WO1999064458A1 - Nouveaux procedes d'identification de modulateurs de l'activite de porteurs d'anions de mitochondries - Google Patents

Nouveaux procedes d'identification de modulateurs de l'activite de porteurs d'anions de mitochondries Download PDF

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Publication number
WO1999064458A1
WO1999064458A1 PCT/US1999/012623 US9912623W WO9964458A1 WO 1999064458 A1 WO1999064458 A1 WO 1999064458A1 US 9912623 W US9912623 W US 9912623W WO 9964458 A1 WO9964458 A1 WO 9964458A1
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mac
carrier
substrate
assay
vesicle
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Barbara E. Corkey
James A. Hamilton
Paul F. Pilch
Stephen R. Farmer
James L. Kirkland
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Boston University
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Boston University
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • Type II diabetes also known as adult-onset or non-insulin dependent diabetes mellitus (NIDDM).
  • Type II diabetes is a chronic and incurable disease that accounts for 90% to 95% of all diagnosed cases of diabetes. It is a leading cause of death in the United States.
  • This condition is a complex disorder involving a combination of factors, including the inability of certain tissues to respond to insulin, referred to as "insulin resistance" and an inability of the pancreas to produce appropriate levels of insulin. Insulin resistance occurs when insulin-stimulated uptake of glucose into peripheral cells is impaired and the inhibitory effect of insulin on glucose production in the liver is reduced. This condition can lead to excessive blood levels of glucose (hyperglycemia).
  • Hyperglycemia in most diabetics is accompanied by, and may even be preceded by, increases in the blood levels of triglycerides, free fatty acids, and other lipids. Hyperglycemia over an extended period of time is believed to damage the walls of blood vessels, causing complications characteristic of diabetes, including blindness from microvascular deterioration in the retina, loss of circulation in the extremities leading to amputation, and kidney failure. In addition, the high blood levels of triglycerides, free fatty acids, and total cholesterol pose serious health risks to the diabetic and are believed to lead to cardiovascular disease including coronary heart disease.
  • Obesity is also a significant health problem in the United States. About 60 million Americans, or 45% of the adult population, are overweight and about 35 million, or 26% of the adult population, are obese. Approximately 30% of obese Americans are Type II diabetics, and many of the others are considered to be in pre-diabetic states with varying levels of insulin resistance or impaired glucose tolerance. Obesity is also considered a risk factor for cardiovascular disease, gall bladder disease and osteoarthritis.
  • Type II diabetes There is no cure for obesity or diabetes.
  • the first course of therapy is to try to control hyperglycemia through diet and exercise. If this dietary and exercise approach fails, patients typically begin medication to achieve glycemic control. Most patients initially take sulfonylureas, which work by stimulating the production of insulin in the pancreas. However, over time, many Type II diabetics taking sulfonylureas lose their ability to control glucose levels, and others lose their ability to secrete insulin. This occurs mainly in patients who are obese and do not lose weight following diagnosis of the disease. When sulfonylureas become ineffective, the Type II diabetic must then begin daily insulin injections.
  • MACs mitochondrial anion carriers
  • ANT adenine nucleotide translocator
  • PiC phosphate carrier
  • the pyruvate carrier supplies pyruvate to the mitochondrial matrix during the catabolic phase of cell metabolism to fuel the citric acid cycle, and during the anabolic phase to initiate glucose synthesis in the liver and kidney.
  • the aspartate/glutamate carrier (AGC) and the ⁇ -ketoglutarate carrier (KGC), also known as the oxoglutarate carrier (OGC) play a vital role in glucose catabolism in cytosolic-mitochondrial shuttle systems responsible in some systems for regenerating NAD + for glycolysis. They also participate in glucose synthesis and in nitrogen metabolism.
  • the dicarboxylate carrier (DIC) is best known for its role in glucose synthesis but also participates in urea synthesis, whereas the citrate carrier (CIC) plays an essential role in fatty acid and lipid biosynthesis.
  • the glutamate carrier (GC) plays a role in urea synthesis and in some other processes related to nitrogen metabolism, mainly in liver.
  • the ATPMg/P j carrier (APC) is evidently responsible for "charging" the mitochondrial matrix with adenine nucleotides during early stages of development, thus inducing a variety of processes including ATP and glucose synthesis.
  • MACs which have a secondary role as proton transporters.
  • the phosphate carrier serves a dual role as a carrier of inorganic phosphate as well as catalyzing P,/H + symport (or P,/OH" antiport).
  • UCPs uncoupling proteins
  • FAA free fatty acid anions
  • This gradient which includes contributions from both a membrane potential and a pH difference is in turn used by a protein complex in the inner membrane to synthesize ATP (respiration, or oxidation, is said to be coupled to the synthesis of ATP.)
  • mitochondrial uncoupling proteins create a pathway that allows dissipation of the proton electrochemical gradient across the inner mitochondrial membrane without coupling to any other energy-consuming process. As a result of this uncoupling, respiratory energy is dissipated as heat rather than being stored as high energy ATP.
  • UCP-2 is a close homologue of UCP-1 which is widely expressed in adult human tissues, including tissues rich in macrophages, and is upregulated in white fat in response to fat feeding. As there is little brown adipose tissue present in adult large-size animals and humans, it is believed that UCP-2 (having 59% amino acid identity to UCP-1) functions in human adult tissues in heat generation, thermoregulation, and body weight regulation. (Fleury et al. (1997) Nature Genetics, 15:269-272.) This belief is strengthened by the knowledge that the UCP-2 gene maps to regions of chromosome 11 and mouse chromosome 7 that have been linked to hyperinsulinemia and obesity.
  • UCP-2 mRNA is upregulated in the white adipose tissue of ob/ob and db/db mice, two genetic mouse models of obesity. (Gimeno et al. (1997) Diabetes, 46:900-906.) This increased expression is believed to be a compensatory change in which UCP-2 induction is attempting to act against powerful genetically-induced obesity.
  • UCP-3 has also been identified as a new member of the mitochondrial transport protein family having 73% amino acid identity with UCP-2 and 57% identity with UCP- 1 (Boss et al. (1997) FEBS Letters, 408(l):39-42 and Vidal-Puig et al. (1997) Biochem. Biophys. Res.
  • UCP-3 is highly expressed in skeletal muscle in humans as well as in brown adipose tissue in rodents.
  • UCP-3 mRNA levels are regulated by both hormonal (e.g., dexamethasone, leptin, or hypo/hyperthyroidism) and dietary manipulations (e.g., starvation).
  • hormonal e.g., dexamethasone, leptin, or hypo/hyperthyroidism
  • dietary manipulations e.g., starvation
  • the present invention is based on the understanding that regulation of the activity of mitochondrial anion carriers (MACs) may be more influential than regulation of their abundance. As such, the present invention provides methods for identifying pharmaceutically effective compounds that modulate the activity of mitochondrial anion carriers (MACs). The invention is based on the knowledge that compounds that modulate such mitochondrial anion carriers (MACs) may also modulate energy efficiency and provide a means for controlling body mass.
  • MACs mitochondrial anion carriers
  • a method of the present invention involves contacting a MAC with a test compound and a MAC substrate which is capable of being transported by the MAC; determining the ability of the test compound to modulate transport of the MAC substrate; and identifying the test compound as a modulator of MAC activity.
  • the MAC is formulated within a barrier separating a first and a second compartment (e.g., a biological or structural barrier) under conditions such that transport of the MAC substrate across the barrier occurs.
  • the barrier includes more than one MAC.
  • the first or second compartment further comprises an indicator of MAC activity.
  • the first and the second compartment include an indicator of MAC activity and/or an indicator of MAC-independent activity (e.g., barrier "leakiness").
  • a method of the present invention involves contacting an assay vesicle, preferably an alkaline assay vesicle, which includes at least one MAC and an indicator of MAC activity (e.g., a pH indicator or a potentiometric probe) with a test compound and a MAC substrate which is capable of being transported across the lipid bilayer of the assay vesicle; determining the ability of the test compound to modulate transport of the MAC substrate across the lipid bilayer of the assay vesicle; and identifying the test compound as a modulator of MAC activity.
  • an assay vesicle preferably an alkaline assay vesicle, which includes at least one MAC and an indicator of MAC activity (e.g., a pH indicator or a potentiometric probe) with a test compound and
  • the MAC can be selected, for example, from the group consisting of an ADP/ATP carrier (AAC), a phosphate carrier (PiC), a pyruvate carrier (PYC), an aspartate/glutamate carrier (AGC), an ⁇ -ketoglutarate carrier or oxoglutarate carrier (KGC/OGC), a dicarboxylate carrier (DIC), a citrate carrier (CIC), a glutamate carrier (GC), an ATPMg/Pj carrier (APC), and an uncoupling protein (UCP).
  • AAC ADP/ATP carrier
  • PiC phosphate carrier
  • PYC pyruvate carrier
  • AGC aspartate/glutamate carrier
  • KGC/OGC ⁇ -ketoglutarate carrier or oxoglutarate carrier
  • DIC dicarboxylate carrier
  • CIC citrate carrier
  • GC glutamate carrier
  • APC ATPMg/Pj carrier
  • UCP uncoupling protein
  • the MAC is a phosphate carrier (PiC) and the MAC substrate is inorganic phosphate (Pi).
  • the MAC is a glutamate carrier (GC) and the MAC substrate is glutamate.
  • the MAC is a mitochondrial uncoupling protein (UCP) and the MAC substrate is a free fatty acid anion (FAA " ).
  • the mitochondrial UCP can be selected from the group consisting of uncoupling protein 1 (UCP1), uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3).
  • the present invention also involves a method for identifying a compound which modulates the activity of a mitochondrial anion carrier (MAC) comprising contacting an assay vesicle which includes at least one MAC, a first MAC substrate, and an indicator of MAC activity (e.g., a pH indicator or a potentiometric probe) with a test compound and a second MAC substrate, wherein the first and second MAC substrates are capable of being transported across the lipid bilayer of the assay vesicle; determining the ability of the test compound to modulate transport of the MAC substrates across the lipid bilayer of the assay vesicle, wherein determining the ability of the test compound to modulate transport of the MAC substrates across the lipid bilayer of the assay vesicle comprises determining a change in the indicator of MAC activity; and identifying the test compound as a modulator of MAC activity.
  • MAC mitochondrial anion carrier
  • the MAC is an ADP/ATP carrier (AAC), the MAC substrates are ADP and ATP.
  • the MAC is an aspartate/glutamate carrier (AGC), and the MAC substrates are aspartate and glutamate.
  • the MAC is an ⁇ -ketoglutarate carrier or oxoglutarate carrier (KGC/OGC) and the MAC substrates are ⁇ -ketoglutarate and malate.
  • the MAC is a dicarboxylate carrier (DIC) and the MAC substrates are inorganic phosphate (Pi) and a dicarboxylate (e.g. malate or succinate).
  • the MAC is a citrate carrier (CIC) and the MAC substrates are malate and citrate.
  • the MAC is an ATPMg/P j carrier (APC) and the MAC substrates are ATP-Mg ++ and inorganic phosphate (Pi).
  • the MAC is an uncoupling protein (UCP) and the MAC substrates are a free fatty acid anion (FAA ⁇ ) and a free fatty acid.
