EP1387692A2 - Molecular interactions in hematopoietic cells - Google Patents
Molecular interactions in hematopoietic cellsInfo
- Publication number
- EP1387692A2 EP1387692A2 EP01987086A EP01987086A EP1387692A2 EP 1387692 A2 EP1387692 A2 EP 1387692A2 EP 01987086 A EP01987086 A EP 01987086A EP 01987086 A EP01987086 A EP 01987086A EP 1387692 A2 EP1387692 A2 EP 1387692A2
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- EP
- European Patent Office
- Prior art keywords
- pdz
- protein
- amino acid
- binding
- carboxy
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5064—Endothelial cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
Definitions
- the present invention relates to peptides and peptide analogues, and methods for using such compositions to regulate activities of cells of the hematopoietic system.
- the invention provides methods of modulating metabolism (e.g., activation) of hematopoietic cells (e.g., T cells and B cells) by antagonizing an interaction between a PDZ domain containing protein and a protem that binds a PDZ domain.
- fusion peptides containing an amino acid sequence corresponding to the carboxyl terminus of a surface receptor expressed by a hematopoietic cell and a transmembrane transporter sequence; such fusion peptides are useful in regulating hematopoietic cells by inhibiting cell activation.
- PDZ dohiains of proteins are named after three prototypical proteins: PSD95, Drosophila large disc protein and Zonula Occludin 1 protein (Gomperts et al., 1996, Cell 84:659- 662).
- PDZ domain-containing proteins are involved in synapse formation by organizing transmembrane neurotransmitter receptors through intracellular interactions.
- PDZ domains contain the signature sequence GLGF (SEQ ID NO: 402).
- typical PDZ domain-containing proteins contain three PDZ domains, one SH3 domain and one guanylate kinase domain.
- Examples of intracellular PDZ doma -containing proteins include LIN-2, LIN-7 and LIN- 10 at the pre-synapse, and PSD95 at the post-synapse.
- PDZ domains have been shown to bind the carboxyl termini of transmembrane proteins in neuronal cells. Songyang et al. reported that proteins capable of binding PDZ domains contain a carboxyl terminal motif sequence of E-S T-X-N/I (Songyang et al., 1997, Science 275:73). X-ray crystallography studies have revealed the contact points between the motif sequence and PDZ domains (Doyle et al., 1996, Cell 88:1067-1076). While the interaction between PDZ domains and ion channels in neurons have been studied extensively, such interactions have had limited studies in other biological systems, especially the hematopoietic system.
- the hematopoietic system is composed of different cell types that perform distinct functions. Many of its diverse functions require coordinated movement of cell surface receptors including ion channels, adhesion surface molecules to coordinate cell-cell interaction, and cytokine receptors. Despite their diverse functional activities, all hematopoietic cells are believed to develop from a multipotent bone marrow hematopoietic stem cell. Such stem cell has been shown to express a surface marker termed CD34. During differentiation, the stem cell gives rise to progenitor cells in each of several specific hematopoietic cell lineages. The progenitor cells then undergo a series of morphological and functional changes to produce mature functionally committed hematopoietic cells.
- lymphocytes which include T cells, B cells and natural killer ( ⁇ K) cells
- monocytes and granulocytes i.e., neutrophils, basophils and eosinophils
- Lymphocytes, monocytes and granulocytes are collectively referred to as white blood cells or leukocytes.
- other hematopoietic cells perform functions that are unrelated to the immune system. For example, erythrocytes are involved in gas transport, and cells of the thrombocytic series are involved in blood clotting.
- T cells and B cells recognize antigens and generate an immune response.
- T cells recognize antigens by heterodimeric surface receptors termed the T cell receptor (TCR).
- TCR T cell receptor
- the TCR is associated with a series of polypeptides collectively referred to as CD3 complex.
- B cells recognize antigens by surface immunoglobulins (lg), which are also secretory molecules.
- lg surface immunoglobulins
- co-stimulatory surface receptors have been identified in T cells and B cells, which augment cellular activation during antigen-induced activation.
