WO1999059560A1

WO1999059560A1 - Glucose transport modulators and uses therefor

Info

Publication number: WO1999059560A1
Application number: PCT/US1999/010718
Authority: WO
Inventors: Steven E. Shoelson
Original assignee: Joslin Diabetes Center Inc
Current assignee: Joslin Diabetes Center Inc
Priority date: 1998-05-15
Filing date: 1999-05-14
Publication date: 1999-11-25
Anticipated expiration: 2000-11-15
Also published as: AU4188399A

Abstract

The invention provides methods and compounds for modulating the number of GLUT4 sorting vesicle resident molecules on the surface of a cell. The invention also provides methods of treating NIDDM in a subject. Methods of evaluating compounds useful in the methods of the invention are also provided.

Description

GLUCOSE TRANSPORT MODULATORS AND USES THEREFOR

The invention relates to molecules which regulate GLUT4 transport sorting vesicle resident proteins distribution in cells and methods for their identification and use.

Background of the Invention Glucose transporter isoform 4, GLUT4, is also known as the insulin- regulatable glucose transporter isoform. GLUT4 is found in adipocytes and muscle cells where, after expression, it is stored in intracellular vesicles. Insulin stimulates the redistribution of these vesicles, and thus GLUT4, to the cell surface where GLUT4 transports glucose into the cells. Upon the removal of the insulin stimulus, GLUT4 vesicles can return to their intracellular locations. The most common glucose regulatory associated disorder, Type II

Diabetes Mellitus, is characterized by a combination of increased hepatic glucose output, reduced skeletal muscle glucose disposal, and impaired β-cell function. Generally, prior to the full onset of the disease, some degree of insulin resistance, e.g., a diminished ability of key targets like muscle, fat and liver to respond to insulin, occurs. In pre-diabetic individuals this insulin resistance is detectable before glucose tolerance can be measured, at a time when insulin secretion may actually be increased (perhaps to compensate for its decreased effectiveness). Thus, insulin resistance is considered by some researchers to be the primary defect not only for non-insulin dependent diabetes mellitus, but for diabetes in general.

Summary of the Invention The inventor has discovered that molecules which regulate GLUT4 sorting vesicle resident proteins distribution in cells can directly modulate glucose uptake in the cells. Accordingly, the invention features, a method of modulating, e.g., increasing or promoting, the number of GLUT4 sorting vesicle resident molecules, e.g., GLUT4 or IRAP molecules, on the surface of a cell, e.g., a cell of a subject. The method includes: administering a treatment which inhibits the interaction of AP-1, AP-2, AP-3 or COP with a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP, thereby increasing or promoting the number of GLUT4 sorting vesicle resident molecules, e.g., GLUT4 molecules, on the cell surface. The method can modulate the activity of GLUT4 sorting vesicle molecule, e.g., by increasing or decreasing its numbers on the cell surface. In another aspect, the invention features, a method of increasingor promoting the number of GLUT4 sorting vesicle resident molecules, e.g., GLUT4 or LRAP molecules, on the surface of a cell, e.g., a cell of a subject. The method includes: administering a treatment which inhibits the interaction of a clathrin associated adaptor complex- 1 molecule (AP-1) with a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP, thereby increasing or promoting the number of GLUT4 sorting vesicle resident molecules, e.g., GLUT4 or IRAP molecules, on the cell surface.

In preferred embodiments, the treatment inhibits endocytotic removal of a GLUT4 sorting vesicle resident molecule, e.g., GLUT4, from the cell surface. In preferred embodiments, the treatment includes administering to a cell, a compound, e.g., a protein, a peptide, ' r a peptidomimetic, or a compound other than a peptide which inhibits the interaction. The inhibition of the interaction can be competitive or non-competitive.

In preferred embodiments, the treatment includes administering a compound, e.g, a molecule which binds to the AP-1 molecule. The molecule can be GLUT4, an AP-1 binding fragment of GLUT4, e.g., a peptide which includes the GLUT4 tail peptide, an AP-1 binding fragment of the GLUT4 tail peptide, a peptide which includes the GLUT4 tail peptide dileucine motif, a peptide which includes the GLUT4 tail peptide diacidic motif, a peptide which includes the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be IRAP, or an AP-1 binding fragment of IRAP, e.g., a fragment which includes the residues_YESSAKLIGMSF.

In preferred embodiments, the molecule is at least 3, 4, 5, 7, 10, 15, 20, 25, 30, amino acids in length. In preferred embodiments, the molecules is less than 100, 50, or 20 amino acids in length.

In preferred embodiments, the method includes administering a compound, e.g., a molecule which binds to the GLUT4 molecule. The molecule can be AP-1, a GLUT4 binding fragment of AP-1, e.g., a peptide which includes the AP- 1 α peptide, a peptide which includes the AP- 1 β 1 peptide. The molecule can be a molecule which binds IRAP, e.g., a fragment of a polypeptide which normally binds IRAP.

In a preferred embodiment, the method is performed in vitro. In a preferred embodiment, the method is performed in vivo. In another aspect, the invention features, a method of modulating, e.g., increasing glucose uptake in a cell, e.g., a cell of a subject. The method includes administering a treatment which inhibits the interaction of an AP-1, AP-2, AP-3 or COP molecule with GLUT4 or LRAP, thereby increasing glucose uptake in a cell, e.g., a cell of a subject. In another aspect, the invention features, a method of increasing glucose uptake in a cell, e.g., a cell of a subject. The method includes administering a treatment which inhibits the interaction of an AP-1 molecule with a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP, thereby increasing glucose uptake in a cell, e.g., a cell of a subject. In preferred embodiments, the treatment inhibits endocytotic removal of

GLUT4 molecules from the cell surface.

In preferred embodiments, the treatment includes administering to a cell a molecule, e.g., a protein, a peptide, a peptidomimetic, or a compound other than a peptide which inhibits the interaction. The inhibition of the interaction can be competitive or non-competitive.

In preferred embodiments, the invention includes administering a compound, e.g. a molecule which binds to the AP-1 molecule. The molecule can be GLUT4, an AP-1 binding fragment of GLUT4, e.g., a peptide which contains the GLUT4 tail peptide, a binding fragment of the GLUT4 tail peptide, e.g., a peptide which contains the GLUT4 tail peptide dileucine motif, a peptide which contains the GLUT4 tail peptide diacidic motif, a peptide which contains the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be IRAP, or an AP-1 binding fragment of IRAP, e.g., a fragment which includes the residues YESSAKLIGMSF. In preferred embodiments, the invention includes administering a compound, e.g., a molecule which binds to the GLUT4 molecule. The molecule can be AP-1, a GLUT4 binding fragment of AP-1, e.g., a peptide which contains the AP-lα peptide, a peptide which contains the AP-lβl peptide. The molecule can be a molecule which binds IRAP, e.g., a fragment of a polypeptide which normally binds IRAP. In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In another aspect, the invention features, a method for treating a subject for an insulin or glucose-related disorder, e.g., glucose intolerance, e.g.,diabetes, e.g., non-insulin dependent diabetes mellitus (NIDDM). The method includes: administering a treatment which inhibits the interaction of AP-1, AP-2, AP-3 or COP with GLUT4 or IRAP, thereby treating the subject for the disorder.

In another aspect, the invention features, a method for treating a subject for an insulin or glucose-related disorder, e.g., glucose intolerance, e.g.,diabetes, e.g., non-insulin dependent diabetes mellitus (NIDDM). The method includes: administering a treatment which inhibits the interaction of AP-1 with a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP, thereby treating the subject for the disorder.

In preferred embodiments, the treatment inhibits endocytotic removal of a GLUT4 molecule from the cell surface.

In preferred embodiments, the treatment includes administering to the cell a compound, e.g., or a protein, a peptide, a peptidomimetic, or a compound other than a peptide, which inhibits the interaction. The inhibition of the interaction can be competitive or non-competitive. In preferred embodiments, the invention includes administering a compound, e.g., a molecule which binds to the AP-1 molecule. The molecule can be GLUT4, an AP-1 binding fragment of GLUT4, e.g., a peptide which includes the GLUT4 tail peptide, an AP-1 binding fragment of the GLUT4 tail peptide, a peptide which includes the GLUT4 tail peptide dileucine motif, a peptide which includes the GLUT4 tail peptide diacidic motif, a peptide which includes the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be LRAP, or an AP-1 binding fragment of IRAP, e.g., a fragment which includes the residues_YESSAKLIGMSF.

In preferred embodiments, the molecule is at least 3, 4, 5, 7, 10, 15, 20, 25, 30, amino acids in length.

In preferred embodiments, the molecules is less than 100, 50, or 20 amino acids in length.

In preferred embodiments, the invention includes administering a compound, e.g, a molecule which binds to the GLUT4 molecule. The molecule can be AP-1, e.g., a GLUT4 binding fragment of AP-1, e.g., a peptide which contains the AP-lα peptide, a peptide which contains the AP-lβl peptide. The molecule can be a molecule which binds IRAP, e.g., a fragment of a polypeptide which normally binds IRAP.