  • the present invention also involves a method for identifying a compound which modulates the activity of a mitichondrial anion carrier (MAC) comprising contacting an assay vesicle which comprises a first MAC, a second MAC, a second MAC substrate, and an indicator of activity of the second MAC (e.g., a pH indicator or a potentiometric probe) with a test compound and a first MAC substrate, wherein the MAC substrates are capable of being transported across the lipid bilayer of the assay vesicle; determining the ability of the test compound to modulate transport of the first MAC substrate across the lipid bilayer of the assay vesicle, wherein determining the ability of the test compound to modulate transport of the first MAC substrate across the lipid bilayer of the assay vesicle comprises determining a change in the indicator of activity of the second MAC; and identifying the test compound as a modulator of MAC activity.
  • MAC mitichondrial anion carrier
  • the first MAC is an aspartate/glutamate carrier
  • the second MAC is a glutamate carrier (GC)
  • the first MAC substrate is aspartate and the second MAC substrate is glutamate.
  • the first MAC is an ATPMg/Pi carrier (APC)
  • the second MAC is a phosphate carrier (PiC)
  • the first MAC substrate is
  • the second MAC substrate is inorganic phosphate (Pi).
  • the first MAC is a dicarboxylate carrier
  • the second MAC is a phosphate carrier
  • the first MAC substrate is a dicarboxylate (e.g., malate or succinate)
  • the second MAC substrate is inorganic phosphate (Pi).
  • the present invention also involves a method for identifying a compound which modulates the activity of a mitichondrial anion carrier (MAC) comprising contacting an assay vesicle which comprises a first MAC, a second MAC, a third MAC, a second MAC substrate, a third MAC substrate, and an indicator of activity of the third MAC (e.g., a pH indicator or a potentiometric probe) with a test compound and a first MAC substrate, wherein the MAC substrates are capable of being transported across the lipid bilayer of the assay vesicle; determining the ability of the test compound to modulate transport of the first MAC substrate across the lipid bilayer of the assay vesicle, wherein determining the ability of the test compound to modulate transport of the first MAC substrate across the lipid bilayer of the assay vesicle comprises determining a change in the indicator of activity of the third MAC; and identifying the test compound as a modulator of MAC activity.
  • the first MAC is an ⁇ - ketoglutarate carrier or oxoglutarate carrier (KGC/OGC)
  • the second MAC is a dicarboxylate carrier
  • the third MAC is a phosphate carrier (PiC)
  • the first MAC substrate is ⁇ -ketoglutarate
  • the second MAC substrate is malate
  • the third MAC substrate is inorganic phosphate (Pi).
  • the first MAC is a citrate transporter (CIC)
  • the second MAC is a dicarboxylate transporter (DIC)
  • the third MAC is a phosphate transporter
  • the first MAC substrate is citrate
  • the second MAC substrate is malate
  • the third MAC substrate is inorganic phosphate (Pi).
  • Assay vesicles are also intended to be within the scope of the present invention.
  • an assay vesicle of the present invention contain a phospholipid, at least one MAC, and an indicator of MAC activity (e.g., a pH indicator or a potentiometric probe).
  • the phospholipid can be selected from the group consisting phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.
  • the MAC can be selected from the group consisting an ADP/ATP carrier (AAC), a phosphate carrier (PiC), a pyruvate carrier (PYC), an aspartate/glutamate carrier (AGC), an oxoglutarate carrier (KGC/OGC), a dicarboxylate carrier (DIC), a citrate carrier (CIC), a glutamate carrier (GC), an ATPMg/Pj carrier (APC), and an uncoupling protein (UCP).
  • AAC ADP/ATP carrier
  • PiC phosphate carrier
  • PYC pyruvate carrier
  • AGC aspartate/glutamate carrier
  • KGC/OGC oxoglutarate carrier
  • DIC dicarboxylate carrier
  • CIC citrate carrier
  • GC glutamate carrier
  • APC ATPMg/Pj carrier
  • UCP uncoupling protein
  • the assay vesicle further comprises a lysophospholipid.
  • the lysophospholipid can be selected from the group consisting of lysophosphotidate, lysophosphatidylcholine (e.g., lyso 1-acyl phosphatidylcholine), lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine.
  • the assay vesicle further includes a fatty acid.
  • the fatty acid can be selected from the group consisting of oleic acid, myristic acid, palmitic acid, lauric acid, stearic acid, and arachidonic acid.
  • vesicle "leakiness” or movement of MAC substrates across the lipid bilayer which occurs in a manner independent of the MAC are intended to be within the scope of the invention.
  • FIG. 1 is a schematic representation of exemplary embodiments of the present invention.
  • the diagram depicts assay vesicles comprising one, two and three mitochondrial anion carriers (MACs) as well as the MAC substrates transported by the enumerated carriers.
  • MACs mitochondrial anion carriers
  • Figure 2 is an alignment of the coding sequences for the human MACs, UCP-1, UCP-2, and UCP-3.
  • the present invention provides, ter ⁇ li ⁇ , rapid, effective assays for screening and identifying pharmaceutically effective compounds that specifically act as modulators of mitochondrial anion carriers (MACs).
  • the present invention is based, at least in part, on the fact that mitochondrial metabolism is dependent on MACs including, for example, the ADP/ATP carrier (AAC), also known as the adenine nucleotide translocator (ANT), the phosphate carrier (PiC), the pyruvate carrier (PYC), the aspartate/glutamate carrier (AGC), the oxoglutarate carrier (KGC/OGC), the dicarboxylate carrier (DIC), the citrate carrier (CIC), the glutamate carrier (GC), and the ATPMg/Pj carrier (APC).
  • AAC ADP/ATP carrier
  • ANT adenine nucleotide translocator
  • PiC phosphate carrier
  • PYC pyruvate carrier
  • AGC aspartate/glutamate carrier
  • the invention is also based on the discovery that a key role of at least one class of MACs, the mitochondrial uncoupling proteins (UCPs), appears to be transport of fatty acid anions to induce cyclical proton movement across the inner mitochondrial membrane. This transport is tightly related to oxidation (i.e. burning) of fatty acids in the mitochondrion, thereby converting fatty acids into energy rather than storing fatty acids.
  • the instant assays provide a convenient format for discovering compounds which have therapeutic potential in the treatment of obesity and diabetes, as well as other diseases and anomalies which result, at least in part, from aberrant cellular metabolism.
  • the assays of the present invention can be utilized to identify compounds which enhance transport activity of the MACs.
  • Compounds which enhance or promote transport are beneficial to increase the energy expenditure of a cell, particularly a cell of an individual that would benefit from enhanced expenditure of energy (e.g., an obese subject).
  • compounds which reduce or inhibit transport are beneficial to decrease the energy expenditure of a cell, particularly a cell of an individual that would benefit from reduced energy expenditure (e.g., an aging subject or subject afflicted with cancer).
  • the present invention involves a method for identifying a compound which modulates the activity of a mitochondrial anion carrier (MAC) which involves contacting a MAC with a test compound and a MAC substrate which is capable of being transported by the MAC; determining the ability of the test compound to modulate transport of the MAC substrate; and identifying the test compound as a modulator of MAC activity.
  • the MAC is formulated within a barrier separating a first and a second compartment (e.g., a biological or structural barrier) under conditions such that transport of the MAC substrate across the barrier occurs.
  • the MAC substrate is transported from the first compartment to the second compartment.
  • the MAC substrate is transported from the second compartment to the first compartment.
  • the first or second compartment further comprises an indicator of MAC activity.
  • the activity of a MAC can be evaluated in an assay system in which a MAC is reconstituted in an assay vesicle and an indicator of MAC activity (e.g., a pH indicator or a membrane potential indicator) is included within the vesicle.
  • an indicator of MAC activity e.g., a pH indicator or a membrane potential indicator
  • the activity of a MAC in a reconstituted assay vesicle of the present invention can be measured by measuring the transport of a MAC substrate across the lipid bilayer of the assay vesicle. For example, the transport of a MAC substrate from the inside of an assay vesicle to the outside of an assay vesicle can be measured.
  • a barrier or assay vesicle can include more than one MAC.
  • a barrier or assay vesicle of the present invention can contain a first MAC and a second MAC which operatively cotransport at least one MAC substrate.
  • a barrier or assay vesicle of the present invention can contain more than two MACs (e.g., a first, second, and third MAC, wherein the first and second MAC operatively co-transport at least one MAC substrate and wherein the second and third MACs operatively cotransport at least one MAC substrate.
  • MACs e.g., a first, second, and third MAC, wherein the first and second MAC operatively co-transport at least one MAC substrate and wherein the second and third MACs operatively cotransport at least one MAC substrate.
  • MACs formulated within barriers as well as assay vesicles reconstituted with at least one MAC and containing an indicator of MAC activity can be used to test the effects of compounds on the transport activity of MAC.
  • the measurements of MAC activity are straightforward and quick, thus allowing the rapid testing of many compounds.
  • Compounds can be tested in the assays of the present invention singly or, more preferably, as members of libraries of compounds.
  • the subject assays enable rapid screening of large numbers of compounds or a library of compounds to identify those compounds which modulate the activity of a MAC.
  • test compound e.g., as in “test compound” is intended to include test agents, usually in the form of a library of agents, which in certain embodiments, will be contacted with the MACs or "assay vesicles” of the invention to determine the ability of such compounds to modulate the activity of a "mitochondrial anion carrier (MAC)" which exists in a barrier or lipid bilayer of an assay vesicle.
  • MAC mitochondrial anion carrier
  • the compounds can be peptidic or non-peptidic in nature. Compounds of the non-peptidic nature often include small organic and/or non-organic molecules.
  • compounds of the present invention can be naturally-occurring compounds such as nucleic acid molecules, proteins, sugars, lipids, or derivatives thereof.
  • Test compounds include compounds for which the structure and/or physical characteristics are known but for which a MAC modulatory function has not been demonstrated, as well as compounds for having a yet undetermined structure and/or physical characteristics.
  • compounds for which an activity other than a MAC modulatory activity is known can be identified as modulators of MAC activity using the methods of the present invention.
  • the term "contacting" i.e., contacting an assay vesicle with a compound
  • contacting is intended to include incubating the compound or agent and the assay vesicle together in vitro (e.g., adding the agent to assay vesicles in a suitable vessel) such that the agent has the potential, for example, to interact with and/or bind to a mitochondrial anion carrier (MAC) which exists in the lipid bilayer of the assay vesicle such that the activity of the MAC is modulated.
  • MAC mitochondrial anion carrier
  • mitochondrial anion carrier is intended to include a class of proteins that exist, in their natural state (e.g., in the intact mitochondria of a cell), in the inner mitochondrial membrane of the mitochondria of a cell.
  • the MACs commonly contain a characteristic 3-fold repeated sequence approximately 100 amino acids in length, each repeated 100 amino acid element being composed of two membrane-spanning alpha-helices linked by an extensive hydrophilic domain.
  • MACs of the present invention include the ADP/ATP carrier (AAC), also known as the adenine nucleotide translocator (ANT) (e.g., isoforms ANT1, ANT2, and ANT3, also known as Tl, T3, and T2), the phosphate carrier (PiC), (e.g., phosphate carrier isoform A (PiC A) and phosphate carrier isoform B (PiC B)), the pyruvate carrier (PYC), the aspartate/glutamate carrier (AGC), the oxoglutarate carrier (KGC/OGC), the dicarboxylate carrier (DIC), the citrate carrier (CIC), the glutamate carrier (GC), the ATPMg/Pj carrier (APC), and the mitochondrial uncoupling proteins (UCPs), (e.g., uncoupling protein 1 (UCP-1), uncoupling protein 2 (UCP-2), and uncoupling protein 3 (UCP-3)).
