WO2002079501A1 - Methods and reagents for identifying weight loss promoters and therapeutic uses therefor - Google Patents
Methods and reagents for identifying weight loss promoters and therapeutic uses therefor Download PDFInfo
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- WO2002079501A1 WO2002079501A1 PCT/US2002/010386 US0210386W WO02079501A1 WO 2002079501 A1 WO2002079501 A1 WO 2002079501A1 US 0210386 W US0210386 W US 0210386W WO 02079501 A1 WO02079501 A1 WO 02079501A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
Definitions
- the controlling step for free fatty acid (FFA) oxidation into CO 2 and partitioning, and esterification into complex lipids and triglycerides (TG), is at the level of the enzyme CPT-1.
- This enzyme has two isoforms, a muscle-type and a liver-type.
- preadipocytes express both isoforms.
- Mature fat cells express primarily the muscle isoform.
- Both isofo ⁇ ns are allosterically regulated by malonyl CoA but the muscle isoform is more sensitive. In aged animals, less FFA is oxidized and more stored. The reason for this is not known, but it could explain the increasing obesity with age.
- CPT-1 Given the important role of CPT-1 in regulating FFA oxidation, there exists a need for understanding in greater detail the mechanism by which CPT-1 is regulated, for identifying modulators of CPT-1 activity which can promote fat oxidation (i.e. fat burning) and/or weight loss.
- the present invention is based; at least in part, on the discovery of a previously unrecognized role for carnitine palmitoyltransferase-1 (CPT-1).
- CPT-1 carnitine palmitoyltransferase-1
- the present invention is based on the discovery that compounds which inhibit malonyl CoA binding to CPT-1 can promote CPT-1 activity and thereby promote free fatty acid (FFA) oxidation and weight loss.
- FFA free fatty acid
- the interaction between CPT-1 and malonyl CoA is important in inhibiting CPT-1 activity, which is important in promoting FFA oxidation.
- Inhibition of the CPT-1 /malonyl CoA interaction is proposed to regulate FFA oxidation and, consequently, weight loss.
- the present inventors are the first to identify the interaction of CPT-1 with malonyl CoA as a target for identifying weight loss inhibitors.
- the invention provides methods for identifying a weight loss promoter comprising assaying the ability of a test compound to inhibit the interaction between CPT-1 and malonyl CoA to thereby inhibit the inhibition of CPT-1 activity and promote free fatty acid (FFA) oxidation.
- the methods are performed in a mitochondrion.
- the level of interaction of CPT-1 and malonyl CoA is measured by measuring the level of CoA-SH produced.
- the invention provides methods for identifying a weight loss promoter comprising assaying the ability of a test compound to inhibit the acetyl CoA carboxylase (ACC) catalyzed production of malonyl CoA.
- the ACC may be purified or in a cytosolic extract.
- the ability of a compound to inhibit malonyl CoA production is determined by measuring pH.
- a weight loss promoter promotes FFA oxidation.
- Other embodiments of the invention provide therapeutic methods for promoting
- FFA oxidation and/or weight loss by administering to a subject a weight loss promoter identified by one of the methods of the invention.
- the present invention is based, at least in part, on the discovery of a previously unrecognized role for carnitine palmitoyltransferase-1 (CPT-1).
- CPT-1 carnitine palmitoyltransferase-1
- the present invention is based on the discovery that compounds which inhibit malonyl CoA binding to CPT-1 can promote CPT-1 activity and thereby promote free fatty acid (FFA) oxidation and weight loss.
- FFA free fatty acid
- the interaction between CPT-1 and malonyl CoA is important in inhibiting CPT-1 activity, which is important in promoting FFA oxidation. Inhibition of the CPT-1 /malonyl CoA interaction regulates FFA oxidation and, consequently, weight loss.
- the present invention features methods of identifying weight loss modulators, in particular weight loss promoters.