- T cell antigen receptor/CD3 complex TCR/CD3
- other molecules expressed by T cells which mediate an activation signal include but are not limited to, CD2, CD4, CD5, CD6, CD8, CD18, CD27, CD28, CD43, CD45, CD152 (CTLA-4), CD154, MHC class I, MHC class ⁇ , CDwl37 (4-lBB), CDwl50, and the like (Barclay et al., The Leucocyte Antigen Facts Book, 1997, Second edition, Academic Press; Leucocyte Typing, 1984,
- T cell surface antigens include antigen-binding antibody derivatives such as variable domains, peptides, superantigens, and their natural ligands such as CD58 (LFA-3) for CD2, fflV gpl20 for CD4, CD27L for CD27,
- Activation molecules expressed by B cells include but are not limited to, surface lg, CD18, CD19, CD20, CD21, CD22, CD23, CD40, CD45, CD80, CD86 and ICAM1.
- natural ligands of these molecules and antibodies directed to them as well as antibody derivatives may be used to deliver an activation signal to B cells.
- the invention provides a method of modulating a biological function of a cell, e.g., an endothelial cell or hematopoietic cell (such as a leukocyte, e.g., T cell or B cell), by introducing into the cell an antagonist that inhibits binding of a PDZ protein and a PL protein in the cell, or a agonist that enhances binding of a PDZ protein and a PL protein in the cell.
- the PL protein is an adhesion protein, an adaptor protein, or an intracellular protein.
- CD6 CD49E, CD49F, CD138i Clasp-1, Clasp-4, VCAM1, Clasp- 2, CD95, DNAM-1 , CD83, CD44, CD4, CD97, CD3n, DOCK2, CD34, FceRIb, or FasLiga ⁇ d.
- the PL protein is characterized by a carboxy-terminal amino acid motif that is X-S-X-A, X-A-D/E-V, X-V/I/L-X*-V, or X-S/T-X-F (where X is any amino acid and X* is any non-aromatic amino acid).
- the PL protein is expressed by T lymphocytes or B lymphocytes.
- the PDZ protein is CASK, MPP1, DLG1, PSD95, NeDLG, SYNla, TAX43, LDP, LIM, LIMK, AF6, FTN-4, prIL16, 41.8, RGS12, DNL1, TAX 40, TIAM1, MI ⁇ T1, K303, TAX2, or KIAA561.
- the cell is a leukocyte and the biological function is cell activation, cell proliferation, maintenance of cell structure, cell metabolic activity, or cytokine production.
- the method further includes detecting a change in leukocyte activation.
- the antagonist is an agent that inhibits the binding of a PL peptide to a PDZ domain polypeptide in an "A" assay, in a "G” assay, or in both an A assay and a G assay.
- the antagonist can be a polypeptide, such as a polypeptide having at the carboxyterminus at least two residues that are the same as the carboxy-terminal two residues of a PL protein, such as a PL protein is expressed in a hematopoietic or endothelial cell, and/or that is an adhesion protein, an adaptor protein, or an intracellular protein.
- the carboxy-terminal four residues of the polypeptide are the same as the carboxy-terminal four residues of the PL protein.
- the PL protein has a carboxy-terminal amino acid motif selected from X-S-X-A, X-A-D/E-V, X-V I/L-X*-V, or X-S T-X-F, where X is any amino acid and X* is any non-aromatic amino acid.
- the PL protein is CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, VCAM1, Clasp-2, CD95, D ⁇ AM-1, CD83, CD44, CD97, CD3n, DOCK2, CD34, FceRIb, or FasLigand.
- the antagonist is a peptide mimetic of a PL inhibitor sequence peptide.
- the antagonist is a fusion polypeptide having a PL sequence and transmembrane transporter amino acid sequence (such as HIN tat, Drosophila antenapedia, herpes simplex virus NP22 or anti-D ⁇ A CDR 2 and 3).
- the invention provides a method of determining whether a test compound is an inhibitor of binding between a PDZ protein and a PL protein by contacting a PDZ domain polypeptide having a sequence from the PDZ protein, and a PL peptide under conditions in which they form a complex, in the presence and in the absence of a test compound, and detecting the formation of the complex in the presence and absence of the test compound, where less complex formation in the presence of the test compound than in the absence of the compound indicates that the test compound is an inhibitor of a PDZ protein -PL protein binding.
- the PL peptide has a sequence that includes the a C-terminal sequence of a PL protein, such as CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, VCAM1, Clasp-2, CD95, D ⁇ AM-1, CD83, CD44, CD97, CD3n, DOCK2, CD34, FceRIb, or FasLigand.
- a PL protein such as CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, VCAM1, Clasp-2, CD95, D ⁇ AM-1, CD83, CD44, CD97, CD3n, DOCK2, CD34, FceRIb, or FasLigand.
- the PDZ domain polypeptide is a fusion polypeptide.