  • AAC ADP/ATP carrier
  • the "activity of a MAC” is to shuttle or transport "MAC substrates" from one side of an inner mitochondrial membrane or lipid bilayer of an assay vesicle (e.g., the outside of the membrane or lipid bilayer) to the other side of an inner mitochondrial membrane or lipid bilayer of an assay vesicle (e.g., the inside of the membrane or lipid bilayer).
  • an inner mitochondrial membrane or lipid bilayer of an assay vesicle e.g., the outside of the membrane or lipid bilayer
  • an inner mitochondrial membrane or lipid bilayer of an assay vesicle e.g., the inside of the membrane or lipid bilayer.
  • a MAC substrate is "capable of being transported across the lipid bilayer of a vesicle" .
  • the activity of a MAC may be coupled to a mitochondrial process such as oxidative phosphorylation that generates cellular energy in the form of ATP (e.g., ACC/ ANT and PiC).
  • a mitochondrial process such as oxidative phosphorylation that generates cellular energy in the form of ATP (e.g., ACC/ ANT and PiC).
  • the activity of a MAC may play a dual role in two or more metabolic processes (e.g. PYC, AGC, KGC/OGC, DIC, CIC, and GC.)
  • the activity of the MAC may be uncoupled (e.g., the UCPs), in which case oxidation results in the generation of heat rather than cellular energy.
  • MAC mitochondrial anion carrier
  • MAC substrate refers to the molecule or molecules which are transported from one side of the inner mitochondrial membrane or "reconstituted lipid bilayer" to the other side of the inner mitochondrial membrane or "reconstituted lipid bilayer".
  • a MAC substrate can be transported from the outside of the inner mitochondrial membrane or vesicle bilayer to the inside of the inner mitochondrial membrane or vesicle bilayer.
  • a MAC substrate can be transported from the inside of the inner mitochondrial membrane or vesicle bilayer to the outside of the inner mitochondrial membrane or vesicle bilayer.
  • a particular MAC is associated with specific MAC substrate, such specificity most likely being determined by unique amino acid sequences attributed to a particular MAC (e.g., the sequences that are not commonly shared among the class of MAC proteins).
  • the PiC specifically transports the MAC substrate, inorganic phosphate (P,- or HPO 4 2 ") across the mitochondrial membrane (e.g., from the outside of the inner mitochondrial membrane to the inside of the inner mitochondrial membrane).
  • the PiC is also referred to herein and in the art as both a Pi/H + symporter (e.g., cotransports Pi and protons (H + s) from the outside of the inner mitochondrial membrane to the inside of the inner mitochondrial membrane) or as a Pi/OH" antiporter (e.g., exchanges Pi for hydroxyl ions (OH”) across the inner mitochondrial membrane).
  • a Pi/H + symporter e.g., cotransports Pi and protons (H + s) from the outside of the inner mitochondrial membrane to the inside of the inner mitochondrial membrane
  • a Pi/OH antiporter e.g., exchanges Pi for hydroxyl ions (OH) across the inner mitochondrial membrane.
  • the ACC or ANT specifically transports the MAC substrate, ADP 3 ", across the inner mitochondrial membrane (e.g., from the outside of the inner mitochondrial membrane to the inside of the inner mitochondrial membrane), and transports the MAC substrate, ATP 4- , across the inner mitochondrial membrane (e.g., from the inside of the inner mitochondrial membrane to the outside of the inner mitochondrial membrane).
  • the PYC specifically transports the MAC substrate, pyruvate, across the inner mitochondrial membrane (e.g., from the outside of the inner mitochondrial membrane to the inside of the inner mitochondrial membrane).
  • the PYC is also referred to herein and in the art as both pyruvate/H + symporter (e.g., cotransports pyruvate and protons (H + s) from the outside of the inner mitochondrial membrane to the inside of the inner mitochondrial membrane) or as a pyruvate/OH" antiporter (e.g., exchanges pyruvate for hydroxyl ions (OH") across the inner mitochondrial membrane).
  • the glutamate carrier specifically transports the MAC substrate, glutamate, across the inner mitochondrial membrane (e.g., from the outside of the inner mitochondrial membrane to the inside of the inner mitochondrial membrane).
  • the GC is also referred to herein and in the art as both glutamate/H + symporter (e.g., cotransports glutamate and protons (H + s) from the outside of the inner mitochondrial membrane to the inside of the inner mitochondrial membrane) or as a glutamate/OH" antiporter (e.g., exchanges glutamate for hydroxyl ions (OH") across the inner mitochondrial membrane).
  • glutamate/H + symporter e.g., cotransports glutamate and protons (H + s) from the outside of the inner mitochondrial membrane to the inside of the inner mitochondrial membrane
  • a glutamate/OH" antiporter e.g., exchanges glutamate for hydroxyl ions (OH) across the inner mitochondrial membrane.
  • the AGC specifically exchanges the MAC substrates, aspartate and glutamate, from across the inner mitochondrial membrane.
  • the KGC/OGC specifically transports the MAC substrate, ⁇ -ketoglutarate (also referred to herein as 2-oxoglutarate), across the inner mitochondrial membrane (e.g., from the outside of the inner mitochondrial membrane to the inside of the inner mitochondrial membrane), and transports the MAC substrate, malate (or other dicarboxylic acids), across the inner mitochondrial membrane (e.g., from the inside of the inner mitochondrial membrane to the outside of the inner mitochondrial membrane).
  • ⁇ -ketoglutarate also referred to herein as 2-oxoglutarate
  • malate or other dicarboxylic acids
  • the DIC specifically transports the MAC substrates, dicarboxylate (e.g., malate and succinate) and inorganic phosphate (Pi), across the inner mitochondrial membrane (e.g., the DIC catalyzes an electroneutral Pi: dicarboxylate or Pi:Pi exchange across the inner mitochondrial membrane).
  • the dicarboxylate exchanger catalyzes the transport of respiratory substrates (e.g., malate and succinate) into the mitochondria and may facilitate translocation of dicarboxylates into the cytoplasm for gluconeogenesis.
  • the CIC specifically transports the MAC substrate, citrate (e.g., tricarboxylate), from one side of the inner mitochondrial membrane (e.g., from the inside of the inner mitochondrial membrane to the outside of the inner mitochondrial membrane.
  • citrate e.g., tricarboxylate
  • the CIC catalyzes a 1 : 1 electroneutral exchange across the inner mitochondrial membrane of a tricarboxylate (e.g., citrate, threo-D s -isocitrate, c/ ' s-aconitate) plus a proton for either another tricarboxylate/H + , a dicarboxylate (e.g., malate, succinate), D- and L- tartarate, or phosphoenolpyruvate.
  • a tricarboxylate e.g., citrate, threo-D s -isocitrate, c/ ' s-aconitate
  • the APC specifically transports the MAC substrate, adenine nucleotides (e.g., ATPMg 2 "), across the inner mitochondrial membrane (e.g., from the outside of the inner mitochondrial membrane to the inside of the inner mitochondrial membrane), and transports the MAC substrate, inorganic phosphate (P f or HPO 4 2" ), across the inner mitochondrial membrane (e.g., from the inside of the inner mitochondrial membrane to the outside of the inner mitochondrial membrane). Transport via the APC is completely reversible and nonproductive exchanges also occur (e.g., ATPMg 2 ": ATPMg 2 " and P 1 :P,).
  • the UCPs specifically transport the MAC substrate, fatty acid anions, across the inner mitochondrial membrane (e.g., from the inside of the inner mitochondrial membrane to the outside of the inner mitochondrial membrane).
  • Transport of palmitate anion and laurate anion has also been demonstrated. (Jezek et al. (1996) J. Biol. Chem. 271 :6199-6205 and references contained therein).
  • Transport of fatty acid anions is also reffered to as fatty acid activation of proton (H + ) transport or proton (H + ) conductance.
  • Transfer of the above-described MAC substrates can be recapitulated in artificial or reconstituted systems.
  • the MAC activity involves transport of the MAC substrate from, for example, the first compartment to the second compartment.
  • activity of the MACs in the inner mitochondrial matrix can be readily reconstituted in lipid bilayer vesicles utilizing methods known in the art as well as those described in detail in Examples 2-3.
  • MACs in a reconstituted lipid bilayer can have either a "right-side-in” orientation (e.g., existing in the reconstituted vesicle in the same orientation as they exist in the intact mitochondria) or, alternatively, can have an "in-side-out” orientation (e.g., existing in a reverse or the opposite orientation as they exist in the intact mitochondria.) Accordingly, a MAC substrate that is transported from the inside of the inner mitochondrial membrane to the outside of the inner mitochondrial membrane in the intact membrane can be transported form the outside of a reconstituted vesicle to the inside when the MAC is inserted in the "in-side-out" orientation.
  • the phrase "modulate transport of the MAC substrate” is intended to include enhancing, promoting, or inducing the transport of a MAC as well as or reducing or inhibiting the transport of the MAC substrate such that the transport of the MAC substrate is detectably enhanced, promoted, induced, reduced, or inhibited after contacting the assay vesicle containing the MAC with a test compound or agent as compared to the transport of the MAC substrate prior to contacting the assay vesicle with a test compound or agent.
  • Determining the ability of a test compound to modulate transport of a MAC substrate includes determining a change in the level of a MAC substrate on one side of the mitochondrial membrane or assay vesicle bilayer which occurs as a result of contacting the MAC-containing mitochondrial membrane or assay vesicle bilayer with a test compound. "Determining the ability of a test compound to modulate transport of a MAC substrate” can involve either a direct determination or an indirect determination of MAC activity.
  • transport of the MAC substrate results in a change in the internal pH of the lipid vesicle (e.g., an acidification or net increase in proton (H + ) concentration, or an alkalinization or net decrease in proton (H + ) concentration).
  • compounds which modulate transport of a MAC substrate can be identified as compounds which effect the change in intravesicular pH caused by transport of the MAC substrate.
  • transport of the MAC substrate results in a change in the internal pH of the lipid vesicle (e.g., an acidification or net increase in proton (H + ) concentration, or an alkalinization or net decrease in proton (H + ) concentration).
  • compounds which modulate transport of a MAC substrate can be identified as compounds which effect the change in intravesicular pH caused by transport of the MAC substrate.
  • transport of the MAC substrate, inorganic phosphate (P ⁇ ) from the outside of an assay vesicle to the inside of a PiC-containing assay vesicle results in a concomitant decrease in intravesicular pH.
  • a test compound which modulates transport of P i by the PiC can be identified as a compound which effects this pH change (e.g., a compound which effects (increases or decreases) either the magnitude or rate of the pH change).
  • transport of the MAC substrate, pyruvate, from the outside of an assay vesicle to the inside of a PYC-containing assay vesicle results in a concomitant decrease in intravesicular pH.
  • a test compound which modulates transport of pyruvate by the PYC can be identified as a compound which effects this pH change (e.g., a compound which effects (increases or decreases) either the magnitude or rate of the pH change).
  • transport of the MAC substrate, glutamate from the outside of an assay vesicle to the inside of a GC-containing assay vesicle results in a concomitant decrease in intravesicular pH.
  • a test compound which modulates transport of glutamate by the GC can be identified as a compound which effects this pH change (e.g., a compound which effects (increases or decreases) either the magnitude or rate of the pH change).
  • transport of the MAC substrate, free fatty acid anion (FAA " ) from the outside of an assay vesicle to the inside of a UCP-containing assay vesicle results in a concomitant decrease in intravesicular pH.
  • a test compound which modulates transport of FAA " by the UCP can be identified as a compound which effects this pH change (e.g., a compound which effects (increases or decreases) either the magnitude or rate of the pH change).
