EP2855532A1 - Identification et expression fonctionnelle du transporteur mitochondrial de pyruvate, mpc1 - Google Patents

Identification et expression fonctionnelle du transporteur mitochondrial de pyruvate, mpc1

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Publication number
EP2855532A1
EP2855532A1 EP13727081.5A EP13727081A EP2855532A1 EP 2855532 A1 EP2855532 A1 EP 2855532A1 EP 13727081 A EP13727081 A EP 13727081A EP 2855532 A1 EP2855532 A1 EP 2855532A1
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Prior art keywords
pyruvate
isolated
modulator
family
import
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German (de)
English (en)
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Sébastien HERZIG
Jean-Claude Martinou
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Universite de Geneve
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Universite de Geneve
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the invention also relates to modulators of the Pyruvate import activity for use in therapy of disorders related to pyruvate uptake.
  • the invention further relates to the screening of drugs for disorders related to pyruvate uptake.
  • pyruvate is converted into acetyl-CoA by the pyruvate dehydrogenase (PDH) complex and participates in the synthesis of branched-chain amino acids (BCAA) in yeast.
  • PDH pyruvate dehydrogenase
  • BCAA branched-chain amino acids
  • Acetyl - CoA donates carbon atoms to the citric acid cycle and participates in the synthesis of octanoic acid, the precursor of lipoic acid.
  • Lipoic acid is an essential cofactor of several miilti-subunit complexes in the mitochondrial matrix, including PDH, a-keloglutarate dehydrogenase (a -K.DH) and the branched-chain keto acid dehydrogenase (BCKDH).
  • Acetyl-CoA which feeds the TCA cycle, generates reductive power that is used for energy production through the respiratory chain.
  • Import of pyruvate across the inner mitochondrial membrane (IMM) requires a specific carrier whose molecular identity has not been established.
  • MPC mitochondrial pyruvate carrier
  • One object of the present invention is to provide an isolated and purified mitochondrial Pyruvate carrier (MPC) family comprising the heterocomplex association of two members of a family of membrane proteins consisting of an isolated and purified MPC1 member (Brp44L) together with an isolated and purified MPC2 member (Brp44), wherein said isolated and purified MPC family is capable of transporting Pyruvate into mitochondria.
  • MPC mitochondrial Pyruvate carrier
  • FIGURES Figure 1 Plienotypes of yeast deleted for MPC genes.
  • A-B Protein immunoblot analysis of cell extracts using antibodies raised against lipoic acid in strains and media as indicated. Ponceau staining is shown as loading control. The blots are representative of at least three independent experiments.
  • C PDH activity of mitochondria isolated from the indicated strains grown in lactate medium. N.D: not detected. Data is expressed as the mean +/- SD of three independent experiments.
  • MPCl and MPC2 are conserved inner mitochondrial membrane proteins.
  • MPC2 NP 001137146.1
  • Pan troglodytes MPCl: XP_0()1137474.1
  • MPC2 XP 001174837.1
  • Bos taurus MPCl: NP_001070510.1, MPC2: Xl ⁇ _001174837.1
  • Mus musculus MPCl NP_061289.1.
  • MPC2 NP_081706.1: Rattus norvegicus MPCl: NP 598245.1; Gallus gallus MPCl: NP 001026695.1, MPC2: XP 001231387.2; Nematostella vectensis MPCl: A7SC70, MPC2: A7SQZ7; Anopheles gambiae MPCl: XP_321716.4, MPC2: XP_314997.3; Drosophila me!anogaster MPCl: NP_650762.1, MPC2:
  • Mpcl is an inner mitochondrial membrane protein in yeast.
  • Figure 8 Model of the fate of pyruvate inside the mitochondria.
  • Pyruvate serves as a precursor for synthesis of BCAA or as a source of acetyl-CoA via PDH for the Krebs cycle and lipoic acid synthesis.
  • Lipoic acid is required for PDH and BCKDH activity, which bypasses PDH and allows generation of acetyl-CoA from leucine.
  • Hfal is the mitochondrial acetyl- CoA carboxylase, which converts acetyl-CoA to malonyl-CoA.
  • Lip5 is the lipoic acid synthase essential for the conversion of octanoic to lipoic acid.
  • Mitochondrial membrane potential was assessed by flow cytometry in the indicated strains grown in lactate medium and stained with 50nM rliodamine 123 (Rh 123) in the absence or presence of 20mM azide (Rhl23 + azide) by flow cytometry. Data are mean +/- SD of three independent experiments.