- the invention provides a method of dete ⁇ nuding whether a test compound is an agonist of binding between a PDZ protein and a PL protein by contacting a PDZ domain polypeptide, and a PL peptide under conditions in which they form a complex, in the presence and in the absence of a test compound, and detecting the formation of the complex in the presence and absence of the test compound, where more complex formation in the presence of the test compound than in the absence of the compound indicates that the test compound is an agonist of a PDZ protein-PL protein binding.
- the invention further provides an inhibitor of binding of a PDZ protein and a PL protein.
- the inhibitor is characterized in that it reduces binding of a peptide selected from the group consisting of a PL peptide selected from the group consisting of CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, NCAM1, Clasp-2, CD95, D ⁇ AM-1, CD83, CD44, CD97, CD3n, DOCK2, CD34, FceRIb, and FasLigand and a PDZ domain polypeptide.
- a peptide selected from the group consisting of a PL peptide selected from the group consisting of CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, NCAM1, Clasp-2, CD95, D ⁇ AM-1, CD83, CD44, CD97, CD3n, DOCK2, CD34, FceRIb, and FasLigand and a PDZ domain polypeptide.
- the inhibitor is a peptide comprising a sequence that is from 3 to about 20 residues of a C-terminal sequence of a PL protein selected from CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, VCAMl, Clasp-2, CD95, DNAM-1, CD83, CD44, CD97, CD3n, DOCK2, CD34, FceRIb, and FasLigand; a peptide having a motif X-S-X-A, X-A-D/E-V, X-N/I/L-X*-V, or X-S/T-X-F, (where X is any amino acid and X* is any non-aromatic amino acid); a peptide mimetic; or a small organic molecule.
- the invention also provides a pharmaceutical composition containing the inhibitor.
- the invention also provides a method for treating a disease characterized by leukocyte activation by administering a therapeutically effective amount of an inhibitor of a PL- PDZ interaction.
- the disease is characterized by an inflammatory or humoral immune response or is an autoimmune disease.
- the invention further provides a method of reducing inflammation in a subject by administering an agent that inhibits binding of a PDZ protein and a PL protein, where the PL protein is an adhesion protein, an adaptor protein, or an intracellular protein.
- the invention also provides use of an inhibitor of the binding of a PDZ protein and a PL protein to inhibit leukocyte activation or to treat a disease mediated by hematopoietic cells, such as a disease is characterized by an inflammatory or humoral immune response.
- the invention also provides use of an inhibitor of the binding of a PDZ protein and a PL protein in the preparation of a medicament for treatment of a disease mediated by hematopoietic cells.
- the invention also provides a method of modulating a biological function of a hematopoietic cell, comprising introducing into the cell an antagonist that inhibits binding of a PDZ protein and a PL protein in the cell as deduced from Table 2, for example, where the PL protein is D ⁇ AM-1 and the PDZ protein is MPP1, MPP2, DLG1, ⁇ eDLG, PSD95, LIM, AF6, 41.8 or RGS12, the PL protein is LPAP and the PDZ protein is DLG1 or ME ⁇ T1, or the PL protein is D ⁇ AM-1 and the PDZ protein is PSD95 or MPP2.
- the present invention also relates to peptides and peptide analogues that bind PDZ domains in hematopoietic cells.
- it relates to fusion peptides and peptide analogues containing a hematopoietic cell surface receptor carboxyl terminal sequence and a transmembrane transporter sequence which facilitates entry of the peptides into a target cell.
- the invention also relates to methods of using such compositions in inhibiting leukocyte activation as measured by cytokine production, cell proliferation, apoptosis and/or cytotoxicity.
- a therapeutically effective amount of the aforementioned fusion peptides, peptide analogues, small molecules and other mediators of PDZ-PL interactions as pharmaceutical compositions, e.g., to a subject to inhibit undesirable cell- mediated (e.g., leukocyte-mediated) events.
- the invention provides a method of determining the apparent affinity (Kd) of binding between a PDZ domain and a ligand by (a) immobilizing a polypeptide comprising the PDZ domain and at least one non-PDZ domain on a surface; (b) contacting the immobilized polypeptide with a plurality of different concentrations of the ligand; (c) determining the amount of binding of the ligand to the immobilized polypeptide at each of the concentrations of ligand; (d) calculating the apparent affinity of the binding from the binding determined in (c).
- the polypeptide is immobilized by binding the polypeptide to an immobilized immunoglobulin that binds the non-PDZ domain.