  • transport of the MAC substrate results in a change in the membrane potential of the assay vesicle (e.g., an increase or decrease in membrane potential).
  • compounds which modulate transport of a MAC substrate can be identified as compounds which effect the change in membrane potential caused by transport of the MAC substrate. For example, transport of the MAC substrates, ADP 3" and ATP 4" , across the lipid bilayer of an AAC-containing assay vesicle results in a concomitant decrease in membrane potential.
  • a test compound which modulates transport of ADP 3" and ATP 4" by the AAC can be identified as a compound which effects this change in membrane potential (e.g., a compound which effects (increases or decreases) either the magnitude or rate of change in membrane potential).
  • transport of the MAC substrates, asparate and glutamate, across the lipid bilayer of an AGC-containing assay vesicle results in a concomitant decrease in membrane potential.
  • a test compound which modulates transport of aspartate and glutamate by the AGC can be identified as a compound which effects this change in membrane potential (e.g., a compound which effects (increases or decreases) either the magnitude or rate of change in membrane potential).
  • transport of the MAC substrate, FAA " from the inside of a UCP-containing assay vesicle to the outside of the assay vesicle results in a concomitant change in membrane potential.
  • a test compound which modulates transport of FAA " by the UCP can be identified as a compound which effects this change in membrane potential (e.g., a compound which effects (increases or decreases) either the magnitude or rate of change in membrane potential).
  • transport of the MAC substrate results in a change in the net charge inside the lipid vesicle (e.g., an increase or decrease in the concentration of a specific ion, for example FAA " ).
  • compounds which modulate transport of a MAC substrate can be identified as compounds which effect the change in charge distribution (e.g., FAA" concentration) resulting from transport of the MAC substrate.
  • charge distribution e.g., FAA
  • transport of the MAC substrate, FAA " from the inside of an assay vesicle to the outside of a UCP-containing assay vesicle results in a concomitant decrease in intravesicular FAA " concentration and a decrease in membrane potential ( ⁇ m).
  • a test compound which modulates transport of FAA " by the UCP can be identified as a compound which affects this change in membrane potential (e.g., a compound which affects (increases or decreases) either the magnitude or rate of the membrane potential).
  • the change in membrane potential is due to a change in charge distribution caused by uncompensated ion movement in one direction (e.g., FAA" is transported out without a compensatory H + or exchange for OH").
  • transport of the MAC substrate by a particular MAC substrate results in neither a change in intravesicular pH or in membrane potential.
  • it may be desirable to couple transport of that MAC substrate to a second MAC wherein transport of the MAC substrate by the second MAC results in a detectable change in intravesicular pH or membrane potential.
  • transport of the MAC substrates, inorganic phosphate (P,) and dicarboxylate (e.g., malate) by a DIC in an assay vesicle can be coupled to transfer of P ; by a PiC in the same assay vesicle, which results in a change in intravesicular pH.
  • transport of the MAC substrates, ATPMg 2 " and inorganic phosphate (P,) by an APC in an assay vesicle can be coupled to transfer of P,- by a PiC in the same assay vesicle, which results in a change in intravesicular pH.
  • transport of the MAC substrates, aspartate and glutamate by an AGC in an assay vesicle can be coupled to transfer of glutamate by a GC in the same assay vesicle, which results in a change in intravesicular pH.
  • Such an embodiment may be preferred when a rapid detection (e.g., change in pH) is desired as compared to the relatively slower change in membrane potential.
  • an assay vesicle comprises three MACs and transport of the respective MAC substrates is effectively coupled resulting in a detectable readout (e.g., a change in pH or membrane potential).
  • a detectable readout e.g., a change in pH or membrane potential.
  • an assay vesicle containing KGC, DIC, and PiC can be utilized to assay for modulators of KGC activity in the following manner. Transport of the MAC substrates ⁇ -ketoglutarate and malate by the KGC is coupled to transport of the MAC substrates malate and inorganic phosphate (P ( ) by the DIC which is, in turn, coupled to transport of the substrate P ( by the PiC which results in a change in pH.
  • an assay vesicle containing CIC, DIC, and PiC can be used to assay for modulators of CIC activity in the following manner. Transport of the MAC substrates, citrate and malate, by the CIC is coupled to transport of the MAC substrates, malate and inorganic phosphate (P,-), by the DIC which is, in turn, coupled to transport of the MAC substrate, P,-, by the PiC which results in a change in pH.
  • barrier is intended to include any biological and/or artificial separator of two compartments which provides a favorable environment for the existence of a MAC.
  • barriers can comprise phospholipids, glycolipids, other lipidic molecules, non-lipid molecules, synthetic molecules, any or all of which may support the function of a transporter which exists, in its natural environment, as a component of an organelle membrane.
  • Structural support such as liquid crystals, films, biological chips, emulsions, for example, are intended to form barriers according to the invention.
  • the phrase “assay vesicles” is intended to encompass unilamellar or multilamellar structures having both an outer lipid monolayer and an inner lipid monolayer.
  • the "assay vesicles" of the present invention can be synthesized by a variety of means as described in Example 1.
  • the assay vesicles of the present invention are reconstituted from purified or, at least, partially-purified components (e.g., phospholipid molecules, protein molecules) and can accordingly be termed "reconstituted lipid bilayer vesicles".
  • the "assay vesicles" of the present invention can include only phospholipid molecules, or can further comprise, for example, one or more of the following: protein molecules (e.g., the MACs of the present invention or other non-MAC proteins, for example); cholesterol; fatty acids (e.g., oleic acid, myristic acid, palmitic acid, lauric acid, stearic acid, or arachidonic acid); phospholipids (e.g., phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and/or phosphatidylserine; glycolipids (e.g., cerebroside or gangliosides); and/or lysophospholipids (e.g., lysophosphidate, lyso 1-acyl phosphatidylcholine (lyso-PC), lysophosphatidylethanolamine, lysophosphatid
  • the assay vesicles of the present invention are “alkaline assay vesicles".
  • an “alkaline assay vesicles” is an assay vesicle in which has an alkaline, intravesicular pH (e.g., a pH of greater than 7.4).
  • alkaline assay vesicles are particularly useful, for example, when utilizing pH change as a determinant of MAC activity.
  • an alkaline assay vesicle has a pH of about 7.4 to 8.0. In another embodiment, an alkaline assay vesicle has a pH of about 8.0 to 9.0.
  • an alkaline assay vesicle has a pH of about 9.0 to 10.0. In yet another embodiment, an alkaline assay vesicle has a pH of about 10.0 to 11.0, 11.0 to 12.0, or greater.
  • pH indicator is intended to include a molecule or composition of molecules which produce a quantitative or qualitative change in response to a change in pH.
  • a “pH indicator” of the present invention can be placed on the inside of a reconstituted lipid bilayer vesicle (an “internal pH indicator”) and/or can be placed on the outside of a reconstituted lipid bilayer vesicle (an “external pH indicator”).
  • a pH indicator dye can be, for example, a fluorescent dye (e.g., pyranine, SNARF, SNAFL, HPTS, BCECF, and/or fluoroscein and its derivatives) or a colorometric inhibitor. Examples of such pH indicators are described in detail in Table 2.
  • membrane potential indicator is intended to include a molecule or composition of molecules which produce a quantitative or qualitative change in response to a change in membrane potential.
  • a “membrane potential indicator” of the present invention can be placed, for example, on the inside of a reconstituted lipid bilayer vesicle (an “internal membrane potential indicator”) . Examples of membrane potential indicators are described in detail in Table 3.
  • an assay vesicle having an indicator of MAC-independent activity (e.g., vesicle "leakiness” or movement of MAC substrates across the lipid bilayer which occurs in a manner independent of the MAC) are intended to be within the scope of the invention.
  • an assay vesicle includes a pH indicator as a determinant of MAC activity (e.g., an assay vesicle including PiC, PYC, GC, or UCP) can have a second pH indicator on the outside of the vesicle to determine movement or "leak" of protons from the inside of the vesicle to the outside of the vesicle.
  • an assay of the present invention may involve contacting an assay vesicle which comprises at least one MAC and an indicator of MAC activity with an inhibitor of MAC activity, a test compound and an MAC substrate, wherein the MAC substrate is capable of being transported across the lipid bilayer of the assay vesicle; determining the ability of the test compound to modulate transport of the MAC substrate across the lipid bilayer of the assay vesicle, wherein determining the ability of the test compound to modulate transport of the MAC substrate across the lipid bilayer of the assay vesicle comprises determining a change in the indicator of MAC activity; and identifying the test compound as a modulator of MAC activity.
  • an "inhibitor of MAC activity" is a compound which, when added to a MAC-containing vesicle, reduces or inhibits the activity of the MAC such that the transport of a MAC substrate from one side of the MAC-containing vesicle to another side of the MAC- containing vesicle is measurably reduced or inhibited.
  • the rate of transport of a MAC substrate from one side of the MAC-containing vesicle to another side of the MAC-containing vesicle may be reduced by an inhibitor of MAC activity.
  • a higher concentration of MAC substrate may be necessary to effect the transport of a MAC substrate from one side of the MAC-containing vesicle to another side of the MAC-containing vesicle.
  • An inhibitor of MAC activity is not restricted by a particular mechanism of inhibition.
  • the inhibitor may function as an allosteric inhibitor of MAC activity, as a competitive inhibitor of MAC activity (e.g., competing with a MAC substrate for binding to a MAC), or may function by inducing a conformational change on a MAC.
  • the inhibitor of MAC activity may be contacted with an assay vesicle of the present invention prior to contacting the assay vesicle with the MAC substrate, concurrent with contacting the assay vesicle with the MAC substrate, and/or subsequent to contacting the assay vesicle with the MAC substrate.
  • the inhibitor of MAC activity can be trapped in an assay vesicle upon formation of the assay vesicle.
  • Inhibitors of MAC activity are intended to include inhibitors that are known in the art as well as compounds whose inhibitory activity remains to be demonstrated.
  • APC activity is inhibited by sulfhydryl reagents (e.g., mersalyl, n- ethylmaleimide (NEM)).
  • AAC activity is inhibited by carboxyatractyloside, atractyloside, and bongkrekate.
  • CIC activity is inhibited by 1,2,3-Benzenetricarboxylic acid (BTA) and is dicarboxylate transport by the CIC is weakly inhibited by n- butylmalonate and/or H-ethyl maleimide (NEM).
  • BTA 1,2,3-Benzenetricarboxylic acid
  • NEM H-ethyl maleimide
  • DIC activity is inhibited by n- butylmalonate or phenylsuccinate (which have no effect on the H+/Pi cotransporter). DIC activity is completely inhibited by mercurial reagents, but is not inhibited by the sulfhydral alkylator NEM. Furthermore, long-chain acyl-CoA acts as an inhibitor of, for example, citrate transport, UCP-1, and ANT. Nucleotides also are known in the art to have inhibitory activity on MACs (e.g., purine nucleotides inhibit the activity of UCP).
  • the methods of the invention generally involve contacting an assay vesicle (e.g., a reconstituted lipid bilayer vesicle having a mitochondrial anion carrier (MAC) as a component of the vesicle bilayer and containing an indicator of MAC activity) with a test compound, determining the ability of the test compound to modulate transport of a MAC substrate, and identifying the test compound as a modulator of MAC activity based on its ability to modulate the transport activity of the MAC.
