candidate drug promotes proton leakage when the level of fluorescence is higher (for example, at least 5% higher) compared to the reference level of fluorescence for the control admixture.

11. The method according to claim 9 or claim 10, wherein the leak channel opener or the candidate drug has been confirmed to be a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener (e.g. has been confirmed to promote proton leakage through the F.sub.1F.sub.o ATP Synthase c-subunit leak channel) by an assay comprising: a. admixing an isolated submitochondrial vesicle (SMV) preparation with the leak channel opener or candidate drug and 9-Amino-6-chloro-2-methoxyacridine (ACMA), to provide an admixture comprising: i. 5-10 ?M leak channel opener or candidate drug; ii. 2 ?M ACMA; and iii. 5 ?g SMV; b. incubating the admixture for 20 minutes; c. contacting the admixture with ATP, to provide the admixture with 1 mM ATP and a volume of 40 ?l, wherein the presence of ATP promotes translocation of protons across the SMV membrane and into the internal space of the SMV(s); d. detecting H.sup.+ external to the SMV(s) by measuring a level of fluorescence (using excitation and emission wavelengths of 410 nm and 483 nm, respectively) from ACMA; e. comparing the level of fluorescence measured at step d) with a reference level of fluorescence for a control admixture lacking the leak channel opener or candidate drug; and f. confirming that the leak channel opener or candidate drug promotes proton leakage when the level of fluorescence is higher (for example, at least 5% higher) compared to the reference level of fluorescence for the control admixture.

12. The method or use according to any one of the preceding claims, further comprising administering the leak channel opener or the candidate drug to a glioma patient when: a. the patient is identified as being suitable for treatment with the medicament comprising leak channel opener; b. the leak channel opener is identified as being suitable for treating a glioma in a patient; c. the candidate drug is identified as being suitable for use as an anti-glioma drug; or d. the drug is identified as a candidate anti-glioma drug.

13. A medicament comprising a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener for use in a method of suppressing a glioma in a patient, by promoting: proton (H.sup.+) leakage through the mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel.

14. A method of suppressing a glioma in a patient by promoting proton (H+) leakage through the mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel, the method comprising administering a medicament comprising a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener to the patient.

15. The medicament for use according to claim 13 or the method according to claim 14, wherein the glioma is temozolomide (TMZ) resistant.

16. The medicament for use or the method according to any one of claims 13-15, wherein the patient has been identified for treatment with the medicament comprising leak channel opener by: detecting the presence of glioma suppression in an isolated sample from the patient subsequent to contact with the medicament comprising leak channel opener, compared to a corresponding control sample that has not been contacted with the leak channel opener.

17. The medicament for use or the method according to any one of claims 13-16, wherein the glioma is glioblastoma multiforme (GBM).

18. The medicament for use or the method according to any one of claims 13-17, wherein cells of the glioma have a mitochondrial transmembrane potential (??m) that is higher than a mitochondrial transmembrane potential in cells of a glioma that is not suppressed by the medicament comprising leak channel opener, and wherein the leak channel opener decreases the mitochondrial transmembrane potential in said cells of the glioma (e.g. by promoting H.sup.+ leakage through the mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel).

19. The medicament for use or the method according to any one of claims 13-18, wherein the leak channel opener suppresses glioma growth by at least 30% compared to glioma growth pre-administration of the leak channel opener.

20. The medicament for use or the method according to any one of claims 13-19, wherein the leak channel opener promotes proton leakage through the F.sub.1F.sub.o ATP Synthase c-subunit leak channel that is at least 5% greater than proton leakage in the absence of the leak channel opener.

21. The medicament for use or method according to claim 20, wherein proton leakage through the F.sub.1F.sub.o ATP Synthase c-subunit leak channel is measurable (e.g. measured) by an assay comprising: a. admixing an isolated submitochondrial vesicle (SMV) preparation with the leak channel opener and 9-Amino-6-chloro-2-methoxyacridine (ACMA), to provide an admixture comprising: i. leak channel opener; ii. ACMA; and iii. SMV preparation; b. contacting the admixture with ATP, wherein the presence of ATP promotes translocation of protons across the SMV membrane and into the internal space of the SMV(s); c. detecting H.sup.+ external to the SMV(s) by measuring a level of fluorescence (e.g. using excitation and emission wavelengths of 410 nm and 483 nm, respectively) from ACMA in the admixture; d. comparing the level of fluorescence measured at step c) with a reference level of fluorescence for a corresponding control admixture lacking the leak channel opener; and e. confirming that the leak channel opener promotes proton leakage when the level of fluorescence is at least 5% greater compared to the reference level of fluorescence for the control admixture.