In another aspect, the invention features, a method of decreasing GLUT4 sorting vesicle resident molecules, e.g., GLUT4 or IRAP molecules, on the surface of a cell, e.g., a cell of a subject. The method includes: administering AP-1 or an agonist thereof to the cell, thereby decreasing the number of GLUT4 sorting vesicle resident molecules on the surface of the cell, e.g., a cell of a subject. In preferred embodiments the treatment promotes endocytotic removal of a GLUT4 sorting vesicle resident molecule, e.g., a GLUT4 or IRAP molecule, from the cell surface.

In preferred embodiments the treatment includes administering to the cell a molecule of AP-1 or an agonist thereof, e.g., a protein, a peptide, a peptidomimetic, of AP-1 or an agonist thereof which promotes the interaction.

In preferred embodiments, the treatment includes administering to a cell, a compound, e.g., a protein, a peptide, or a peptidomimetic, or a compound other than a peptide, which inhibits the interaction of AP-1 and a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP. The inhibition can be competitive or non-competitive.

In a preferred embodiment, the method is performed in vitro. In a preferred embodiment, the method is performed in vivo. In another aspect, the invention features, a method of evaluating a compound, e.g., a protein, a peptide, a peptidomimetic, or a compound other than a peptide, for the ability to inhibit an interaction between AP-1, AP-2, AP-3 or COP and a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or LRAP. The method includes: providing a GLUT4 moiety, contacting the compound with the GLUT4 moiety, determining if the compound binds the GLUT4 moiety, binding of the compound to the GLUT4 moiety, being, e.g., indicative of its ability to inhibit the interaction. Analagous methods can be used to evaluate inhibitions inhibitions of other GLUT4 sorting vesicle resident proteins, e.g., LRAP, with AP-1, AP-2, AP-3, or COP.

In another aspect, the invention features, a method of evaluating a compound, e.g., a protein, a peptide, a peptidomimetic, or a compound other than a peptide, for the ability to inhibit an interaction between AP-1 and GLUT4. The method includes: providing a GLUT4 moiety, contacting the compound with the GLUT4 moiety, determining if the compound binds the GLUT4 moiety, binding of the compound to the GLUT4 moiety, being, e.g., indicative of its ability to inhibit the interaction. In preferred embodiments, the GLUT4 moiety is: GLUT4, an AP-1 binding fragment of GLUT4, e.g., a peptide which includes the GLUT4 tail peptide, a peptide which includes the GLUT4 tail peptide dileucine motif, a peptide which includes the GLUT4 tail peptide diacidic motif, a peptide which includes the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif.

In a preferred embodiment, the method is performed in vitro. In a preferred embodiment, the method is performed in vivo. In preferred embodiments, the method is a two-hybrid assay, a phage display assay, e.g., a filamentous phage assay. In a preferred embodiment, the method further includes contacting the compound with a GLUT4 moiety and an AP-1 moiety, and evaluating the ability of the compound to inhibit an interaction between the GLUT4 moiety and the AP-1 moiety.

In preferred embodiments, the method further includes contacting a cell, or a cell of a subject, with the compound and evaluating its effect on surface levels of a GLUT4 sorting vesicle resident molecule, e.g., GLUT4, or on glucose uptake, endocytosis, exocytosis, glucose metabolism, insulin metabolism, or plasma glucose levels.

In another aspect, the invention features, a method of evaluating a compound, e.g., for the ability to inhibit an interaction between AP-1, AP-2, AP- 3 or COP and a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP. The method includes: providing an AP-1, AP-2, AP-3 or COP moiety, contacting the compound with the AP-1, AP-2, AP-3 or COP moiety, determining if the compound binds the AP-1, AP-2, AP-3 or COP moiety, the binding of the compound, e.g., being indicative of its ability to inhibit the interaction.

In another aspect, the invention features, a method of evaluating a compound, e.g., for the ability to inhibit an interaction between AP-1 and GLUT4. The method includes: providing an AP-1 moiety, contacting the compound with the AP-1 moiety, determining if the compound binds the AP-1 moiety, the binding of the compound, e.g., being indicative of its ability to inhibit the interaction.

In preferred embodiments the AP-1 moiety is: AP-1, a GLUT4 binding fragment of AP-1, e.g., a peptide which contains the AP-lα peptide, a peptide which contains the AP- 1 β 1 peptide.

In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In preferred embodiments, the method is a two-hybrid assay, a phage display assay, e.g., a filamentous phage assay. In a preferred embodiment, the method further includes contacting the compound with a GLUT4 moiety and an AP-1 moiety, and evaluating the ability of the compound to inhibit an interaction between the GLUT4 moiety and the AP-1 moiety

In another aspect, the invention features, a method of evaluating a compound, e.g., for the ability to inhibit an interaction between AP-1, AP-2, AP- 3 or COP and GLUT4 sorting vesicle resident protein, e.g., GLUT4 or LRAP. The method includes: providing a GLUT4 sorting vesicle resident protein, e.g., GLUT4 moiety or IRAP and an AP-1, AP-2, AP-3 or COP moiety, contacting the compound with the GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP moiety and the AP-1, AP-2, AP-3 or COP moiety, determining if the compound binds the GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP moiety or the AP-1, AP-2, AP-3 or COP moiety, the binding of the compound being indicative of its ability to inhibit the interaction.

In a preferred embodiment, the method is performed in vitro. In a preferred embodiment, the method is performed in vivo.

In preferred embodiments, the method is a two-hybrid assay, a phage display assay, e.g., a filamentous phage assay.

In another aspect, the invention features, a method of evaluating a compound for the ability to modulate the interaction between AP-1, AP-2, AP-3 or COP and GLUT4 sorting vesicle resident protein, e.g., GLUT4 or LRAP. The method includes: providing a cell having a reporter gene under the control of an AP-1, AP-2, AP-3, COP or a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP regulatory agent, contacting the cell with the compound, and evaluating the effect of the compound on the expression of the reporter gene. In another aspect, the invention features, a method of evaluating a compound for the ability to modulate the interaction between AP-1 and GLUT4. The method includes: providing a cell having a reporter gene under the control of an AP-1 or a GLUT4 regulatory agent, contacting the cell with the compound, and evaluating the effect of the compound on the expression of the reporter gene. In another aspect, the invention features, a purified preparation of a molecule capable of binding either GLUT4, or AP-1, AP-2, AP-3 or COP and inhibiting binding between GLUT4 and AP-1, AP-2, AP-3 or COP.

In another aspect, the invention features, a purified preparation of a molecule capable of binding either GLUT4, or AP-1, and inhibiting binding between GLUT4 and AP-1.

In preferred embodiments, the preparation includes a compound containing a GLUT4 binding fragment of AP-1, e.g., a peptide containing the AP- lα peptide, a peptide containing the AP-lβl peptide; a peptide which binds AP-1.

In preferred embodiments, the preparation includes a compound containing an AP-1 binding fragment of GLUT4, e.g., a peptide containing the GLUT4 tail peptide, a binding fragment of the GLUT4 tail peptide, e.g., a peptide containing the GLUT4 tail peptide dileucine motif, a peptide containing the GLUT4 tail peptide diacidic motif, a peptide containing the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif; a peptide which binds GLUT4.

In an alternate embodiment, the invention features, a method of increasing or promoting the number of GLUT4 sorting vesicle resident molecules, e.g., GLUT4 or LRAP molecules, on the surface of a cell, e.g., a cell of a subject. The method includes: administering a treatment which inhibits the interaction of a clathrin associated adaptor complex-2 molecule (AP-2) with GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP, thereby increasing or promoting the number of GLUT4 sorting vesicle resident molecules, e.g., GLUT4 molecules, on the cell surface.

In preferred embodiments, the treatment inhibits endocytotic removal of a GLUT4 sorting vesicle resident molecule, e.g., GLUT4, from the cell surface.

In preferred embodiments, the treatment includes administering to a cell, a compound, e.g., a protein, a peptide, or a peptidomimetic, or a compound other than a peptide which inhibits the interaction. The inhibition of the interaction can be competitive or non-competitive.

In preferred embodiments, the treatment includes administering a compound, e.g, a molecule which binds to the AP-2 molecule. The molecule can be GLUT4, an AP-2 binding fragment of GLUT4, e.g., a peptide which includes the GLUT4 tail peptide, an AP-2 binding fragment of the GLUT4 tail peptide, a peptide which includes the GLUT4 tail peptide dileucine motif, a peptide which includes the GLUT4 tail peptide diacidic motif, a peptide which includes the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be IRAP, or an AP-2 binding fragment of IRAP, e.g., a fragment which includes the residues_YESSAKLIGMSF. .

In preferred embodiments, the molecules is less than 100, 50, or 20 amino acids in length. In preferred embodiments, the invention includes administering a compound, e.g., a molecule which binds to the GLUT4 molecule. The molecule can be AP-2, a GLUT4 binding fragment of AP-2, e.g., a peptide which includes the AP-2γ peptide, a peptide which includes the AP-2β2 peptide. The molecule can be a molecule which binds IRAP, e.g., a fragment of a polypeptide which normally binds LRAP.