  • an assay vesicle e.g., a reconstituted lipid bilayer vesicle having a mitochondrial anion carrier (MAC) as a component of the vesicle bilayer and containing an indicator of MAC activity
  • the invention provides a method for identifying a compound which modulates the activity of a mitochondrial anion carrier (MAC) comprising:
  • the assay is based, at least in part, on the fact that a change in intravesicular pH or membrane potential is indicative of MAC activity and a modulation of MAC activity can effect, for example, the pH change (e.g., the magnitude of the pH change or the kinetics of the pH change) or change in membrane potential which results from contacting an assay vesicle with a MAC substrate.
  • a change in intravesicular pH or membrane potential is indicative of MAC activity
  • a modulation of MAC activity can effect, for example, the pH change (e.g., the magnitude of the pH change or the kinetics of the pH change) or change in membrane potential which results from contacting an assay vesicle with a MAC substrate.
  • the MAC can be selected, for example, from the group consisting of an ADP/ATP carrier (AAC), a phosphate carrier (PiC), a pyruvate carrier (PYC), an aspartate/glutamate carrier (AGC), an ⁇ -ketoglutarate carrier or oxoglutarate carrier (KGC/OGC), a dicarboxylate carrier (DIC), a citrate carrier (CIC), a glutamate carrier (GC), an ATPMg/Pj carrier (APC), and an uncoupling protein (UCP).
  • AAC ADP/ATP carrier
  • PiC phosphate carrier
  • PYC pyruvate carrier
  • AGC aspartate/glutamate carrier
  • KGC/OGC ⁇ -ketoglutarate carrier or oxoglutarate carrier
  • DIC dicarboxylate carrier
  • CIC citrate carrier
  • GC glutamate carrier
  • APC ATPMg/Pj carrier
  • UCP uncoupling protein
  • the MAC is a phosphate carrier (PiC) and the MAC substrate is inorganic phosphate (Pi).
  • the MAC is a glutamate carrier (GC) and the MAC substrate is glutamate.
  • the MAC is a mitochondrial uncoupling protein (UCP) and the MAC substrate is a fatty acid anion (FAA " ).
  • the mitochondrial UCP can be selected from the group consisting of uncoupling protein 1 (UCP1), uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3).
  • the invention provides a method for identifying a compound which modulates the activity of a mitochondrial anion carrier (MAC) comprising:
  • the MAC is an ADP/ATP carrier (AAC), the MAC substrates are ADP and ATP.
  • the MAC is an aspartate/glutamate carrier (AGC), and the MAC substrates are aspartate and glutamate.
  • the MAC is an ⁇ -ketoglutarate carrier or oxoglutarate carrier (KGC/OGC) and the MAC substrates are ⁇ -ketoglutarate and malate.
  • the MAC is a dicarboxylate carrier (DIC) and the MAC substrates are inorganic phosphate (Pi) and a dicarboxylate (e.g. malate or succinate).
  • the MAC is a citrate carrier (CIC) and the MAC substrates are Malate and citrate.
  • the MAC is an ATPMg/Pj carrier (APC) and the MAC substrates are ATP-Mg ++ and inorganic phosphate (Pi).
  • the MAC is an uncoupling protein (UCP) and the MAC substrate is a fatty acid anion (FAA " ).
  • the invention provides a method for identifying a compound which modulates the activity of a mitichondrial anion carrier (MAC) comprising:
  • the first MAC is an aspartate/glutamate carrier
  • the second MAC is a glutamate carrier (GC)
  • the first MAC substrate is aspartate and the second MAC substrate is glutamate.
  • the first MAC is an ATPMg/Pi carrier (APC)
  • the second MAC is a phosphate carrier (PiC)
  • the first MAC substrate is ATPMg ++
  • the second MAC substrate is inorganic phosphate (Pi).
  • the first MAC is a dicarboxylate (e.g., malate or succinate)
  • the second MAC substrate is inorganic phosphate (Pi).
  • the invention provides a method for identifying a compound which modulates the activity of a mitichondrial anion carrier (MAC) comprising:
  • an assay vesicle which comprises a first MAC, a second MAC, a third MAC, a second MAC substrate, a third MAC substrate, and an indicator of activity of the third MAC with a test compound and a first MAC substrate, wherein the MAC substrates are capable of being transported across the lipid bilayer of the assay vesicle;
  • the first MAC is an ⁇ -ketoglutarate carrier or oxoglutarate carrier (KGC/OGC)
  • the second MAC is a dicarboxylate carrier
  • the third MAC is a phosphate carrier (PiC)
  • the first MAC substrate is ⁇ -ketoglutarate
  • the second MAC substrate is malate
  • the third MAC substrate is inorganic phosphate (Pi).
  • the first MAC is a citrate transporter (CIC)
  • the second MAC is a dicarboxylate transporter (DIC)
  • the third MAC is a phosphate transporter
  • the first MAC substrate is citrate
  • the second MAC substrate is malate
  • the third MAC substrate is inorganic phosphate (Pi).
  • the present invention involves a method for identifying a compound which modulates the activity of a mitichondrial anion carrier (MAC) comprising:
  • an assay vesicle which comprises a first MAC, a second MAC, and an indicator of activity of the first and second MAC (e.g., a pH indicator or a potentiometric probe) with a test compound and a MAC substrate, wherein the MAC substrate is capable of being transported across the lipid bilayer of the assay vesicle by the first and the second MAC;
  • an indicator of activity of the first and second MAC e.g., a pH indicator or a potentiometric probe
  • determining the ability of the test compound to modulate transport of the MAC substrate across the lipid bilayer of the assay vesicle comprises determining a change in the indicator of MAC activity
  • an assay vesicle including UCP-1 and UCP-2, UCP-2 and UCP-3, or UCP-1 and UCP-3 can be utilized according to the present embodiment.
  • an assay vesicle can comprise more than two MACs (e.g., UCP-1, UCP-2, and UCP-3).
  • identifying the test compound as a modulator of MAC activity can further include the step of comparing the effect of the test compound as a modulator of the activity of a first MAC in a first assay vesicle to the effect of the test compound as a modulator of at least the activity of a second MAC in a second assay vesicle.
  • identifying the test compound as a modulator of UCP-1 activity can include comparing the effect of the compound as a modulator of UCP-1 activity in a first assay vesicle to the activity of the compound as a modulator of, for example, UCP-2 or UCP-3 activity, in a second assay vesicle.
  • a compound which has activity in the first UCP-1 assay vesicle but shows no activity in the second UCP-2 or UCP-3 containing assay vesicle can be said to act "specifically" on UCP-1.
  • Such a comparison facilitates the identification of specific MAC modulators from among, for example, tens of thousands of compounds being assayed for modulatory activity.
  • the methods of the invention are carried out using a lipid vesicle having as a component of its lipid bilayer at least one MAC.
  • the vesicles of the present invention can be generated utilizing a variety of methods known in the art.
  • the assay vesicles can be prepared by sonication (see, for example, Hamilton and Small (1981) Proc. Nat'l Acad. Sci. USA 78:878-6882), by extrusion through membranes (see, for example, Kamp et al. (1995) Biocehmistry 34:11928-11957), by detergent dialysis (see, for example, Garlid et al.
  • the reconstituted lipid vesicles can have a weight ratio of protein/phospholipid of, for example, 0.01-0.40, preferably 0.02-0.30, more preferably 0.05-0.25, and more preferably 0.10-0.20; a molar ratio of MAC to phospholipid of, for example, 0.01-1.00, preferably 0.02-0.50, more preferably 0.05-0.25, more preferably 0.10-0.15, and more preferably 0.12 ⁇ mol MAC/mol phospholipid; an internal volume of, for example, 0.4- 10, preferably 0.8-8, more preferably 1.0-5.0, and more preferably 1.5-2 ⁇ l/mg phospholipid; a vesicle diameter of, for example, 250-2000A, preferably 300-1750A, more preferably 350-1500A, more preferably 400-1250A, more preferably 500-lOO ⁇ A and even more
  • the methods of the invention can further be carried out using essentially any MAC as a component of the reconstituted lipid vesicle bilayer.
  • Preferred MACs are
  • MACs which produce a change in intravesicular pH as a "read-out" of activity (e.g., PiC, GC, PYC, and/or UCP) due to the fact that change in pH as an indicator of MAC activity is a more rapid response when compared to change in membrane potential as an indicator of MAC activity.
  • Other examples of suitable MACs include, but are not limited to, AAC, AGC, OCG, DIC, CIC, and APC.
  • modulation of the activity of a MAC can be determined directly using a membrane potential indicator as an indicator of MAC activity.
  • the activity of a MAC can be indirectly determined in an assay vesicle comprising a second MAC which produces a change in intravesicular pH as an indicator of MAC activity, wherein the first and second MAC operatively cotransport at least one MAC substrate.
  • the first MAC is an aspartate/glutamate carrier
  • the second MAC is a glutamate carrier (GC)
  • the first MAC substrate is aspartate
  • the second MAC substrate is glutamate.
  • the first MAC is an ATPMg/Pi carrier (APC)
  • the second MAC is a phosphate carrier (PiC)
  • the first MAC substrate is ATPMg ++
  • the second MAC substrate is inorganic phosphate (Pi).
  • the first MAC is a dicarboxylate (e.g., malate or succinate)
  • the second MAC substrate is inorganic phosphate (Pi).
  • the activity of such a MAC can be indirectly determined in an assay vesicle comprising a second MAC which produces a change in intravesicular pH as an indicator of MAC activity, wherein the first and second MAC operatively cotransport at least one MAC substrate.
  • the first MAC is an ⁇ -ketoglutarate carrier or oxoglutarate carrier (KGC/OGC)
  • the second MAC is a dicarboxylate carrier
  • the third MAC is a phosphate carrier (PiC)
  • the first MAC substrate is ⁇ -ketoglutarate
  • the second MAC substrate is malate
  • the third MAC substrate is inorganic phosphate (Pi).
  • the first MAC is a citrate transporter (CIC)
  • the second MAC is a dicarboxylate transporter (DIC)
  • the third MAC is a phosphate transporter
  • the first MAC substrate is citrate
  • the second MAC substrate is malate
  • the third MAC substrate is inorganic phosphate (Pi).
  • MACs of the present invention can be obtained, at least for example, as purified protein preparations from appropriate biological sources or as products of recombinant protein expression technologies.
  • a “purified” or “isolated” protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the MAC is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of MAC protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of MAC protein having less than about 30% (by dry weight) of non-MAC protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-MAC protein, still more preferably less than about 10% of non-MAC protein, and most preferably less than about 5% non-MAC protein.
  • non-MAC protein also referred to herein as a "contaminating protein”
  • contaminating protein also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%), and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of MAC protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of MAC protein having less than about 30% (by dry weight) of chemical precursors or non-MAC chemicals, more preferably less than about 20% chemical precursors or non-MAC chemicals, still more preferably less than about 10% chemical precursors or non-MAC chemicals, and most preferably less than about 5% chemical precursors or non-MAC chemicals.
  • a general MAC purification scheme involves solubilization of MACs with a non-ionic detergent, chromatography on hydroxylapatite, followed in some cases, by SH-affinity chromatography, See, for example, Wohlrab, H. (1986) Biochim. Biophys. Ada 853:8170-8173; Wehrle, J. P. and Pedersen, P. L. (1989) J. Membr. Biol. 111 :199- 213; Kramer, R. and Palmeri, F. (1989) Biochim. Biophys. Ada 974:1-23; Kolbe, H. V. J. et al. (1984) J. Biol. Chem. 259:9115-9120; Kaplan, R. S. et al.