22. The medicament for use or method according to claim 20 or claim 21, wherein proton leakage through the F.sub.1F.sub.o ATP Synthase c-subunit leak channel is measurable (e.g. measured) by an assay comprising: a. admixing an isolated submitochondrial vesicle (SMV) preparation with the leak channel opener and 9-Amino-6-chloro-2-methoxyacridine (ACMA), to provide an admixture comprising: i. 5-10 ?M leak channel opener; ii. 2 ?M ACMA; and iii. 5 ?g of a SMV preparation; b. incubating the admixture for 20 minutes; c. contacting the admixture with ATP, to provide the admixture with 1 mM ATP and a volume of 40 ?l, wherein the presence of ATP promotes translocation of protons across the SMV membrane and into the internal space of the SMV(s); d. detecting H.sup.+ external to the SMV(s) by measuring a level of fluorescence (using excitation and emission wavelengths of 410 nm and 483 nm, respectively) from ACMA; e. comparing the level of fluorescence measured at step d) with a reference level of fluorescence from ACMA for a corresponding control admixture lacking the leak channel opener; and f. confirming that the leak channel opener promotes proton leakage when the level of fluorescence is at least 5% greater compared to the reference level of fluorescence for the control admixture.

23. The medicament for use or the method according to any one of claims 13-22, wherein the leak channel opener is a compound selected from the group consisting of: Donepezil-HCl, Salmeterol, Nitazoxanide, Efavirenz, Duloxetine-HCl, Febuxostat, Colistin Sulfate, Sulfadiazine, Clotrimazole, Dexchlorpheniramine Maleate, Hydroxyzine Dihydrochloride, Procarbazine-HCl, Mitoxantrone-HCl, Amiodarone-HCl, Dihydroergotamine Mesylate, Sertaconazole, Propranolol-HCl, Darifenacin-HBr, Fluvoxamine Maleate, Doxepin-HCl, Iloperidone, Telmisartan, Malathion, Acitretin, Tolterodine Tartrate, Vinblastine Sulfate, Dactinomycin (=Actinomycin D), Rifapentine, Irinotecan-HCl, Gefitinib, Dasatinib, Amlodipine, Clomipramine-HCl, Sunitinib Malate, Loxapine Succinate, Perphenazine, Tamoxifen Citrate, Thioridazine-HCl, and Cyproheptadine-HCl Sesquihydrate.

24. The medicament for use or the method according to any one of claims 13-23, wherein the leak channel opener is selected from the group consisting of: Donepezil-HCl, Salmeterol, Nitazoxanide, Efavirenz, and Duloxetine-HCl.

25. The medicament for use or the method according to any one of the claims 13-24, wherein the method comprises administering at least two such leak channel opener(s) in combination.

26. The medicament for use or the method according to any one of claims 13-25, further comprising administering temozolomide.

27. The medicament for use, or the method according to any one of claims 13-26, wherein the leak channel opener or the candidate drug has been confirmed to be a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener (e.g. has been confirmed to induce proton leakage through the F.sub.1F.sub.o ATP Synthase c-subunit leak channel) by an assay.

