WO2003099994A2 - Method of detecting cholesterol transporting proteins - Google Patents

Method of detecting cholesterol transporting proteins Download PDF

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WO2003099994A2
WO2003099994A2 PCT/US2003/015624 US0315624W WO03099994A2 WO 2003099994 A2 WO2003099994 A2 WO 2003099994A2 US 0315624 W US0315624 W US 0315624W WO 03099994 A2 WO03099994 A2 WO 03099994A2
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photocholesterol
mammalian
accordance
cell
contacted
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WO2003099994A3 (en
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Patricia A. Detmers
Vinay S. Bansal
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Merck & Co., Inc.
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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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells

Definitions

  • the present invention relates to a method for detecting proteins that are involved in the cholesterol transport process.
  • the method utilizes commercially available reagents and is particularly useful in elucidating the possible mechanisms of action for drugs that affect the absorption and transport of cholesterol from the gastrointestinal tract.
  • Compounds of distinct structural classes have been identified, including azetidinones and sterol glycosides, that inhibit the absorption of cholesterol in many animal species. From a variety of studies in animals, the compounds are thought to work within the small intestine to block the uptake and transport of cholesterol by absorptive epithelial cells. While the overall mechanism of action of these compounds has been demonstrated, the molecular target for cholesterol absorption inhibitors has yet to be identified.
  • one objective is to provide a method of detecting proteins that are involved in the cholesterol uptake, trafficking and transport process. Another objective is to provide proteins that are useful in the drug discovery process.
  • a method of identifying a protein that is involved in cholesterol uptake, trafficking or transport comprising contacting a mammalian cell, a mammalian cellular or acellular component or a mammalian subcellular fraction with photocholesterol, and characterizing any protein that is identified as containing, complexed or bound to the photocholesterol.
  • the invention encompasses the use of a radiolabeled photoactivatable analog of cholesterol, [3 - H]6-azi-5 a -cholestan-3 -ol ( H-photocholesterol or photocholesterol) and has the following structure:
  • This compound is a synthetic derivative of cholesterol and is disclosed in Thiele, C. et al, Nat. Cell Biol. 2:42-9. The method for synthesis is included among the Chemical Syntheses section disclosed therein. The compound is also available commercially from American Radio Chemical, St. Louis, MO.
  • Photocholesterol is approximately 6% absorbed from the gastrointestinal tract of test mammals, as measured by recovery from the liver and plasma. In contrast, cholesterol is approximately 20 percent absorbed in the same species.
  • Photocholesterol is useful as described herein to identify and characterize the proteins that are involved in the uptake, transport and trafficking of cholesterol in mammalian cells.
  • photocholesterol is useful in characterizing proteins from mammalian gastrointestinal cells, and more particularly, from particular gastrointestinal cells, such as jejunal enterocytes, where cholesterol uptake from the gastrointestinal tract occurs.
  • Such proteins can be used to identify and characterize the molecular target of cholesterol absorption inhibitors, such as the azetidinone, sterol glycoside and other drug classes.
  • cholesterol uptake, trafficking and transport is used in the conventional sense to refer to the absorption and reabsorption of cholesterol from the mammalian gastrointestinal tract.
  • subcellular fraction refers to any fraction of mammalian cells, such as the brush border, cytosolic, plasma membrane and vesicular fractions, obtainable via known methods.
  • photocholesterol comes in contact with the molecular target of cholesterol absorption inhibitors, it can be reacted to cause it to associate with the molecule of interest.
  • the photocholesterol forms covalent cross-linkages with the molecule(s) through the use of an appropriate activating or cross-linking agent.
  • an appropriate activating or cross-linking agent is ultraviolet light.
  • the target is radioactively labeled, by virtue of the radioactivity associated with the cross-linked 3 H-photocholesterol.
  • the radioactive target can then be analyzed using standard biochemical techniques, for example, polyacrylamide gel electrophoresis and amino acid sequencing and similar techniques.
  • a method of identifying a protein that is involved in cholesterol uptake, trafficking or transport is described which is comprised of contacting a mammalian cell, a mammalian cellular or acellular component or a mammalian subcellular fraction with photocholesterol, and characterizing any protein that is detected as containing, associated with, complexed with or bound to the photocholesterol.