- an assay of the present invention is a cell-free assay in which a CPT-1 polypeptide, or biologically active portion thereof, is contacted with a test compound in the presence of malonyl CoA and the ability of the test compound to inhibit binding of malonyl CoA to the CPT-1 polypeptide or bioactive fragment thereof is determined.
- binding of malonyl CoA to the CPT-1 polypeptide can be accomplished, for example, by coupling the CPT-1 polypeptide or malonyl CoA with a radioisotope or enzymatic label such that binding of malonyl CoA to the CPT-1 polypeptide can be determined, e.g., by detecting labeled malonyl CoA or polypeptide in a complex.
- malonyl CoA or CPT-1 polypeptides can be labeled with 1 5 I, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
- substrates or polypeptides can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
- Binding of malonyl CoA to the CPT-1 polypeptide can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705.
- BIOA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g. , BIAcoreTM). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
- the assay includes contacting the CPT-1 polypeptide or biologically active portion thereof with malonyl CoA to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to modulate binding between the CPT-1 polypeptide and malonyl CoA.
- the assay includes contacting the CPT-1 polypeptide or biologically active portion thereof with malonyl CoA to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a CPT-1 polypeptide, wherein determining the ability of the test compound to interact with a CPT-1 polypeptide comprises determining the ability of the test compound to preferentially bind to CPT-1 or the bioactive portion thereof, for example, as compared to malonyl CoA.
- the assay is a cell-free assay in which a CPT-1 polypeptide or bioactive portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the CPT-1 polypeptide or biologically active portion thereof is determined.
- determining the activity of the test compound to modulate the activity of CPT-1 is accomplished in the presence of malonyl CoA and the ability of CPT-1 to catalyze the following reaction: long chain acyl CoA + carnitine - CoA-SH + acylcarnitine is determined.
- the level of the products of the above reaction can be measured.
- the level of CoA-SH can be measured by adding 5,5'- Dithiobis(2-nitrobenzoic acid) (DTNB) to the composition and determining the level of color as measured at 412 nm (Extinction Coefficient 13.6x10 3cm l ).
- determining the ability of the test compound to modulate the activity of a CPT-1 polypeptide can be accomplished, for example, by determining the ability of the CPT-1 polypeptide to modulate a downstream CPT-1 target molecule, e.g., an indirect assay for CPT-1 activity modulation.
- CPT-1 target molecule includes any non-CPT-1 molecule with which CPT-1 interacts, for example, in vitro or in vivo.
- CPT-1 :"CPT-1 target molcule” interaction is a significant element of CPT-1 biological activity (e.g., enzymatic activity) or function (e.g., cellular function).
- exemplary CPT-1 target molecules include, but are not limited to acyl CoA binding protein, acyl CoA synthase, fatty acid binding protein and CPT-1 products.
- the cell-free assay involves contacting a CPT-1 polypeptide or biologically active portion thereof with a CPT-1 target molecule CPT-1 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to preferentially modulate the activity of a CPT-1 target molecule, as compared to the CPT-1.
- the assay is a cell free assay for identifying a compound which can modulate the production of malonyl CoA in which ACC polypeptide or a biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of ACC (or a biologically active fragment thereof) is determined.
- determining the ability of the test compound to modulate ACC is determined by detecting the following reaction, which is catalyzed by ACC:
- a composition comprising ACC is contacted with a test compound in the presence of an ACC substrate (e.g., acetyl CoA), and readout of the reaction is measured by measuring the pH of the composition.
- an ACC substrate e.g., acetyl CoA
- the above described assays may be performed using purified proteins or cytosolic extracts.
- assay reagents e.g. , test compounds, malonyl CoA, CPT- 1 , CPT-1 substrates, CPT-1 target molecules, indicator reagents (e.g., fluorescent dyes, ACC and the like) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro- centrifuge tubes.
- a fusion protein can be provided which adds a domain that allows protein reagents to be bound to a matrix.
