Title of the Invention

COUPLED ENZYME ASSAY FOR MEASURINGACETYL COA CARBOXYLASE AND MALONYL COA DECARBOXYLASE ACTIVITY

Field of the Invention

[0001] The subject matter described and claimed herein relates to assays for measuring acetyl CoA carboxylase (hereinafter "ACC"), or malonyl CoA decarboxylase (hereinafter "MCD"), activity. More particularly, the assays concern rapid, sensitive, reliable and robust assays for measuring ACC or MCD activity comprising a coupled enzyme assay suitable for high-throughput screening. Further described are methods for 1) identifying modulators of ACC or MCD activity; 2) methods for measuring the IC50 of a test compound modulating via the carboxyl transferase domain of ACC; 3) methods for differentiating a competitive inhibitor of ACC or MCD from a non-competitive inhibitor of ACC or MCD; and 4) methods for assaying ACC and MCD catalytic activity.

Background of the Invention

[0002] ACC is the rate-determining enzyme of fatty acid biosynthesis in plants and animals. ACC is a biotin containing enzyme which catalyzes the carboxylation of acetyl CoA to form malonyl CoA in a two-step reaction (Beaty & Lane, (1982). J. Biol. Chem. 257:924-929). The first step is the ATP-dependent carboxylation of biotin covalently linked to the enzyme. The second step is a carboxyltransferase step wherein a carboxyl group is transferred to the substrate, acetyl CoA, to form malonyl CoA (Figure 1). Malonyl CoA is the C2 donor for de novo synthesis of long chain fatty acids. ATP-dependent carboxylation of a biotin is catalyzed by the biotin carboxylase domain. The carboxyltransferase (CT) domain of ACC catalyzes the transfer of the activated carboxyl group to acetyl CoA to form malonyl CoA. The CT domain comprises approximately one-third of the C-terminal domain in multi-domain eukaryotic ACCs (Zhang et al 2004). The CT domain (9OkD) comprises about 800 residues of the C-terminal portion of the ACC molecule. ACCs decarboxylate malonyl CoA by biotin-dependent and -independent mechanisms. ADP, inorganic phosphate ("Pi") and divalent metal ions are not required for these decarboxylations. The biotin-dependent malonyl CoA decarboxylation by ACC is stimulated by acetyl CoA and citrate (a potent allosteric activator). The biotin-independent

decarboxylation is unaffected (Moss and Lane 1972). Such activity is thought to be associated with the CT domain of ACC.

[0003] In mammals, there are two subtypes of ACC-ACCl and ACC2. ACCl is primarily localized in lipogenic tissues such as adipose tissue and liver. ACC2 is found primarily in non-lipogenic tissues such as skeletal muscle and heart muscle. [0004] Malonyl CoA allosterically inhibits carnitine palmitoyl transferase 1 ("CPT 1 "), which is an enzyme that transfers long chain fatty acids into the mitochondria for β-oxidation. ACC2 is co-localized with CPT-I, and it is postulated that the primary role of malonyl CoA synthesized by ACC2 is regulation of the rate of β-oxidation in the mitochondria.

[0005] ACC is a potential target in metabolic diseases for the treatment of the metabolic syndrome including obesity, insulin resistance, and dyslipidemia. Increased rates of muscle fatty acid oxidation, a reduced fat content, and a reduction in total body fat were observed in ACC-2 knock-out mice (Abu-Elheiga et al, (2001) Science 291 :2613-2616; Abu-Elheiga et al, (2003) Proc. Natl. Acad. ScL USA. 100: 10207-10212). Harwood et al. reported that ACC inhibitors caused reduction in fatty acid synthesis, increase in fatty acid oxidation, and reduction of respiratory quotient in rats (Harwood et al, (2003) J. Biol. Chem. 278:37099-37111). Chronic dosing of these compounds resulted in the reduction of whole body fat mass and improvement of insulin sensitivity (Harwood et al, (2003) J. Biol. Chem. 278:37099- 37111). These observations further validated the enzyme as a drug target. [0006] ACC is a desirable drug target due to its role(s) in a variety of metabolic processes and related disease states. However, technical difficulties associated with assaying ACC activity have hindered progress in identifying ACC-related drugs. One of the technical challenges has been the lack of a convenient, robust, and economical enzyme assay that would facilitate high-throughput screening (HTS) for ACC inhibitors.

[0007] There are known non-HTS ACC assays. One example, is a Cθ ₂ -fixation assay, which is the most commonly used ACC assay (see Figure 2, scheme 1). In the CO ₂ fixation assay, [ ¹⁴ C]-NaHCO ₃ , acetyl CoA, Mg-ATP, citrate and ACC are incubated at 37°C. The reaction mixture is quenched with acid, followed by heating to remove bicarbonate as ¹⁴ CO ₂ . Scintillant is then added and the acid-stable malonyl

CoA remaining in the vial is counted in a scintillation counter (Waite, M., and Wakil, SJ. (1962) J. Biol. Chem. 237:2750-2757, Tanabe et al, (1981) Methods Enzymol 71 Pt C, 5-16). The CO ₂ fixation assay is a multi-step radioactive assay, which is time consuming and labor-intensive. The assay requires large amounts of radioactivity, and special laboratory equipment to trap liberated ¹⁴ CC^. Although the assay may be run using a 96-well microtiter plate format, it is at best a low-throughput assay format, given the above-identified drawbacks, and not suitable for HTS. [0008] Another assay is a continuous ATP regeneration-coupled spectrophotometric assay. In this type of assay, the ADP generated in the ACC enzyme reaction is converted to ATP by a pyruvate kinase/lactate dehydrogenase coupled enzyme system, and NADH disappearance is followed spectrophotometrically or fluorometrically (Tanabe et ah, (1981) Methods Enzymol. 71 Pt C, 5-16; Figure 2, scheme 2). The ATP-regeneration system is very sensitive to the presence of ATPases. Since ATPase is highly abundant in tissue or cell culture extract, a disadvantage of this assay is that it demands highly pure ACC protein and is less sensitive. Additionally, colored compounds with certain absorption spectra (e.g., 340 nm) may yield false negatives.

