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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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• • 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

• CPTl carnitine palmitoyltransferase 1
• the present invention also contemplates compound screening using a variety of assay formats.
• the present invention contemplates a method for compound screening, comprising: a) providing: i) a purified preparation comprising malonyl CoA decarboxylase, ii) a substrate, and iii) a test compound; b) mixing said malonyl CoA decarboxylase and said substrate under conditions such that said malonyl CoA decarboxylase can act on said substrate to produce product, wherein said mixing is done in the presence and absence of said test compound; and c) measuring directly or indirectly the amount of said product produced in the presence and absence of said test compound.
• the present invention contemplates a method of screening for tumors, said method comprising: a) providing in any order: i) microassay microchips wherein said microchip comprises cDNA encoding at least a portion ofthe oligonucleotide sequence of SEQ ID NOS: 1,2 or 3, ii) DNA from at least one tissue sample suspected of having mutations in genes similar to SEQ LD NOS:l, 2 or 3; b) contacting said microassay microchips with said DNA; and c) detecting hybridization of said cDNA with said tissue sample DNA. Isolated DNA would then be sequenced to assay for genetic mutations.
• the gene sequences of the present invention may also be used to screen for homologous.
• the present invention may also be used to identify novel or mutant constituents ofthe MCD pathway.
• antibodies generated to translation products of the invention may be used in immunoprecipitation experiments to isolate novel MCD pathway constituents or natural mutations thereof.
• the invention may be used to generate fusion proteins that could also be used to isolate novel MCD pathway constituents or natural mutations thereof.
• screens may be conducted using the yeast two-hybrid system.
• the present invention also contemplates screening for homologues using standard molecular procedures. In one embodiment screens are conducted using Northern and Southern blotting.
• the present invention contemplates a method of screening a compound, said method comprising: a) providing in any order: i) a first group of cells comprising a recombinant expression vector, wherein said vector comprises at least a portion of the oligonucleotide sequence of SEQ LD NOS:l, 2 or 3, ii) a second group of cells comprising a recombinant expression vector, wherein said vector comprises the plasmid used above without any portion ofthe oligonucleotide from SEQ ID NOS:l, 2, or 3, and iii) at least one compound suspected of having the ability to modulate MCD pathway activity; b) contacting said first and second groups of cells with said compound; and c) detecting inhibition or enhancement of MCD activity of said compound.
• FIGURE 3 graphically depicts Malonyl CoA decarboxylase activity in aerobic, ischemic and reperfused ischemic rat hearts. Values are the mean + S.E. of 5-10 hearts for all groups.
• FIGURE 4 is a representative photograph of immunoblot analysis using anti MCD antibody depicting the levels of malonyl CoA decarboxylase protein in aerobic, ischemic and reperfused ischemic rat hearts.
• FIGURE 10 shows the characterization of malonyl CoA decarboxylase antibodies.
• A the polyclonal antibodies directed against rat liver MCD were tested using protein from a rat liver mitochondrial fraction.
• the MCD239 antibodies (lane 1) recognized MCD only, while MCD240 (lane 2) recognized both catalase and MCD. MCD and catalase are indicated at the left.
• B immuno-inhibition studies were performed using both the MCD antibodies.
• the MCD antibody or pre-immune serum mixtures was measured for MCD activity and expressed as a % of MCD pre-incubated without serum for an identical period of time (30 minutes).
• FIGURE 14 shows rat liver malonyl CoA decarboxylase activity and protein levels in control fasted, and refed rats.
• MCD activity (A) and MCD protein quantification (B) was performed on rat livers from control, fasted and refed rats.
• MCD protein is located in the bottom panel of each group and catalase protein levels are included as loading and transfer controls in the top panels.
• the Western blots underwent densitometry analysis and the levels of MCD protein were quantified (C).
• the substrate is Malonyl CoA.
• the terms “complementary” or “complementarity” when used in reference to polynucleotides refer to polynucleotides which are related by the base-pairing rules. For example, for the sequence 5'-AGT-3' is complementary to the sequence 5'-ACT-3'.