  • a method of identifying a protein that is involved in cholesterol uptake, trafficking or transport is described wherein the photocholesterol is cross-linked to the protein via exposure to ultraviolet light. More particularly, a method of identifying a protein that is involved in cholesterol uptake, trafficking or transport is described wherein a mammalian cell is contacted with photocholesterol.
  • a method of identifying a protein that is involved in cholesterol uptake, trafficking or transport is described wherein the mammalian cell is an intestinal cell.
  • a method of identifying and characterizing proteins involved in the cholesterol uptake, trafficking and transport process is described, which is comprised of contacting mammalian gastrointestinal cells or components thereof with photocholesterol and characterizing any proteins that are detected as complexed with the photocholesterol.
  • the intestinal cell is a jejunal enterocyte.
  • a method of identifying and characterizing proteins involved in the cholesterol uptake, trafficking and transport process is described in which a mammalian acellular component is contacted with photocholesterol.
  • the mammalian acellular component is plasma, bile or the contents of the intestinal lumen.
  • a method is disclosed in which the mammalian acellular component is contacted with photocholesterol for a time period and at a temperature that is effective for causing the photocholesterol to interact with the uptake, trafficking or transport protein.
  • a method of identifying and characterizing proteins involved in the cholesterol uptake, trafficking and transport process is described in which a mammalian subcellular fraction is contacted with photocholesterol.
  • a method in which the mammalian subcellular fraction is contacted with photocholesterol for a time period and at a temperature that is effective for causing the photocholesterol to interact with the uptake, trafficking or transport protein.
  • the mammalian subcellular fraction is selected from the brush border, cytosolic, plasma membrane and vesicular fractions.
  • a method in which the mammalian cell is contacted with photocholesterol for a time period and at a temperature that is effective for causing the photocholesterol to interact with the uptake, trafficking or transport protein.
  • a method wherein after the mammalian cell is contacted with photocholesterol for a period and at a temperature effective to cause the photocholesterol to interact with the uptake, trafficking or transport protein, the cell is exposed to a crosslinking agent to cause the photocholesterol to covalently bind to the protein.
  • the crosslinking agent is ultraviolet light.
  • a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal is addressed that is comprised of: contacting a mammalian cell, a mammalian cellular or acellular component or a mammalian subcellular fraction with photocholesterol in the presence of a drug candidate, and comparing the amount of photocholesterol complexed or bound to the cell or cellular or acellular component to a standard.
  • a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal is addressed wherein a mammalian cell is contacted with photocholesterol.
  • the method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal is disclosed wherein the mammalian cell is an intestinal cell.
  • a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal is addressed identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal, is addressed wherein the intestinal cell is a jejunal enterocyte.
  • a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal is addressed wherein the mammalian cell is a hepatocyte.
  • a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal is addressed wherein a mammalian acellular component is contacted with photocholesterol.
  • a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal wherein the mammalian acellular component is plasma, bile or the contents of the intestinal lumen.
  • a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal is addressed wherein a mammalian subcellular fraction is contacted with photocholesterol.
  • a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal wherein the mammalian subcellular fraction is a brush border, cytosolic, plasma membrane or vesicular fraction.
  • cells can be analyzed for their involvement in cholesterol uptake, trafficking and transport, by using such cells in the assay described above. Jejunal enterocytes are an example of cells that can be used in the above method and demonstrated to have significant cholesterol absorption, trafficking and transport involvement.
  • compounds can be evaluated for activity inhibiting the absorption, trafficking and transport of cholesterol, using the method described herein.
  • the assay is conducted in the presence and absence of compounds, and the results compared to determine whether the compounds are active in affecting the absorption, trafficking or transport of cholesterol from the gastrointestinal tract.
  • Photocholesterol is used by incubating with the appropriate cellular components, e.g., the lumenal surface of the small intestine, cells isolated from the small intestine, for example enterocytes, or subcellular fractions isolated from such cells.
  • the intestinal source can be any mammalian or other species that is capable of absorbing cholesterol in the small intestine or in an analogous organ.