- the polypeptide comprising the PDZ domain is a fusion protein, for example a GST-PDZ domain fusion protein.
- the invention provides a method of determining the Ki of an inhibitor or suspected inhibitor of binding between a PDZ domain and a ligand, by (a) immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a surface; (b) contacting the immobilized polypeptide with a plurality of different mixtures of the ligand and inhibitor, wherein the different mixtures comprise a fixed amount of ligand, at least a portion of which is detectably labeled, and different concentrations of the inhibitor; (c) determining the amount of ligand bound at the different concentrations of inhibitor; (d) calculating the Ki of the inhibitor from the binding determined in (c).
- the polypeptide is immobilized by binding the polypeptide to an immobilized immunoglobulin that binds the non-PDZ domain.
- the fixed amount of ligand- is between about 0.01 Kd and about 2 Kd.
- the invention provides a method of identifying an agent that enhances the binding of a PDZ domain to a ligand, by immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a surface; (b) contacting the immobilized polypeptide with the ligand in the presence of a test agent and determining the amount of ligand bound; and, (c) comparing the amount of ligand bound in the presence of the test agent with the amount of ligand bound by the polypeptide in the absence of the test agent, wherein at least two-fold greater binding in the presence of the test agent compared to the absence of the test agent indicates that the test agent is an agent that enhances the binding of the PDZ domain to the ligand.
- the invention provides a method of determining the potency (Ke ⁇ h a n cer) of an enhancer of binding between a PDZ domain and a ligand, by (a) immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a surface; (b) contacting the immobilized polypeptide with a plurality of different mixtures of the ligand and enhancer, wherein the different mixtures comprise a fixed amount of ligand, at least a portion of which is detectably labeled, and different concentrations of the enhancer; (c) determining the amount of ligand bound at the different concentrations of enhancer; (d) calculating the potency (Ke ⁇ hancer) of the enhancer from the binding determined in (c).
- the polypeptide is immobilized by binding the polypeptide to an immobilized immunoglobulin that binds the non- PDZ domain.
- the fixed amount of ligand is between about 0.01 Kd and about 0.5 Kd.
- the invention provides a method of identifying a high specificity interaction between a particular PDZ domain and a ligand known or suspected of binding at least one PDZ domain, by (a) providing a plurality of different immobilized polypeptides, each of said polypeptides comprising a PDZ domain and a non-PDZ domain; (b) determining the affinity of the ligand for each of said polypeptides; (c) comparing the affinity of binding of the ligand to each of said polypeptides.
- an interaction between the ligand and a particular PDZ domain is deemed to have high specificity when the ligand binds an immobilized polypeptide comprising the particular PDZ domain with at least 2-fold higher affinity than to immobilized polypeptides not comprising the particular PDZ domain in (a).
- the polypeptide is immobilized by binding the polypeptide to an immobilized immunoglobulin that binds the non- PDZ domain.
- the invention provides a method for determining the PDZ-PL inhibition profile of a compound by (a) providing (i) a plurality of different immobilized polypeptides, each of said polypeptides comprising a PDZ domain and a non-PDZ domain; (ii) a plurality of corresponding ligands, wherein each ligand binds at least one PDZ domain in (i); (b) contacting each of said immobilized polypeptides in (i) with a corresponding ligand in (ii) in the presence and absence of a test compound; (c) determining for each polypeptide-ligand pair in (b) whether the test compound inhibits binding between the immobilized polypeptide and the corresponding ligand thereby determining the PDZ-PL inhibition profile of the test compound.
- the invention provides an array comprising a plurality of different immobilized polypeptides, each of said polypeptides comprising a PDZ domain and a non-PDZ domain.
- the array is situated in a plastic multiwell plate.
- the array has at least 12 different polypeptides comprising at least 12 different PDZ domains, for example, at least 12 different PDZ domains are from PDZs expressed in lymphocytes.
- the PDZs are selected from those listed in Table 2 or 6.
- the invention provides an assay device comprising a plurality of different immobilized PDZ-containing proteins organized in an array. In one embodiment, the device has at least 25 different PDZ-containing proteins.
- the invention provides a method for identifying an interaction between a PDZ domain and a PL by contacting a PL to a plurality of PDZ containing polypeptides and detecting binding of at least one PL to a PDZ.
- the contacting occurs on an assay device comprising a plurality of different immobilized PDZ- containing proteins organized in an array.
- the device has at least 25 different PDZ-containing proteins.