  • SEQ ID NO: 1 represents the amino acid sequence of M PCI in Human (Homo sapiens)
  • SEQ ID NO: 2 represents the amino acid sequence of MPC2 in Human (Homo sapiens)
  • SEQ ID NO: 3 represents the amino acid sequence of MPC3 in Saccharomyces cerevisiae
  • SEQ ID NO: 4 represents the DNA sequence of hMPCl in Human (Homo sapiens)
  • SEQ ID NO: 5 represents the DNA sequence of hMPC2 in Human (Homo sapiens)
  • SEQ ID NO: 6 represents the artificial shRNA sequence against MPC! .
  • subject or “patient” are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human,
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures.
  • Those in need of treatment include those already with the disorder, for example cancer, as well as those in which the disorder, for example cancer, is to be prevented.
  • the mammal to be treated herein may have been diagnosed as having the disorder, for example cancer, or may be predisposed or susceptible to the disorder, for example cancer.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals or pet animals, such as dogs, horses, cats, cows, monkeys etc. Preferably, the mammal is human.
  • the term "therapeutically effective amount” refers to an amount of a drag effective to treat a disease or disorder in a mammal.
  • the therapeutically effective amount of the drag may reduce the number of cancer cells; reduce the tumour size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; inhibit, to some extent, tumour growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • the drag may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to prevent, or preferably reduce by at least about 30 percent, preferably by at least 50 percent, preferably by at least 70 percent, preferably by at least 80 percent, preferably by at least 90%, a clinically significant change in the growth or progression or mitotic activity of a target cellular mass, group of cancer cells or tumour, or other feature of pathology.
  • cancer refers to or describe the physiological condition in mammals mammals that is typically characterized by unregulated cell growth. According to the present invention, cancer refers preferably to.
  • MPC Mitochondrial Pyruvate Carrier
  • the present invention provides an isolated and purified mitochondrial Pyruvate carrier (MPC ) family comprising the heterocomplex association of two members of a family of membrane proteins consisting of an isolated and purified MPCl member (Brp44L) together with an isolated and purified MPC2 member (Brp44), wherein said isolated and purified MPC family is capable of transporting Pyruvate into mitochondria.
  • MPC mitochondrial Pyruvate carrier
  • the mitochondrial pyruvate carrier consists in the mandator)' association of a heterodimer of MPC1/MPC2.
  • MPC mitochondrial Pyruvate carrier
  • the isolated and purified mitochondrial Pyruvate carrier (MPC) is a heterodimer of human MPC1/MPC2.
  • the isolated and purified mitochondrial Pyruvate carrier (MPC) family further comprises the association of a third member of a family of membrane proteins consisting of an isolated and purified MPC3 member.
  • the modulation of the transport of Pyruvate into mitochondria has direct consequences in the treatment or prevention of cancer or diabetes as well as in treating or preventing ischemia reperfusion injury.
  • Cancers to be treated according to the present invention include osteosarcoma, Breast cancer, Small lung cell cancer, Gliomas and glioblastoma, Colon cancer as well as leukemia
  • the term "diabetes” or “insulin resistance” refers to a state in which a normal amount of insulin produces a subnormal biologic response relative to the biological response in a subject that does not have insulin resistance.
  • An “insulin resistance disorder” refers to any disease or condition that is caused by or contributed to by insulin resistance.
  • protein refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not refer to, or exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, natural amino acids, etc.), polypeptides with substituted linkages as well as other modifications known in the art, both naturally and non-natural ly occurring.
  • a homologous sequence is taken to include an amino acid sequence which is at least 60, 70, 80 or 90% identical, preferably at least 95 or 98% identical at the amino acid level over at least 20, 50, 100, 200, 300 or 400 amino acids with the amino acid sequences of Brp44L and Brp44.
  • homology should typically be considered with respect to those regions of the sequence known to be essential for the function of the protein rather than non-essential neighbouring sequences.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • sequence identity i.e. amino acid residues having similar chemical properties/functions
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences. Percentage (%) homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
  • isolated is substantially purified when at least about 60 to 75% of a sample exhibits a single polypeptide sequence.
  • a substantially purified protein will typically comprise about 60 to 90% W/W of a protein sample, more usually about 95%, and preferably will be over about 99% pure.
  • Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel. For certain memeposes, higher resolution may be provided by using HPLC or other means well known in the art which are utilized for application.
  • a MPC protein is substantially free of naturally associated components when it is separated from the native contaminants that accompany it in its natural state.
  • a polypeptide that is chemically synthesised or synthesised in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
  • a protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • a polypeptide produced as an expression product of an isolated and manipulated genetic sequence is an "isolated polypeptide," as used herein, even if expressed in a homologous cell type.
  • Synthetically made forms or molecules expressed by heterologous cells are inherently isolated molecules.
  • the isolated and purified mitochondrial Pyruvate carrier (MPC) family of the invention is capable of transporting Pyruvate into mitochondria of mammalian cells, preferably human cells.
  • the isolated and purified mitochondrial Pyruvate carrier (MPC) family of the invention is also capable of transporting Pyruvate into mitochondria of yeast.
  • Mpcl Ygl OSOw
  • Mpc2 Yhrl62w
  • the modulator of the pyruvate import can be either a shRNA directed against MPC 1 or MPC2 or a small molecule.
  • a modulator compound of the invention can include, but is not limited to: nucleic acids, peptides, proteins such as antibodies, sugars, polysaccharides, glycoproteins, lipids, and small organic molecules.
  • a “modulator compound” is a compound that can be tested in the screening assay of the present invention.
  • small molecule or small organic molecule typically refers to molecules of a size comparable to those organic molecules generally used in pharmaceuticals. Small organic molecules generally exclude biological polymers (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da., more preferably up to 2000 Da., and most preferably up to about 1000 Da.
  • the modulator compound can be at least one of a metal, a peptide, a protein, a lipid, a polysaccharide, a nucleic acid, a library of small organic molecules, and a drug.
  • the modulator compound of the invention can include a fluorescent molecule, a radionuclide, a protein tag or combination thereof.
  • modulator of the Pyruvate import activity of the invention can be an inhibitor in the form of a nucleic acid.
  • the nucleic acids modulator of the invention encompasses siRNA molecules, shRNA molecules, miRNA molecules, and antisense nucleic acid molecules.
  • a shRNA nucleic acid molecule refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • a preferred modulator of the Pyruvate import activity of the invention acting as an inhibitor is a sliRNA of SEQ ID NO: 6 directed against MPCl .
  • said shRNA is capable of reducing the growth of osteosarcoma cells.
  • a lentiviras expressing a shRNA against MPCl is capable of reducing the growth of osteosarcoma cells.
  • orthologs As used herein, orthologs, homologs, functional fragments, or mutant forms of shRNA of SEQ ID NO:
  • nucliec acids having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100%, identity to, or homologous with the SEQ ID NO: 6 or nucleic acid sequence that hybridizes to the coding region of the nucleic acid sequence in SEQ II ) NO: 6 or complementary sequences thereof under "stringent hybridization conditions" as is defined herein and commonly used in the art of molecular biology, for example, in: Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridisation with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).
  • the modulator of the Pyruvate import activity of the invention when acting as an inhibitor may be used in a method of treating or preventing cancer or diabetes (see example 4.3 ).
  • Cancers to be treated according to the present invention include osteosarcoma, Breast cancer, Small lung cell cancer, Gliomas and glioblastoma, Colon cancer as well as leukemia.
  • the modulator of the Pyruvate import activity of the invention when acting as an inhibitor, may be used in a method of treating or preventing ischemia rcperfusion injury (see example 4.4).
  • shRNAs directed against MPC1 or MPC2 can alter expression of the MFC and thereby decrease the activity of the carrier. Expression of these shRNA is mediated by lenti viruses (see example 4.3).
  • Another object of the invention is to provide an in vitro method (or screening method) of identifying modulator candidates of the Pyruvate import activity comprising the steps of:
  • modulator candidates that decrease the import of pyruvate are inhibitors of the MPC activity whereas modulator candidates that increase the import of pyruvate are activators of the MPC activity.
  • the added labeled pyruvate of the invention can include a fluorescent molecule, a radionuclide, a protein tag or combination thereof.
  • the added labeled pyruvate is radioactive.
  • the modulator candidates acting as inhibitors of the MPC activity are used in a method of treating or preventing cancer or diabetes as well as in a method of treating or preventing ischemia reperfusion injury.
  • Preferred cancers to be treated according to the present invention include osteosarcoma, Breast cancer, Small lung cell cancer, Gliomas and glioblastoma, Colon cancer as well as leukemia.