  • the concentration of photocholesterol depends on the protein content of the sample to be exposed, but can be on the order of about 10 ⁇ Ci 3 H- photocholesterol per sample.
  • the sample is incubated with photocholesterol for a suitable period, e.g., about 15 minutes at 37°C, although the time may vary depending on the sample.
  • the photocholesterol is covalently cross-linked to the proteins by exposure to ultraviolet light at a suitable wavelength, such as about 365 nm.
  • a suitable wavelength such as about 365 nm.
  • One suitable light is the Stratagene UV exposure box equipped with appropriate bulbs. Exposure can be for about 10 minutes at 4°C. Again, the time for cross-linking may be varied within wide limits, depending on the sample.
  • Paniculate fractions of irradiated samples are collected by centrifugation, with time and force dependent on the sample to be collected. Soluble fractions, are collected as acellular components or as the supematants of heterogenous samples containing particulate and soluble material. Collection methods are standard, such as by centrifugation, with time and force dependent on the sample to be collected. Samples are then dissolved in an appropriate buffer for analysis, such as by polyacrylamide gel electrophoresis (PAGE), either in one or two dimensions. Bands or spots from the PAGE are excised and subjected to amino acid analysis or mass spectroscopy to identify the proteins that have become radioactively labeled following exposure to photocholesterol.
  • PAGE polyacrylamide gel electrophoresis
  • the procedures described herein are additionally useful for screening drug candidates for reducing cholesterol trafficking or transport.
  • the mammalian gastrointestinal cells or the components thereof are contacted with photocholesterol as described above, in the presence and absence of the drug candidate (absence of the drug candidate serves as a control).
  • the level of photocholesterol bound to the sample cells, or cellular or acellular components in the presence of the drug candidate is compared to the control.
  • Photocholesterol (6%) is absorbed less efficiently than cholesterol (20%).
  • Compound A @ 20 MPK inhibited photocholesterol (84%) absorption as efficiently as cholesterol (72%).
  • Photocholesterol is demonstrated as useful for identifying proteins in the example provided below.
  • BBM Brush border membranes
  • [ 3 H]photocholesterol- methyl- ⁇ -cyclodextrin (M ⁇ CD) inclusion complex was prepared as described by Thiele et al [(2000) Nature Cell Biol., 2, 42-49].
  • Brush border membranes were suspended in 2 mM Tris-HCl, pH 7, 5 ml EGTA, 50 mM D-mannitol buffer containing protease inhibitors, and incubated with 20 ⁇ Ci of [ 3 H]photocholesterol- M ⁇ CD complex for 30 min on a shaker at 37°C.
  • the membranes were cross-linked in a UV Stratalinker at 365 nm for 15 min to covalently link [ 3 H]photocholesterol to BBM proteins.
  • Membranes were pelleted in an Eppendorf centrifuge, and the pellet was washed once in the BBM suspension buffer. The two supematants were combined and defined as Wash. The pellet was re-suspended in 1% Triton X-100 solution made in the above Tris buffer for 30 min at 4°C and centrifuged at 14,000 rpm. The supernatant was defined as TX-S for the 1% Triton X-100 soluble fraction, and the pellet was re-suspended in 1% Triton X-100 solution and called TX-P.
  • the proteins in all three samples were acetone precipitated and suspended in 1% Triton X-100 solution at ⁇ 2 ⁇ g/ml.
  • the samples were diluted in SDS buffer at 1 ⁇ g/ml, boiled for 10 min, spun at 14,000 rpm and loaded on a 4-20% SDS gel.
  • the proteins were resolved by SDS-PAGE electrophoresis, and the gel was amplified, dried and exposed for autoradiography to detect [ 3 H]photocholesterol covalently linked to protein bands. After 30 days, the autoradiography film was developed and a radioactive band was observed in the Triton X-100 soluble fraction (TX-S).