[0009] Another ACC assay is an ACC/FAS coupled assay (see Figure 2, scheme 3). In this assay, malonyl CoA is formed from acetyl CoA. Malonyl CoA can then be used as a substrate for FAS with NADPH as the cofactor. The reaction may be monitored by the rate of utilization of NADPH spectrophotometrically (Wakil et ah, (1959) Biochem. Biophys. Acta 34:227-233). However, this ACC/FAS coupled assay requires large amounts of pure ACC and FAS enzymes, making this assay potentially expensive and time/resource intensive. Moreover, the rate of NADPH utilization has to be measured kinetically making the assay less amenable for HTS. [0010] The assays discussed above are not practical for HTS, because HTS requires a robust, reliable homogeneous assay using only small amounts of the enzymes. To fill this long- felt need, the inventors developed a homogenous coupled enzyme assay method using ACC and chloramphenicol acetyl transferase (CAT). The assay involves the reverse ACC reaction, in which the ACC CT domain catalyzes decarboxylation of malonyl-CoA to form acetyl CoA. CAT catalyzes the formation of acetylchloramphenicol from acetyl CoA and chloramphenicol (Figure 3). This

assay may be particularly powerful for HTS of potential ACC modulators. The present assay may be used routinely for calculation of IC50 values and for differentiating non-competitive inhibitors of ACC from competitive inhibitors of ACC.

[0011] Tissue malonyl CoA content is regulated by ACC (synthesis) and MCD (degradation). A regulatory role for MCD in mitochondrial fatty acid oxidation both in heart and skeletal muscle has been shown (Dyck et al ., (1998) Am. J. Physiol. 275:H2122-H2129, Goodwin, G. W. and Taegtmeyer, H. (1999) Am. J. Physiol. 277: E772-E777, Sakamoto et al, (2000) Am. J. Physiol. 278: Hl 196-H1204, Young et al, (2001) Am. J. Physiol. 280: E471-E479, Saha et al, (2000) J. Biol. Chem. 275:24279-24283)

[0012] Like ACC, a number of non HTS assays for measuring MCD activity have been described. For example, Fox describes a direct radiochemical assay wherein the substrate [3- ¹⁴ C]malonyl CoA is converted to acetyl CoA and ¹⁴ CO ₂ and the evolved ¹⁴ CO ₂ is trapped and counted (Fox (1971) Anal. Biochem 41:578-588) (Figure 3, Scheme 1). Sherwin and Natelson describe a fluorometric malatedehydrogenase assay in which the product formed acetyl CoA is monitored by a coupled assay with citrate synthase and malatedehydrogenase measuring NADH (Sherwin and Natelson (1975) Clin.Chem. 21 :230-234) (Figure 3, Scheme T). Dyck et al describe a coupled radiochemical assay wherein the acetyl CoA produced is coupled to [ ¹⁴ C] -oxalacetate to produce [ ¹⁴ C]-citrate by citrate synthase and the final product [ ¹⁴ C]-citrate was separated on a Dowex column, scintillant added and counted {Dyck et al, (1998) Am. J. Physiol. Ti '5:H2122-H2129) (Figure 3, Scheme 3). Recently, Kerner and Hoppel described a radiochemical assay wherein [ ¹⁴ C]-acetyl CoA formed from [2- ¹⁴ C]-malonyl CoA is converted to [2- ¹⁴ C]acetylcarnitine in the presence of L-carnitine and carnitine acetyl transferase. The charged [2- ¹⁴ C]acetylcarnitine is separated from [2- ¹⁴ C]-malonyl CoA on Dowex resin and radioactivity is measured by scintillation counting (Kerner and Hoppel (2002) Anal. Biochem. 306:283-289) (Figure 3, Scheme

4).

[0013] The above-described MCD assays are not practical for HTS. To fill this long-felt need, the inventors developed a homogenous coupled enzyme assay method using MCD and CAT. The assay involves monitoring a MCD reaction, in which the

enzyme catalyzes decarboxylation of [2- ¹⁴ C]-malonyl-CoA to form [ ¹⁴ C]-acetyl CoA. CAT catalyzes the formation of [ ¹⁴ C] -acetylchloramphenicol from [ ¹⁴ C] -acetyl CoA and chloramphenicol. This assay is amenable to HTS and may be used to identify MCD modulators.

Summary of the Invention

[0014] The subject matter described and claimed herein involves assays for measuring enzymatic activity. More particularly, the described subject matter relates to a rapid, sensitive, reliable and robust homogeneous assay for measuring ACC or MCD activity which comprises a coupled enzyme assay useful in a high-throughput screen.

[0015] Generally, the methods may be used to measure ACC or MCD activity; identify modulators of ACC or MCD, identify competitive inhibitors of ACC or MCD, and identify allosteric modulators of ACC or MCD. [0016] For example, one embodiment may be a method for identifying a compound that modulates ACC or MCD activity, depending on which enzyme is used in the assay. The method comprises: (a) providing a reaction mixture comprising chloramphenicol, CAT, ACC or MCD, and a test compound; (b) adding labeled malonyl CoA to the reaction mixture; (c) incubating the reaction mixture; (d) terminating the reaction; (e) measuring the amount of labeled acetyl chloramphenicol in the terminated reaction mixture; and (f) determining if the test compound modulates ACC or MCD activity by comparing the amount of labeled acetyl chloramphenicol from the terminated reaction mixture with the amount of labeled acetyl chloramphenicol from a control reaction mixture to which no test compound was added.