  • the surprising advantage of the in vitro method (or screening method) of identifying modulator candidates resides in the use of Lactococcus lactis as an expression system and in the expression of said two human proteins in this artificial system. Importantly, these two human proteins are able to reconstitute a functional pyruvate earner in the bacteria.
  • the screening method according to the present invention requires the expression of both MPCl and MPC2 in the membrane of bacteria. Thus the use of the entire heterodimer according to the invention is required for the screening method to be functional and effective.
  • the screening method according to the present invention requires that the heterodimer be expressed in a membrane, such as the cell membrane of a bacterium.
  • the assay cannot function with purified soluble proteins as the mitochondrial pyruvate transporter heterodimer has to reside in a membrane to be functional and as pyruvate transport can only be measured across a membrane.
  • This method is also characterized by the use of bacteria that do not have mitochondria and do not express the mitochondrial pyruvate carrier endogeneously.
  • the screening method according to the invention is based on the expression of the human mitochondrial pyruvate carrier in bacteria that naturally do not contain the latter (i.e. introduction into a cell that does not contain it). This allows the measurement of pyruvate transport by the exogeneously introduced mitochondrial pyruvate transporter with insignificant background noise from endogeneous pyruvate transport.
  • the bacteria or prokaryotic cells able to express both MPCl and MPC2 members at their surface and preferably expressing a human MPC1/MPC2 heterodimer in their cell membrane is lactococcus lactis.
  • bacteria i.e. Lactoccus lactis
  • MPCl a cell-derived protein
  • MPC2 a cell-derived protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based protein-based on the bacteria.
  • the bacteria are isolated from the culture medium by filtration and the radioactivity is measured. The higher the radioactivity, the higher the import of pyruvate into the bacteria.
  • the goal is to identify chemical compounds that can modulate pyruvate import.
  • Modulator candidates i.e.
  • the screening assay of the present invention includes the screening of one or more modulator candidate compounds in the form of a library of compounds.
  • the libraiy of compounds comprises a combinatorial chemical library.
  • Combinatorial chemical libraries can include a plurality of small organic molecules.
  • the combinatorial chemical library can contain at least 1000 candidate compounds.
  • the candidate compound is a small organic molecule.
  • a further object of the invention is a kit for identifying modulator candidates of the Pyruvate import activity comprising bacteria able to express both MPC1 and MPC2 members at their surface and optionally radioactive pyruvate.
  • the bacteria able to express both MPC1 and MPC2 members at their surface is lactococcus lactis.
  • the identified modulator candidates acting as inhibitors of the MFC activity are useful in the treatment or prevention of cancers or diabetes as well as in the treatment or prevention of ischemia reperfusion injury.
  • Preferred cancers to be treated according to the present invention include osteosarcoma, Breast cancer, Small lung cell cancer, Gliomas and glioblastoma, Colon cancer as well as leukemia.
  • the kit comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds the compound's composition or the pro-drug composition or pharmaceutically acceptable salts thereof that are effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the label or package insert indicates that the composition is used for treating the condition of choice, such as cancer.
  • the present invention also includes pharmaceutical compositions and formulations that include the modulator compounds of the invention.
  • the pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • Applicants have identified a family of membrane proteins (pfam UPF0041) whose members are necessary and sufficient for the transport of pyruvate into mitochondria. Applicants have renamed this family the Mitochondrial Pyruvate Carrier (MPC) family.
  • MPC Mitochondrial Pyruvate Carrier
  • MPC1 (formerly Brp44L) in a proteomic analysis of the IMM of mouse liver (J).
  • MPC1 and its paralog MPC2 (formerly Brp44) have unknown function. Both are IMM proteins with three predicted transmembrane a -helices on the basis of secondary structure predictions and hydropathy profiling (Fig. 5). They share sequence similarity with yeast Mpcl (YglOSOw), Mpc2 (Yhrl62w) and Mpc3 (Ygr243w) (Fig. 5 C and D).
  • yeast Mpcl yeast Mpcl
  • Mpc2 Yhrl62w
  • Mpc3 Ygr243w
  • MPC3 had no visible effect on growth.
  • I .ipoie acid is covalently attached to the E2 subunits of PDH (Latl) and a -KDH (Kgd2), the E3- binding protein of PDH (Pdxl) and the H subunit of the glycine cleavage system (Gcv3).