In a preferred embodiment, the method is performed in vitro. In a preferred embodiment, the method is performed in vivo. In another aspect, the invention features, a method of increasing glucose uptake in a cell, e.g., a cell of a subject. The method includes administering a treatment which inhibits the interaction of an AP-2 molecule with a GLUT4 sorting vesicle resident prtein, e.g., GLUT4 or IRAP, thereby increasing glucose uptake in a cell, e.g., a cell of a subject.

In preferred embodiments, the treatment inhibits endocytotic removal of GLUT4 molecules from the cell surface. In preferred embodiments, the treatment includes administering to a cell a compound, e.g., a protein, a peptide, a peptidomimetic, or a compound other than a peptide which inhibits the interaction. The inhibition of the interaction can be competitive or non-competitive.

In preferred embodiments, the invention includes administering a compound, e.g. a molecule which binds to the AP-2 molecule. The molecule can be GLUT4, an AP-2 binding fragment of GLUT4, e.g., a peptide which contains the GLUT4 tail peptide, a binding fragment of the GLUT4 tail peptide, e.g., a peptide which contains the GLUT4 tail peptide dileucine motif, a peptide which contains the GLUT4 tail peptide diacidic motif, a peptide which contains the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be IRAP, or an AP-2 binding fragment of IRAP, e.g., a fragment which includes the residues_YESSAKLIGMSF.

In preferred embodiments, the invention includes administering a compound, e.g., a molecule which binds to the GLUT4 molecule. The molecule can be AP-2, a GLUT4 binding fragment of AP-2, e.g., a peptide which contains the AP-2γ peptide, a peptide which contains the AP-2β2 peptide. The molecule can be a molecule which binds IRAP, e.g., a fragment of a polypeptide which normally binds IRAP.

In another aspect, the invention features, a method for treating a subject for an insulin or glucose-related disorder, e.g., glucose intolerance, e.g.,diabetes, e.g., non-insulin dependent diabetes mellitus (NIDDM). The method includes: administering a treatment which inhibits the interaction of AP-2 with A GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP, thereby treating the subject for the disorder.

In preferred embodiments, the treatment includes administering to the cell a compound, e.g., a protein, or a peptide, a peptidomimetic, or a compound other than a peptide, which inhibits the interaction. The inhibition of the interaction can be competitive or non-competitive.

In preferred embodiments, the invention includes administering a compound, e.g., a molecule which binds to the AP-2 molecule. The molecule can be GLUT4, an AP-2 binding fragment of GLUT4, e.g., a peptide which includes the GLUT4 tail peptide, an AP-2 binding fragment of the GLUT4 tail peptide, a peptide which includes the GLUT4 tail peptide dileucine motif, a peptide which includes the GLUT4 tail peptide diacidic motif, a peptide which includes the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be IRAP, or an AP-2 binding fragment of IRAP, e.g., a fragment which includes the residues_YESSAKLIGMSF. In preferred embodiments, the molecule is at least 3, 4, 5, 7, 10, 15, 20, 25, 30, amino acids in length.

In preferred embodiments, the invention includes administering a compound, e.g, a molecule which binds to the GLUT4 molecule. The molecule can be AP-2, e.g., a GLUT4 binding fragment of AP-2, e.g., a peptide which contains the AP-2γ peptide, a peptide which contains the AP-2β2 peptide. The molecule can be a molecule which binds IRAP, e.g., a fragment of a polypeptide which normally binds IRAP. In another aspect, the invention features, a method of decreasing GLUT4 sorting vesicle resident molecules, e.g., GLUT4 molecules, on the surface of a cell, e.g., a cell of a subject. The method includes: administering AP-2 or an agonist thereof to the cell, thereby decreasing the number of GLUT4 sorting vesicle resident molecules on the surface of the cell, e.g., a cell of a subject.

In preferred embodiments the treatment promotes endocytotic removal of a GLUT4 sorting vesicle resident molecule, e.g., a GLUT4 molecule, from the cell surface. In preferred embodiments the treatment includes administering to the cell a molecule of AP-2 or an agonist thereof, e.g., a protein, a peptide, a peptidomimetic, of AP-2 or an agonist thereof which promotes the interaction.

In preferred embodiments, the treatment includes administering to a cell, a compound, e.g., a protein, a peptide, or a peptidomimetic, or a compound other than a peptide, which inhibits the interaction of AP-2 and GLUT4. The inhibition can be competitive or non-competitive.

In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo. In another aspect, the invention features, a method of evaluating a compound, e.g., a protein, a peptide, a peptidomimetic, or a compound other than a peptide, for the ability to inhibit an interaction between AP-2 and a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP. The method includes: providing a GLUT4 moiety, contacting the compound with the GLUT4 or IRAPmoiety, determining if the compound binds the GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP moiety, binding of the compound to the GLUT4 sorting vesicle resident protein, e.g., GLUT4 moiety, being, e.g., indicative of its ability to inhibit the interaction.

In preferred embodiments, the GLUT4 moiety is: GLUT4, an AP-2 binding fragment of GLUT4, e.g., a peptide which includes the GLUT4 tail peptide, a peptide which includes the GLUT4 tail peptide dileucine motif, a peptide which includes the GLUT4 tail peptide diacidic motif, a peptide which includes the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In a preferred embodiment, the method further includes contacting the compound with a GLUT4 moiety and an AP-2 moiety, and evaluating the ability of the compound to inhibit an interaction between the GLUT4 moiety and the AP-2 moiety.

In another aspect, the invention features, a method of evaluating a compound, e.g., for the ability to inhibit an interaction between AP-2 and GLUT4 sorting vesicle resident protein, e.g., GLUT4 or LRAP. The method includes: providing an AP-2 moiety, contacting the compound with the AP-2 moiety, determining if the compound binds the AP-2 moiety, the binding of the compound, e.g., being indicative of its ability to inhibit the interaction.

In preferred embodiments the AP-2 moiety is: AP-2, a GLUT4 binding fragment of AP-2, e.g., a peptide which contains the AP-2γ peptide, a peptide which contains the AP-2β2 peptide.

In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In a preferred embodiment, the method further includes contacting the compound with a GLUT4 moiety and an AP-2 moiety, and evaluating the ability of the compound to inhibit an interaction between the GLUT4 moiety and the AP-2 moiety. In preferred embodiments, the method further includes contacting a cell, or a cell of a subject, with the compound and evaluating its effect on surface levels of a GLUT4 sorting vesicle resident molecule, e.g., GLUT4, or on glucose uptake, endocytosis, exocytosis, glucose metabolism, insulin metabolism, or plasma glucose levels. In another aspect, the invention features, a method of evaluating a compound, e.g., for the ability to inhibit an interaction between AP-2 and a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP. The method includes: providing a GLUT4 sorting vesicle resident protein, e.g., GLUT4 moiety or LRAP and an AP-2 moiety, contacting the compound with a GLUT4 sorting vesicle resident protein, e.g.,GLUT4 or LRAP moiety and the AP-2 moiety, determining if the compound binds a GLUT4 sorting vesicle resident protein, e.g.,GLUT4 or IRAP moiety or the AP-2 moiety, the binding of the compound being indicative of its ability to inhibit the interaction.

In another aspect, the invention features, a method of evaluating a compound for the ability to modulate the interaction between AP-2 and a GLUT4 sorting vesicle resident protein, e.g.,GLUT4 or IRAP. The method includes: providing a cell having a reporter gene under the control of an AP-2 or a GLUT4 sorting vesicle resident protein, e.g.,GLUT4 or LRAP regulatory agent, contacting the cell with the compound, and evaluating the effect of the compound on the expression of the reporter gene. In another aspect, the invention features, a purified preparation of a molecule capable of binding either a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP, or AP-2, and inhibiting binding between a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP and AP-2.

In preferred embodiments, the preparation includes a compound containing a GLUT4 binding fragment of AP-2, e.g., a peptide containing the AP- 2γ peptide, a peptide containing the AP-2β2 peptide; a peptide which binds AP-2.

In preferred embodiments, the preparation includes a compound containing an AP-2 binding fragment of GLUT4, e.g., a peptide containing the GLUT4 tail peptide, a binding fragment of the GLUT4 tail peptide, e.g., a peptide containing the GLUT4 tail peptide dileucine motif, a peptide containing the

GLUT4 tail peptide diacidic motif, a peptide containing the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif; a peptide which binds GLUT4.

In an alternate embodiment, the invention features,a method of increasing or promoting the number of GLUT4 sorting vesicle resident molecules, e.g.,

GLUT4 or LRAP molecules, on the surface of a cell, e.g., a cell of a subject. The method includes: administering a treatment which inhibits the interaction of a clathrin associated adaptor complex-3 molecule (AP-3) with a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or LRAP, thereby increasing or promoting the number of GLUT4 sorting vesicle resident molecules, e.g., GLUT4 or IRAP molecules, on the cell surface.