- an interaction between a PDZ and more than one PL, or between a PL and more than one PDZ, is detected.
- the invention provides method for identifying a modulator of an interaction between a PDZ and a PL by conducting any of the aforementioned assays in the presence and absence of a test compound and detecting a difference in at least one PDZ-PL interaction in the presence and absence of the test compound.
- the modulator is an enhancer of the interaction. In other embodiments, the modulator is an inhibitor of the interaction.
- any of the aforementioned methods or devices (as further described herein) comprising a plurality of PDZ-domain containing polypeptides comprises at least one, usually at least 2, typically at least 5 and often at least 10 different PDZ-containing polypeptides comprising PDZ sequences from proteins selected from: MPP1 (p55), K303, K807, DLG1, PSD95, NeDLG, TAX IP43, LDP, LIM, K545, TIP1, PTN-4, CBP, AF6, PDZK1, DLG5, Syntenin, WWP3, K561.
- the invention provides a method of modulating a biological function of an endothelial cell or hematopoietic cell (e.g., a leukocyte such as a T cell or a B cell), comprising introducing into the cell an agent that inhibits binding of a PDZ protein and a PL protein in the cell, wherein any of the following (T 0 apply:
- the PL is CD105, VCAM1, CD95, Spectrin ⁇ , KN1.3, D ⁇ AM1, ⁇ euroligin 3, CD44, CD38, CD3 ⁇ , LPAP, CD46, CDwl28B, DOCK2, PAG, CD34, or BLR-1;
- the PDZ is MPP1, K303, K807, DLG1, PSD95, ⁇ eDLG, IP43, LDP, LIM,
- the PDZ protein is MPP 1 and the PL protein has a carboxy-terminal amino acid motif X-S/T/Y I-X-N; the PDZ protein is LIMK1 and the PL protein has a carboxy-terminal amino acid motif X-S/T/Y-X-V; the PDZ protein is K303 and the PL protein has a carboxy- terminal amino acid motif X-S-X-V; the PDZ protein is K807 and the PL protein has a carboxy- terminal amino acid motif XI -S/T-X2-N/I/IJF; the PDZ protein is DLG1, PSD95, or ⁇ eDLG and the PL protein has a carboxy-terminal amino acid motif X-S/T/Y/A E-X-V/I/L; the PDZ protein is S ⁇ Tal and the PL protein has a carboxy-terminal amino acid motif X-S/T/Y-D/Y- N/I/L;
- the agent is a peptide comprising a sequence of at least the carboxy- terminal two or three residues of the PL protein;
- the agent is a small molecule or peptide mimetic of the carboxy-terminus of the PL protein;
- the invention provides a method for determining whether a test compound is an inhibitor of binding between a PDZ protein and a PL protein by contacting a PDZ domain polypeptide having a sequence from the PDZ protein, and a PL peptide, wherein the PL peptide comprises a C-terminal sequence of a PL protein under conditions in which they form a complex, where the contacting is carried out in the presence and in the absence of a test compound, and detecting the formation of the complex in the presence and absence of the test compound.
- the PL protein is CD105, VCAMl, CD95, Spectrin ⁇ , KV1.3, DNAM1, Neuroligin 3, TAX, CD44, CD38, CD3 ⁇ , LPAP, CD46, CDwl28B, DOCK2, PAG, CD34, or BLR-1 and less complex formation in the presence of the test compound than in the absence of the compound indicates that the test compound is an inhibitor of a PDZ protein -PL protein binding.
- the invention also contemplates the inhibitor identified by this method.
- the inhibitor is (a) a peptide comprising a sequence that is from 3 to about 20 residues of a C-terminal sequence of CD105, VCAMl, CD95, Spectrin ⁇ , KV1.3, DNAM1, Neuroligin 3, TAX, CD44, CD38, CD3 ⁇ , LPAP, CD46, CDwl28B, DOCK2, PAG, CD34, or BLR-1 ; (b) a peptide mimetic of such a peptide; or (c) a small organic molecule with a molecular weight less than 1 kD.
- the invention further contemplates a pharmaceutical composition containing the inhibitor, as well as a method for treating a disease characterized by leukocyte activation by administering a therapeutically effective amount of the inhibitor.
- the disease is characterized by an inflammatory or humoral immune response, e.g., an autoimmune disease.