  • MAC purification scheme involves solubilization of MACs with a non-ionic detergent, chromatography on hydroxylapatite, followed by sequential chromatography on Matrex Gel Orange ATM, Matrix Gel Blue BTM, and Affi-Gel 501TM. See Kaplan, R. S. et al. (1990) J Biol. Chem. 265:13379-13385.
  • MAC proteins of the present invention can also be produced using recombinant protein expression technologies.
  • the MAC proteins of the present invention can be produce in organisms including bacteria (e.g., Escherichia col ⁇ ) and yeast (e.g., Saccharomyces cerevisiae or Candida par apsilosis).
  • bacteria e.g., Escherichia col ⁇
  • yeast e.g., Saccharomyces cerevisiae or Candida par apsilosis
  • Production of MAC proteins in E. coli can been accomplished with slight modification of standard recombinant expression procedures e.g., those used to recombinantly produce globular proteins) taking into account the fact that MACs are integral membrane proteins. For instance, it has been demonstrated that abundant expression of the MACs, KGC/OGC and PiC, can be obtained in E. coli.
  • the anionic detergent, sarkosyl can be further utilized to enable the extraction of the expressed transporter from inclusion bodies in a form that can be functionally reconstituted into lipid vesicles. See, for example, Fiermonte, G. et al. (1993) Biochem. J. 294:293-299 and Wohlrab, H. and Briggs, C. (1994) Biochemistry 33:9371-9375. Furthermore, one of skill in the art can apply the procedures of Kaplan, R. S. (1996) J. Bioenerg. Biomembr. 28:41-47, to express other members of the MAC protein family.
  • the gene encoding DIC from the yeast Saccharomyces cerevisiae was recently cloned and the protein recombinantly expressed in E. coli. Kakhniashvili, et al. (1997) J. Biol. Chem. 272(7):4516-4521.
  • the MAC of the present invention is selected from the group consisting of UCP-1, UCP-2, and UCP-3.
  • the MAC of the present invention is selected from the group consisting of human UCP-1 (SEQ ID NO:2), UCP- 2 (SEQ ID NO:4), and UCP-3 (SEQ ID NO:6).
  • recombinant production of MACs can be accomplished by inserting the coding sequence of the gene encoding a MAC into a recombinant expression vector in the context of the appropriate regulatory elements, such that expression of the recombinant MAC protein is achieved.
  • a procedure is described in detail, for example, in Example 2.
  • the MAC proteins of the present invention can be recombinantly expressed at high levels and purified from mammalian cells.
  • Preferred mammalian cells for recombinant expression are those that can be efficiently transfected and express high levels of recombinant protein.
  • Such cells include, but are not limited to, baby hamster kidney (BHK) cells (ATCC No. CCL 10); Ltk" cells (ATCC No. CCL 1.3); DG44 cells (see e.g., Chasin (1986) Cell. Molec. Genet. 12:555); human embryonic kidney (HEK) cells (ATCC No. CRL 1573); Chinese hamster ovary (CHO) cells (ATCC Nos.
  • CRL 9618, CCL 61, CRL 9096 PC 12 cells (ATCC No. CRL 1721); CV-1 cells (ATCC No. CCL 70); and the COS-7 monkey kidney cell line (see e.g., Gluzman (1981) Cell 23:175) (ATCC No. CRL 1651).
  • Purification of MAC proteins from mammalian cells can be accomplished according to the procedures described herein for the isolation of MACs from appropriate biological sources.
  • the MACs of the present invention can have the amino acid sequences of any of the human UCPs, (e.g., human UCP-1, human UCP-2, or human UCP-3); the human PiCs, (e.g., PiC A, PiC B, or PiC C); the human glutamate carrier; or the human pyruvate carrier.
  • the nucleic acid sequence that encodes human UCP-1 can be found, at least for example, as GenBank Accession Number U28480.
  • the nucleic acid sequence that encodes human UCP-2 can be found, at least for example, as GenBank Accession Number U82819 or as GenBank Accession Number U76367.
  • the nucleic acid sequence that encodes human UCP-3 can be found, at least for example, as GenBank Accession Number AF001787 or as GenBank Accession Number U84763.
  • the nucleic acid sequence that encodes the human PiCs, (e.g., PiC A, PiC B, and PiC C; three alternatively spliced forms of PiC) can be found, at least for example, as GenBank Accession Number X77337.
  • the MACs of the present invention can have the amino acid sequences of any of AAC, AGC, OCG, DIC, CIC, and APC.
  • the nucleic acid sequences that encode the electroneutral carrier proteins can be found, at least for example, as GenBank Accession Numbers gi: 339919 (ANT1), gi: 178660 (ANT2), gi: 339722 (ANT3), gi: 23843 (OGC), gi: 950003 (CIC), for example.
  • the exemplary MACs of described herein can be encoded by nucleic acid sequences other than those specified herein, due to the degeneracy of the genetic code.
  • the MACs of the present invention can have an amino acid sequence having sufficient homology to one of the amino acid sequences described herein.
  • the term "sufficiently homologous" refers to a first amino acid sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues to a second amino acid sequence such that the first and second amino acid sequences share a common functional activity.
  • amino acid sequences that share at least 40%, preferably 50%, more preferably 60, 70, or 80% homology and share a common functional activity are defined herein as sufficiently homologous.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%), or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the UCP-1 amino acid sequence of SEQ ID NO:2 having 307 amino acid residues, at least 92, preferably at least 123, more preferably at least 154, even more preferably at least 176, and even more preferably at least 215, 246 or 276 amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity”).
  • the comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77.
  • Such an algorithm is inco ⁇ orated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids
  • a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • a PAM120 weight residue table a gap length penalty of 12
  • a gap penalty of 4 can be used.
  • MACs having amino acid substitutions at "non-essential" amino acid residues can be used according to the present invention.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of the exemplary MACs described herein without altering the functional activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the MAC proteins of the present invention are predicted to be particularly unamenable to alteration. Such conserved amino acids can be determined, at least for example, from an alignment of the amino acid sequences of the exemplary MAC proteins described herein.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • an “activity” or “functional activity” refers to an activity exerted by a protein of the present invention, (e.g., a MAC protein), as determined in vivo or in vitro, according to standard techniques.
  • a functional activity of a MAC protein can be the transport of a MAC substrate across an inner mitochondrial membrane or, alternatively, across a reconstituted lipid bilayer vesicle.
  • the activity of a MAC can be determined by measuring at least one component of MAC transporter activity (e.g., a change in membrane potential or change in intravesicular pH).
  • MAC transporter activity e.g., a change in membrane potential or change in intravesicular pH.
  • chromogenic indicator refers to a compound which produces a colorometric readout as an indicator of, for example, pH or membrane potential. A change in the colorometric readout of such a chromogenic indicator is indicative of a change in, for example, pH or membrane potential.
  • the change in the colorometric readout of such a chromogenic indicator is a quantitative measurement of the change in, for example, pH or membrane potential.
  • chromogenic indicators which change their color in response to a change in pH include, but are not limited to, litmus, phenolphthalein, and phenol red.
  • the Mg 2+ dye, Magfure is used to measure citrate entry via CIC due to the ability of citrate to chelate Mg 2+ .
  • a fluorescent indicator can be utilized in accordance with the present invention.
  • fluorescent indicator refers to a compound which produces a fluorescent readout as an indicator of, for example, pH or membrane potential.
  • a change in the fluorescent readout of such an indicator is indicative of a change in, for example, pH or membrane potential.
  • the change in the fluorescent readout of such an indicator is a quantitative measurement of the change in, for example, pH or membrane potential.
  • fluorescent indicators which produce a change in fluorescence in response to a change in pH include, but are not limited to, fluorescein and fluorescein derivatives (e.g., fluorescein diacetate (FDA), carboxyfluorescein diacetate (CFDA), the polar fluorescein derivative, BCECF, and the AM ester of BCECF, methylated fluorescein and/or fluorescein diacetate, and fluorescein sulfonic acid and/or fluorescein sulfonic acid diacetate); the seminaphthorhodafluors (SNARF dyes) and seminapthofluoresceins (SNAFL dyes); and/or pyranin.
  • fluorescent indicators can be further
  • fatty acids may be desirable to incorporate free fatty acids as a component of the assay vesicles.
  • FFAs free fatty acids
  • fatty acid molecules can be inco ⁇ orated into the assay vesicles of the present invention during the process of forming the assay vesicles.
  • the assay vesicles of the present invention can be contacted with fatty acid molecules subsequent to the formation of the assay vesicles and prior to contacting the assay vesicles with a test compound and/or a prior to contacting the vesicle with a test compound.
  • test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • biological libraries include biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
  • Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, and peptidomimetics.
  • peptidomimetic is intended to encompass compounds that are comprised, at least in part, of molecular structures different from naturally-occurring L-amino acid residues linked by natural peptide bonds.
  • Peptidomimetics are intended to include compounds composed, in whole or in part, of structures such as D-amino acids, non-naturally-occurring L-amino acids, modified peptide backbones and the like, as well as compounds that are composed, in whole or in part, of molecular structures unrelated to naturally-occurring L-amino acid residues linked by natural peptide bonds.
  • compounds identified in the subject assay can be formulated in pharmaceutical preparations for in vivo administration to an animal, preferably a human.
  • Such preparations comprise a therapeutically (or prophylactically) effective amount of the mitochondrial transport modulator, and a pharmaceutically acceptable carrier or excipient.
  • the preparations may be formulated for administration with a pharmaceutically acceptable carrier except insofar as any conventional media or agent is incompatible with the activity of the compound.
  • compositions can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • Other carriers include water, saline, saline buffered with phosphate or citrate at suitable concentrations so as to obtain at all times isotonic pharmaceutical preparations of the desired pH, (for example, neutral pH), dextrose, ethanol, mannitol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • the use of other such media for pharmaceutically active substances is known in the art.
  • the optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists.
  • the carrier and composition can be sterile. The formulation should suit the mode of administration.
  • Single and multiple (e.g., 5 to 7 days) dose investigative toxicology studies are typically performed in the efficacy test species using the intended route of administration for the efficacy study. These investigative toxicology studies are performed to identify maximum tolerated dose, subjective bioavailability from the intraperitoneal or oral routes of administration , and estimation of an initial safety margin. Initial bioavailability and pharmacokinetics (blood clearance) of the compounds may be determined, with standard cold or radioactive assay methods, to assist in defining appropriate dosing regimens for the compounds in the animal models.
  • the effective dose of the mitochondrial transport modulator will typically be in the range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 m g/kg of mammalian body weight, administered in single or multiple doses.
  • the mitochondrial transport modulator may be administered to patients in need of such treatment in a daily dose range of about 0.5 to about 2000 mg per patient.
  • the amount of the mitochondrial transport modulator which will be effective in the treatment or prevention of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the precise dosage level of the mitochondrial transport modulator, as the active component(s) should be determined by the attending physician or other health care provider and will depend upon well known factors, including route of administration, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the disease; and the use (or not) of concomitant therapies.
  • Suitable delivery vehicles are known and can be used to administer the mitochondrial transport modulator, e.g., encapsulation in liposomes, microparticles, injectable "deposit formulations", microcapsules, etc.
  • Materials and methods for producing the various formulations are well known in the art [see e.g. US Patent Nos. 5,182,293 and 4,837,311 (tablets, capsules and other oral formulations as well as intravenous formulations)].
  • Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). Administration can be systemic or local.