In preferred embodiments, the treatment includes administering a compound, e.g, a molecule which binds to the AP-3 molecule. The molecule can be GLUT4, an AP-3 binding fragment of GLUT4, e.g., a peptide which includes the GLUT4 tail peptide, an AP-3 binding fragment of the GLUT4 tail peptide, a peptide which includes the GLUT4 tail peptide dileucine motif, a peptide which includes the GLUT4 tail peptide diacidic motif, a peptide which includes the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be IRAP, or an AP-3 binding fragment of IRAP, e.g., a fragment which includes the residues_YESSAKLIGMSF.

In preferred embodiments, the invention includes administering a compound, e.g., a molecule which binds to the GLUT4 molecule. The molecule can be AP-3, a GLUT4 binding fragment of AP-3. The molecule can be a molecule which binds IRAP, e.g., a fragment of a polypeptide which normally binds IRAP.

In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In another aspect, the invention features, a method of increasing glucose uptake in a cell, e.g., a cell of a subject. The method includes administering a treatment which inhibits the interaction of an AP-3 molecule with GLUT4 or LRAP, thereby increasing glucose uptake in a cell, e.g., a cell of a subject.

In preferred embodiments, the treatment inhibits endocytotic removal of GLUT4 molecules from the cell surface.

In preferred embodiments, the treatment includes administering to a cell a compound, e.g., a protein, a peptide, a peptidomimetic, or a compound other than a peptide,which inhibits the interaction. The inhibition of the interaction can be competitive or non-competitive.

In preferred embodiments, the invention includes administering a compound, e.g. a molecule which binds to the AP-3 molecule. The molecule can be GLUT4, an AP-3 binding fragment of GLUT4, e.g., a peptide which contains the GLUT4 tail peptide, a binding fragment of the GLUT4 tail peptide, e.g., a peptide which contains the GLUT4 tail peptide dileucine motif, a peptide which contains the GLUT4 tail peptide diacidic motif, a peptide which contains the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be IRAP, or an AP-3 binding fragment of IRAP, e.g., a fragment which includes the residues_YESSAKLIGMSF.

In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In another aspect, the invention features, a method for treating a subject for an insulin or glucose-related disorder, e.g., glucose intolerance, e.g., diabetes, e.g., non-insulin dependent diabetes mellitus (NIDDM). The method includes: administering a treatment which inhibits the interaction of AP-3 with a GLUT4 sorting vesicle resident protein, e.g.,GLUT4 or IRAP, thereby treating the subject for the disorder. In preferred embodiments, the treatment inhibits endocytotic removal of a

GLUT4 molecule from the cell surface.

In preferred embodiments, the treatment includes administering to the cell a compound, e.g., a protein, a peptide, a peptidomimetic, or a compound other than a peptide, which inhibits the interaction. The inhibition of the interaction can be competitive or non-competitive.

In preferred embodiments, the invention includes administering a compound, e.g., a molecule which binds to the AP-3 molecule. The molecule can be GLUT4, an AP-3 binding fragment of GLUT4, e.g., a peptide which includes the GLUT4 tail peptide, an AP-3 binding fragment of the GLUT4 tail peptide, a peptide which includes the GLUT4 tail peptide dileucine motif, a peptide which includes the GLUT4 tail peptide diacidic motif, a peptide which includes the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be LRAP, or an AP-3 binding fragment of LRAP, e.g., a fragment which includes the residues YESSAKLIGMSF. In preferred embodiments, the molecule is at least 3, 4, 5, 7, 10, 15, 20, 25, 30, amino acids in length.

In preferred embodiments, the molecules is less than 100, 50, or 20 amino acids in length. In preferred embodiments, the invention includes administering a compound, e.g, a molecule which binds to the GLUT4 molecule. The molecule can be AP-3, e.g., a GLUT4 binding fragment of AP-3. The molecule can be a molecule which binds IRAP, e.g., a fragment of a polypeptide which normally binds IRAP. In another aspect, the invention features, a method of decreasing GLUT4 sorting vesicle resident molecules, e.g., GLUT4 or IRAP molecules, on the surface of a cell, e.g., a cell of a subject. The method includes: administering AP-3 or an agonist thereof to the cell, thereby decreasing the number of GLUT4 sorting vesicle resident molecules on the surface of the cell, e.g., a cell of a subject.

In preferred embodiments the treatment promotes endocytotic removal of a GLUT4 sorting vesicle resident molecule, e.g., a GLUT4 molecule from the cell surface.

In preferred embodiments the treatment includes administering to the cell a molecule of AP-3 or an agonist thereof, e.g., a protein, a peptide, a peptidomimetic, of AP-3 or an agonist thereof which promotes the interaction.

In preferred embodiments, the treatment includes administering to a cell, a compound, e.g., a protein, a peptide, a peptidomimetic, or a compound other than a peptide, which inhibits the interaction of AP-3 and GLUT4. The inhibition can be competitive or non-competitive.

In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In another aspect, the invention features, a method of evaluating a compound, e.g., a protein, a peptide, a peptidomimetic, or a compound other than a peptide, for the ability to inhibit an interaction between AP-3 and a GLUT4 sorting vesicle protein, e.g., GLUT4 or IRAP. The method includes: providing a GLUT4 moiety, contacting the compound with a GLUT4 sorting vesicle protein, e.g., GLUT4 or LRAP moiety, determining if the compound binds the GLUT4 moiety, binding of the compound to a GLUT4 sorting vesicle protein, e.g., GLUT4 or IRAP moiety, being, e.g., indicative of its ability to inhibit the interaction.

In preferred embodiments, the GLUT4 moiety is: GLUT4, an AP-3 binding fragment of GLUT4, e.g., a peptide which includes the GLUT4 tail peptide, a peptide which includes the GLUT4 tail peptide dileucine motif, a peptide which includes the GLUT4 tail peptide diacidic motif, a peptide which includes the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif.

In a preferred embodiment, the method further includes contacting the compound with a GLUT4 moiety and an AP-3 moiety, and evaluating the ability of the compound to inhibit an interaction between the GLUT4 moiety and the AP-3 moiety.

In another aspect, the invention features, a method of evaluating a compound, e.g., for the ability to inhibit an interaction between AP-3 and a GLUT4 sorting vesicle protein, e.g., GLUT4 or IRAP. The method includes: providing an AP-3 moiety, contacting the compound with the AP-3 moiety, determining if the compound binds the AP-3 moiety, the binding of the compound, e.g., being indicative of its ability to inhibit the interaction.

In preferred embodiments the AP-3 moiety is: AP-3, a GLUT4 binding fragment of AP-3. In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In a preferred embodiment, the method further includes contacting the compound with a GLUT4 moiety and an AP-3 moiety, and evaluating the ability of the compound to inhibit an interaction between the GLUT4 moiety and the AP-3 moiety

In another aspect, the invention features, a method of evaluating a compound, e.g., for the ability to inhibit an interaction between AP-3 and a GLUT4 sorting vesicle protein, e.g., GLUT4 or IRAP. The method includes: providing a GLUT4 moiety and an AP-3 moiety, contacting the compound with a GLUT4 sorting vesicle protein, e.g., GLUT4 or IRAP moiety and the AP-3 moiety, determining if the compound binds a GLUT4 sorting vesicle protein, e.g., GLUT4 or IRAP moiety or the AP-3 moiety, the binding of the compound being indicative of its ability to inhibit the interaction.

In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In preferred embodiments, the method is a two-hybrid assay, a phage display assay, e.g., a filamentous phage assay. In preferred embodiments, the method further includes contacting a cell, or a cell of a subject, with the compound and evaluating its effect on surface levels of a GLUT4 sorting vesicle resident molecule, e.g., GLUT4, or on glucose uptake, endocytosis, exocytosis, glucose metabolism, insulin metabolism, or plasma glucose levels. In another aspect, the invention features, a method of evaluating a compound for the ability to modulate the interaction between AP-3 and a GLUT4 sorting vesicle protein, e.g., GLUT4 or IRAP. The method includes: providing a cell having a reporter gene under the control of an AP-3 or a GLUT4 sorting vesicle protein, e.g., GLUT4 or IRAP regulatory agent, contacting the cell with the compound, and evaluating the effect of the compound on the expression of the reporter gene.

In another aspect, the invention features, a purified preparation of a molecule capable of binding either a GLUT4 sorting vesicle protein, e.g., GLUT4 or LRAP or AP-3, and inhibiting binding between GLUT4 and AP-3. In preferred embodiments, the preparation includes a compound containing a GLUT4 binding fragment of AP-3.

In preferred embodiments, the preparation includes a compound containing an AP-3 binding fragment of GLUT4, e.g., a peptide containing the GLUT4 tail peptide, a binding fragment of the GLUT4 tail peptide, e.g., a peptide containing the GLUT4 tail peptide dileucine motif, a peptide containing the GLUT4 tail peptide diacidic motif, a peptide containing the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif; a peptide which binds GLUT4. In an alternate embodiment, the invention features, a method of increasing or promoting the number of GLUT4 sorting vesicle resident molecules, e.g., GLUT4 or IRAP molecules, on the surface of a cell, e.g., a cell of a subject. The method includes: administering a treatment which inhibits the interaction of a coatomer complex (COP) with a GLUT4 sorting vesicle protein, e.g., GLUT4 or IRAP, thereby increasing or promoting the number of GLUT4 sorting vesicle resident molecules, e.g., GLUT4 or IRAP molecules, on the cell surface.