- the invention provides a method of modulating a biological function in a cell (e.g., a hematopoietic cell) by introducing into the cell an antagonist that inhibits binding of a PDZ protein and a PL protein in the cell, wherein, the PDZ protein is MPP1 (p55) and the PL is Spectrin ⁇ ; the PDZ protein is K303 and the PL is Spectrin ⁇ ; the PDZ protein is K807 and the PL VCAMl, Spectrin ⁇ , KV1.3, Neuroligin 3, CD38, CD3 ⁇ , LPAP, CD46 (form 1), CDwl28B, DOCK2, PAG, CD34, or BLR-1; the PDZ protein is DLG1 and the PL is Spectrin; the PDZ protein is PSD95 and the PL is Spectrin ⁇ , CD34, or CD38; the PDZ protein is NeDLG and the PL is Spectrin ⁇ or CD38; the PDZ
- the invention also provides the use of an inhibitor of the binding of a PDZ protein and a PL protein described herein or identified according to a method of the invention to inhibit leukocyte activation,, or for preparation of a medicament for treatment of a disease mediated by a PDZ-PL interaction, e.g., in hematopoietic cells or in viral infection.
- CD105 is described at GenBank accession no. X72012; VCAMl is described at GenBank accession no. M73255; CD95 is described at GenBank accession no. M67454; Spectrin ⁇ is described at GenBank accession no. NM000347; KV1.3 is described at GenBank accession no. AAC31761; DNAMl is described at GenBank accession no. U56102; Neuroligin 3 is described at GenBank accession no. NMO 18977; TAX is described at GenBank accession no. AB038239; CD44 is described at GenBank accession no.
- CD49E (4) is described at GenBank accession no. X06256; CD49F is described at GenBank accession no. X53586; CD97 is described at GenBank accession no. X84700; CD98 is described at GenBank accession no. J02939; CD138 is described at GenBank accession no. J05392; CD148 is described at GenBank accession no. D37781; CD166 is described at GenBank accession no. L38608; CDwl37 (4-lBB) is described at GenBank accession no. NM001561; FasL is described at GenBank accession no. U11821; FceRIb is described at GenBank accession no.
- Galectin3 is described at GenBank accession no. J02921; CD114 is described at GenBank accession no. NM000760; 5 CDW125 (TL5R) is described at GenBank accession no. X62156; CDW128A (TL8RA) is described at GenBank accession no. M68932; Mannose Receptor is described at GenBank accession no. NM002438; NMDA is described at GenBank accession no. NP000824; Glycophorin C is described at GenBank accession no. AAA52574; Neurexin is described at GenBank accession no. ABOl 1150; Syndecan-2 is described at GenBank accession no. A33880;
- CC CKR-IR is described at GenBank accession no. L09230; CC CKR-2 is described at GenBank accession no. U03882; CC CKR-3 is described at GenBank accession no. HSU28694; CC CKR- 4 is described at GenBank accession no. X85740; Volt. Gated Ca2+ is described at GenBank accession no. Q00975; CD83 is described at GenBank accession no. Z11697; CD62E is described at GenBank accession no. M30640; CD5 is described at GenBank accession no.
- FIGURES 1A-1D show the results of exemplary assays in which the binding of 20 biotinylated peptides having a sequence of the carboxyl-terminus ("c-terminus") of various leukocyte proteins to PDZ domains (i.e., GST-PDZ domain fusion proteins) was determined using the "G" assay described infra.
- the PDZ domains are: PSD95 (Fig. 1 A ); NeDLG (Fig. IB); DLG1 (Fig IC); and 41.8 (Fig. ID). These and other PDZ domain fusion proteins are described infra (e.g., TABLE 2).
- peptides 1-31 refer to the biotinylated PL 25.
- peptides used in the assay and are identified in the Key, infra.
- “Peptide IDs” are defined in TABLE 3. Key:
- FIGURES 2A and 2B show the Apparent Affinity Determination for PDZ- Ligand Interactions. Varying concentrations of biotinylated CLASP-2 (Fig.2 A; TABLE 4) or Fas (Fig. 2B; TABLE 4) C-teraiinal peptides were reacted with immobilized (plate bound) GST polypeptide or GST-PDZ fusion proteins (GST-DLGl, GST-NeDLG, and GST-PSD95). The binding to GST alone ( ⁇ 0.2 OD units) was subtracted from the binding to the fusion proteins to obtain the signal at each peptide concentration.