  • Methods of introduction include but are not limited to pulmonary administration, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes, for example by infusion or bolus injection, by abso ⁇ tion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the side of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • Topical administration to an individual of an effective amount of the biased agonist can be accomplished by administering the compound(s) directly to the affected area of the skin of the individual.
  • the mitochondrial transport modulator is administered or applied in a composition including a pharmacologically acceptable topical carrier, such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils.
  • topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water.
  • Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.
  • the mitochondrial transport modulator may be administered locally to the area in need of treatment by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by means of a catheter, by means of a suppository, or by means of a skin patch or implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers which release the compound into the skin, by either passive or active release mechanisms.
  • Vesicles can be prepared by a variety of methods. For example, vesicles can be prepared according to the following techniques: RAPID METHOD
  • This method involves preparing lipid bilayer vesicles comprising FAA and lysophospholipids.
  • the vesicle preparations can be analyzed by NMR and electron microscopy.
  • Small unilamellar vesicles can be formed spontaneously from equimolar mixtures of lyso 1-acyl phosphatidylcholine (lyso-PC) and oleic acid.
  • lyso-PC lyso 1-acyl phosphatidylcholine
  • a dried lipid film is hydrated in 50 mM Tris buffer, pH 7, 20 °C and vortexed vigorously.
  • the resulting lipid dispersion appears translucent with a slight bluish tinge typical of small unilamellar vesicles.
  • the ⁇ lp NMR spectra of this dispersion is also characteristic of such vesicles, showing partially resolved signals for outer and inner monolayer lyso-PC.
  • Egg phosphatidylcholine, cardiolipin (optional), and phosphatidic acid are mixed at a weight ratio of 1 :2.65:60 with octyl-POE and aqueous buffer.
  • Octyl-POE is used during reconstitution of these vesicles to dissolve the lipids and to achieve the optimal detergent:Bio-Beads ratio.
  • Cardiolipin a natural component of inner membrane, causes a reproducibly larger liposomal volume. The lipids are dissolved by stirring and heating in a 50° water bath and the resulting clear solution cooled. Liposomes are formed by detergent removal using Bio-Beads SM-2, as follows.
  • the lipid-detergent-buffer mixture is incubated for 90 min at 4° with Bio-Beads SM-2 that have been preequilibrated with internal medium.
  • the mixture is loaded onto a column or centrifuged to collect eluate. This can be followed by a second 30-min incubation with fresh Bio-Beads to remove residual detergent from the formed liposomes. Successful formation of liposomes is readily apparent from the milky, opalescent appearance of the eluate. (Garlid et al. (1995) Method Enzymol. 260:331-348).
  • the composition of the bilayer membrane can be varied from phosphatidylcholine alone to mixtures of phospholipids resembling those in the inner mitochondrial membrane.
  • aqueous KCl 0.05 M KBr/0.01 M potassium phosphate/0.1 mM EDTA/0.1% sodium azide, pH 7.4, or 0.5-2.0%) (wt/vol) aqueous KCl is added to the flask.
  • buffer 0.05 M KBr/0.01 M potassium phosphate/0.1 mM EDTA/0.1% sodium azide, pH 7.4
  • aqueous KCl aqueous KCl
  • distilled, deionized water is boiled to bring the pH to neutrality, and all subsequent steps are done under N2 to keep the pH above 6.5.
  • the sample is agitated for 1 hr at room temperature on a Vortex mixer and then transferred to a centrifuge tube, using 0.1 ml of buffer and 0.1 ml of ⁇ H2 ⁇ to rinse the flask.
  • the sample compositions are given as % triolein by weight of tltal lipid (i.e., 5% triolein, 95%) PtdCho), with the PtdCho concentration ranging between 10 and 100 mg/ml.
  • the sample is sonicated by using a Branson sonifier with a micro-tip, at power level 3 in a pulsing mode with a 30% duty cycle.
  • samples are analyzed for composition and purity. No ( ⁇ 1%) unesterified fatty acid or lysolecithin is detected by thin-layer chromatography.
  • the phosphatidylcholine concentration of the samples in aqueous KCl is determined by a modified Bartlett method.
  • the methods can be further modified to facilitate the formation of vesicles including an internal pH indicator or potentiometric indicator (e.g., a fluorescent dye).
  • an internal pH indicator or potentiometric indicator e.g., a fluorescent dye
  • Vesicles e.g., small unilamellar vesicles, SUVs
  • SUVs small unilamellar vesicles
  • LUVs large unilamellar vesicles
  • a hydrated egg phosphatidylcholine (Avanti Polar Lipids) suspension 19 times through an extrusion apparatus.
  • Untrapped pyranin is removed by placing 1.0 ml of the vesicle suspension on a gel filtration column (20 ml of Sephadex G-25, medium grade), washing with 100 mM Hepes/KOH (pH 7.4), and using a UV lamp to monitor the separation. Accordingly, the fluorophore is removed from the external membrane by passing the vesicles over a column, and the dye remains trapped inside the vesicle, where it reports internal pH.
  • the fluorescent probe can be added after the lipid solution has been cooled.
  • 250- ⁇ l aliquots of the proteoliposome suspension are passed twice through 5-ml Sephadex G-25-300 columns, which have been preequilibrated with internal medium lacking probe. Note: This resin contains high amounts of K + , which is removed with extensive washing in storage buffer (normally identical to internal medium lacking fluorescent probe).
  • Ideal lipid vesicles have a low permeability to ions and the pH gradient returns to equilibrium very slowly. Inco ⁇ oration of a fluorescent dye into an assay vesicle, can be used as an indicator of the success of generating a "non-leaky" vesicle preparation. For example, the ability of these vesicles to retain trapped contents can be established using pyranine, a membrane impermeant fluorescent dye. In particular, when FAA are added to non-leaky vesicles, the pH drop is sustained over long time periods (minutes). It returns slowly to equilibrium as K + from the external buffer exchanges with + H inside the vesicle.
  • This example describes the preparation and purification of the MAC, UCP, for use in the preparation of assay vesicles.
  • exemplary methods are provided for bacterial expression of UCP-1, overexpression of UCP-1 in yeast, and purification of UCP from hamster mitochondria or, alternatively, from yeast overexpressing UCP-1.
  • the open reading frame (ORF) encoding the MAC (e.g., UCP-1 (SEQ ID NO:l), UCP-2 (SEQ ID NO:3), or UCP-3 (SEQ ID NO:5)) plus additional 3' untranslated sequence is amplified by PCR.
  • ORF open reading frame
  • a forward primer is utilized which contains an restriction endonuclease site plus additional 5' sequence (approximately 10-20 nucleotides) in order to allow efficient restriction digestion. If necessary, modifications are introduced into the forward primer in order to eliminate existing rare codons at the beginning of the ORF which might minimize the level of express in E.
  • a reverse primer is designed which corresponds to sequence following the end of the ORF. The location of this primer is chosen to correspond to a region of the template that is both near the end of the ORF and will allow specific priming.
  • the reverse primer contains also a restrictionendonuclease site plus additional 5' bases (approximately 10-20 nucleotides). Amplifications are performed using standard conditions that have been previously described in detail (Kaplan et al. (1995) J. Biol. Chem. 270:4108-4144).
  • the product is purified using, for example, the Geneclean II Kit (Bio 101, Inc.).
  • the purified product is sequentially digested with the appropriate restriction endonucleases (depending on the sites added via the primers.
  • the resulting digest is extracted and purified, again, for example, with the Geneclean II Kit.
  • the purified fragment is then directionally cloned into either an appropriate expression plasmid.
  • this exemplary expression procedure is described in detail in Kaplan (1996) J. Bioenerg. Biomembr.
  • Transformants are screened for the presence of the UCP insert via: (i) direct colony PCR (using the same primer combination as above); and/or (ii) restriction analysis of purified plasmid DNA using the appropriate restriction endonucleases.
  • BL21(DE3) competent cells Novagen
  • the expression host are then transformed with l ⁇ l of a 50-fold dilution of plasmid DNA, purified (via the Wizard Minipreps DNA Purification System (Promega)) from NovaBlue cells, the storage host.
  • Transformants are screened for inserts as described above.
  • the cloned UCP fragment is then sequenced in its entirety, using plasmid DNA purified from BL21(DE3) cells as the template, in order to ensure that no mutations have occurred.
  • the suspension is centrifuged at 12,000 X g for 15 min, and the pellet is resuspended in 2 ml of Buffer B (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.0; 1.0 mM dithioerythritol) and recentrifuged.
  • the resulting pellet is resuspended in 2 ml of Buffer B and the inclusion body fraction isolated by centrifugation at 131 ,000 x g for 4.5 h through a step gradient consisting of 12.4 ml of 40%) (w/v) sucrose and 18.6 ml of 53% sucrose (Fiermonte et al. (1993) Biochem. J. 294:293-299) (the sucrose solutions are prepared in Buffer B).
  • the inclusion body pellet is resuspended in 30 ml of Buffer B and centrifuged at 12,000 x g for 15 min.
  • the UCP is then extracted by resuspension of the inclusion body pellet in 2 ml of 1.2% (w/v) sarkosyl dissolved in Buffer B (Fiermonte et al. (1993)). Following centrifugation at 314,000 x g for 30 min, the resulting supernatant contains the solubilized UCP.
  • the S. cerevisiae strain JB 516 (MAT a, ura3, adel, leu2, his4, gal + ) is transformed with the Escherichia coli/Saccharomyces cerevisiae multicopy shuttle vector pCGSl 10 (Collaborative Research Inc.) containing the coding region of human UCP cDNA (e.g., the coding region of SEQ ID NO:l, SEQ ID NO:3, or SEQ ID NO:5) inserted at the unique BamHl site beside the inducible Gall promoter.
  • the vector contains the ura3 gene as the yeast selectable marker.
  • the altered cDNA can be constructed in a bacterial plasimd vector (e.g., pGEM5Zf (Promega)) and subsequently subcloned into pCGSl 10 prior to transformion into yeast. Yeast transformants are grown at 30°C in selective medium containing
  • yeast nitrogen base 0.5% (NH ⁇ SO and 0.005% each of adenine, leucine, and histidine.
  • the carbon source is 2% lactate, 0.05% glucose (Daum et al. (1982) J. Biol. Chem. 257:13028-13033).
  • Mitochondrial Isolation Mitochondrial Isolation. Mitochondria are isolated from yeast transformants as described by Gasser (1983) Methods Enzymol. 97:329-336 from cells induced with 0.2%) galactose for 8-10 h. The nuclear fraction is recovered at 3500 ⁇ m for 5 min, and the mitochondrial fraction, at 9000 ⁇ m for 10 min. Mitochondria are resuspended in a solution containing 0.6 M mannitol, 20 mM Hepes-KOH, pH 7.4, and 0.1%) fatty acid- free bovine serum albumin. Inner Membrane Preparation.
  • Yeast mitochondria are fractionated in a 1.0-ml volume using a 0.4:1.0 ratio of digitonin to mitochondrial protein.
  • the resulting mitoplasts are resuspended in 500 ⁇ l of hypotonic medium containing 10 mM Tris, 1 mM EDTA, pH 7.2, and sonicated 3 x 20 s in a bath sonicator to release the matrix contents.
  • Membranes are collected at 150,000 x g for 20 min and resuspended in 500 ⁇ l of the mitochondrial resuspension buffer described above.
  • UCP can purified from yeast mitochondria, for example, using the same protocols that have been described for tissue mitochondria (See Jezek et al. (1990) J Biol. Chem. 265:19296- 19302).
  • UCP from eukaryotic (e.g., mammalian or yeast) mitochondria can be achieved using the following exemplary procedure.