[0017] Another embodiment is a method for measuring the potency (e.g., IC50 value) of a test compound using either MCD, ACC, or the carboxyl transferase (CT) domain of ACC. The method involves essentially the same steps (a) through (e) described above with respect to the methods for identifying ACC/MCD modulators. However, ACC CT may be used as the assay enzyme and the sixth step "(f)" comprises determining the IC50 value of the test compound using MCD, ACC or the CT domain of ACC, based upon the amount of labeled acetyl chloramphenicol in each reaction mixture.

[0018] Yet another embodiment is a method for differentiating a competitive inhibitor of ACC or MCD (depending on the enzyme used) from a non-competitive inhibitor of ACC or MCD. This method involves essentially the same steps (a) through (e) described above with respect to the methods for identifying ACC/MCD/ACC CT modulators. However, ACC or MCD may be used as the assay enzyme, a known enzyme inhibitor is used in the assay rather than a "test compound", and the sixth step "(f)" is different. Step "(f)" comprises determining if the test inhibitor is a competitive inhibitor of the enzyme under scrutiny or a non-competitive inhibitor of the enzyme based upon the amount of labeled acetyl chloramphenicol in each reaction mixture.

[0019] Another embodiment is a method for a method for assaying ACC or MCD catalytic activity. This method involves essentially the same steps (a) through (d) described above with respect to the methods for identifying ACC/MCD/ACC CT modulators. However, the fifth step "(e)" is different. Step "(e)" comprises measuring the amount of labeled acetyl chloramphenicol in the terminated reaction mixture, wherein the presence of labeled acetyl chloramphenicol is indicative of ACC or MCD catalytic activity.

[0020] There are many experimental conditions that may be used in the methods described and claimed herein. For example, in some embodiments, the label used is a radioactive, fluorogenic, chromogenic or enzymatic label. Preferred radioactive labels are ¹⁴ C or ³ H. The malonyl CoA used may be labeled at the C2 position. If ACC is selected for measurement, an ACC CT domain may be used rather than a full- length ACC peptide. The reaction may be terminated by addition of scintillant, or acid, or by heat quench. The incubation period for the reaction may be for about 1-4 hours, and the incubation temperature may be at about 37°C. The reaction mixture may comprise about 0.05 - 4 μg of ACC, ACC CT domain, or MCD, and may further comprise about 0.05 - 1 unit CAT. In preferred embodiments, the methods are employed in a high throughput screen format.

[0021] Further contemplated are kits useful for practicing the methods disclosed and claimed herein. For example, one embodiment may be a kit used for identifying one or more compounds that modulate ACC activity, comprising chloramphenicol,

CAT, ACC, ACC CT, or MCD, labeled malonyl CoA and instructions for identifying one or more compounds that modulate enzymatic activity.

[0022] The assays described herein offer many advantages over known methods for assaying ACC and MCD activity. First, the instant assay is not cumbersome as are known methods. The ¹⁴ CCh fixation assay, for example, requires specialized equipment for working with gases, which may be expensive and technically challenging to operate. In contrast, the instant assay may be performed in a single reaction vessel and does not require any transfer of materials. [0023] The instant assays are simple, homogenous assays involving very few steps. The assay reagents are added and incubated, the reaction is terminated, e.g., by addition of scintillant, and then read in a TOPCOUNT™. In contrast, the ¹⁴ CO ₂ fixation assay, for example, is a multi-step process, which may be tedious and is not feasible for HTS operations.

[0024] Additionally, the instant assays provide rapid results, making it suitable for use in a HTS setting. Most known methods for assaying for ACC or MCD are time- consuming and cannot be employed or adapted to perform adequately in a HTS operation.

[0025] Furthermore, the instant assays: 1) require only low levels of radiation, making the assays preferable to those employing higher levels of radiation; 2) are relatively inexpensive to perform; 3) employ small amounts of enzymes that may be isolated as described herein, or may be acquired from commercial sources; 4) facilitate establishment of a color quench curve using a TOPCOUNT™, which results in minimal interference from colored compounds; and 5) are robust and reproducible- -statistical analysis indicates that the signal to background ratio is >40 and the Z' value is about 0.77.

[0026] Finally, the assay is adaptable. For example, partially purified (e.g., avidin-Sepharaose affinity chromatography) ACC enzyme or MCD enzyme may be used. Thus there is no need for highly purified enzyme in the instant assay which saves time and reduces expense.

Brief Description of the Drawings [0027] Figure 1 provides an exemplary ACC reaction. [0028] Figure 2 provides methods for determining ACC activity, (schemes 1-3).

[0029] Figure 3 provides methods for determining MCD activity, (schemes 1-4). [0030] Figure 4 provides a diagram reflecting exemplary ACC/CAT and MCD/CAT coupled enzyme assays.

[0031] Figure 5 provides a flowchart reflecting a method for purifying ACC enzymes.

[0032] Figures 6A and 6B show the time course of a reaction conducted at room temperature (RT) (closed circles and solid line) and at 37°C (open circles and dotted line). Figure 6A shows the net cpm and Figure 6B shows the signal/background. [0033] Figures 7A and 7B show ACC enzyme titration at room temperature and 37°C, respectively. Closed circles represent 0.5 units CAT, open circles represent 0.1 units CAT, closed triangles represent 0.05 units CAT, open triangles represent 0.01 units CAT and closed squares represent no CAT.