• purified or “to purify” refers to the removal of contaminants from a sample.
• the present invention contemplates purified compositions
• the present invention relates generally to compositions and methods of identifying and testing Malonyl CoA decarboxylase (MCD) inhibitors, and in particular, compositions comprising a novel cardiac isoform of MCD, identified to be a key regulator of fatty acid oxidation in the heart. Additionally, the invention relates to compositions and methods of identifying MCD pathway agonists and antagonists, and in particular, compositions comprising novel DNA sequences of rat heart, liver and pancreatic DNA
• MCD Malonyl CoA decarboxylase
• inhibition of 5'-AMP-activated protein kinase and/or stimulation of acetyl CoA carboxylase may be a pharmacologic approach to inhibiting myocardial fatty acid oxidation during reperfusion. Decreasing fatty acid oxidation is accompanied by a parallel increase in glucose oxidation that results in an improvement in both cardiac function and efficiency in the reperfused ischemic heart [Lopaschuk, Am JCardiol 80:11A-16A (1997)].
• MCD Partial purification of cardiac MCD revealed a protein of approximately 45 kDa, that runs as a tetrameric complex when subjected to polyacrylamide gel electrophoresis.
• MCD has previously been described as a mitochondrial enzyme which is involved in protecting certain mitochondrial enzymes such as methylmalonyl CoA mutase and propionyl CoA from inhibition by mitochondrial derived malonyl CoA [Kim and Kolattukudy, Arch. Biochem. Biophys. 190:234-246 (1978); Scholte, Biochim. Biophys. Acta. 178:137-144 (1969); Landriscina et al, Eur. J. Biochem.
• the liver is thought of as mainly a biosynthetic organ, however, it also oxidizes fatty acids as a source of energy [Goodridge, Fatty acid synthesis in eucaryotes In Biochemistry of lipids, liposomes and membranes Ed Vance and Vance 111-139 (1991)] Malonyl CoA is important in this process, since it inhibits carnitine palmitoyltransferase 1 (CPTl), the rate- limiting enzyme involved in the mitochondrial uptake of fatty acids [McGarry and Brown, Eur J Biochem 244 1-14 (1997), Alam and Saggerson, Biochem J 224 233-241 (1998), Bird and Saggerson, Biochem 222 639-647 (1984)] By inhibiting CPTl, mitochondrial uptake of fatty acids is decreased, thereby reducing mitochondrial fatty acid oxidation [, Lopaschuk et al, J Biol Chem 269 25871-25878(1994)] During times of nutritional deficiency
• Preferred embodiments include binding assays which use cardiac MCD which are produced by recombinant methods or chemically synthesized.
• the methods of screening employs, in addition to MCD and the substrate malonyl CoA, the use of C- labeled oxaloacetate to produce C-labeled citrate.
• the screening assays ofthe present invention will, among other things, identify agents that will inhibit MCD activity.
• Such inhibitors are contemplated to be useful in the treatment of ischemia (e.g., Pharmacological inhibition of MCD should result in an increase in myocardial levels of malonyl CoA in the reperfused ischemic heart), as well as other disorders. This will lower the fatty oxidation rates, increase glucose oxidation and improve contractile function of reperfused ischemic hearts.
• the present invention contemplates compound screening using a variety of assay formats.
• the present invention contemplates a method for compound screening, comprising: a) providing: i) a purified preparation comprising malonyl CoA decarboxylase, ii) a substrate, and iii) a test compound; b) mixing said malonyl CoA decarboxylase and said substrate under conditions such that said malonyl CoA decarboxylase can act on said substrate to produce product, wherein said mixing is done in the presence and absence of said test compound; and c) measuring directly or indirectly the amount of said product produced in the presence and absence of said test compound.
• inhibition is measured by detection of acetyl CoA formation as estimated by reduction of [ C] citrate levels.
• Transfection assays allow for a great deal of flexibility in assay development.
• the wide range of commercially available transfection vectors will permit the expression ofthe MCD genes ofthe present invention in a extensive number of cell types.