- glutathione- S-transferase/ ACC fusion proteins or glutathione- S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound and appropriate reagents (or the test compound and either the non- adsorbed CPT-1 or ACC), and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
- the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
- the complexes can be dissociated from the matrix, and the level of CPT-1 binding or activity or ACC binding or activity determined using standard techniques.
- a CPT-1 polypeptide or an ACC polypeptide can be immobilized utilizing conjugation of biotin and streptavidin.
- Biotinylated polypeptides can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
- antibodies reactive with CPT-1 polypeptide or ACC polypeptides but which do not interfere with polypeptide binding or activity can be derivatized to the wells of the plate, and unbound polypeptide trapped in the wells by antibody conjugation.
- Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the CPT-1 polypeptide or ACC polypeptide, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the CPT-1 polypeptide or ACC polypeptide.
- the CPT-1 polypeptides or ACC polypeptides can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
- binding proteins or "target molecules”
- target molecules proteins which bind to or interact with CPT-1 or ACC
- target molecules are also likely to be involved in the regulation of cellular activities modulated by the CPT-1 or ACC polypeptides.
- the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
- the assay utilizes two different DNA constructs.
- the gene that codes for a CPT-1 or ACC polypeptide is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
- a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey” proteins are able to interact, in vivo, forming a CPT-1- or
- the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the CPT-1 or ACC polypeptide.
- a reporter gene e.g., LacZ
- an assay is a cell-based assay in which a cell which expresses a CPT-1 polypeptide, or biologically active portion thereof, is contacted with a test compound in the presence of malonyl CoA and/or CPT-1 substrates and the ability of the test compound to modulate the activity of the CPT-1 polypeptide, or biologically active portion thereof determined.
- the cell for example, can be of mammalian origin or a yeast cell.
- the CPT-1 polypeptide for example, can be expressed heterologously or native to the cell.
- the assay is performed in a cellular organelle, e.g., a mitochondrion.
- an assay is a mitochondria-based assay in which a mitochondria comprising a CPT-1 polypeptide (e.g., having a CPT-1 polypeptide, or bioactive portion thereof, appropriately expressed the mitochondrial membrane) is contacted with a test compound in the presence of malonyl CoA and/or CPT-1 substrates and the ability of the test compound to modulate the activity of the CPT-1 polypeptide, or biologically active portion thereof determined. Determining the ability of the test compound to modulate the activity of a CPT-1 polypeptide, or biologically active portion thereof, can be accomplished by assaying for any of the activities of a CPT-1 polypeptide described herein.
- Determining the ability of the test compound to modulate the activity of a CPT-1 polypeptide, or biologically active portion thereof can also be accomplished by assaying for the activity of a CPT-1 target molecule. In one embodiment, determining the ability of the test compound to modulate the activity of a CPT-1 polypeptide, or biologically active portion thereof, is accomplished by assaying for the ability of the test compound to preferentially bind CPT-1, e.g., as compared to malonyl CoA. In another embodiment, determining the ability of the test compound to modulate the activity of a CPT- 1 polypeptide, or biologically active portion thereof, is accomplished by assaying for production of CoA- SH as described above.
- the cell or the mitochondrion which expresses the CPT-1 polypeptide, or biologically active portion thereof is contacted with the test compound in the presence of malonyl CoA.
- the cell or mitochondria is contacted with a compound which stimulates or inhibits a CPT-1 -associated activity (e.g., FFA oxidation) and the ability of a test compound to modulate the CPT-1 -associated activity is determined.
- a CPT-1 -associated activity e.g., FFA oxidation
- an assay is a cell-based assay in which a cell which expresses a CPT-1 polypeptide, or biologically active portion thereof, is contacted with a bioactive peptide derived from a CPT-1 target molecule and a test compound and the ability of the test compound to modulate the activity of the CPT-1 polypeptide, or biologically active portion thereof, determined.
- a bioactive peptide for use in the methodology of the instant derived from the amino acid sequence of ACC.
- the bioactive peptide corresponds to a bioactive domain of ACC (e.g., a CPT-1 interacting domain).