[0034] Figure 8 demonstrate the effect of DMSO on ACC activity. Figure 8A provides net cpm and Figure 8B provides the signal/background. [0035] Figure 9 reflects the K _m for the substrate malonyl CoA. The K _m was determined by assaying the activity at various concentrations of malonyl CoA up to 300 μM

[0036] Figure 10 reflects the dose dependent inhibition of ACC activity by Compound A (closed circles) and palmitoyl CoA (open circles). Detailed Description of the Invention I. Definitions

[0037] The terms "a" and "an" mean "one or more" when used in this application, including the claims.

[0038] The term ACC (acetyl CoA carboxylase) is not limited to enzyme derived from a particular species. Thus, it is intended that all ACC orthologs are encompassed by the term ACC, including human ACCl and ACC2 enzymes and rat ACCl and ACC2 enzymes. The term also encompasses fragments of ACC that exhibit catalytic activity, such as the ability to convert acetyl CoA to malonyl CoA. An exemplary fragment of ACC is an ACC CT domain. An example of ACC2 is human ACC2. An example of ACCl is rat ACCl. The term ACC also encompasses ACC sequences comprising one or more mutations in the amino acid sequence. Such

mutations may be naturally occurring or may be engineered using standard mutagenesis techniques known in the art.

[0039] The term "ACC CT domain" means a fragment of ACC comprising approximately 800 amino acid residues of the C-terminal end of the ACC molecule. An ACC CT domain has carboxyl transferase activity. Non-limiting examples of ACC CT domains are amino acid residues 1516-2345 of rat ACCl (SEQ ID NO: 1); amino acid residues 1633-2456 of rat ACC2 (SEQ ID NO: 2); amino acid residues 1517-2346 of human ACCl (SEQ ID NO: 3); and amino acid residues 1636-2458 of human ACC2 (SEQ ID NO: 4).

[0040] The term "MCD" means the enzyme Malonyl CoA Decarboxylase (EC 4.1.1.9). The term encompasses all forms of MCD. Further, the term MCD is not limited to enzyme derived from a particular species. Thus, it is intended that all MCD orthologs are encompassed by the term MCD, including human and rat MCD. [0041] The term "CAT" means the enzyme chloramphenicol acetyltransferase. The term encompasses all forms of CAT. Further, the term CAT is not limited to enzyme derived from a particular species. Thus, it is intended that all CAT orthologs are encompassed by the term CAT, including human and rat CAT. The term also encompasses fragments of CAT that exhibit catalytic activity, namely the ability to convert chloramphenicol and acetyl CoA to acetylchloramphenicol. Thus the term includes segments of CAT that are shorter than full-length CAT, but which still retain catalytic activity. Exemplary sources of CAT are E. coli, rat, human, mouse and chicken. The term CAT also encompasses CAT sequences comprising one or more mutations in the amino acid sequence. Such mutations may be naturally occurring or may be engineered using standard mutagenesis techniques known in the art. [0042] The term "high-throughput screening" means any method or operation by which a plurality of test samples are analyzed for one or more properties, such as ACC or MCD modulation. A high-throughput operation is preferably automated. [0043] The term "modulator" and derivatives thereof means the ability of a compound to alter the function of ACC or MCD. The alteration may be enhancement, diminishment, activation and/or inactivation of ACC or MCD activity. Exemplary modulators of ACC or MCD comprise, but are not limited to, compounds that activate or inhibit ACC activity.

[0044] The term "test compound" means an agent known or suspected to interact with an ACC polypeptide or a fragment thereof. Representative test compounds include "xenobiotics", such as drugs and other therapeutic agents, carcinogens and environmental pollutants, natural products and extracts, as well as "endobiotics", such as steroids, fatty acids and prostaglandins. Other examples of test compounds include, but are not restricted to, activators and inhibitors of ACC, hormones (e.g., opioid peptides, steroids, etc.), peptides, enzyme substrates, co-factors, lectins, sugars, oligonucleotides, proteins, small molecules and antibodies including monoclonal antibodies, and fragments thereof such as ScFV and Fv fragments, domain antibodies, Fab fragments, and the like.

II. Preparation OfACC. MCD And CAT Enzymes

[0045] The ACC, MCD and/or CAT enzymes used in the assays described herein may be obtained from commercial sources or they may be prepared using the methods described herein or those known to the skilled artisan. ACC, MCD and/or CAT, may be isolated from any suitable animal source, for example from mammalian (e.g., rat) or chicken livers, mammalian or chicken adipose tissue, mammalian or chicken skeletal muscle and/or mammalian or chicken heart muscle. ACC, MCD and/or CAT may be isolated from a biological sample using standard protein purification methodology known to those of the art (see, e.g., Janson, Protein Purification: Principles, High Resolution Methods, and Applications, (2 ^nd ed.) Wiley, New York, (1997); Rosenberg, Protein Analysis and Purification: Benchtop Techniques, Birkhauser, Boston, (1996); Walker, The Protein Protocols Handbook, Humana Press, Totowa, New Jersey, (1996); Doonan, Protein Purification Protocols, Humana Press, Totowa, New Jersey, (1996); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York, (1994); Harris, Protein Purification Methods: A Practical Approach, IRL Press, New York, (1989), all of which are incorporated in their entireties herein by reference). Guidance in the isolation of an ACC and/or CAT is provided herein, (see Example 1) and in the Drawings (see Figure 5). Other methods for purifying active enzymes may be known to those of skill in the art and any such methods may be employed.