• cells are transiently transfected with an expression construct comprising in operable combination 1) nucleic acid encoding MCD and 2) an inducible promotor. Cells would be exposed to the agent suspected of modulating MCD activity, MCD expression would be turned on and MCD activity would be measured. Rates of MCD activity in cells expressing recombinant MCD are compared to rates of MCD activity in cells transfected with a control expression vector (e.g., an empty expression vector).
• a control expression vector e.g., an empty expression vector
• Rates of MCD activity can be quantitated by any of a number of ways reported in the literature and known to those practiced in the art.
• MCD interactive molecules There are several different approaches to identifying MCD interactive molecules.
• the invention would allow the identification of proteins that may only associated with nonactive (or reduced activity) MCD or constitutively active MCD molecules. Such proteins may regulate MCD function. Techniques that may be used are, but not limited to, immunoprecipitation of MCD with antibodies generated to the transcription product ofthe invention. This would also bring down any associated bound proteins.
• Another method is to generate fusion proteins containing the mutant form of MCD connected to a generally recognized pull-down protein such as glutathione S-transferase. Bound proteins can then be eluded and analyzed.
• a method similar to immunoprecipitation is to construct fusion proteins of MCD and glutathione S-transferase (GST).
• GST glutathione S-transferase
• the MCD fusion proteins are then incubated with cell extracts and then removed with glutathione Sepharose beads. Any bound, MCD proteins are then characterized.
• Standard molecular biological techniques can be used to identify MCD homologues in rat, human or other species.
• the present invention contemplates a variety of approaches including, but are not limited to, DNA-DNA hybridization techniques (e.g., Southern blots) and DNA-RNA hybridization techniques (e.g., Northern blots). Additional techniques may include, for example, immunoscreening of proteins made from library stocks with antibodies generated to translation products of SEQ ID NOS: l, 2 or 3. Furthermore, immunoprecipitation of known or suspected interactive proteins of MCD can be followed by the identification of possible mutant MCD homologues with antibodies generated to translation products of
• Fresh or frozen hearts (10-15 mg of tissue) used for MCD measurements were homogenized for 2 x 15 seconds in a buffer consisting of KC1 (75 mM), Sucrose (20 mM), HEPES (10 mM), EGTA (1 mM), with or without NaF (50 mM) and NaPPi (5 mM). A fraction of this homogenate corresponding to 2 mg of tissue was used in the MCD assay.
• Rat hearts were excised and separated from their atrias. The ventricles were then minced into 1 mm cubes and rinsed with ice cold mannitol/sucrose/EGTA
• MSE buffer 225 mM mannitol; 75 mM sucrose; 1 mM EGTA, pH 7.5.
• a ratio of 2: 1 of MSE buffer to wet tissue weight was prepared and then homogenized using 5 strokes of a glass homogenizer with a teflon pestle. The homogenate was then diluted with MSE to 10 ml/g of wet tissue and centrifuged at 480 x g for 5 minutes. The supernatant was filtered through two layers of cheese cloth and then centrifuged at 10,000 x g for 30 minutes.
• the pellet was then resuspended in 10 mM sodium phosphate buffer, pH 7.6, containing 0.5 mM dithioerythritol (DTE) (50 mL/g of wet mitochondrial pellet).
• Triton X-100 was added to the suspension (0.1 %) and then the slurry was stirred at 4°C for 16 hours to lyse the mitochondria.
• the solution was then centrifuged at 24,000 x g for 10 minutes, the supernatant saturated to 40 % with powdered ammonium sulfate and stirred for 1 hour on ice. The precipitated protein was removed by centrifugation at 24,000 x g for 10 minutes.
• CoA esters were extracted from powdered tissue into 6 % perchloric acid and measured using the modified high performance liquid chromatography (HPLC) procedure described earlier [Lopaschuk et al. , J. Biol. Chem.