  • the rats were fasted overnight. They were gavaged with 1 ml of MC solution (saline containing 0.25% methylcellulose, 0.6% Tween 80 and 10% ethanol) and test compounds, A (20 mg/kg) and B (10 mg/kg) in MC solution. Compound B potently blocked cholesterol absorption in hamsters, rats and dogs and was used as a positive control inhibitor for cholesterol absorption. After 10 min, the rats were gavaged with 1 ml of liquid diet containing 0.1% photocholesterol/cholesterol, 0.5% cholic acid and 2 ⁇ Ci [ 3 H ] -photocholesterol (sp. activity - 20 Ci/mmol, ARC, St. Louis, MO)/ [ 3 H ]-cholesterol (sp. activity - 40-60 Ci/mmol, PE-Life Sciences, Boston, MA).
  • MC solution saline containing 0.25% methylcellulose, 0.6% Tween 80 and 10% ethanol
  • test compounds A (20 mg/kg) and B (10 mg/kg

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Abstract

A method of identifying a protein that is involved in cholesterol uptake, trafficking or transport is disclosed which comprises contacting a mammalian cell, or a mammalian cellular or acellular component with photocholesterol, and characterizing any protein that is identified as containing, complexed or bound to the photocholesterol.

Description

METHOD OF DETECTING CHOLESTEROL TRANSPORTING PROTEINS
BACKGROUND OF THE INVENTION The present invention relates to a method for detecting proteins that are involved in the cholesterol transport process. The method utilizes commercially available reagents and is particularly useful in elucidating the possible mechanisms of action for drugs that affect the absorption and transport of cholesterol from the gastrointestinal tract. Compounds of distinct structural classes have been identified, including azetidinones and sterol glycosides, that inhibit the absorption of cholesterol in many animal species. From a variety of studies in animals, the compounds are thought to work within the small intestine to block the uptake and transport of cholesterol by absorptive epithelial cells. While the overall mechanism of action of these compounds has been demonstrated, the molecular target for cholesterol absorption inhibitors has yet to be identified.
Consequently one objective is to provide a method of detecting proteins that are involved in the cholesterol uptake, trafficking and transport process. Another objective is to provide proteins that are useful in the drug discovery process.
These and other objectives will be apparent from the description contained herein.
SUMMARY OF THE INVENTION A method of identifying a protein that is involved in cholesterol uptake, trafficking or transport, comprising contacting a mammalian cell, a mammalian cellular or acellular component or a mammalian subcellular fraction with photocholesterol, and characterizing any protein that is identified as containing, complexed or bound to the photocholesterol. DETAILED DESCRIPTION OF THE INVENTION
The invention encompasses the use of a radiolabeled photoactivatable analog of cholesterol, [3 - H]6-azi-5a-cholestan-3 -ol ( H-photocholesterol or photocholesterol) and has the following structure:
Figure imgf000003_0001
This compound is a synthetic derivative of cholesterol and is disclosed in Thiele, C. et al, Nat. Cell Biol. 2:42-9. The method for synthesis is included among the Chemical Syntheses section disclosed therein. The compound is also available commercially from American Radio Chemical, St. Louis, MO.
Photocholesterol is approximately 6% absorbed from the gastrointestinal tract of test mammals, as measured by recovery from the liver and plasma. In contrast, cholesterol is approximately 20 percent absorbed in the same species.
Photocholesterol is useful as described herein to identify and characterize the proteins that are involved in the uptake, transport and trafficking of cholesterol in mammalian cells. In particular, photocholesterol is useful in characterizing proteins from mammalian gastrointestinal cells, and more particularly, from particular gastrointestinal cells, such as jejunal enterocytes, where cholesterol uptake from the gastrointestinal tract occurs. Such proteins can be used to identify and characterize the molecular target of cholesterol absorption inhibitors, such as the azetidinone, sterol glycoside and other drug classes.
As used herein, the term "cholesterol uptake, trafficking and transport" is used in the conventional sense to refer to the absorption and reabsorption of cholesterol from the mammalian gastrointestinal tract.
The term "subcellular fraction" refers to any fraction of mammalian cells, such as the brush border, cytosolic, plasma membrane and vesicular fractions, obtainable via known methods. When photocholesterol comes in contact with the molecular target of cholesterol absorption inhibitors, it can be reacted to cause it to associate with the molecule of interest. In one aspect of the invention, the photocholesterol forms covalent cross-linkages with the molecule(s) through the use of an appropriate activating or cross-linking agent. One such agent is ultraviolet light. By exposure to ultraviolet light of the appropriate wavelength, the target is radioactively labeled, by virtue of the radioactivity associated with the cross-linked 3H-photocholesterol. The radioactive target can then be analyzed using standard biochemical techniques, for example, polyacrylamide gel electrophoresis and amino acid sequencing and similar techniques.