[0046] In some situations, it may be desirable to recombinantly express the enzymes described herein. Conventional molecular biology, microbiology,

recombinant DNA, and protein chemistry techniques known to those of ordinary skill of the art may be employed to produce a DNA sequence encoding an enzyme to be used in the instant assay(s). Such techniques are explained in the literature (see, e.g., Sambrook et ah, Molecular Cloning: A Laboratory Manual, (3 ^rd ed.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (2001); Glover, DNA Cloning: A Practical Approach, (2 ^nd ed.) IRL Press, New York, USA (1995); Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Press, New York, USA (1984); Hames & Higgins, Nucleic Acid Hybridisation: A Practical Approach, IRL Press, Washington, D. C, USA (1985); Hames & Higgins, Protein Expression: A Practical Approach, Oxford University Press, New York, USA, (1999); Masters, Animal Cell Culture: A Practical Approach, Oxford University Press, New York, USA (2000); Bickerstaff, Immobilization of Cells And Enzymes, Humana Press, Totowa, New Jersey, USA (1997); Perbal, A Practical Guide To Molecular Cloning (2 ^nd ed.) Wiley, New York, New York, USA (1988); Current Protocols in Molecular Biology, (Ausubel et ah, eds.), Greene Publishing Associates and Wiley-Interscience, New York (2002); Ausubel, Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, (4 ^l ed.) John Wiley & Sons, New York, New York, USA (1999)).

[0047] A DNA sequence encoding an ACC or MCD peptide and/or CAT polypeptide (including mutants, analogs, and functional equivalents), may be prepared by various molecular biology methods known in the art. Vectors comprising such sequences may be used as well. See e.g., (Ha et ah, (1994) J. Biol. Chem. 269: 22162-22168); MCD sequence (Gao et al. J. Lipid. Res. 40: 178-182); CAT sequence (Shaw, W. V. Chloramphenicol acetyltransferase: Enzymology and Molecular Biology. CRC Crit. Rev. Biochem, (1983) 14: 1-46).

III. Methods For Identifying A Compound That Modulates ACC or MCD Activity [0048] As described above, one embodiment described herein is a method for identifying a compound that modulates ACC or MCD activity. If ACC is the target enzyme, then ACC, or a fragment thereof is added to the assay mixture. If MCD is the target enzyme, then MCD, or a fragment thereof is added to the assay mixture. In preferred embodiments, the methods are conducted as a high throughput screen. For

example, the reaction may be carried in a welled plate, such as a 96- or 384-welled plate.

[0049] The method comprises: (a) providing a reaction mixture comprising chloramphenicol, CAT, ACC or MCD, and a test compound; (b) adding labeled malonyl CoA to the reaction mixture; (c) incubating the reaction mixture; (d) terminating the reaction; (e) measuring the amount of labeled acetyl chloramphenicol in the terminated reaction mixture; and (f) determining if the test compound modulates ACC or MCD activity by comparing the amount of labeled acetyl chloramphenicol from the terminated reaction mixture with the amount of labeled acetyl chloramphenicol from a control reaction mixture to which no test compound was added.

[0050] The first step "(a)" involves providing a reaction mixture comprising chloramphenicol, CAT, ACC or MCD, and a test compound in an assay buffer. The reaction mixture comprises about 0.05 - 4 μg ACC or MCD, and preferably comprises about 0.5 μg of ACC or MCD. IfACC is used in the assay, the ACC may be further defined as an ACC CT domain. The enzyme used may be partially purified. The reaction mixture comprises about 0.05-1 units of CAT and preferably comprises about 0.5 units of CAT.

[0051] Determining the amount of chloramphenicol and assay buffer used in the reaction mixture is a matter within the skill of an ordinary artisan. An exemplary amount of chloramphenicol is about 1 mM and an exemplary assay buffer comprises about 50 mM Tris-HCl, pH 7.6, 1 mM EDTA.

[0052] The second step "(b)" involves adding labeled malonyl CoA to the reaction mixture. In a preferred embodiment, the malonyl CoA is labeled at the C2 position. The labeled malonyl CoA may be labeled with any agent that allows for subsequent detection of labeled acetyl chloramphenicol. Exemplary labels are radioactive, fluorogenic, chromogenic or enzymatic labels. It is within the skill of an ordinary artisan to determine an appropriate label and how to make malonyl CoA containing the selected label (e.g., at the C-2 position). In a preferred embodiment, the label is a radioactive label selected from the group consisting of ³ H and ¹⁴ C. [0053] The third step ("c") involves incubating the reaction mixture. The incubation step allows for the reagents react and generate enzymatic products. The

reaction mixture may be incubated for about 1-4 hours, preferably for about 1 hour. The incubation may take place at about 37°C or about room temperature. [0054] The fourth step ("d") involves termination of the reaction. Exemplary methods for terminating the reaction include, but are not limited to, addition of acid or scintillation fluid to the reaction mixture, and heat quench. In a preferred embodiment, when malonyl CoA is labeled with a radioactive label, scintillation fluid is used to terminate the reaction. Exemplary heat quench conditions are about 95°C for about 5 minutes.

[0055] The fifth step ("e") involves measuring the amount of labeled acetyl chloramphenicol in the terminated reaction mixtures. Any procedure for measuring the amount of a labeled molecule may be used. Exemplary detection methods, include, but are not limited to, scintillation counting, and scintillation proximity, spectroscopy. The reaction may be terminated by addition of scintillation fluid which facilitates extraction of the labeled acetyl chloramphenicol into the scintillant layer. The acetyl chloramphenicol may be labeled with a radioactive label, the reaction is terminated by addition of scintillation fluid and the amount of radiolabeled acetyl chloramphenicol may be detected by scintillation counting. In other embodiments, the amount of radiolabeled acetyl chloramphenicol present in the scintillant layer is determined in the same reaction vessel in which the reaction was carried out. In such embodiments, it is not necessary to remove any of the components of the reaction mixture prior to scintillation counting because the product is selectively extracted into the scintillant. For example, radiolabeled acetyl chloramphenicol may be extracted into the scintillation layer while the radiolabeled malonyl CoA and radiolabeled acetyl CoA are not extracted into the scintillant layer.