- This signal was then normalized by dividing the signal at each peptide concentration by the maximum signal observed for each peptide-PDZ pair (i.e. the signal obtained at 30 uM Clasp 2 peptide or 100 uM Fas peptide; 0.4 - 1.0 OD units for Clasp 2 and 1.2 - 2.0 OD units for Fas).
- the normalized signals were then plotted and fit to a saturation binding curve, yielding an apparent affinity of 21 uM for DLG1- Clasp 2 interaction, 7.5 uM for NeDLG-Clasp 2 interaction, 45 uM for PSD95-Clasp 2 interaction, 54 uM for DLGl-Fas interaction, 54 uM for NeDLG-Fas interaction, and 85 uM for PSD95-Fas interaction.
- Data are means of duplicate data points, with standard errors between duplicate data points ⁇ 20%.
- FIGURES 3A-3F show inhibition of PDZ - PL peptide interactions.
- a fixed concentration of biotinylated C-terminal peptide having a sequence based on the C-terminal sequence of a cell surface receptor protein (Clasp 2, CD46, Fas, and KV1.3; see TABLE 4) was bound to immobilized GST polypeptide or the GST-fusion protein indicated at the top left of each frame, in the presence or absence of the competitor peptides indicated in the legend of each frame and the level of inhibition determined.
- the competitor peptides are present at 100 uM
- Figs. 3C-F the competitor is present at the indicated concentration.
- FIGURES 4 A and 4B shows the results of introduction of a Tat-CD3 fusion peptide on T cell activation.
- Antigen-specific T cell activation was measured by cytokine production.
- Fusion peptides containing tat and a T cell surface molecule carboxyl terminus inhibited ⁇ -interferon (IFN) production by a T cell line in response to myelin basic protein (MBP) stimulation.
- IFN ⁇ -interferon
- MBP myelin basic protein
- FIGURES 5 A and 5B show TIP1-RFP overexpression enhances anti-CD95 induced apoptosis in Jurkat T cells.
- Jurkat E6 T cells were transfected with either DsRED (RFP), TIP1-RFP, or PAR6(N-P)-RFP. 24 hours post- transfection, cells were treated for 2 hours with anti-CD95 and then incubated with annexin V-FITC and analyzed using flow cytometry.
- Fig. 5 A shows the results of FACS analysis.
- Fig. 5B shows a 30% increase in apoptosis of RFP-TIP1 positive compared to RFP negative apoptotic (annexin V positive) cells.
- FIGURE 6 Binding of a 20-mer peptide (20 uM) corresponding to the C- terminus of CD95 (Fas) to TIP-1 can be inhibited by an 8-mer peptide corresponding to the C- terminus of TAX. 50% inhibition can be achieved by 20-100 uM of inhibitor.
- FIGURE 7 Binding of a 20-mer peptide (20 uM) corresponding to the C- terminus of TAX to TIP-1 can be inhibited by an 8-mer peptide corresponding to the C- terminus of CD95 (Fas). 50% inhibition can be achieved by 500 uM of inhibitor.
- FIGURE 8 Binding of a 20-mer peptide (1 uM) corresponding to the C- terminus of BLR-1 (CXCR5) to KIAA0807 (PDZ domain)-GST fusion protein can be inhibited by an 8-mer peptide corresponding to the C-terminus of BLR-1 and a small molecule inhibitor
- acetyl-LTTF 50% inhibition can be achieved by greater than 100 uM of the 8-mer peptide and 1 uM of the small molecule inhibitor.
- FIGURE 9 Binding of a 20-mer peptide (10 uM) corresponding to the C- terminus of DOCK2 to KIAA0807 (PDZ domain)-GST fusion protein can be inhibited by an 8-mer peptide corresponding to the C-terminus of DOCK2 and a small molecule inhibitor (acetyl-STDL). 50% inhibition can be achieved by 250 uM of the 8-mer peptide and less than 250 uM of the small molecule inhibitor.
- acetyl-STDL small molecule inhibitor
- a "fusion protein” or “fusion polypeptide” as used herein refers to a composite protein, i.e., a single contiguous amino acid sequence, made up of two (or more) distinct, heterologous polypeptides which are not normally fused together in a single amino acid sequence.
- a fiision protein can include a single amino acid sequence that contains two entirely distinct amino acid sequences or two similar or identical polypeptide sequences, provided that these sequences are not normally found together in the same configuration in a single amino acid sequence found in nature.
- Fusion proteins can generally be prepared using either recombinant nucleic acid methods, i.e., as a result of transcription and translation of a recombinant gene fusion product, which fusion comprises a segment encoding a polypeptide of the invention and a segment encoding a heterologous protein, or by chemical synthesis methods well known in the art.