In preferred embodiments, the treatment includes administering a compound, e.g, a molecule which binds to the COP molecule. The molecule can be GLUT4, a COP binding fragment of GLUT4, e.g., a peptide which includes the GLUT4 tail peptide, an COP binding fragment of the GLUT4 tail peptide, a peptide which includes the GLUT4 tail peptide dileucine motif, a peptide which includes the GLUT4 tail peptide diacidic motif, a peptide which includes the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be IRAP, or an COP binding fragment of IRAP, e.g., a fragment which includes the residues_YESSAKLIGMSF.

In preferred embodiments, the molecules is less than 100, 50, or 20 amino acids in length. In preferred embodiments, the invention includes administering a compound, e.g., a molecule which binds to the GLUT4 molecule. The molecule can be COP, a GLUT4 binding fragment of COP, e.g., a peptide which includes the COPβ' peptide. The molecule can be a molecule which binds IRAP, e.g., a fragment of a polypeptide which normally binds IRAP.

In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In another aspect, the invention features, a method of increasing glucose uptake in a cell, e.g., a cell of a subject. The method includes administering a treatment which inhibits the interaction of an COP molecule with GLUT4 or IRAP, thereby increasing glucose uptake in a cell, e.g., a cell of a subject.

In preferred embodiments, the treatment includes administering to a cell a compound, e.g., a protein, a peptide, a peptidomimetic, or a compound other than a peptide, which inhibits the interaction. The inhibition of the interaction can be competitive or non-competitive.

In preferred embodiments, the invention includes administering a compound, e.g. a molecule which binds to the COP molecule. The molecule can be GLUT4, an COP binding fragment of GLUT4, e.g., a peptide which contains the GLUT4 tail peptide, a binding fragment of the GLUT4 tail peptide, e.g., a peptide which contains the GLUT4 tail peptide dileucine motif, a peptide which contains the GLUT4 tail peptide diacidic motif, a peptide which contains the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be LRAP, or an COP binding fragment of LRAP, e.g., a fragment which includes the residues_YESSAKLIGMSF.

In preferred embodiments, the invention includes administering a compound, e.g., a molecule which binds to the GLUT4 molecule. The molecule can be COP, a GLUT4 binding fragment of COP, e.g., a peptide which contains the COPβ' peptide. The molecule can be a molecule which binds IRAP, e.g., a fragment of a polypeptide which normally binds LRAP.

In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In another aspect, the invention features, a method for treating a subject for an insulin or glucose-related disorder, e.g., glucose intolerance, e.g., diabetes, e.g., non-insulin dependent diabetes mellitus (NIDDM). The method includes: administering a treatment which inhibits the interaction of COP with a GLUT4 sorting vesicle protein, e.g., GLUT4 or IRAP, thereby treating the subject for the disorder. In preferred embodiments, the treatment inhibits endocytotic removal of a

GLUT4 molecule from the cell surface.

In preferred embodiments, the invention includes administering a compound, e.g., a molecule which binds to the COP molecule. The molecule can be GLUT4, an COP binding fragment of GLUT4, e.g., a peptide which includes the GLUT4 tail peptide, an COP binding fragment of the GLUT4 tail peptide, a peptide which includes the GLUT4 tail peptide dileucine motif, a peptide which includes the GLUT4 tail peptide diacidic motif, a peptide which includes the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be IRAP, or an COP binding fragment of IRAP, e.g., a fragment which includes the residues_YESSAKLIGMSF. In preferred embodiments, the molecule is at least 3, 4, 5, 7, 10, 15, 20,

25, 30, amino acids in length.

In preferred embodiments, the invention includes administering a compound, e.g, a molecule which binds to the GLUT4 molecule. The molecule can be COP, e.g., a GLUT4 binding fragment of COP, e.g., a peptide which contains the COPβl' peptide The molecule can be a molecule which binds IRAP, e.g., a fragment of a polypeptide which normally binds IRAP.

In another aspect, the invention features, a method of decreasing GLUT4 sorting vesicle resident molecules, e.g., GLUT4 or IRAP molecules, on the surface of a cell, e.g., a cell of a subject. The method includes: administering COP or an agonist thereof to the cell, thereby decreasing the number of GLUT4 sorting vesicle resident molecules on the surface of the cell, e.g., a cell of a subject. In preferred embodiments the treatment promotes endocytotic removal of a GLUT4 sorting vesicle resident molecule, e.g., a GLUT4 molecule from the cell surface.

In preferred embodiments the treatment includes administering to the cell a molecule of COP or an agonist thereof, e.g., a protein, a peptide, a peptidomimetic, of COP or an agonist thereof which promotes the interaction.

In preferred embodiments, the treatment includes administering to a cell, a compound, e.g., a protein, a peptide, a peptidomimetic, or a compound other than a peptide, which inhibits the interaction of COP and GLUT4. The inhibition can be competitive or non-competitive.

In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In another aspect, the invention features, a method of evaluating a compound, e.g., a protein, a peptide, a peptidomimetic, or a compound other than a peptide, for the ability to inhibit an interaction between COP and a GLUT4 sorting vesicle protein, e.g., GLUT4 or IRAP. The method includes: providing a GLUT4 sorting vesicle protein, e.g., GLUT4 or IRAP moiety, contacting the compound with the GLUT4 moiety, determining if the compound binds a GLUT4 sorting vesicle protein, e.g., GLUT4 moiety, binding of the compound to a GLUT4 sorting vesicle protein, e.g., GLUT4 moiety, being, e.g., indicative of its ability to inhibit the interaction.

In preferred embodiments, the GLUT4 moiety is: GLUT4, a COP binding fragment of GLUT4, e.g., a peptide which includes the GLUT4 tail peptide, a peptide which includes the GLUT4 tail peptide dileucine motif, a peptide which includes the GLUT4 tail peptide diacidic motif, a peptide which includes the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. The molecule can be IRAP, or an COP binding fragment of LRAP, e.g., a fragment which includes the residues_YESSAKLIGMSF.

In a preferred embodiment, the method further includes contacting the compound with a GLUT4 moiety and an COP moiety, and evaluating the ability of the compound to inhibit an interaction between the GLUT4 moiety and the COP moiety. The molecule can be a molecule which binds IRAP, e.g., a fragment of a polypeptide which normally binds IRAP.

In another aspect, the invention features, a method of evaluating a compound, e.g., for the ability to inhibit an interaction between COP and a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP. The method includes: providing an COP moiety, contacting the compound with the COP moiety, determining if the compound binds the COP moiety, the binding of the compound, e.g., being indicative of its ability to inhibit the interaction.

In preferred embodiments the COP moiety is: COP, a GLUT4 binding fragment of COP, e.g., a peptide which contains the COPβ' peptide.

In a preferred embodiment, the method is performed in vitro.

In a preferred embodiment, the method is performed in vivo.

In preferred embodiments, the method is a two-hybrid assay, a phage display assay, e.g., a filamentous phage assay. In a preferred embodiment, the method further includes contacting the compound with a GLUT4 moiety and a COP moiety, and evaluating the ability of the compound to inhibit an interaction between the GLUT4 moiety and the COP moiety.

In another aspect, the invention features, a method of evaluating a compound, e.g., for the ability to inhibit an interaction between COP and a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or LRAP. The method includes: providing a GLUT4 sorting vesicle resident protein, e.g., a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or LRAP moiety and a COP moiety, contacting the compound with a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or LRAP moiety and the COP moiety, determining if the compound binds a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP moiety or the COP moiety, the binding of the compound being indicative of its ability to inhibit the interaction.

In another aspect, the invention features, a method of evaluating a compound for the ability to modulate the interaction between COP and a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP. The method includes: providing a cell having a reporter gene under the control of an COP or a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP regulatory agent, contacting the cell with the compound, and evaluating the effect of the compound on the expression of the reporter gene. In another aspect, the invention features, a purified preparation of a molecule capable of binding either GLUT4, or COP, and inhibiting binding between GLUT4 and COP.

In preferred embodiments, the preparation includes a compound containing a GLUT4 binding fragment of COP, e.g., a peptide containing the COPβ' peptide; a peptide which binds COP.

In preferred embodiments, the preparation includes a compound containing an COP binding fragment of GLUT4, e.g., a peptide containing the GLUT4 tail peptide, a binding fragment of the GLUT4 tail peptide, e.g., a peptide containing the GLUT4 tail peptide dileucine motif, a peptide containing the GLUT4 tail peptide diacidic motif, a peptide containing the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif; a peptide which binds GLUT4.

As used herein, the term "glucose transport modulator" refers to a molecule which modulates the interaction of a transport vesicle coating complex with the glucose transporter isoform 4 GLUT4, or a fragment thereof. As used herein, the term "GLUT4 sorting vesicle" refers to a specialized recycling vesicle capable of sequestering GLUT4 sorting vesicle resident protein intracellularly and transporting them to the cell surface in response to certain stimulants, e.g., insulin. As used herein, the term "GLUT4 sorting vesicle resident protein" refers to any one of the group of proteins, e.g., insulin-responsive aminopeptidase (IRAP), sortilin, mannose 6-phospate/IGF2 receptor, acyl CoA synthetase, and fragments thereof, which are found in GLUT4 sorting vesicles.