28. The medicament for use, or the method according to claim 27, wherein the leak channel opener or the candidate drug has been confirmed to be a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener (e.g. has been confirmed to promote proton leakage through the F.sub.1F.sub.o ATP Synthase c-subunit leak channel) by an assay comprising: a. admixing an isolated submitochondrial vesicle (SMV) preparation with the leak channel opener or candidate drug and 9-Amino-6-chloro-2-methoxyacridine (ACMA), to provide an admixture comprising: i. leak channel opener or candidate drug; ii. ACMA; and iii. SMV preparation; b. contacting the admixture with ATP, wherein the presence of ATP promotes translocation of protons across the SMV membrane and into the internal space of the SMV(s); c. detecting H.sup.+ external to the SMV(s) by measuring a level of fluorescence (using excitation and emission wavelengths of 410 nm and 483 nm, respectively) from ACMA; d. comparing the level of fluorescence measured at step c) with a reference level of fluorescence for a corresponding control admixture lacking the leak channel opener or candidate drug; and e. confirming that the leak channel opener or candidate drug promotes proton leakage when the level of fluorescence is higher (for example, at least 5% higher) compared to the reference level of fluorescence for the control admixture.

29. The medicament for use, or the method according to claim 27 or claim 28, wherein the leak channel opener or the candidate drug has been confirmed to be a mitochondrial F.sub.1F.sub.o ATP Synthase c-subunit leak channel opener (e.g. has been confirmed to promote proton leakage through the F.sub.1F.sub.o ATP Synthase c-subunit leak channel) by an assay comprising: a. admixing an isolated submitochondrial vesicle (SMV) preparation with the leak channel opener or candidate drug and 9-Amino-6-chloro-2-methoxyacridine (ACMA), to provide an admixture comprising: i. 5-10 ?M leak channel opener or candidate drug; ii. 2 ?M ACMA; and iii. 5 ?g SMV; b. incubating the admixture for 20 minutes; c. contacting the admixture with ATP, to provide the admixture with 1 mM ATP and a volume of 40 ?l, wherein the presence of ATP promotes translocation of protons across the SMV membrane and into the internal space of the SMV(s); d. detecting H.sup.+ external to the SMV(s) by measuring a level of fluorescence (using excitation and emission wavelengths of 410 nm and 483 nm, respectively) from ACMA; e. comparing the level of fluorescence at step d) with a reference level of fluorescence for a control admixture lacking the leak channel opener or candidate drug; and f. confirming that the leak channel opener or candidate drug promotes proton leakage when the level of fluorescence is higher (for example, at least 5% higher) compared to the reference level of fluorescence for the control admixture.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0400] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples, in which:

[0401] FIG. 1 shows the effect of hypoxia and mitochondrial metabolism on proliferation of primary glioma cells. A) Hypoxia decreases the rate of proliferation of primary cells from glioma with a high proliferation rate (grade IV (GBM) cells), while it has no effect on the proliferation of primary cells from glioma with a comparatively low proliferation rate (grade III cells). B) Inhibition of pyruvate entry into the mitochondria by UK5099 or inhibition of oxidative phosphorylation by rotenone or oligomycin decreases the proliferation of Grade IV (GBM) cells.

[0402] FIG. 2 shows correlation of mitochondrial membrane potential with proliferation of primary glioma cells. The mitochondrial membrane potential (measured by TMRE) is significantly higher in the primary cells from glioma with a high proliferation rate (Grade IV (GBM) cells, left), than that of the primary cells from glioma with a comparatively low proliferation rate (Grade III cells, right), demonstrating a correlation between the rate of proliferation and mitochondria activity. TMRE=tetramethylrhodamine, ethyl ester (a cell-permeant, cationic, red-orange fluorescent dye that is readily sequestered by active mitochondria).

[0403] FIG. 3 shows a schematic of the ACMA assay. The H+ fluorescent indicator 9-Amino-6-chloro-2-methoxyacridine (ACMA) and pig brain submitochondrial vesicles (SMVs) enriched in mitochondrial F.sub.1F.sub.o ATP synthase are used to measures the movement of protons into the SMVs in response to ATP hydrolysis. After the addition of ATP to the SMVs, ATPase activity results in decreased H+ concentration in the buffer solution surrounding the vesicles measured by a decrease in fluorescence intensity of the SMV-excluded H+ indicator, ACMA. The screen identified drugs that increase leak currents in the membrane and opening of the leak channel/s, reducing the ATP quenching effect.