  • Mitochondria from brown adipose tissue or S. cerevisiae are washed with extraction buffer (e.g., 250 mM sucrose, 10 mM TEA-TES, 1 mM K-EGTA, pH 6.7, containing 2 mg BSA/ml) and centrifuged.
  • extraction buffer e.g. 250 mM sucrose, 10 mM TEA-TES, 1 mM K-EGTA, pH 6.7, containing 2 mg BSA/ml
  • Extraction buffer contains 25 mM SOI/2. 1.5 mM EGTA (TEA" salts), and 50 mM phosphate. pH 7.2 (Tris salt).
  • To 36 mg of a dried lipid mixture whose composition is given in Table 1 add 0.550 ml of extraction buffer and 70 ⁇ l of Octyl-POE. Lipids are solubilized by heating to 50°, then cooled to 0°. The mitochondrial pellet, containing 10 mg of protein, is gently homogenized with this buffer-lipid-detergent mixture.
  • the extraction buffer and 0.45g of dry hydroxylapatite (Bio-Gel HTP. Bio-Rad) are placed in a 3 -ml polyethylene syringe with silicon fibers in the outlet and centrifuged for 2 min at 800 g to remove free buffer.
  • the protein-lipid-detergent mixture is loaded onto the column, mixed with the hydroxylapatite, incubated for 10 min at 20° to inactivate the ATP/ ADP exchanger, and then incubated for 25 min at 0°.
  • the column is centrifuged for 2 min at 800g.
  • the flow-through eluate from the hydroxylapatite column contains predominately (>95%) UCP and is used directly for reconstitution.
  • Fractions are identified by the presence of a 32-kDa protein on SDS-PAGE gels, by Western blot analysis using antibodies raised against the purified uncoupling protein, and by reconstitutive activity.
  • the method can be further modified to facilitate the formation of vesicles including a particular MAC, for example, UCP-1.
  • a particular MAC for example, UCP-1.
  • purified or recombinant MACs can be included during vesicle formation using the well-established methods of Jezek et al. (1994) J. Biol. Chem. 269:26184-190; Jekek et al. (1990) J. Biol. Chem. 265:19296-302; Garlid et al. (1996) J. Biol. Chem. 271 :2615-2620; and Garlid et al. (1995) Methods Enzymol. 260:331-348.
  • the composition of vesicles can be optimized for reproducibility of vesicle formation, ease of preparation and ultimately effective insertion of the MAC into the membrane.
  • the topography of MAC insertion into the bilayer e.g., the "sidedness" or orientation of the MAC
  • orientation-specific antibodies such as those described in Brandolin at al. (1989), Biochemistry 28(3):1093- 1100; Capobianco et al. (1991) Biochemistry 30(20): 4963-4969; Ferreira et al. (1990) J. Biol. Chem. 265(34): 21202-21206; and Miroux et al. (1992) J. Biol Chem.
  • antibodies directed to portions of the MAC which face the cytoplasm in the intact cell can be reacted with vesicle preparations containing a MAC as a component of the lipid bilayer and compared, for example, to vesicles containing no MAC. Interaction of the antibody with the vesicle containing the MAC which is detectable when compared to the interaction of the antibody with the vesicle containing no MAC indicated inco ⁇ oration of the at least some MAC molecules having the correct sidedness or orientation.
  • a lack of interaction of antibodies directed to portions of a MAC which face the matrical side of an intact mitochondria as compared to the interaction of such antibodies with control vesicles containing no MAC, can also provide a positive indicator of the correct sidedness of MAC inco ⁇ oration.
  • orientation can be determined using know inhibitors of MAC activity added to the vesicle preparation either extravesicularly or inco ⁇ orated internally, during the formation of the assay vesicles.
  • the nucleotide inhibitor of UCP-1 GPD
  • GPD can be added extravesicularly to a vesicle preparation, and the effect of GDP addition on proton conductance determined.
  • External addition of GDP to UCP-containing vesicles should inhibit the activity of correctly-oriented UCP (the inhibitory binding site for GDP occurs on the cytoplasmic face of the mitochondria in an intact cell) but should have no effect on UCP molecules which have inco ⁇ orated "in- side-out".
  • Assay sensitivity can be enhanced by determining the ratio of internal to external changes.
  • the data presented in Example 1 demonstrates the feasibility of combining two dyes with differing fluorescence characteristics that monitor different aspects of a related change. A reasonable balance between the two dyes is necessary to assure adequate sensitivity of the measurement. This can be achieved by lowering the ratio of vesicles to external medium, by placing the higher yield fluorescent indicator on the outside where it will be more dilute and by adjusting the concentration of external dye.
  • pyranine with its lower fluorescence yield (excitation 450 nm, emission at 509 nm), can be trapped inside of vesicles and SNARF (Molecular Probes, Eugene, OR) can be added to the outside (excitation 534 nm, emission 640 nm).
  • SNARF Molecular Probes, Eugene, OR
  • the rationale for using a different dye on the outside with different spectral characteristics than the internal dye is to permit continuous assessment of vesicular leakiness.
  • a satisfactory combination of dyes gives a linear rate of internal acidification and external alkalinization in response to FAA addition during the time interval of the measurement. It may be desirable to begin with a more alkaline internal pH in order to prolong the linear phase of acidification.
  • the vesicles reconstituted with UCPs and containing the trapped pH dye are designed to be used to test the effects of biomolecules or drugs on the transport function of the UCPs. Not only can the influence of drugs on the FAA induced pH gradient across the bilayer but also the effects of drugs on inhibition of FAA transport by the endogenous regulators of UCPs, nucleotides can be studied.
  • the assays of Examples 1-3 can be adapted to a multiwell plate and automated plate reader. In such a format, the fluorescence measurements are straightforward and quick, thus allowing rapid testing of many drugs.

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Abstract

L'invention porte sur des procédés de criblage de composés thérapeutiques de traitement des troubles du métabolisme, tels que le diabète. L'invention porte en particulier sur des procédés d'identification de composés modulant l'activité de porteur d'anions de mitochondries (MAC) consistant: à mettre en contact les vésicules d'essai de l'invention avec des composés d'essai; à déterminer la capacité des composés d'essai à moduler le transport d'un substrat de MAC à travers la double couche de lipide de la vésicule d'essai; puis à identifier le composé d'essai comme présentant une activité de modulateur d'un MAC. Les vésicules d'essai de l'invention comportent par exemple un, deux, ou trois MAC, l'activité de l'un d'eux au moins étant couplée à une lecture détectable (par exemple une modification du potentiel ou du pH membranaire). L'invention porte également sur de nouvelles vésicules d'essai et sur leurs nouvelles méthodes de préparation.
PCT/US1999/012623 1998-06-08 1999-06-08 Nouveaux procedes d'identification de modulateurs de l'activite de porteurs d'anions de mitochondries Ceased WO1999064458A1 (fr)

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Cited By (9)

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WO2001040289A1 (fr) * 1999-11-30 2001-06-07 Bioroad Gene Development Ltd. Shanghai Nouveau polypeptide, support mitochondrial 12, et polynucleotide codant pour ce polypeptide
WO2001079508A1 (fr) * 2000-03-28 2001-10-25 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine porteuse mitochondriale humaine 18, et polynucleotide codant pour ce polypeptide
US6365796B1 (en) 2000-02-16 2002-04-02 Beth Israel Deaconess Medical Center Transgenic UCP2 knockout mouse and use thereof
WO2001098512A3 (fr) * 2000-06-22 2003-01-03 Genentech Inc Equilibrage du metabolisme a l'aide de compositions du porteur de 2-oxoglutarate humain
EP1475439A3 (fr) * 2000-06-22 2005-01-05 Genentech, Inc. Contrôle du métabolisme avec des compositions contenant le transporteur de 2-oxoglutarate
WO2005012570A1 (fr) * 2003-07-31 2005-02-10 Banyu Pharmaceutical Co., Ltd. Methode d'evaluation d'un compose efficace pour traiter l'obesite au moyen de slc25a10
WO2006066599A3 (fr) * 2004-12-22 2006-08-17 Rheoscience As Cibles de medicaments
US7118873B2 (en) 2000-12-12 2006-10-10 The University Of Connecticut Polynucleotides encoding cellular transporters and methods of use thereof
WO2022020559A1 (fr) * 2020-07-24 2022-01-27 The Regents Of The University Of California Méthodes et compositions pour le traitement de troubles métaboliques

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HALESTRAP A.P.: "Pyruvate and Ketone-Body Transport Across the Mitochondrial Membrane. Exchange Properties, pH-Dependence and Mechanism of the Carrier", BIOCHEM. J., vol. 172, no. 3, June 1978 (1978-06-01), pages 377 - 387, XP002927022 *
JABUREK M. ET AL.: "Transport Function and Regulation of Mitochondrial Uncoupling Proteins 2 and 3", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 37, 10 September 1999 (1999-09-10), pages 26003 - 26007, XP002927026 *
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JEZEK P. ET AL.: "Reconstituted Plant Uncoupling Mitochondrial Protein Allows for Proton Translocation via Fatty Acid Cycling Mechanism", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 39, 26 September 1997 (1997-09-26), pages 24272 - 24278, XP002927025 *
JEZEK P. ET AL.: "Transport of Anions and Protons by the Mitochondrial Uncoupling Protein and its Regulation by Nucleotides and Fatty Acids, A New Look at Old Hypotheses", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 42, 21 October 1994 (1994-10-21), pages 26184 - 26190, XP002927021 *
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001040289A1 (fr) * 1999-11-30 2001-06-07 Bioroad Gene Development Ltd. Shanghai Nouveau polypeptide, support mitochondrial 12, et polynucleotide codant pour ce polypeptide
US6365796B1 (en) 2000-02-16 2002-04-02 Beth Israel Deaconess Medical Center Transgenic UCP2 knockout mouse and use thereof
WO2001060152A3 (fr) * 2000-02-16 2002-04-18 Beth Israel Hospital Mammifere knockout transgenique non humain a gene ucp2 desactive
WO2001079508A1 (fr) * 2000-03-28 2001-10-25 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine porteuse mitochondriale humaine 18, et polynucleotide codant pour ce polypeptide
WO2001098512A3 (fr) * 2000-06-22 2003-01-03 Genentech Inc Equilibrage du metabolisme a l'aide de compositions du porteur de 2-oxoglutarate humain
EP1475439A3 (fr) * 2000-06-22 2005-01-05 Genentech, Inc. Contrôle du métabolisme avec des compositions contenant le transporteur de 2-oxoglutarate
US7141421B2 (en) 2000-06-22 2006-11-28 Genentech, Inc. Control of metabolism with human 2-oxoglutarate carrier
US7118873B2 (en) 2000-12-12 2006-10-10 The University Of Connecticut Polynucleotides encoding cellular transporters and methods of use thereof
WO2005012570A1 (fr) * 2003-07-31 2005-02-10 Banyu Pharmaceutical Co., Ltd. Methode d'evaluation d'un compose efficace pour traiter l'obesite au moyen de slc25a10
JPWO2005012570A1 (ja) * 2003-07-31 2007-09-27 萬有製薬株式会社 Ｓｌｃ２５ａ１０による肥満治療に有効な化合物の評価方法
WO2006066599A3 (fr) * 2004-12-22 2006-08-17 Rheoscience As Cibles de medicaments
WO2022020559A1 (fr) * 2020-07-24 2022-01-27 The Regents Of The University Of California Méthodes et compositions pour le traitement de troubles métaboliques

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