In one aspect of the invention, a method of identifying a protein that is involved in cholesterol uptake, trafficking or transport, is described which is comprised of contacting a mammalian cell, a mammalian cellular or acellular component or a mammalian subcellular fraction with photocholesterol, and characterizing any protein that is detected as containing, associated with, complexed with or bound to the photocholesterol.
More particularly, a method of identifying a protein that is involved in cholesterol uptake, trafficking or transport is described wherein the photocholesterol is cross-linked to the protein via exposure to ultraviolet light. More particularly, a method of identifying a protein that is involved in cholesterol uptake, trafficking or transport is described wherein a mammalian cell is contacted with photocholesterol.
In particular, a method of identifying a protein that is involved in cholesterol uptake, trafficking or transport is described wherein the mammalian cell is an intestinal cell.
In one aspect of the invention, a method of identifying and characterizing proteins involved in the cholesterol uptake, trafficking and transport process is described, which is comprised of contacting mammalian gastrointestinal cells or components thereof with photocholesterol and characterizing any proteins that are detected as complexed with the photocholesterol.
More particularly, the intestinal cell is a jejunal enterocyte.
In another aspect of the invention, a method of identifying and characterizing proteins involved in the cholesterol uptake, trafficking and transport process is described in which a mammalian acellular component is contacted with photocholesterol.
In a particular aspect of the invention, the mammalian acellular component is plasma, bile or the contents of the intestinal lumen. In particular, a method is disclosed in which the mammalian acellular component is contacted with photocholesterol for a time period and at a temperature that is effective for causing the photocholesterol to interact with the uptake, trafficking or transport protein.
In another aspect of the invention, a method of identifying and characterizing proteins involved in the cholesterol uptake, trafficking and transport process is described in which a mammalian subcellular fraction is contacted with photocholesterol.
In particular, a method is disclosed in which the mammalian subcellular fraction is contacted with photocholesterol for a time period and at a temperature that is effective for causing the photocholesterol to interact with the uptake, trafficking or transport protein.
More particularly, a method of identifying and characterizing proteins involved in the cholesterol uptake, trafficking and transport process is described wherein the mammalian subcellular fraction is selected from the brush border, cytosolic, plasma membrane and vesicular fractions.
In particular, a method is disclosed in which the mammalian cell is contacted with photocholesterol for a time period and at a temperature that is effective for causing the photocholesterol to interact with the uptake, trafficking or transport protein.
Even more particularly, a method is disclosed wherein after the mammalian cell is contacted with photocholesterol for a period and at a temperature effective to cause the photocholesterol to interact with the uptake, trafficking or transport protein, the cell is exposed to a crosslinking agent to cause the photocholesterol to covalently bind to the protein.
In a preferred aspect of the invention, the crosslinking agent is ultraviolet light.
In a different aspect of the invention, a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal, is addressed that is comprised of: contacting a mammalian cell, a mammalian cellular or acellular component or a mammalian subcellular fraction with photocholesterol in the presence of a drug candidate, and comparing the amount of photocholesterol complexed or bound to the cell or cellular or acellular component to a standard.
More particularly, a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal, is addressed wherein a mammalian cell is contacted with photocholesterol.
Even more particularly, the method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal, is disclosed wherein the mammalian cell is an intestinal cell.
Still more particularly, a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal, is addressed identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal, is addressed wherein the intestinal cell is a jejunal enterocyte.
Alternatively, a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal, is addressed wherein the mammalian cell is a hepatocyte.
In another embodiment of the invention, a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal, is addressed wherein a mammalian acellular component is contacted with photocholesterol.
In another aspect of the invention, a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal is described wherein the mammalian acellular component is plasma, bile or the contents of the intestinal lumen.
In another embodiment of the invention, a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal, is addressed wherein a mammalian subcellular fraction is contacted with photocholesterol.