As used herein, the term "peptidomimetic" refers to molecules which mimic the chemical structure of a peptide and retain biological properties of the peptide.

As used herein, the term "modulates the interaction" refers to an alteration or modification, e.g., either an increase or promotion or a decrease or depression, of the interaction of a transport vesicle coating complex with GLUT4 or a fragment thereof.

As used herein, the term "transport vesicle coating complex" refers to a clathrin associated adaptor complex or to a coatomer complex.

As used herein, the terms "glucose transporter isoform 4" and "GLUT4" are used interchangeably, and refer to the insulin responsive transporter protein. As used herein, the term "GLUT4 tail peptide" refers to a fragment of

GLUT4 which includes amino acids 467-509.

As used herein, the term "GLUT4 moiety" refers to a fragment of GLUT4 which includes a sequence of amino acids which interact with a fragment of a vesicle coating complex. As used herein, the term "GLUT4 moiety" refers to a fragment of GLUT4 which includes a sequence of amino acids which interact with a fragment of a vesicle coating complex.

As used herein, the term "AP-1 moiety" refers to a fragment of AP-1 which includes a sequence of amino acids which interact with a fragment of GLUT4.

As used herein, the term "AP-2 moiety" refers to a fragment of AP-2 which includes a sequence of amino acids which interact with a fragment of GLUT4. As used herein, the term "AP-3 moiety" refers to a fragment of AP-3 which includes a sequence of amino acids which interact with a fragment of GLUT4.

As used herein, the term "COP moiety" refers to a fragment of COP which includes a sequence of amino acids which interact with a fragment of GLUT4.

As used herein, the term "subject" refers to a human, an experimental animal, e.g., a rat or a mouse, a domestic animal, e.g., a dog, cow, sheep, pig or horse, a non-human primate, e.g. a monkey. As used herein, the term "purified preparation" of a protein or a peptide refers to a protein or a peptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the protein or peptide is also separated from substances, e.g. antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Detailed Description The drawings are first briefly described. Brief Description of the Drawings

Figure I is a schematic representation of the insulin signaling cascade. Figure 2 depicts the amino acid sequences of the cytoplasmic tail portions of GLUT4 sorting vesicle-resident proteins.

Figure 3 is a graphic representation of the comparison of the uptake of [3H]-2-deoxyglucose in 3T3-L1 adipocytes stimulated by insulin and GLUT4. Figure 4 is a schematic representation of BPA/Biotin/Vector/GLUT4 crosslinking.

Figure 5 is a schematic representation of the adaptor protein complexes sorting functions. Figure 6 is depicts the peptide sequences used in the to investigate the of the binding specificity and identity of the sorting receptor.

Figure 7 is a schematic representation of the insulin exocytosis/endocytosis cycle. I. Glucose Transport Modulators

Glucose transport modulators of the invention include proteins, peptides, peptidomimetics and other molecules capable of modulating GLUT4 glucose transport in cells. These glucose transport modulators can bind to transport vesicle coating complexes or to the GLUT4 tail peptide. Preferred glucose transporter compounds include at least a portion of the amino acid sequence of the GLUT4 tail peptide, which includes amino acids 467-509 and adjacent portions of the protein, as well as portions of the peptide and peptidomimetics which retain the binding specificity of the peptide. Preferred glucose transporter compounds contain the dileucine binding motif and or the diacidic binding motif of the GLUT4 tail peptide. Upon interaction with the transport vesicle coating complexes, the glucose transport compounds of the invention are capable of modulating the number of GLUT4 sorting vesicle resident molecules on the surface of the cell, thereby modulating glucose transport in the cell. II. Methods for Preparing Glucose Transport Modulators The modulating compounds of the invention can be prepared by purifying the parent molecule, e.g., by purifying the parent molecule from a natural source, such as a cell which expresses GLUT4, e.g., adipocytes or skeletal muscle cells, by cleavage of the parent molecule, or they can be synthetically or recombinantly produced using standard methods for peptide synthesis, recombinant peptide production, and peptide modification.

Proteins can be produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding GLUT4 can be cloned into an expression vector. The expression vector can be introduced into a host cell, and the protein can be expressed in the host cell. The GLUT4 protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Gene and Cell Therapy

The nucleic acid constructs which encode molecules of the invention can also be used as a part of a gene or cell therapy protocol to deliver nucleic acids encoding glucose transport modulating peptides. The invention features expression vectors for transfection and expression of a glucose transport modulating polypeptide in particular cell types so as to reconstitute the function of, or alternatively, modulate the function of glucose transport modulating polypeptide in a cell. Expression constructs of glucose transport modulating polypeptides, may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the glucose transport modulating encoding nucleic acids to cells. Approaches include insertion of the subject nucleic acid construct in viral vectors including recombinant retro viruses, adenovirus, adeno-associated virus, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes

(lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4 precipitation.

A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding a glucose transport modulating polypeptide. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271). A replication defective retrovirus can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology. Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZLP, pWE and pEM which are known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ψCrip, ψCre, ψ2 and ψAm. Retroviruses have been used to introduce a variety of genes and nucleic acids into many different cell types, including epithelial cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014- 3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802- 1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573) which are hereby incorporated by reference.

Another viral gene delivery system useful in the present invention utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in the art. Recombinant adeno viruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al. (1992) cited supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267).

Yet another viral vector system useful for delivery of the subject gene is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr. Topics in Micro, and Immunol. (1992) 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790) which are hereby incorporated by reference.

In addition to viral transfer methods, such as those illustrated above, non- viral methods can also be employed to cause expression of a glucose transport modulating polypeptide in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non- viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject glucose transport modulating nucleic acid by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes. In a representative embodiment, a nucleic acid encoding a glucose transport modulating polypeptide can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:541 -551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075) which are hereby incorporated by reference.

In clinical settings, the gene delivery systems for the therapeutic glucose transport modulating nucleic acid can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant nucleic acid is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by Stereotactic injection (e.g. Chen et al. (1994) PNAS 91 : 3054-3057) which are hereby incorporated by reference.

The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g. refroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system. Antisense Therapy

Another aspect of the invention relates to the use of an glucose transport modulating encoding nucleic acid in "antisense" therapy. As used herein, "antisense" therapy refers to administration or in situ generation of oligonucleotides or their derivatives which specifically hybridize under cellular conditions, with cellular mRNA and/or genomic DNA so as to inhibit expression of the encoded protein, e.g. by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, "antisense" therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a glucose transport modulating peptide. Alternatively, the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of an glucose transport modulating peptide encoding nucleic acid. Such oligonucleotide probes are preferably modified oligonucleotide which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and is therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668 which are hereby incorporated by reference.

The antisense constructs of the present invention, by antagonizing the normal biological activity of the glucose transport modulating peptide, can be used in the manipulation of glucose homeostasis, both in vitro and in vivo. Production of Molecules Which Modulate the GLUT4 Sorting Vesicle Resident Protein and Clathrin Associated Adaptor or Coatomer Complex Interaction

The inventor has discovered that molecules which regulate GLUT4 sorting vesicle resident protein distribution in cells can directly modulate glucose uptake in the cells. Therefore, molecules which promote or inhibit the interaction of GLUT4 sorting vesicle resident proteins with the clathrin associated adaptor or coatomer complexes are useful. Such molecules include GLUT4, e.g., the

GLUT4 tail peptide, a binding fragment of the GLUT4 tail peptide, e.g., a peptide containing the GLUT4 tail peptide dileucine motif, a peptide containing the GLUT4 tail peptide diacidic motif, a peptide containing the GLUT4 tail peptide dileucine motif and the GLUT4 tail peptide diacidic motif. One skilled in the art can produce additional glucose transport modulators of the invention, e.g., molecules which promote or inhibit the interaction of GLUT4 sorting vesicle resident proteins with the clathrin associated adaptor or coatomer complexes, by producing fragments or analogs of GLUT4, AP-1, AP-2, AP-3 and COP, and testing the newly produced structures for activity. One skilled in the art can also produce additional glucose transport modulators of the invention by making other GLUT4, AP-1, AP-2, AP-3, and COP binding proteins and producing their fragments and analogs. Examples of prior art methods which allow the production and testing of fragments and analogs are discussed below. These, or other methods, can be used to make and test compounds useful in the methods of the invention.

Generation of Fragments

Fragments of a protein can be produced in several ways, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end- nibbling" endonucleases can thus generate DNA's which encode an array of fragments. DNA's which encode fragments of a protein can also be generated by random shearing, restriction digestion or a combination of the above-discussed methods.

Fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, peptides of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.