[0404] FIG. 4 shows the effect of the ACMA screen-identified FDA approved drugs on the mitochondrial inner membrane leak currents and the proliferation of primary cells from glioma with a high proliferation rate (GBM cells). A) The positive and negative regulators of the mitochondrial leak channels identified using the FDA-approved drug library. Negative values represent reduction in ATP response as a result of drug treatment (@ 10 ?M n=4 p<0.01). B) the effect of hits from the ACMA screen on proliferation of primary cells from glioma with a high proliferation rate (GBM cells) (treatment for 5 days @10 ?M). C) Correlation of the effect of the FDA-approved drugs on leak channel opening and proliferation (Pearson correlation coefficient r=0.465). D, E) An example of the effect of the drugs on two primary cells lines from glioma with a high proliferation rate (GBM cell lines), one resistant to TMZ (D) and another that was responsive to TMZ (E).

EXAMPLES

Materials and Methods

Protocol for Isolation of Mitochondria and Preparation of Submitochondrial Vesicles (SMVs) From Pig Brains

Chemicals:

Isolation Buffer (IB):

[0405]

TABLE-US-00001 Final Concentration Volume in 500 mL 1M Sucrose 250 mM 125 mL 1M HEPES (pH 7.2) 20 mM 10 mL 0.1M EDTA 1 mM 5 mL 5% BSA 0.5% 50 mL ddH.sub.2O 310 mL 500 mL bottle; filter and store at 4? C.

Ficoll Centrifugation Buffer (FB):

[0406]

TABLE-US-00002 Final Concentration Volume (for 2 tubes) 20% Ficoll-400 12.79% 22 mL 1M Sucrose 349 mM 12 mL 1M Tris-HCl (pH 7.4) 11 mM 375 ?L 0.1M EDTA 55 ?M 18.75 ?L 50 mL tube; Store at 4? C.

Protocol:

[0407] 1. Isolation Buffer (IB) and Ficoll Centrifugation Buffer (FB) were prepared from stock solutions and were stored at 4? C. or on ice on the day of use. [0408] 2. Whole pig brains were placed in chilled IB in a petri dish on ice to rinse off blood. [0409] 3. Brains were transferred to a second petri dish with 7 mL IB for mincing (with scissors) and then transferred to 10 mL homogenising vessel chilled on ice. Minced tissue was subjected to 100 strokes with a Teflon coated pestle, rotated at 500 rpm, using a beaker of ice-water to chill the homogenising vessel during this step. [0410] 4. SPIN 1 & 2: Homogenate (with 0.5 g tissue per ml of isolation buffer) was transferred to a 50 mL tube, making up to 25 mL with IB, and centrifugated at 1,500? g, 4? C. for 10 mins. The supernatant was recovered (S1; contains cytosolic, ER, mitochondrial and synaptosomal fractions) to a 50 ml tube and was kept on ice. The pellet was resuspended (undisrupted cells, nuclei and cell debris) with 5 mL IB for a second round of homogenisation (repeat steps 4-5). [0411] 5. SPIN 3: The S1 supernatants were pooled and split between two 50 mL tubes (made up to 50 mL with IB) and centrifugated at 10,000? g, 4? C. for 15 mins. The number of tubes was increased depending on the number of batches. The pellet(s) (P2; mitochondrial and synaptosome fractions) were resuspended in 250 ?L IB (final volume should be ?500 ?L) and transferred to 1.5 ml tube(s). [0412] 6. The 1.5 mL tube(s) were placed upright and un-capped in the high-pressure vessel (max. of 6 tubes). The vessel was pressurised to 1,200 psi (?83 bar) with N.sub.2 gas. When charged, the vessel was sealed and incubated on ice for 15-20 mins. [0413] 7. During this step, the Ficoll gradient was prepared by diluting the FB with IB:

TABLE-US-00003 Ficoll % 10% 7.5% IB 2.5 mL 6 mL FB 15.5 mL 10 mL
in a pair of 13 mL ultracentrifuge tubes, 6 mL of the 10% solution was aliquoted before layering 5 mL of the 7.5% solution on top. [0414] 8. Quickly release the pressure in the vessel was quickly released; the 1.5 ml tube(s) were retrieved and the samples were pooled and mixed and placed onto the Ficoll gradient. [0415] 9. SPIN 4 (ULTRACENTRIFUGATION): The balanced 12 mL tubes were placed in the sample buckets, sealed and placed into the TH-641 rotor and centrifugated at 126,500? g, 4? C. for 20 mins. [0416] 10. Following the spin, the 7.5% Ficoll layer (including the top layer), the synaptosome layer at the 7.5%/10% gradient boundary and the 10% Ficoll layer were removed. [0417] 11. SPIN 5 (WASH): The pellets of purified mitochondria were resuspended in 100 ?L IB, using a chilled 3 mL glass pestle. The suspension was triturated, transferred to a 1.5 mL tube and centrifugated at 16,000? g, 4? C. for 10 mins. [0418] 12. The supernatant was discarded, and the pellet(s) were re-suspended in IB at 5? the biomass then incubated with an equal volume of 1% digitonin, on ice, for 15 mins. The samples were mixed at 5 min intervals. [0419] 13. SPIN 6 & 7 (WASH): The solution(s) were made up to 1 mL with IB and centrifugated at 16,000? g, 4? C. for 10 mins. The supernatant was discarded, and the pellets were resuspended in 1 mL IB and centrifugated at 16,000? g, 4? C. for 10 mins. [0420] 14. The supernatant was discarded, and the pellets were resuspended in IB at 5? the biomass then incubated with a 1/100 dilution of 20% Lubrol PX (C12E9) solution, on ice for 15 mins. The samples were mixed at 5 min intervals. [0421] 15. SPIN 8 (ULTRACENTRIFUGATION): The sample(s) were layered onto the IB and centrifugated at 182,000? g, 4? C. for 60 mins. [0422] 16. SPIN 9 (WASH): The supernatant was discarded, and the pellet of submitochondrial vesicles (SMVs) were resuspended in 100 ?L IB, using the chilled 3 mL glass pestle to break up the pellet before triturating the suspension. The samples were centrifugated at 16,000? g, 4? C. for 10 mins. [0423] 17. The supernatant was discarded, and the pellet(s) were resuspended in IB at 5? the biomass. The samples were aliquoted in 50-100 ?L volumes, snap frozen in liquid N.sub.2 and stored at ?80? C.

Cells

[0424] The cell lines employed were low-passage lines, derived from tumour tissue. The cells were obtained from tumour tissue of a highly proliferative glioma (which was a grade IV/glioblastoma multiforme glioma). For comparison, cells were also obtained from a tumour tissue of a comparatively slow-growing glioma (which was a grade III glioma).

ACMA (9-amino-6-chloro-2-methoxy acridine) Assay Protocol

Equipment and Materials:

[0425] ACMA assay buffer (concentrations used throughout the assay):

TABLE-US-00004 HEPESKOH 10 mM MgCl.sub.2 5 mM KCl 100 mM pH (HCl) 7.5
ACMA solution: [0426] Stock solution of 1 mg/ml (3.87 mM) ACMA in 100% ethanol [0427] Diluted to working solution (3 ?M) in assay buffer [0428] Aliquots of 10 ?L to be added to wells

Protocol:

[0429] 1. Sub-mitochondrial vesicles (SMVs) isolated from pig brains were thawed and prepared to 0.17 ?g/?L in ACMA assay buffer. [0430] 2. Drug solutions were added to 386-well plates at a final concentration of 10 ?M. [0431] 3. Sub-mitochondrial vesicles (SMVs), isolated from pig brains, were thawed and prepared to 0.17 ?g/?L in ACMA assay buffer. 30 ?L of this solution (containing 5 ?g of polypeptide, as measured via the QuickStart Bradford Protein Assay (Bio-Rad, UK)) was added to each well. [0432] 4. Stock solution of 1 mg/ml (3.87 mM) ACMA (9-amino-6-chloro-2-methoxyacridine) were diluted to working solution 3 ?M in assay buffer. Aliquots of 10 ?L were added to each well (final concentration of 0.75 ?M). [0433] 5. The samples were mixed and incubated at room temperature for 20 mins. [0434] 6. The ACMA fluorescence was measured at 1 min intervals using a Tecan Infinite M200 plate readerexcitation 412 nm, emission 480 nm. [0435] 7. Working solution (40 mM) of ATP was prepared in assay buffer just prior to use and added at a final concentration of 1 mM to the wells, following the first 2 baseline recordings. [0436] 8. Fluorescence was measured quickly after addition of ATP for another 7 mins (1 min intervals).