More particularly, in another embodiment of the invention, a method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal is described wherein the mammalian subcellular fraction is a brush border, cytosolic, plasma membrane or vesicular fraction. In another aspect of the invention, cells can be analyzed for their involvement in cholesterol uptake, trafficking and transport, by using such cells in the assay described above. Jejunal enterocytes are an example of cells that can be used in the above method and demonstrated to have significant cholesterol absorption, trafficking and transport involvement. In another aspect of the invention, compounds can be evaluated for activity inhibiting the absorption, trafficking and transport of cholesterol, using the method described herein. In this embodiment, the assay is conducted in the presence and absence of compounds, and the results compared to determine whether the compounds are active in affecting the absorption, trafficking or transport of cholesterol from the gastrointestinal tract.
The function of these proteins is inhibited by cholesterol absorption inhibitors. Photocholesterol is used by incubating with the appropriate cellular components, e.g., the lumenal surface of the small intestine, cells isolated from the small intestine, for example enterocytes, or subcellular fractions isolated from such cells. The intestinal source can be any mammalian or other species that is capable of absorbing cholesterol in the small intestine or in an analogous organ.
The concentration of photocholesterol depends on the protein content of the sample to be exposed, but can be on the order of about 10 μCi 3H- photocholesterol per sample. The sample is incubated with photocholesterol for a suitable period, e.g., about 15 minutes at 37°C, although the time may vary depending on the sample. After incubation of the photocholesterol with the source of potential target proteins, the photocholesterol is covalently cross-linked to the proteins by exposure to ultraviolet light at a suitable wavelength, such as about 365 nm. One suitable light is the Stratagene UV exposure box equipped with appropriate bulbs. Exposure can be for about 10 minutes at 4°C. Again, the time for cross-linking may be varied within wide limits, depending on the sample.
Paniculate fractions of irradiated samples are collected by centrifugation, with time and force dependent on the sample to be collected. Soluble fractions, are collected as acellular components or as the supematants of heterogenous samples containing particulate and soluble material. Collection methods are standard, such as by centrifugation, with time and force dependent on the sample to be collected. Samples are then dissolved in an appropriate buffer for analysis, such as by polyacrylamide gel electrophoresis (PAGE), either in one or two dimensions. Bands or spots from the PAGE are excised and subjected to amino acid analysis or mass spectroscopy to identify the proteins that have become radioactively labeled following exposure to photocholesterol.
The procedures described herein are additionally useful for screening drug candidates for reducing cholesterol trafficking or transport. In this aspect of the invention, the mammalian gastrointestinal cells or the components thereof are contacted with photocholesterol as described above, in the presence and absence of the drug candidate (absence of the drug candidate serves as a control). The level of photocholesterol bound to the sample cells, or cellular or acellular components in the presence of the drug candidate is compared to the control.
Several compounds that have an effect on the absorption, trafficking or transport of cholesterol can be mentioned as examples. Cholesterol absorption inhibition can be demonstrated using known compounds. Two such compounds are as follows:
Figure imgf000008_0001
Figure imgf000008_0002
Compound B
These compounds are reported in Hernandez, M. et al. Biochim Biophys Acta 1486 (2000) 232-242, 234.
Photocholesterol (6%) is absorbed less efficiently than cholesterol (20%). Compound A @ 20 MPK inhibited photocholesterol (84%) absorption as efficiently as cholesterol (72%).
Photocholesterol is demonstrated as useful for identifying proteins in the example provided below.