Generation of Analogs: Production of Altered DNA and Peptide Sequences by Random Methods

Amino acid sequence variants of a protein can be prepared by random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. (Methods for screening proteins in a library of variants are described elsewhere herein.) PCR Mutagenesis In PCR mutagenesis, reduced Taq polymerase fidelity is used to introduce random mutations into a cloned fragment of DNA (Leung et al., 1989, Technique 1:11-15). This is a very powerful and relatively rapid method of introducing random mutations. The DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g., by using a dGTP/dATP ratio of five and adding Mn^⁺ to the PCR reaction. The pool of amplified DNA fragments are inserted into appropriate cloning vectors to provide random mutant libraries. Saturation Mutagenesis

Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242). This technique includes generation of mutations, e.g., by chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complimentary DNA strand. The mutation frequency can be modulated by modulating the severity of the treatment, and essentially all possible base substitutions can be obtained. Because this procedure does not involve a genetic selection for mutant fragments both neutral substitutions, as well as those that alter function, are obtained. The distribution of point mutations is not biased toward conserved sequence elements. Degenerate Oligonucleotides

A library of homologs can also be generated from a set of degenerate oligonucleotide sequences. Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S. Patents Nos. 5,223,409, 5,198,346, and 5,096,815) which are hereby incorporated by reference. Generation of Analogs: Production of Altered DNA and Peptide Sequences by Directed Mutagenesis

Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3. Alanine Scanning Mutagenesis

Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244:1081-1085, 1989). In alanine scanning, a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine). Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity. Oligonucleotide-Mediated Mutagenesis

Oligonucleotide-mediated mutagenesis is a useful method for preparing substitution, deletion, and insertion variants of DNA, see, e.g., Adelman et al., (DNA 2:183, 1983). Briefly, the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the desired protein. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the desired protein DNA. Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. (Proc. Natl. Acad. Sci. USA, 75: 5765 [1978]) which is hereby incorporated by reference. Cassette Mutagenesis

Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al. (Gene, 34:315[1985]). The starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated. The codon(s) in the protein subunit DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the desired protein subunit DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3' and 5' ends that are comparable with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated desired protein subunit DNA sequence. Combinatorial Mutagenesis

Combinatorial mutagenesis can also be used to generate mutants. E.g., the amino acid sequences for a group of homologs or other related proteins are aligned, preferably to promote the highest homology possible. All of the amino acids which appear at a given position of the aligned sequences can be selected to create a degenerate set of combinatorial sequences. The variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences. Analogs

Analogs can differ from naturally occurring glucose transport modulators in amino acid sequence or in ways that do not involve sequence, or both. Non- sequence modifications include in vivo or in vitro chemical derivatization of glucose transport modulators. Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation.

Preferred analogs include GLUT4 (or biologically active fragments thereof) and vesicle coating complexes (or biologically active fragments thereof) whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish the glucose transport modulator biological activity. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions can be taken from the table below.

TABLE 1 CONSERVATIVE AMINO ACID REPLACEMENTS

Other analogs within the invention are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L- amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids; and cyclic analogs. Peptide Mimetics

The invention also provides mimetics, e.g. peptide or non-peptide mimetics, of GLUT4, GLUT4 tail peptide, or GLUT4 moieties and vesicle coating complex peptides, GLUT4 binding fragments thereof and vesicle coating complex moieties. Peptide mimetics can modulate binding of GLUT4 to a vesicle coating complexpeptide. The critical residues of a subject glucose transport modulator polypeptide which are involved in molecular recognition of a polypeptide can be determined and used to generate GLUT4-derived peptidomimetics which competitively or noncompetatively inhibit binding of the vesicle coating complex peptide with a ligand (see, for example, "Peptide inhibitors of human papillomavirus protein binding to retinoblastoma gene protein" European patent applications EP-412,762A and EP-B31,080A). For example, scanning mutagenesis can be used to map the amino acid residues of a particular GLUT4 polypeptide involved in binding a vesicle coating complex polypeptide, and peptidomimetic compounds (e.g. diazepine or isoquinoline derivatives) generated which mimic those residues in binding to the ligand. These products can inhibit binding of a GLUT4 polypeptide to a ligand and thereby interfere with the function of GLUT4 or the ligand. Non-hydrolyzable peptide analogs of critical residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986) JMed Chem 29:295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), and β-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun\26:4\9; and Dann et al. (1986) Biochem Biophys Res Commun 134:71) hereby incorporated by reference.

Accordingly, the polypeptides, nucleic acids, and other compounds of the invention are useful in therapeutic, diagnostic, and research contexts. The polypeptides and nucleic acids of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous for injection, the polypeptides and nucleic acids of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the polypeptides and nucleic acids may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included in the invention. The polypeptides and nucleic acids can be administered orally, or by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives, and detergents. Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the polypeptides and nucleic acids are formulated into conventional oral administration forms such as capsules, tablets, and tonics. For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as known in the art. III. Methods of Identifying Glucose Transport Modulators

The invention provides methods for evaluating a compound for the ability to modulate glucose transport. One method includes: providing the GLUT4 tail peptide or a GLUT4 binding peptide of a clathrin associated adaptor complex or a coatomer complex, contacting the compound with the GLUT4 tail peptide or the clathrin associated adaptor complex or coatomer complex, determining if the compound binds the GLUT4 tail peptide or the clathrin associated adaptor complex or coatomer complex, the binding of the compound being indicative of its ability to inhibit the interaction.

A second method includes: providing a cell having a reporter gene under the control of a clathrin associated adaptor complex or coatomer complex regulatory agent, contacting the cell with the compound, and evaluating the effect of the compound on the reporter gene.

Primary Hi h-Through-Put Methods for Screening Libraries of Peptide Fragments or Homologs Libraries of compounds can also be screened to determine whether any members of the library have the desired glucose transport modulating activity, and, if so, to identify the most active species. Various techniques are known in the art for screening generated mutant gene products. Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, e.g., in this case, the interaction, e.g., binding, of GLUT4 to a vesicle coating peptide, or the interaction, e.g., binding of a candidate polypeptide with a GLUT4 binding fragment or a vesicle coating binding fragment facilitate relatively easy isolation of the vector encoding the gene whose product was detected. Each of the techniques described below is amenable to high throughput analysis for screening large numbers of sequences created, e.g., by random mutagenesis techniques. Two Hybrid Systems

Two hybrid assays such as the system described above (as with the other screening methods described herein), can be used to identify fragments or analogs of a glucose transport modulator which binds to GLUT4, e.g., the GLUT4 tail peptide or a vesicle coating binding fragment thereof or to a vesicle coating complex, e.g., AP- 1 , AP-2, AP-3, COP or a GLUT4 binding fragment thereof. These may include agonists, superagonists, and antagonists. A GLUT4 polypeptide, e.g., the GLUT4 tail peptide or a vesicle coating binding fragment thereof, can be used as the bait protein and the library of variants of vesicle coating complex fragments are expressed as fish fusion proteins.) In an analogous fashion, a two hybrid assay (as with the other screening methods described herein), can be used to find fragments and analogs of GLUT4 peptides e.g., peptides which bind vesicle coating complex fragments. Display Libraries

Once libraries containing large numbers of fragments or analogs of compounds, are made, they can be tested for the ability to interact with GLUT4, AP-1, AP-2, AP-3 and COP, or fragments thereof.

In one approach to screening assays, the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay". For example, the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140) which are hereby incorporated by reference. In a similar fashion, a detectably labeled ligand can be used to score for potentially functional peptide homologs. Fluorescently labeled ligands, e.g., receptors, can be used to detect homolog which retain ligand-binding activity. The use of fluorescently labeled ligands, allows cells to be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, to be separated by a fluorescence-activated cell sorter.

A gene library can be expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can be applied to affinity matrices at concentrations well over 10 3 phage per milliliter, a large number of phage can be screened at one time. Second, since each infectious phage displays a gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages M13, fd., and fl are most often used in phage display libraries. Either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle. Foreign epitopes can be expressed at the NH2- terminal end of pill and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBOJ 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461) which are hereby incorporated by reference.

A common approach uses the maltose receptor of E. coli (the outer membrane protein, LamB) as a peptide fusion partner (Charbit et al. (1986) EMBO 5, 3029-3037). Oligonucleotides have been inserted into plasmids encoding the LamB gene to produce peptides fused into one of the extracellular loops of the protein. These peptides are available for binding to ligands, e.g., to antibodies, and can elicit an immune response when the cells are administered to animals. Other cell surface proteins, e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392), PhoE (Agterberg, et al. (1990) Gene 88, 37-45), and PAL (Fuchs et al. (1991) Bio/Tech 9, 1369-1372), as well as large bacterial surface structures have served as vehicles for peptide display. Peptides can be fused to pilin, a protein which polymerizes to form the pilus-a conduit for interbacterial exchange of genetic information (Thiry et al. (1989) Appl. Environ. Microbiol. 55, 984-993). Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular environment. Another large surface structure used for peptide display is the bacterial motive organ, the flagellum. Fusion of peptides to the subunit protein flagellin offers a dense array of may peptides copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083). Surface proteins of other bacterial species have also served as peptide fusion partners. Examples include the Staphylococcus protein A and the outer membrane protease IgA o Neisseria (Hansson et al. (1992) J. Bacteriol. 174, 4239-4245 and Klauser et al. (1990) EMBO J. 9, 1991 - 1999) which are hereby incorporated by reference.