[0056] Any type of scintillation fluid may be used. An exemplary scintillation fluid is toluene containing 10 % 2,5-diphenyloxazole and 0.125% l,4-bis(5- phenyloxazol-2-yl)benzene or Microscint E (PerkinElmer).

[0057] In some embodiments, a scintillation counter may be used for the detection step. The method of detection may vary. When partially purified enzymes are used, a color quench curve may be established in a TOPCOUNT™ to reduce interference from colored compounds. When the reaction vessel is a biotinylated chloramphenicol coated streptavidin FLASHPLATE™ or other welled plate, the scintillation signal

may be detected using an automated plate reader, such as a TOPCOUNT™, MICROBET A™, LEADSEEKER™ or VIEWLUX™ plate reader. Alternatively, biotinylated chloramphenicol may be used as CAT substrate with malonyl CoA containing the selected label (preferably at the C2 position) as a substrate for ACC or MCD. The labeled acetyl biotinyl chloramphenicol product may be bound to streptavidin coated scintillation proximity beads, the scintillation signal may be detected using an automated plate reader, such as a TOPCOUNT™, MICROBET A™, LEAOSEEKER™ or VIEWLUX™ plate reader. A scintillation signal is indicative of enzymatic (e.g., ACC) catalytic activity. When the assay is performed, detectable scintillation signal will be generated if ACC is present. The amount of scintillation signal may be expressed in counts per minute (cpm) or in intrinsic optical density (IOD) units.

[0058] The final step "(f)" involves comparing the amount of labeled acetyl chloramphenicol from the terminated reaction mixture with the amount of labeled acetyl chloramphenicol from a control reaction mixture to which no test compound was added. The control reaction mixture is prepared following the steps described above, except no test compound is added. If the amount of labeled acetyl chloramphenicol from the terminated reaction mixture is more or less than that in the control reaction mixture, this suggests that the test compound modulates ACC activity.

IV. Additional Methods

[0059] The methods described above in section III ( i.e., Methods For Identifying A Compound That Modulates ACC or MCD Activity) can be readily adapted to assay other enzymatic characteristics. For example, the methods of section III can be modified to produce methods for: measuring the potency (e.g., IC50) of a test compound; differentiating a competitive inhibitor from a non-competitive inhibitor; and for assaying enzymatic (e.g., ACC or MCD) catalytic activity. [0060] Embodiments of such methods are described below.

A) Method for Measuring The IC50 Value Of A Test Compound At The CT Domain Of ACC

[0061] One embodiment is a method for measuring the IC50 value of a test compound at the CT domain of ACC. As is well known, IC50 is the concentration of a compound that yields 50% inhibition of enzymatic activity. [0062] The method comprises: (a) providing reaction mixtures comprising chloramphenicol, CAT, CT domain of ACC and increasing amounts of a test compound; (b) adding labeled malonyl CoA to the reaction mixtures; (c) incubating the reaction mixtures; (d) terminating the reactions; (e) measuring the amount of labeled acetyl chloramphenicol in each reaction mixture; and (f) determining the IC50 value of the test compound at the CT domain of ACC based upon the amount of labeled acetyl chloramphenicol in each reaction mixture.

[0063] Steps (a) through (e) are essentially the same steps as steps (a) through (e) described above with respect to the methods for identifying ACC/MCD/ACC CT modulators. However, ACC CT is used as the assay enzyme and the sixth step "(f)" is different from above.

[0064] Step "(f)" comprises determining the IC50 value of the test compound at the CT domain of ACC based upon the amount of labeled acetyl chloramphenicol in each reaction mixture. The amount of test compound that may be used is any amount that enables a determination of the IC50 value. Determining the amount of test compound for use in the method is within the level of skill of an ordinary artisan. For example, the activity in the presence of 8-11 half-log concentrations of the compound may be determined. The control activity in the absence of the compound (subtracted from no enzyme blank) serves as 100% activity and the percent inhibition at each compound concentration is calculated [1- activity with compound/control activity] x 100. The percent inhibition is plotted against the log of the compound concentration and the data fitted to the following equation Y = A + ((B-(A) /((l+((C/x) ^λ (D))). [0065] Note that the above-described method can be adapted to assay the IC50 of test compounds for other enzymes such as ACC and MCD.

B) Method For Differentiating A Competitive Inhibitor From A Non-Competitive Inhibitor

[0066] Another embodiment is a method for differentiating a competitive inhibitor of ACC or MCD (carboxyl transfer reaction) from a non-competitive inhibitor of ACC or MCD. The method comprises: (a) providing a reaction mixture comprising chloramphenicol, CAT, CT domain of ACC or MCD, and increasing amounts of a known inhibitor compound; (b) adding labeled malonyl CoA to the reaction mixtures; (c) incubating the reaction mixture; (d) terminating the reaction; (e) measuring the amount of labeled acetyl chloramphenicol in each reaction mixture; and (f) determining if the test inhibitor is a competitive or non-competitive inhibitor of ACC or a competitive or non-competitive inhibitor of MCD (depending on which is used in the reaction), based upon the amount of labeled acetyl chloramphenicol. [0067] Steps (a) through (e) are essentially the same steps as steps (a) through (e) described above with respect to the methods for identifying ACC/MCD/ACC CT modulators. ACC or MCD may be used as the assay enzyme, a known ACC or MCD inhibitor is used in the assay rather than a "test compound", and the sixth step "(f)" is different. The test ACC or MCD inhibitor is preferably a compound known to inhibit enzymatic activity and is present in the reaction mixture at about 0.015-30 μM. [0068] Step "(f)" comprises determining if the test inhibitor is a competitive or non-competitive inhibitor of ACC or MCD, based upon the amount of labeled acetyl chloramphenicol present in the reaction mixture. For example, as illustrated in Example 7, known competitive inhibitors of ACC, Compound A and palmitoyl CoA, inhibited ACC activity with an IC50 of 0.85 μM. However, a known non-competitive inhibitor of ACC, Compound B did not inhibit the malonyl CoA decarboxylation activity of ACC significantly (data not shown). One skilled in the art would be able to evaluate data obtained from known competitive or non-competitive inhibitors and compare that data with putative inhibitors and determine whether the putative inhibitor is a competitive or non-competitive inhibitor.