EXAMPLE 1
PHOTO AFFINITY LABELING OF RAT BRUSH BORDER MEMBRANES TO IDENTIFY CHOLESTEROL BINDING PROTEINS Brush border membranes (BBM) from the jejunum of the rat small intestine were isolated as described (Hauser H. et al (1980) Biochim Biophys Acta, 602, 567-577 ) and sucrase activity was used as a marker to determine the enrichment of BBM. Based on the assay, a ~ 15 fold enrichment of the sucrase activity was obtained in the BBM fraction relative to crude homogenate. [3H]photocholesterol- methyl-β-cyclodextrin (MβCD) inclusion complex was prepared as described by Thiele et al [(2000) Nature Cell Biol., 2, 42-49]. Brush border membranes were suspended in 2 mM Tris-HCl, pH 7, 5 ml EGTA, 50 mM D-mannitol buffer containing protease inhibitors, and incubated with 20 μCi of [3H]photocholesterol- MβCD complex for 30 min on a shaker at 37°C. The membranes were cross-linked in a UV Stratalinker at 365 nm for 15 min to covalently link [3H]photocholesterol to BBM proteins. Membranes were pelleted in an Eppendorf centrifuge, and the pellet was washed once in the BBM suspension buffer. The two supematants were combined and defined as Wash. The pellet was re-suspended in 1% Triton X-100 solution made in the above Tris buffer for 30 min at 4°C and centrifuged at 14,000 rpm. The supernatant was defined as TX-S for the 1% Triton X-100 soluble fraction, and the pellet was re-suspended in 1% Triton X-100 solution and called TX-P. The proteins in all three samples (Wash, TX-S and TX-P) were acetone precipitated and suspended in 1% Triton X-100 solution at ~ 2 μg/ml. The samples were diluted in SDS buffer at 1 μg/ml, boiled for 10 min, spun at 14,000 rpm and loaded on a 4-20% SDS gel. The proteins were resolved by SDS-PAGE electrophoresis, and the gel was amplified, dried and exposed for autoradiography to detect [3H]photocholesterol covalently linked to protein bands. After 30 days, the autoradiography film was developed and a radioactive band was observed in the Triton X-100 soluble fraction (TX-S). EXAMPLE 2
IN VIVO ABSORPTION OF PHOTOCHOLESTEROL The absorption of photocholesterol was compared to cholesterol in rats. Rats (Sprague Dawley rats from Taconic, male, 170 g body weight) were divided into five groups. Each group consisted of six rats except group 3 which had four rats. Details pertaining to each group are set forth below.
Figure imgf000009_0001
Figure imgf000010_0002
The rats were fasted overnight. They were gavaged with 1 ml of MC solution (saline containing 0.25% methylcellulose, 0.6% Tween 80 and 10% ethanol) and test compounds, A (20 mg/kg) and B (10 mg/kg) in MC solution. Compound B potently blocked cholesterol absorption in hamsters, rats and dogs and was used as a positive control inhibitor for cholesterol absorption. After 10 min, the rats were gavaged with 1 ml of liquid diet containing 0.1% photocholesterol/cholesterol, 0.5% cholic acid and 2 μCi [3H ] -photocholesterol (sp. activity - 20 Ci/mmol, ARC, St. Louis, MO)/ [3H ]-cholesterol (sp. activity - 40-60 Ci/mmol, PE-Life Sciences, Boston, MA).
After 24 h, plasma and liver samples were collected from each rat and radioactivity was counted. The amount of [3H ]-photocholesterol/[3H ]-cholesterol in plasma and liver was calculated based on the assumption that plasma volume and liver weight are 4% of body weight. The results are shown below in the table provided.
Figure imgf000010_0001
Compound A @ 20 MPK and Compound B @ 10 MPK, inhibited cholesterol absorption by 75%. The absorption of photocholesterol (6%) was significantly lower than cholesterol (20%) in rats. Compound A @ 20 MPK inhibited photocholesterol (84%) absorption as efficiently as cholesterol (72%). The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Various publications are cited herein, including patents, patent applications and scientific publications. All such publications are hereby incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED IS:
1. A method of identifying a protein that is involved in cholesterol uptake, trafficking or transport, comprising contacting a mammalian cell, a mammalian cellular or acellular component or a mammalian subcellular fraction with photocholesterol, and characterizing any protein that is detected as containing, complexed or bound to the photocholesterol.
2. A method in accordance with claim 1 wherein the photocholesterol is crosslinked to the protein via ultraviolet light.
3. A method in accordance with claim 1 wherein a mammalian cell is contacted with photocholesterol.
4. A method in accordance with claim 3 wherein the mammalian cell is an intestinal cell.
5. A method in accordance with claim 4 wherein the intestinal cell is a jejunal enterocyte.
6. A method in accordance with claim 1 wherein a mammalian acellular component is contacted with photocholesterol.
7. A method in accordance with claim 4 wherein the mammalian acellular component is plasma, bile or the contents of the intestinal lumen.