• the newborn rabbit hearts were perfused with Krebs'-Henseleit solution containing 3 % bovine serum albumin, 0.4 mM [9,10- C]palmitate, 11 mM glucose and
• the one bearing the highest enzymatic activity was sequenced in both directions using the deletion technique (Erase-a-Base, Promega) and the dye-primer sequencing strategy (Autoread Sequencing Kit and ALF DNA sequencer from Pharmacia). The size ofthe DNA was 2020 bp. An additional positive clone was sequenced to confirm the sequence obtained with that bering high enzymatic activity upon transfection in 293 cells. DNA transfection ofthe ⁇ -galactosidase gene under the control ofthe CMV promoter served as a negative control for MCD activity and as positive control for the efficiency of transfection (about 20 %).
• Ins-1 cells [Asfarai et al, Endocrinology 130:167-178 (1992)]and human kidney 293 cells [Becker et al, J Biol Chem 269:21234-21238 (1994)] were cultured as described in the quoted references.
• the assay was optimized for use in heart homogenates, as well as for isolated mitochondrial preparations.
• a tissue protein standard curve using heart homogenates demonstrated that 2 mg of tissue produced MCD activity which was linear for up to 20 minutes of incubation.
• a time course standard curve using a 10 minute incubation time produced rates that were linear when 0 to 10 mg of tissue were used. Therefore 2 mg of tissue homogenate and a 10 minute incubation period was used. This gave acetyl CoA values that were in the middle ofthe acetyl CoA standard curve. Similar experiments for time and protein dependence were also performed for the isolated mitochondrial preparations.
• Heart homogenates were assayed for MCD activity in the presence or absence of NaF and NaPPi and with or without alkaline phosphatase treatment.*, indicates significant differences between groups of hearts.
• the cytoplasmic form of MCD is approximately 55 kDa while the mitochondrial form is processed post-translationally into a 50 kDa molecular weight protein [Courchesne-Smith et al, Arch. Biochem. Biophys. 298:576-586 (1992)].
• Western blot analysis suggested that similar processing may occur in the rat heart.
• the present invention demonstrates that the heart expresses a 45 kDa isoform of MCD which is important in regulating myocardial malonyl CoA levels.
• An increase or maintained MCD activity in conjunction with a decrease in ACC activity can result in a decrease in malonyl CoA levels and an increase in fatty acid oxidation in the heart.
• drug screening by using the novel MCD composition and assay ofthe present invention will help to identify such potential compounds.
• Such inhibitors are contemplated to be useful in the treatment of ischemia.
• the kinases or phosphatases tested were casein kinase II, cyclic AMP dependent protein kinase protein kinase C, 5'AMP-activated protein kinase, protein phosphatase 2 A and protein phosphatase 2C.
• Another group of pooled fractions from the affinity elution ofthe SP-sepharose column was subjected to SDS-PAGE, coomassie stained and the 52 kDa band was cut from the gel. The protein was eluted from the gel fragment and injected into rabbits for the generation of MCD antibodies.
• another fraction from the affinity elution of the S-sepharose column was injected into rabbits without undergoing the gel separation step.
• EXAMPLE 9 The cloning ofthe rat pancreatic ⁇ -cell cDNA was performed in a two step approach using degenerated oligonucleotides. One ofthe selected pairs tested on INS cells mRNA generated a single product of an expected size of 580 bp as predicted by comparison with the goose cDNA ( Figure 15). Sequencing of this fragment indicated 67% identity with the goose nucleotide sequence and no match with any other sequence available on the blast server. This fragment was then used as a probe to screen an INS-1 cell cDNA library. Among the 4 in vzvo-excised clones, only one showed substantial MCD activity after transfection into 293 cells.
• 293 cells did not express the enzyme at a detectable level either under untransfected conditions or following transfection with the ⁇ -galactosidase gene. Sequencing of this clone of 2020 bp revealed an open reading frame of 1473 bases corresponding to an amino acid sequence of 491 residues and a predicted protein of about 52 KDa. A poly-A tail with a polyadenylation signal at the end ofthe 3' non-coding region was also present.
• Previous studies with the MCD enzyme(s) purified from various mammalian tissues evaluated a global molecular weight of about 170 KDa suggesting that the enzyme might multimerize in vivo.

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