C) Method For Assaying ACC or MCD Catalytic Activity [0069] Another embodiment is a method for assaying ACC or MCD catalytic activity. The method comprises: (a) providing a reaction mixture comprising chloramphenicol, CAT, and a sample known or suspected to comprise ACC or MCD; (b) adding labeled malonyl CoA to the reaction mixture; (c) incubating the reaction mixture; (d) terminating the reaction; and (e) measuring the amount of labeled acetyl

chloramphenicol in the terminated reaction mixture, wherein the presence of labeled acetyl chloramphenicol is indicative of enzymatic catalytic activity.

[0070] Steps (a) through (d) are essentially the same steps as steps (a) through (d) described above with respect to the methods for identifying ACC/MCD/ACC CT modulators. However, the reaction mixture comprises a sample known to or believed to comprise ACC or MCD and the fifth step "(e)" is different.

[0071] Step "(e)" comprises measuring the amount of labeled acetyl chloramphenicol in the terminated reaction mixture, wherein the presence of labeled acetyl chloramphenicol is indicative of enzymatic catalytic activity. When the sample is known to contain ACC (such as ACC or ACC CT) or MCD, the assay may be performed to measure the amount and/or activity of the enzyme present in the sample.

When it is not known whether a sample contains ACC or MCD, the assay may be performed to identify the presence and/or activity of ACC or MCD in that sample.

Such a sample could be the product of an ACC or MCD purification procedure, for example.

V. Kits

[0072] Other embodiments disclosed and claimed herein are kits that facilitate the use of the methods described herein. For example, the kits may be used to perform methods for: identifying a compound that modulates ACC or MCD activity; measuring the IC50 value of a test compound; differentiating a competitive inhibitor of

ACC from a non-competitive inhibitor of ACC; and for assaying ACC catalytic activity.

[0073] The kit comprises chloramphenicol, CAT, ACC, labeled malonyl CoA, such as C2 labeled malonyl CoA, and instructions for performing the methods described herein.

[0074] In some embodiments, a container is provided for carrying out the reaction. The contents of the kit may be provided in concentrated solutions or in ready-to-use aliquots.

Examples

[0075] The following Examples have been included to illustrate preferred embodiments of the methods/assays disclosed and claimed herein. The following Examples are illustrative and changes, modifications or alterations may be employed by those of ordinary skill in the art without departing from the spirit and scope of the subjected matter disclosed and claimed herein.

Example 1 Enzyme Preparation

[0076] ACC was purified by a rapid purification procedure (see Figure 5) from frozen livers of rats that were fasted for 36 hours and fed with fat- free carbohydrate diet for 48 hours (Seethala & Benjamin, (1983) Preparative Biochem. 13:475-488; Jamil & Madsen, (1987) J. Biol. Chem. 262:630-637). The livers were thawed and washed with PBS buffer and minced into small pieces in 3 volumes of buffer A (50 mM potassium phosphate buffer, pH 7.5, 1 mM EDTA, 0.1 mM EGTA, 10 mM β mercaptoethanol, 5 mM benzamidine, protease inhibitor cocktail (10 μM TLCK, lμg/ml of leupeptin, aprotinin, pepstatin, AEBSF, pepstatin A and trypsin inhibitor) and 250 mM sucrose) and homogenized in a Polytron (4 x 1 min.). All the work was performed at 4°C, or on ice.

[0077] The homogenate was centrifuged at 5000 x g for 5 minutes. The supernatant (1) was saved and the pellet suspended in one volume of buffer A and re-homogenized in a Polytron (4 x 1 min.), and centrifuged at 5000 x g for 5 minutes. The pellet was discarded and the supernatant (2) was combined with supernatant (1) and centrifuged at 25,000 x g for 30 minutes. The pellet was discarded and the supernatant 3 was centrifuged at 100,000 x g for 60 minutes. Ammonium sulfate was added to the high-speed supernatant to 44% saturation (25 g/100 ml). The pH was adjusted with ammonia/lM KOH and centrifuged at 25,000 x g for 20 min. The pellet was dissolved (one-fifth original volume) in Buffer B (100 mM Tris-HCl buffer, pH 7.5, 0.5 M NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, protease inhibitor cocktail, and 5% glycerol) and centrifuged at 25,000 x g for 20 minutes, monomeric avidin Sepaharose equilibrated in Buffer B was added to the supernatant in a roller bottle and mixed on a bottle roller overnight. The mixture was filtered on a sintered funnel.

[0078] Avidin Sepaharose was washed with 30 volumes of Buffer B and loaded onto a column. ACC was eluted with 5 mM biotin in Buffer B in 3 ml fractions. The fractions containing ACC activity were pooled, ammonium sulfate was added to 44% saturation and centrifuged at 25,000 x g for 20 min. The pellet was dissolved in Buffer C (100 mM Tris-HCl buffer, pH 7.5, 1 mM EDTA, 0.5 mM dithiothreitol, protease inhibitor cocktail, and 20% glycerol) and dialyzed against 100 volumes of Buffer C with an additional change of buffer. The dialyzed ACC enzyme was aliquoted into small volumes and stored at -80 ⁰ C.