- the bioactive peptide corresponds to a trafficking motif of ACC.
- weight loss promoters Compounds identified in the screening assays of the invention as weight loss promoters can be further tested for the ability to modulate (e.g., promote) FFA oxidation in adipocytes or animals (e.g., animal models for obesity) using standard methods known to those skilled in the art.
- Assay reagents e.g., promote FFA oxidation in adipocytes or animals (e.g., animal models for obesity) using standard methods known to those skilled in the art.
- 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 are 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).
- the library is a natural product library.
- CPT-1 or ACC biologically active portions (i.e., bioactive fragments) of CPT-1 or ACC, including polypeptide fragments suitable for use as immunogens to raise anti-CPT-1 antibodies or ACC antibodies or to make CPT-1 or ACC fusion proteins.
- CPT-1 or ACC immunogens or bioactive fragments can be generated from CPT-1 or ACC isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
- CPT-1 or ACC immunogens or bioactive fragments are produced by recombinant DNA techniques.
- a CPT- lor ACC immunogens or bioactive fragments can be synthesized chemically using standard peptide synthesis techniques.
- An immunogen, bioactive fragment or fusion protein, as used herein is preferably “isolated” or “purified”.
- isolated and purified are used interchangeably herein.
- isolated or purified means that the immunogen, bioactive fragment or fusion protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the polypeptide is derived, substantially free of other protein fragments , for example, non-desired fragments in a digestion mixture, or substantially free from chemical precursors or other chemicals when chemically synthesized.
- the language “substantially free of cellular material” includes preparations in which the polypeptide is separated from other components of the cells from which it is isolated or recombinantly produced.
- the language “substantially free of cellular material” includes preparations of CPT-1 polypeptide having less than about 30% (by dry weight) of non-CPT-1 polyepptide (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-CPT-1 polypeptide, still more preferably less than about 10% of non-CPT-1 polypeptide, and most preferably less than about 5% non-CPT-1 polypeptide.
- the language "substantially free of cellular material” includes preparations of ACC polypeptide having less than about 30% (by dry weight) of non-ACC polyepptide (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-ACC polypeptide, still more preferably less than about 10% of non-ACC polypeptide, and most preferably less than about 5% non-ACC polypeptide.
- non-ACC polyepptide also referred to herein as a "contaminating protein”
- the immunogen, bioactive portion or fusion protein is recombinantly produced, it is 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 polypeptide preparation.
- the preparation is preferably free of enzyme reaction components or chemical reaction components and is free of non- desired CPT-1 or ACC fragments, i.e., the desired polypeptide represents at least 75% (by dry weight) of the preparation, preferably at least 80%, more preferably at least 85%, and even more preferably at least 90%, 95%, 99% or more or the preparation.
- the language “substantially free of chemical precursors or other chemicals” includes preparations of polypeptide in which the polypeptide is separated from chemical precursors or other chemicals which are involved in the synthesis of the polypeptide.
- the language “substantially free of chemical precursors or other chemicals” includes preparations having less than about 30% (by dry weight) of chemical precursors or reagents, more preferably less than about 20% chemical precursors or reagents, still more preferably less than about 10% chemical precursors or reagents, and most preferably less than about 5% chemical precursors or reagents.
- Bioactive fragments of CPT-1 or ACC include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the CPT-1 protein or the ACC protein, respectively, which include less amino acids than the full length protein, and exhibit at least one biological activity of the full-length protein.
- biologically active portions comprise a domain or motif with at least one activity of the full-length protein.
- a biologically active portion of a CPT-1 or ACC can be a polypeptide which is, for example, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more amino acids in length.
- a bioactive portion of a CPT-1 protein or ACC protein comprises at least a catalytic domain.
- a bioactive portion of a CPT-1 protein comprises at least a transferase domain domain.
- a bioactive portion of an ACC protein comprises at least a carboxyl transferase domain.