Protocol for Sulforhodamine B (SRB) Colorimetric Assay: Cell Density Measurement, Based on the Quantity of Cellular Protein Content

Materials:

[0437] 50% (wt/vol) trichloroacetic acid (TCA)

[0438] 1% (vol/vol) acetic acid

[0439] 0.4% SRB in 1% (vol/vol) acetic acid

[0440] 10 mM Tris base solution (pH 10.5) (121.14 g/mol; 1.21 g in 1 litre)

Assay:

[0441] 1. Without removing the cell culture medium, gently add 50% cold TCA to each well (the final concentration of TCA should be 10%), and incubate the plates at 4? C. for 1 h.
For 150 ?L medium volume in each well, add 30 ?L of 50% TCA. [0442] 2. Wash the plates 4 times with slow-running tap water. Then allow the plates to air-dry at RT.
After fixing and drying, the plates can be stored indefinitely at RT. [0443] 3. Add 0.4% SRB solution to each well (e.g. 80 ?L/well). Leave at room temperature for 1 h (no shaking) and then quickly rinse the plates 4 times with 1% acetic acid to remove unbound dye. Then allow the plates to dry at RT.
Stained and dried plates can be stored indefinitely at RT. [0444] 4. Add 10 mM Tris base solution to each well (e.g. 80 ?L/well) and shake the plate for 5 min to solubilize the protein-bound dye. Alternatively, if a shaker is not available, SRB can be solubilized after 30 min in 10 mM Tris base solution. [0445] 5. Measure the OD at 490 nm in a microplate reader. The suboptimal wavelengths should be 490-530 nm.

Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE) Assay

[0446] TMRE (Thermo Fisher Scientific, UK) was added directly to the GBM and astrocytoma cultures at a concentration of 10 nM. The cultures were then incubated for 20 min. at 37? C. and imaged, using a confocal microscope (Ex/Em=550/575 nm). The intensity of signal was quantified using ImageJ.

Glioma Suppression Assay

[0447] 1000 Grade IV cells (cell line 1 in FIG. 2) were plated in cell culture medium (10% FBS in DMEM) per each well in 96-well plates. On day 1, the cells were treated with 10 ?M of the indicated drugs and incubated at 37? C. for 5 days. SRB assay was performed prior to addition of the drugs and after 5 days. The values were normalized against those of the control (untreated cultures).

EXAMPLE 1

Demonstrating a Subgroup of Glioma Having Enhanced Mitochondrial Coupling and Efficiency in ATP Synthesis Through the Closure of the Inner Membrane Leak Channels

[0448] The inventors compared the metabolic and mitochondrial energetic profile of slow-growing glioma/astrocytoma cells (chosen in particular due to their relatively slow growth rate) to that in cells of a highly proliferative glioma primary tumour cells by performing hypoxia experiments with primary tumour cells, in which primary tumour cells were deprived of adequate oxygen supply and compared with control cells not so deprived (normoxia). The relatively lower rate of proliferation-cells were chosen as controls to demonstrate that higher efficacy of the drugs is seem in the more proliferative cells. The slow growing cells were selected from a grade III glioma, and the highly proliferative cells were chosen from a grade IV glioma i.e. glioblastoma multiforme cells. Interestingly, it was found that the rate of proliferation in glioblastoma multiforme (GBM, grade IV astrocytoma) primary tumour cells was significantly reduced under hypoxia, suggesting that they rely more on their mitochondria than the less proliferative glioma cells (FIG. 1A).

[0449] To further investigate this, primary tumour cells were incubated (both under hypoxia and normoxia) with the toxins rotenone (100 nM), oligomycin (50 nM), a combination of rotenone and oligomycin (100 nM and 50 nM, respectively) or UK5099 (10 ?M). These toxins, which act by inhibiting mitochondrial respiratory complexes and oxidative phosphorylation, impeded the proliferation of GBM cells, without a significant effect on the grade III astrocytoma cells (FIG. 1B). Furthermore, a direct correlation was observed between mitochondrial membrane potential (measured by TMRE) and the rate of cellular proliferation, demonstrating that highly proliferative glioma cells employ efficient mitochondrial metabolism to achieve their higher proliferation rates (FIG. 2). These results demonstrate that enhanced mitochondrial coupling and efficiency in ATP synthesis through closure of the inner membrane leak channels contribute to the proliferation of high energy demand glioma cells.