[0079] MCD in a similar manner. For example, MCD may be obtained and prepared from rat liver, heart and skeletal muscle by homogenization in a buffer (50 mM potassium phosphate buffer, pH 7.5, 1 mM EDTA, 0.1 mM EGTA, 10 mM β mercaptoethanol, 5 mM benzamidine, protease inhibitor cocktail (10 μM TLCK, lμg/ml of leupeptin, aprotinin, pepstatin, AEBSF, pepstatin A and trypsin inhibitor). The mitochondrial fraction may be prepared by differential centrifugation and the mitochondrial MCD is solubilized with 1% octylglucoside (Kim and Kolattukudy (1978) Arch. Biochem. Biophys. 190:234-246). CAT may be purchased from Sigma (C-8413).

Example 2

Solution Preparation and Assay Conditions

[0080] Table 1 provides examples of solutions made and techniques used in the methods described and claimed herein.

Table 1

[0081] The reagents were added to each well of a microtiter plate in the following sequence:

Step 1 : Add 39.5 μl of enzyme mix in assay buffer.

Step 2: Add 0.5 μl of 100% DMSO or 1OX compound in 100 % DMSO.

Step 3: Incubate at room temperature for 15 min

Step 4: Add 10 μl of 5X [ ¹⁴ C]-Malonyl CoA mix to start the reaction.

Step 5: Incubate the plate for 1 hour at 37°C.

Step 6: Add 200 μl of scintillant and shake plate on a plate shaker.

Step 7: Count the plate in a TOPCOUNT™ NXT for 2 min/well.

Example 3

Reaction Time Course

[0082] In a representative optimization of the ACC/CAT coupled enzyme assay, malonyl CoA and CAT were added to assay buffer and the reaction initiated by addition of ACC. For determining compound inhibition CAT, ACC and a test compound were added to assay buffer and the reaction initiated by addition of [ ¹⁴ C]- Malonyl CoA.

[0083] The time course of the reaction was studied at room temperature (RT; 21 ⁰ C) and at 37°C. The reaction was linear up to 4 hours at room temperature. The reaction increased linearly up to 2 hours and then leveled off at 37°C (Figure 6). The maximum signal to background S/B ratio was higher at 37°C than at RT (S/B = 20 at RT and S/B = 45 at 37°C at 2 hours.). The S/B ratio decreased slightly at 3, 4 and 5 hours at 37°C and the S/B ratio was similar to that at room temperature incubation after four hours. Based on these studies, the preferred incubation conditions were determined to be 37°C for 1 hour.

Example 4

Enzyme Titration

[0084] To determine the optimal concentrations of ACC and CAT to obtain the best signal, the reaction was titrated with ACC (purified through the avidin-Sepharose step) at different CAT concentrations at room temperature and also at 37°C (Figure 7). When conducted at room temperature, at all CAT concentrations tested, the signal increased linearly with increasing ACC enzyme up to 2.0 μg/assay and leveled off at higher ACC concentrations. At a 37°C incubation however, the signal increased rapidly up to 1 μg ACC/assay and the signal remained constant at higher ACC concentrations. For all the 96-well assays in this study a combination of 0.5 μg ACC and 0.5 Units CAT were used per assay and the reaction was incubated for one hour at 37°C.

Example 5 Effect of DMSO

[0085] For screening in drug discovery, compounds are routinely dissolved in 100% DMSO then further diluted to 1% or lower DMSO in the final assay. The effect of DMSO on the ACC/CAT coupled assay was studied (Figure 8). DMSO up to 10% (v/v), had no significant effect on the activity. A final DMSO concentration of 1% was used for the assays described herein.

Example 6

Malonyl CoA Concentration Dependence

[0086] The K _m for the substrate malonyl CoA was determined by assaying the activity at various concentrations of malonyl CoA up to 300 μM (Figure 9). The enzyme activity gave a hyperbolic response with malonyl CoA. The K _m for malonyl CoA was determined as about 15 μM.

[0087] The ACC/CAT coupled assay has an average Z' value of 0.77 which favors its use as a HTS assay. The signal window is >20 and the S/B is about 41. These values suggest that this assay is a very robust, and highly reproducible assay, which is amenable to HTS.

Example 7

Dose Dependent Inhibition of ACC Activity by Known Competitive ACC

Inhibitors (Compound A and palmitoyl CoA) and Differentiation Of A Known

Non-Competitive ACC Inhibitor (Compound B)

[0088] To further characterize and validate the assays described herein, the IC50 of known ACC competitive inhibitors, Compound A and palmitoyl CoA, was determined (Figure 10). Compound A inhibited ACC activity with an IC50 of 0.85 μM and palmitoyl CoA inhibited the ACC activity with an IC50 of 3.12 μm. A known non-competitive potent inhibitor of ACC, Compound B, did not inhibit the malonyl CoA decarboxylation activity of ACC significantly (data not shown). This result confirms that the disclosed assay(s) can provide a method for gathering data to differentiate competitive inhibitors from non competitive inhibitors with respect to acetyl CoA substrate for ACC.

[0089] The references cited herein are incorporated herein by reference to the extent that they supplement, explain, provide a background for or teach methodology, techniques and/or compositions employed herein. All cited patents, including patent applications, and publications referred to in this application are herein expressly incorporated by reference. Also expressly incorporated herein by reference are the contents of all citations of GenBank accession numbers, LocusID, and other computer database listings, as well as the contents of any Sequence Listing associated herewith. [0090] It will be understood that various details of the subject matter disclosed and claimed herein may be changed.