8. A method in accordance with claim 1 wherein a mammalian subcellular fraction is contacted with photocholesterol.
9. A method in accordance with claim 8 wherein the mammalian subcellular fraction is a brush border, cytosolic, plasma membrane or vesicular subcellular fraction.
10. A method in accordance with claim 1 wherein the mammalian cell is contacted with photocholesterol for a time period and at a temperature that is effective for causing the photocholesterol to interact with the uptake, trafficking or transport protein.
11. A method in accordance with claim 10 wherein after the mammalian cell is contacted with photocholesterol for a period and at a temperature effective to cause the photocholesterol to interact with the uptake, trafficking or transport protein, the cell is exposed to a crosslinking agent to cause the photocholesterol to covalently bind to the protein.
12. A method in accordance with claim 11 wherein the crosslinking agent is ultraviolet light.
13. A method of identifying a compound that reduces cholesterol absorption, trafficking or transport in a mammal, comprising: contacting a mammalian cell, a mammalian cellular or acellular component or a mammalian subcellular fraction with photocholesterol in the presence of a drug candidate, and comparing the amount of photocholesterol complexed or bound to the cell or cellular or acellular component to a standard.
14. A method in accordance with claim 13 wherein a mammalian cell is contacted with photocholesterol.
15. A method in accordance with claim 14 wherein the mammalian cell is an intestinal cell.
16. A method in accordance with claim 15 wherein the intestinal cell is a jejunal enterocyte.
17. A method in accordance with claim 14 wherein the mammalian cell is a hepatocyte.
18. A method in accordance with claim 13 wherein a mammalian acellular component is contacted with photocholesterol.
19. A method in accordance with claim 17 wherein the mammalian acellular component is plasma, bile or the contents of the intestinal lumen.
20. A method in accordance with claim 13 wherein a mammalian subcellular fraction is contacted with photocholesterol.
21. A method in accordance with claim 20 wherein the mammalian subcellular fraction is a brush border, cytosolic, plasma membrane or vesicular subcellular fraction.
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Citations (4)

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Publication number Priority date Publication date Assignee Title
NL8901732A (en) * 1989-07-06 1991-02-01 Nii Fiz Khim Meditsiny METHOD FOR PREPARING AFFINO-ENZYMATIC COMPOUNDS FOR VISUAL INDICATION OF SKIN SURFACE CHOLESTEROL BASED ON A DETECTIVE WITH CHOLESTEROL AND VISUALIZING AGENT AND USE THEREOF
US5681819A (en) * 1994-12-01 1997-10-28 Oklahoma Medical Research Foundation Method and compositions for reducing cholesterol absorption
US5872141A (en) * 1997-02-18 1999-02-16 Umbreit; Jay N. Method of inhibiting cholesterol transport
US6555323B2 (en) * 2000-02-08 2003-04-29 Pfizer Inc. Assay for ABCA1

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Publication number Priority date Publication date Assignee Title
NL8901732A (en) * 1989-07-06 1991-02-01 Nii Fiz Khim Meditsiny METHOD FOR PREPARING AFFINO-ENZYMATIC COMPOUNDS FOR VISUAL INDICATION OF SKIN SURFACE CHOLESTEROL BASED ON A DETECTIVE WITH CHOLESTEROL AND VISUALIZING AGENT AND USE THEREOF
US5681819A (en) * 1994-12-01 1997-10-28 Oklahoma Medical Research Foundation Method and compositions for reducing cholesterol absorption
US5872141A (en) * 1997-02-18 1999-02-16 Umbreit; Jay N. Method of inhibiting cholesterol transport
US6555323B2 (en) * 2000-02-08 2003-04-29 Pfizer Inc. Assay for ABCA1

Non-Patent Citations (1)

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Title
MINTZER E.: 'Behavior of a photoactivatable analog of cholesterol, 6-photocholesterol, in model membranes' FEBS LETTERS vol. 510, no. 3, 2002, pages 181 - 184, XP004332637 *

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