  • This modification can be readily assessed by protein immunoblotting with antibodies to lipoic acid (10).
  • WT, mpcl A, mpc2A, mpc3A and mpc2A mpcSA cells had similar amounts of lipoylated complexes (Fig. 2 A).
  • MFC proteins appeared to act upstream of PDH and may function in the transport of pyruvate into mitochondria.
  • Applicants measured uptake of !4 C pyruvate in mitochondria isolated from WT, mpcl A, mpc2A, mpcSA and mpc2A mpc3A cells grown in lactate medium (Fig. 3 A). The specificity of uptake was assessed by the use of UK5099, an non specific inhibitor of the mitochondrial pyruvate carrier (4). Uptake of pyruvate in WT mitochondria was sensitive to the proton ionophore CCCP (Fig. 3B). Mitochondria from mpcl A and mpc2A mpc3A cells showed decreased pyruvate uptake (Fig.
  • mMPCl murine MPC1
  • MPC2 MPC2
  • mMPC2 murine MPC1
  • mMPC2 failed to restore growth of the double deletion strain of its orthologous genes MFC 2 and MFC 3.
  • growth of the triple deletion strain m pc I ⁇ mpc2 ⁇ cells was restored by coexpression of both mMPCl and mMPC2 (Fig. 4A).
  • mMPCl and mMPC2 together functionally complement the absence of pyruvate transport.
  • Fig. 4C bacterium Lactococcus lactis
  • Fig. 4D and E bacterium Lactococcus lactis
  • the mitochondrial pyruvate carrier is composed of Mpcl and either Mpc2 or Mpc3 in yeast and of MPC 1 and MPC2 in mammals.
  • Yeast strains used in this study are derived from BY4741 requiring only uracil (ura) or uracil and histidine (his ura) and are listed in table SI .
  • Cells were grown according to standard procedure. SD medium was supplemented with 20 mg/1 uracil and 100 mg/1 valine or leucine when indicated (SD when indicated (SD +V and SD + L respectively).
  • SD medium was supplemented with 20 mg/1 uracil and 100 mg/1 valine or leucine when indicated (SD when indicated (SD +V and SD + L respectively).
  • SD +V and SD + L for cells containing a plasmid, uracil was oinitted.
  • SC -V -L contains all amino acids (at 50 mg/l) except valine and leucine.
  • MPC1 (ygl080w), MPC2 (yhrl62w) and MPC3 (ygr243w) genes were cloned by PCR from BY4741 yeast genomic DNA into p2U vector (2 ⁇ , GPD promoter).
  • Mouse MPC1 (NM_018819.4) and mouse MPC2 (NM 027430.2) coding sequences were optimized for yeast codon usage and synthesized by GenScript Inc (USA) and cloned downstream up the GPD promoter into pRS423 and pRS426 vectors respectively. Wild type his ura cells were cotransfonned with two vectors to generate cells expressing no protein, either protein alone or both proteins together.
  • BCAA decarboxylation Briefly, 500 ⁇ of exponentiaily growing cells were incubated for 15 min with 20 ⁇ 1-14C valine or leucine in SD medium at 30°C during which 14 C02 was trapped by a filter paper pre- soaked in NaOH. The reaction was terminated with acetic acid and the incubation was continued for 1 hour to trap all the 14 C02. The filters were then transferred to a scintillation vial and radioactivity measured by liquid scintillation counting. The reaction proved to be linear over this time frame. The decarboxylation is expressed as nmol C02 released per OD unit per hour.
  • Yeast mitochondria were isolated from cells grown in lactate medium, unless otherwise stated. Mammalian mitochondria were isolated from 293T cells using a Dounce homogenizer. Nuclei and cell debris were removed (2000g) and mitochondria were recovered by centrifugation at 10'OOOg.
  • ⁇ ⁇ of mitochondria (5mg/ml) isolated from yeast or 293T cells were added to 90 ⁇ 1 of isolation buffer or 90 ⁇ 1 of 2()m.Vl Hepes (swelling) or 90 ⁇ 1 of isolation buffer containing 1% Triton XI 00. 5 ⁇ ig proteinase K was added when required and incubated on ice for 30 minutes. ImM PMSF was 1 mM PMSF was added to inhibit the proteinase K and all samples were precipitated with 12% TCA and resolved on SDS-PAGE followed by protein immunoblotting. For sodium carbonate extraction ⁇ ⁇ mitochondria were mixed with 90 ⁇ ] 20 mM Hepes and incubated for 20 minutes on ice.