- a preferred activity of a catalytic domain is an enzymatic activity possessed by the full length CPT-1 or ACC protein. Additional preferred CPT-1 domains include, but are not limited to, a cytoplasmic domain, transmembrane or mitochindrial intermenbrane domain and a mitochondrial domain.
- biologically active portions in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native CPT-1 or ACC protein.
- the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment).
- the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position.
- the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
- the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity i.e., a local alignment.
- a preferred, non-limiting example of a local alignment 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 incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol Biol 215:403-10.
- the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment).
- Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Research 25(17):3389-3402.
- the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment).
- a preferred, non-limiting example of a mathematical algorithm utilized for the global 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.
- a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
- the invention also provides CPT-1 and ACC chimeric or fusion proteins.
- a CPT-1 or ACC "chimeric protein” or “fusion protein” comprises a CPT-1 or ACC polypeptide operatively linked to a non-CPT-1 polypeptide or non-ACC polypeptide, respectively.
- a "CPT-1 polypeptide” or “ACC polypeptide” refers to a polypeptide having an amino acid sequence corresponding to the CPT-1 or ACC protein, respectively
- a "non-CPT-1 polypeptide” or “non-ACC polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially identical to the CPT-1 protein or ACC protein.
- the CPT-1 or ACC polypeptide can correspond to all or a portion of a CPT-1 or ACC protein.
- a CPT-1 or ACC fusion protein comprises at least one biologically active portion of a CPT-1 or ACC protein, respectively.
- a CPT-1 or ACC fusion protein comprises at least two biologically active portions of a CPT-1 or ACC protein, respectively.
- the term "operatively linked" is intended to indicate that the CPT-1 or ACC polypeptide and the non-CPT-1 polypeptide or non-ACC polypeptide are fused in-frame to each other.
- the non-CPT-1 polypeptide or non-ACC polypeptide can be fused to the N-terminus or C-terminus of the CPT-1 polypeptide or ACC polypeptide, respectively.
- the fusion protein is a GST-fusion protein in which the CPT-1 or ACC sequences are fused to the C-terminus of the GST sequences.
- the fusion protein is a chitin fusion protein in which the CPT-1 or ACC sequences are fused to the N-terminus of chitin sequences.
- Such fusion proteins can facilitate the purification of recombinant CPT-1 or ACC.
- a chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
- DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
- the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
- PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
- anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
- many expression vectors are commercially available that already encode a fusion moiety.
- a CPT-1- or ACC-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the CPT- 1 or ACC polypeptide when expressed.
- a CPT-1 polypeptide or ACC polypeptide, or a portion or fragment of CPT-1 or ACC can also be used as an immunogen to generate antibodies that bind CPT-1 or ACC or that block CPT-1 /ACC binding using standard techniques for polyclonal and monoclonal antibody preparation.
- a full-length polypeptide can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens.
- an antigenic fragment comprises at least 8 amino acid residues of the amino acid sequence of CPT-1 (as set forth in GenBank Accession no. P50416) or ACC (e.g., ACC1 as set forth in GenBank Accession no. ACC501139 or ACC2 as set forth in GenBank Accession no.
- AAB58382 encompasses an epitope of CPT-1 or ACC such that an antibody raised against the peptide forms a specific immune complex with CPT-1 or ACC, respectively.
- the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
- Preferred epitopes encompassed by the antigenic peptide are regions of CPT-1 or ACC that are located on the surface of the protein, e.g., hydrophilic regions.
- a CPT-1 or ACC immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
- An appropriate immunogenic preparation can contain, for example, recombinantly expressed CPT-1 or ACC polypeptide or a chemically synthesized CPT-1 or ACC polypeptide.
- the preparation can further include an adjuvant, such as Freund' s complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic CPT-1 or ACC preparation induces a polyclonal anti-CPT-1 or anti-ACC antibody response, respectively.
- antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as CPT-1 or ACC.
- immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
- the invention provides polyclonal and monoclonal antibodies that bind CPT-1 or ACC.
- monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of CPT-1 or ACC.
- a monoclonal antibody composition thus typically displays a single binding affinity for a particular CPT-1 or ACC polypeptide with which it immunoreacts.
- Polyclonal anti-CPT-1 or anti-ACC antibodies can be prepared as described above by immunizing a suitable subject with a CPT-1 or ACC immunogen, respectively.
- the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized CPT-1 or ACC.
- ELISA enzyme linked immunosorbent assay
- the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
- antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al (1981) J. Immunol 127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J.
- an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a CPT-1 or ACC immunogen as described above, and the culture supematants of the resulting hybridoma cells are screened , to identify a hybridoma producing a monoclonal antibody that binds CPT-1 or ACC, respectively.
- lymphocytes typically splenocytes
- Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-CPT-1 or anti-ACC monoclonal antibody (see, e.g., G.
- the immortal cell line e.g. , a myeloma cell line
- murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
- Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG").
- PEG polyethylene glycol
- Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
- Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supematants for antibodies that bind CPT-1 or ACC, e.g., using a standard ELISA assay.
- a monoclonal anti-CPT-1 or anti-ACC antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with CPT-1 or ACC to thereby isolate immunoglobulin library members that bind CPT-1 or ACC, respectively.
- Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
- examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No.
- An anti-CPT-1 or anti-ACC antibody can be used to isolate CPT-1 or ACC, bioactive portions thereof, or fusion proteins by standard techniques, such as affinity chromatography or immunoprecipitation. Detection of anti CPT-1 or anti-ACC antibodies can be facilitated by coupling (t.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
- suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase;
- suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
- suitable fluorescent materials include umbelliferone, fluorescein, fiuorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
- an example of a luminescent material includes luminol;
- examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
- vectors for producing fusion protein reagents of the instant invention.
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- a preferred vector is a "plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
- plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
- the recombinant expression vectors of the invention comprise a nucleic acid that encodes, for example CPT-1 or ACC or a bioactive fragment of CPT-1 or ACC, in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
- operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
- regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals).
- the expression vectors can be introduced into host cells to thereby produce proteins, including fusion proteins or peptides.
- the recombinant expression vectors of the invention can be designed for expression of CPT-1 or ACC polypeptides in prokaryotic or eukaryotic cells.
- CPT-1 or ACC polypeptides can be expressed in bacterial cells such as E coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
- Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
- Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
- a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
- Purified fusion proteins are particularly useful in the cell-free assay methodologies of the present invention.
- a CPT-1 or ACC-encoding nucleic acid is expressed in mammalian cells, for example, for use in the cell-based assays described herein.
- the expression vector's control functions are often provided by viral regulatory elements.
- the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
- An assay cell can be prokaryotic or eukaryotic, but preferably is eukaryotic.
- Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al (Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
- An assay cell of the invention can be contacted with a test compound and assayed for any CPT-1 and/or ACC biological activity (by methods described herein or as known in the art) in order to identify the compound as an weight loss modulator.
- Weight loss modulators identified by the above- described screening assays can be tested in an appropriate animal model.
- an weight loss modulator identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such a modulator.
- a modulator identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
- this invention pertains to uses of weight loss modulators identified by the above-described screening assays for therapeutic treatments as described infra.
- compositions suitable for administration typically comprise the nucleic acid molecule, protein, antibody, or modulatory compound and a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifiingal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
- the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
- a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
- routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
- Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
- a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
- antibacterial agents such as benzyl alcohol or methyl parabens
- antioxidants
- compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
- suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
- the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- Prevention of the action of microorganisms can be achieved by various antibacterial and antifiingal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
- isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
- Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
- a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
- the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
- a binder such as microcrystalline cellulose, gum tragacanth or gelatin
- an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
- a lubricant such as magnesium stearate or Sterotes
- a glidant such as colloidal silicon dioxide
- the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
- a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
- Systemic administration can also be by transmucosal or transdermal means.