EXAMPLE 2

Screening for Drugs That Induce Proton Leakage Through the F.SUB.1.F.SUB.o .ATP Synthase c-Subunit, and Thus Suppress Highly Proliferative Glioma Subgroups

[0450] The results of Example 1 suggest modulation of the mitochondrial leak channels (to induce proton leakage) as a therapeutic approach for treatment of highly proliferative glioma tumours. More particularly, it was postulated that inducing proton (H.sup.+) leakage through F.sub.1F.sub.o ATP synthase c-subunit may perturb the surprisingly high level of mitochondrial efficiency in this highly proliferative glioma subgroup. The inventors thus investigated whether modulation of this leak channel (inducing leakage) causes glioma suppression.

ACMA assayIdentifying Candidate Drugs

[0451] To investigate this, the inventors first conducted a screen to identify candidate anti-glioma drugs. In more detail, to identify the pharmacological agents that may reduce the efficiency of mitochondrial metabolic processes through increasing the ion leak currents associated with the F.sub.1F.sub.o ATP synthase (e.g. within the c-subunit), the inventors developed a high throughput fluorometric assay, using the H.sup.+ fluorescent indicator 9-Amino-6-chloro-2-methoxyacridine (ACMA) and isolated swine brain submitochondrial vesicles (SMVs), which are enriched for the F.sub.1F.sub.o ATP synthase. This assay measures the movement of H.sup.+ ions into the SMVs in response to ATP hydrolysis. After the addition of ATP to the SMVs, ATPase activity results in a decrease in the H.sup.+ concentration in the bath surrounding the vesicles which is measured by a decrease in fluorescence intensity of the SMV-excluded H.sup.+ indicator, ACMA (FIG. 3) (Alavian, K. N. et al .; Nat Cell Biol 13, 1224-1233, doi:10.1038/ncb2330 (2011). As negative control the ATP response was attenuated by either inhibition of H.sup.+ ion import through the F.sub.o pump (by addition of oligomycin) or by leakage of H.sup.+ out of the SMVs (by carbonylcyanide p-trifluoromethoxyphenylhydrazone, FCCP). Using this assay, the effect of 786 drugs (approved by the FDA for clinical use) were tested, as well as a library of 12,000 new small molecules on the inner membrane leak current activity. The screen identified 40 FDA-approved drugs as regulators of proton leak conductance (n=4; p<0.01) (FIG. 4A).

Glioma Suppression Assay

[0452] Having identified candidate anti-glioma drugs via the above-mentioned screen, the inventors proceeded to validate the suppressive effect of these drugs on isolated, patient-obtained glioma biopsies.

[0453] In more detail, the inventors examined the effect of the positive hits, as well as TMZ (the standard chemotherapeutic agent for treatment of adult and paediatric gliomas), on proliferation of GBM cells in vitro. In this hit confirmation assay, 35 of the 40 drugs reduced the proliferation of GBM1 cells by 12-96% (n=4, p<0.01) (FIG. 4B). These results provided validation of the assay, as the vast majority of the drugs that attenuated the effect of ATP in the ACMA screen reduced the proliferation of GBM cells (Pearson correlation coefficient r=0.465) (FIG. 4C). Interestingly, TMZ was effective in slowing down proliferation of a subset of GBM cell lines and had no effect on the cell line (GBM1) with the highest rate of proliferation, while selected candidate drugs significantly reduced the rate of proliferation in this specific cell line (FIG. 4D,E). These results demonstrate that metabolic reprogramming through modulation of mitochondrial ion leak currents (as a standalone therapy and/or as part of a combination therapy) to be an effective method for treatment of malignant glioma. Advantageously, this two-step method (ACMA plus glioma suppression assays) allows identification of the most effective pharmacological agents and establishes a pathway for optimal treatment of GBM in future clinical trials.

[0454] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.