  • mitochondria were solubilized in IP buffer (150 mM NaCl, 10 mM I ris pH 7.4, 10% glycerol, 1 % digitonin) on ice for 30 minutes and centrifuged at 100 ⁇ 00 g for 1 hour.
  • IP buffer 150 mM NaCl, 10 mM I ris pH 7.4, 10% glycerol, 1 % digitonin
  • Protein G beads (GE healthcare) were incubated with 2 ⁇ anii HA (Covance Inc.) for 2 hours at
  • PDH and a-KDH activities were assessed using 3-Acetylpyridine adenine dinucleotide (APAD) in place of NAD+ to avoid reoxidation. Reactions were initiated by the addition of 1 mM substrate (pyruvate or u-ketoglutarate respectively). Activities are expressed as nmol reduced APAD per minute per mg of mitochondrial protein (mU/mg).
  • APAD 3-Acetylpyridine adenine dinucleotide
  • Pyruvate import was assessed in isolated mitochondria by the inhibitor-stop method as described previously (I) with minor modifications. Briefly, mitochondria were suspended at 10 mg/ml in import buffer (120 mM KG, 5m M NaCl, 2 mM potassium phosphate pf 17.4) and transferred to the same buffer at pi I 6.8 containing 50 uM 2-14C pyruvate to initiate import. UK5099 was used to terminate the reaction or was present before mitochondria as control. Mitochondria were spun down, washed with import buffer containing 50 mM cold pyruvate and radioactivity was quantified in the pellet by liquid scintillation counting. Radioactivity in control sample was subtracted from ail time points to determine the inhibitor-sensitive import. Rate of pyruvate import were calculated from the first two minutes of the reaction. Pyruvate import in bacteria
  • Bacteria were grown overnight in Ml 7 containing 1% glucose, 1 Opg/ml chloramphenicol and lO ⁇ tg/ml erythromycin when needed, diluted to an 01)600 of 0.1 , further grown for 3 hours before induction of expression with lng/1 Nisin A. Import was measured after 20 hours of induction by incubating bacteria in PBS with 37.5 ⁇ [2- ,4 C] pyruvate for 10, 20, 30 or 60 minutes at room temperature followed by filtration over nitrocellulose ester filters. The filters were washed twice with cold PBS, transferred to a scintillation vial followed by liquid scintillation counting. Protein immunoblotting was performed on the same cells to assess the expression level of the proteins.
  • Multicopy suppressor screen MCS
  • mpclA cells were transformed with a homemade gDNA library in YEP 13 vector and plated on medium lacking leucine, in order to select for both transformation events and rescue of growth without leucine. 23 independent clones were isolated, which all contained a plasmid with MPC1 gene.
  • mpclA cells were transformed with a pool of 1588 plasm ids covering 97% of the S. cere vis iae genome (Open Biosystems). Cells were plated on SD plate containing isoleucine, a condition where the growth defect of mpclA was stronger and allowed better identification of rescued clones. In this screen, 38 independent clones were isolated and all contained the WT MFC 7 ORF.
  • Human MPC1 (hMPCl) and MPC2 (hMPC2) are codon optimized for expression in L. lactis.
  • KMPC2 is subcloncd downstream of the nisin promoter in pNZ8148 vector that contains a chloramphenicol resistance cassette.
  • hMPCl is subcloncd downstream of the nisin promoter in a modified pNZ8148 vector, in which the chloramphenicol cassette is replaced by the erythromycin resistance cassette of the vector pi 1, 253.
  • Bacteria are grown overnight in Ml 7 Broth containing 1 % glucose, 10 ug/ml chloramphenicol and 10 t ug/ml erythromycin, diluted to an OD600 of 0.1, further grown for 3 hours before induction of expression with lng/1 Nisin A.
  • Import is measured after 20 hours of induction by incubating bacteria for 60 minutes at room temperature in PBS containing 37.5 ⁇ [2- !4 C
  • a compound of a chemical library is added at 20 ⁇ in 50 or 25 ⁇ Mix before addition of 50 or 25 ⁇ bacteria (10 OD/ml).