In the filamentous phage systems and the LamB system described above, the physical link between the peptide and its encoding DNA occurs by the containment of the DNA within a particle (cell or phage) that carries the peptide on its surface. Capturing the peptide captures the particle and the DNA within. An alternative scheme uses the DNA-binding protein Lad to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89:1865-1869). This system uses a plasmid containing the Lad gene with an oligonucleotide cloning site at its 3 '-end. Under the controlled induction by arabinose, a Lacl-peptide fusion protein is produced. This fusion retains the natural ability of Lad to bind to a short DNA sequence known as LacO operator (LacO). By installing two copies of LacO on the expression plasmid, the Lacl-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide-DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides. The associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands. As a demonstration of the practical utility of the method, a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dynorphin B. A cohort of peptides was recovered, all related by a consensus sequence corresponding to a six-residue portion of dynorphin B. (Cull et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89-1869) which is hereby incorporated by reference This scheme, sometimes referred to as peptides-on-plasmids, differs in two important ways from the phage display methods. First, the peptides are attached to the C-terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini. Both of the filamentous phage coat proteins, pill and pVIII, are anchored to the phage through their C- termini, and the guest peptides are placed into the outward-extending N-terminal domains. In some designs, the phage-displayed peptides are presented right at the amino terminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6378-6382) A second difference is the set of biological biases affecting the population of peptides actually present in the libraries. The Lad fusion molecules are confined to the cytoplasm of the host cells. The phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles. The peptides in the Lad and phage libraries may differ significantly as a result of their exposure to different proteolytic activities. The phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to incorporation into phage. Certain peptides exert a deleterious effect on these processes and are underrepresented in the libraries (Gallop et al. (1994) J. Med. Chem. 37(9):1233- 1251). These particular biases are not a factor in the Lad display system.

The number of small peptides available in recombinant random libraries is enormous. Libraries of 10^-10^ independent clones are routinely prepared. Libraries as large as 10* * recombinants have been created, but this size approaches the practical limit for clone libraries. This limitation in library size occurs at the step of transforming the DNA containing randomized segments into the host bacterial cells. To circumvent this limitation, an in vitro system based on the display of nascent peptides in polysome complexes has recently been developed. This display library method has the potential of producing libraries 3- 6 orders of magnitude larger than the currently available phage/phagemid or plasmid libraries. Furthermore, the construction of the libraries, expression of the peptides, and screening, is done in an entirely cell-free format.

In one application of this method (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251), a molecular DNA library encoding 10*2 decapeptides was constructed and the library expressed in an E. coli S2> in vitro coupled transcription/translation system. Conditions were chosen to stall the ribosomes on the mRNA, causing the accumulation of a substantial proportion of the RNA in polysomes and yielding complexes containing nascent peptides still linked to their encoding RNA. The polysomes are sufficiently robust to be affinity purified on immobilized receptors in much the same way as the more conventional recombinant peptide display libraries are screened. RNA from the bound complexes is recovered, converted to cDNA, and amplified by PCR to produce a template for the next round of synthesis and screening. The polysome display method can be coupled to the phage display system. Following several rounds of screening, cDNA from the enriched pool of polysomes was cloned into a phagemid vector. This vector serves as both a peptide expression vector, displaying peptides fused to the coat proteins, and as a DNA sequencing vector for peptide identification. By expressing the polysome-derived peptides on phage, one can either continue the affinity selection procedure in this format or assay the peptides on individual clones for binding activity in a phage ΕLISA, or for binding specificity in a completion phage ΕLISA (Barret, et al. (1992) Anal. Biochem 204,357-364). To identify the sequences of the active peptides one sequences the DNA produced by the phagemid host. Secondary Screens The high through-put assays described above can be followed by secondary screens in order to identify further biological activities which will, e.g., allow one skilled in the art to differentiate agonists from antagonists. The type of a secondary screen used will depend on the desired activity that needs to be tested. For example, an assay can be developed in which the ability to inhibit an interaction between a protein of interest and its respective ligand can be used to identify antagonists from a group of peptide fragments isolated though one of the primary screens described above.

Therefore, methods for generating fragments and analogs and testing them for activity are known in the art. Once the core sequence of interest is identified, it is routine to perform for one skilled in the art to obtain analogs and fragments. Drug Screening Assays

By making available purified and recombinant-glucose transport modulator polypeptides, the present invention provides assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function, in this case, of the subject GLUT4 and vesicle coating complex polypeptides. In one embodiment, the assay evaluates the ability of a compound to modulate binding between a GLUT4 polypeptide and a naturally occurring ligand, e.g., a vesicle coating complex peptide. A variety of assay formats will suffice and, in light of the present inventions, will be comprehended by skilled artisan.

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi- purified proteins, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or change in enzymatic properties of the molecular target.

The invention is further illustrated in the following non-limiting examples. Examples

Example 1: Cell Preparation

3T3-L-1 adipocytes were treated with biotin- vector/GLUT4-peptide. Following 1-2 hour incubations with 1-50 μM compound, cells were washed, fixed and treated with FITC-streptavidin. Cells treated with biotin- vector/GLUT4-peptide were clearly stained in a cytoplasmic pattern.

Quantitative fluorescence and radiolabeling indicate that about 5% of the applied compounds entered the cells. As assessed by MTT assays of mitochondrial integrity, the vector-peptides were not cytotoxic. Example 2: Mapping the Molecular Target

The photactive amino acid benzoylphenylalanine (Bpa) was substituted into the GLUT4-vector peptides at various positions. Two peptides were added to cells at 37 C. At 1-2 after the Bpa peptides entered the cells, the period when bioeffects are greatest, the cells were chilled to 4 C and flashed with ultraviolet light (340 nm) for 5-60 min. The cells were placed 1 cm from the light source.

A single protein was cross-linked with very high efficiency (see Figure 4). It has an apparent sized of 104 kDA, which includes 4 kDA for the peptide. Cross-linking is sequence specific and inhibitable. No cross-linking has been seen with alternative sequences, and addition of excess unreactive peptide blocks cross-linking. Example 3: In Vitro Modulation of Glucose Transport

Fibroblast cells were differentiated into fat cells according to established protocols. The 3T3-L-1 cells were differentiated for 10-15 days prior to use, and were serum starved for 3 hours. 100 μM insulin for 10 minutes provided a 3-5 fold stimulation of 3[H]-2-deoxyglucose (DOG) uptake. 50 μM vector/biotin- GLUT4 peptides for 1-2 hours provided 4-7 fold stimulation of ³[H]-2- deoxyglucose (DOG) uptake.

Stimulation by insulin and vector/biotin-GLUT4 peptides were both suppressed by 10 μM cytochalasin B, a specific inhibitor of glucose transporters. Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Other embodiments are within the following claims.

What is claimed is:

Claims

1. A method of modulating the number of GLUT4 sorting vesicle resident molecules on the surface of a cell comprising administering a treatment which inhibits the interaction of AP- 1 , AP-2, AP-3 or COP with a GLUT4 sorting vesicle resident protein, thereby increasing or promoting the number of GLUT4 sorting vesicle resident molecules on the cell surface.

2. The method of claim 1 , wherein the number is increased.

3. The method of claim 1 , wherein the number is decreased.

4. The method of claim 1, wherein the GLUT4 sorting vesicle resident protein is GLUT4.

5. The method of claim 1, wherein the GLUT4 sorting vesicle resident protein is IRAP.

6. The method of claim 1, wherein, wherein an interaction with AP-1 is inhibited.

7. The method of claim 1 , wherein the method includes administering a compound which binds to the AP-1 molecule.

8. The method of claim 1, wherein the method includes administering an AP-1 binding fragment of the GLUT4 tail peptide.

9. The method of claim 1, wherein the method includes administering an AP-1 binding fragment of the IRAP.

10. The method of claim 1 , wherein the method is performed in vitro.

11. The method of claim 1 , wherein the method is performed in vivo.

12. The method of claim 1, wherein a the treatment is administered to a subject a having an insulin or glucose-related disorder.

13. The method of claim 11, wherein the disorder non-insulin dependent diabetes mellitus.

14. A method of modulating glucose uptake in a cell comprising administering a treatment which inhibits the interaction of an AP-1, AP-2, AP-3 or COP molecule with a GLUT4 sorting vesicle resident protein, thereby modulating glucose uptake in a cell, e.g., a cell of a subject.

15. The method of claim 11 , wherein a the treatment is administered to a subject a having an insulin or glucose-related disorder.

16. The method of claim 12, wherein the disorder non-insulin dependent diabetes mellitus.

17. A method of evaluating a compound for the ability to inhibit an interaction between AP-1, AP-2, AP-3 or COP and a GLUT4 sorting vesicle resident protein, e.g., GLUT4 or IRAP comprising providing a GLUT4 moiety, contacting the compound with the GLUT4 moiety, determining if the compound binds the GLUT4 moiety, binding of the compound to the GLUT4 moiety.