- penetrants appropriate to the barrier to be permeated are used in the formulation.
- penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
- Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
- the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
- the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
- the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
- Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
- the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
- Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
- Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
- the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
- Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
- the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
- Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
- the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
- the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
- the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
- the therapeutically effective dose can be estimated initially from cell culture assays.
- a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
- IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
- levels in plasma may be measured, for example, by high performance liquid chromatography.
- compositions can be included in a container, pack, or dispenser together with instructions for administration.
- the present invention further provides for therapeutic methods of treating a subject having an weight disorder, for example, obesity, overweight, reduced insulin sensitivity, insulin resistance, diabetes (e.g., Type II diabetes), cachexia, or anorexia.
- the therapeutic methods a particularly useful for treating obese subjects and/or obese diabetics.
- a preferred aspect of the invention pertains to methods of modulating CPT- 1/substrate (e.g, CPT-1/malonyl CoA) interactions for therapeutic purposes. Accordingly, in an exemplary embodiment, the therapeutic method of the invention involves.
- CPT- 1/substrate e.g, CPT-1/malonyl CoA
- the effectiveness of treatment of a subject with an weight loss modulator can be accomplished by (i) detecting the level of FFA oxidation, blood insulin levels or, alternatively, body weight in the subject prior to treating with an appropriate modulator; (ii) detecting the level of FFA oxidation, blood insulin or, alternatively, body weight in the subject post treatment with the modulator; (iii) comparing the levels pre- administration and post administration; and (iv) altering the administration of the modulator to the subject accordingly.
- Increased administration of the modulator may be desirable if the subject continues to demonstrate overweight or obesity, or reduced FFA oxidation.
- Obese subjects for example, typically exhibit normal blood glucose, high insulin levels and increased levels of blood FFA.
- Effective treatment is evidenced by decreased blood FFA (due to increased FFA oxidation). Decreased levels of blood insulin are also indicative of effective treatment (due, for example, to increased insulin sensitivity).
- the pre- and post-treatment profiles of an obese-diabetic are similar, except that they typically exhibit high blood glucose levels, as well, when detection is performed pre-treatment. Decreased blood glucose is further indicative of effective treatment in these subjects.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1471906A1 (en) * | 2002-02-08 | 2004-11-03 | Johns Hopkins University School of Medicine | Stimulation of cpt-1 as a means to reduce weight |
US7150969B2 (en) | 2004-06-04 | 2006-12-19 | Rosetta Inpharmatics Llc | Alternatively spliced isoform of acetyl-CoA carboxylase 2 (ACC2) |
-
2002
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Non-Patent Citations (2)
Title |
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KIM J.-Y. ET AL.: "Lipid oxidation is reduced in obese human skeletal muscle", AM. J. PHYSIOL. ENDOCRINOL. METAB., vol. 279, November 2000 (2000-11-01), pages E1039 - E1044, XP002954391 * |
THUPARI ET AL.: "C75 increases peripheral energy utilization and fatty acid oxidation in diet-induced obesity", PROC. NATL. ACAD. SCI. USA, vol. 99, no. 14, 9 July 2002 (2002-07-09), pages 9498 - 9502, XP002954392 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1471906A1 (en) * | 2002-02-08 | 2004-11-03 | Johns Hopkins University School of Medicine | Stimulation of cpt-1 as a means to reduce weight |
EP1471906A4 (en) * | 2002-02-08 | 2006-02-01 | Univ Johns Hopkins Med | Stimulation of cpt-1 as a means to reduce weight |
US7459481B2 (en) | 2002-02-08 | 2008-12-02 | The Johns Hopkins University School Of Medicine Licensing And Technology Development | Stimulation of CPT-1 as a means to reduce weight |
US7150969B2 (en) | 2004-06-04 | 2006-12-19 | Rosetta Inpharmatics Llc | Alternatively spliced isoform of acetyl-CoA carboxylase 2 (ACC2) |
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