  • the mitochondrial pyruvate inhibitor UK5099 (Sigma), is used as a control. After incubation for 1 hour, the plate is transferred to a MultiScreen HT S vacuum manifold and the bacteria isolated by filtration. 50 or 25 ⁇ scintillation fluid is added to each well before counting using a microplate reader. IC50s are determined for each compound that shows an inhibitory activity in the first screen. Compounds with an IC50 ⁇ 1 mM (Hits) are selected for further assays.
  • Osteosarcoma cells (143 B cells) are cultured in 96 well plates. Compounds are tested at 10 ⁇ and the pi 1 and oxygen consumption measured during a 12-hour period using a Seahorse XF-96 apparatus. Compounds that significantly decrease the pH and oxygen consumption are tested for their specific activity on the mitochondrial pyruvate carrier using isolated mitochondria.
  • Mitochondria from 143B cells are isolated using a standard procedure and used to test the activity of the compounds on pyruvate import and oxygen consumption.
  • 500 iig mitochondria are incubated with 37.5 ⁇ [2- 14 C] pyruvate in 120 mM KC1, 5 mM NaCl and 2 mM KH 2 P0 4 , pH 6.8 (KC1 buffer) for 2 min.
  • Mitochondria are eentrifuged at 16 ⁇ 00 g for 1 min.
  • the pellet is washed in 500 ml KC1 buffer containing 50 mM cold pyruvate.
  • Mitochondria are eentrifuged as above and the pellet resuspended in 200 ml 3 ⁇ 40 before addition of 3 ml of scintillation fluid and counting using a scintillation counter.
  • Oxygen consumption is measured on isolated mitochondria in the presence or absence of compounds, using a Seahorse XF-96 apparatus. 5 mM Pyruvate or succinate is used as a respiratory substrate.
  • lactate produced in the hypoxic region of the tumor has been suggested to feed the respiratory chain of cells in more oxygenated regions of the tumor and this effect was shown to be essential for tumor cell viability.
  • pyruvate oxidation was reported to be was reported to be essential for tumor proliferation in breast cancers (6)
  • MPC1 upregulation was shown to be a marker of tumorigenesis and metastasis in breast cancer, where MPC1 mRNA and protein levels were higher in cancer cells than in control cells (7). Therefore, the observation that pyruvate fuels mitochondrial respiration in breast cancer and that MPC1 is overexpressed in tumorigenic and metastatic breast cancer cells strongly suggest that mitochondrial pyruvate transport is a key determinant in breast cancer progression. Specific inhibition of MFC proteins could be used as a therapeutic strategy for this type of cancer.
  • Applicants transduced 143B osteosarcoma cells with lentiviruses expressing a doxycycline- inducible shRNA against MPC1 (shMPCl) or carrying an empty vector as a control.
  • Cells were then seeded at a density of 20 ⁇ 00 cells per well in a 6- well plate and grown for 6 days in the absence (0) or with the indicated concentration of doxycycline. The doxycycline was changed every 48 hours. The number of cells in the wells was then counted. As shown in Fig 10A, there was a decrease in the number of cells per well in the presence of doxycycline. Applicants did not observe cell death in these wells and thus concluded that the proliferation of these cells decreased.
  • the proliferation was not altered by doxycycline alone and can thus be attributed to the induction of the MPC1 shRNA.
  • the level of MPC1 protein after 6 days of shRNA induction with the different doses of doxycycline is shown in Fig. l OB.
  • a strong reduction in the amount of MPC1 (arrow) was observed for all the doses tested compared to no doxycycline or to the empty vector in the presence of the highest doxycycline dose.
  • downregulation of MPC1 correlated with a decrease in the proliferation of 143 B osteocarcoma cells.
  • ROS reactive oxygen species
  • Mitochondrial pyruvate inhibitors are expected to be of key interest in myocardial infarction and in stroke.
  • A. P. Halestrap The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors. Biochem J 148, 85 (Apr, 1975).

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Abstract

La présente invention concerne la caractérisation d'une nouvelle famille de transporteurs mitochondriaux de pyruvate isolés et purifiés, pouvant transporter du pyruvate dans des mitochondries. L'invention concerne également des modulateurs de l'activité d'importation de pyruvate destinés à la thérapie de troubles associés à l'absorption du pyruvate. L'invention concerne en outre le criblage de médicaments destinés à des troubles associés à l'absorption du pyruvate.
EP13727081.5A 2012-05-24 2013-05-24 Identification et expression fonctionnelle du transporteur mitochondrial de pyruvate, mpc1 Withdrawn EP2855532A1 (fr)

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