- a "fusion protein construct” as used herein is a polynucleotide encoding a fusion protein.
- PDZ domain refers to protein sequence (i.e., modular protein domain) of approximately 90 amino acids, characterized by homology to the brain synaptic protein PSD-95, the Drosophila septate junction protein Discs-Large (DLG), and the epithelial tight junction protein ZOl (ZOl).
- PDZ domains are also known as Discs-Large homology repeats ("DHRs") and GLGF (SEQ ID NO: 402) repeats. PDZ domains generally appear to maintain a core consensus sequence (Doyle, D. A., 1996, Cell 85: 1067-76).
- PDZ domains are found in diverse membrane-associated proteins including members of the MAGUK family of guanylate kinase homologs, several protein phosphatases and kinases, neuronal nitric oxide synthase, and several dystrophin-associated proteins, collectively known as syntiophins.
- PDZ domain-containing proteins and PDZ domain sequences are shown in TABLE 3.
- the term "PDZ domain” also encompasses variants (e.g., naturally occuring variants) of the sequences of TABLE 3 (e.g., polymorphic variants, variants with conservative substitutions, and the like).
- PDZ domains are substantially identical to those shown in TABLE 3, e.g., at least about 70%, at least about 80%, or at least about 90% amino acid residue identity when compared and aligned for maximum correspondence.
- PDZ protein refers to a naturally occurring protein containing a PDZ domain, e.g., a human protein.
- exemplary PDZ proteins include CASK,
- Exemplary PDZ proteins are listed in TABLE 2 and TABLE 3.
- PDZ-domain polypeptide refers to a polypeptide containing a PDZ domain, such as a fusion protein including a PDZ domain sequence, a naturally occurring PDZ protein, or an isolated PDZ domain peptide.
- PL protein or "PDZ Ligand protein” refers to a naturally occurring protein that forms a molecular complex with a PDZ-domain, or to a protein whose carboxy-terminus, when expressed separately from the full length protein (e.g., as a peptide fragment of 4-25 residues, e.g., 16 residues), forms such a molecular complex.
- the molecular complex can be observed in vitro using the "A assay” or "G assay” described infra, or in vivo.
- Exemplary PL proteins listed in TABLE 2 are demonstrated to bind specific PDZ proteins. This definition is not intended to include anti-PDZ antibodies and the like.
- a "PL sequence” refers to the amino acid sequence of the C- te ⁇ ninus of a PL protein (e.g., the C-terminal 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20 or 25 residues) ("C-terminal PL sequence") or to an internal sequence known to bind a PDZ domain (“internal PL sequence).
- a "PL peptide” is a peptide of having a sequence from, or based on, the sequence of the C-terminus of a PL protein. Exemplary PL peptides (biotinylated) are listed in TABLE 4.
- a "PL fusion protein” is a fusion protein that has a PL sequence as one domain, typically as the C-terminal domain of the fusion protein.
- An exemplary PL fusion protein is a tat-PL sequence fusion.
- PL inhibitor peptide sequence refers to PL peptide amino acid sequence that (in the form of a peptide or PL fusion protein) inhibits the interaction between a PDZ domain polypeptide and a PL peptide (e.g., in an A assay or a G assay).
- a "PDZ-domain encoding sequence” means a segment of a polynucleotide encoding a PDZ domain.
- the polynucleotide is DNA, RNA, single stranded or double stranded.
- the terms "antagonisf ' and "inhibitor,” when used in the context of modulating a binding interaction are used interchangeably and refer to an agent that reduces the binding of the, e.g., PL sequence (e.g., PL peptide) and the, e.g., PDZ domain sequence (eig., PDZ protein, PDZ domain peptide).
- PL sequence e.g., PL peptide
- PDZ domain sequence eig., PDZ protein, PDZ domain peptide
- the terms “agonist” and “enhancer,” when used in the context of modulating a binding interaction are used interchangeably and refer to an agent that increases the binding of the, e.g., PL sequence (e.g., PL peptide) and the, e.g., PDZ domain sequence (e.g., PDZ protein, PDZ domain peptide).
- PL sequence e.g., PL peptide
- PDZ domain sequence e.g., PDZ protein, PDZ domain peptide.
- the terms “peptide mimetic, " “peptidomimetic,” and “peptide analog” are used interchangeably and refer to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of an PL inhibitory or PL binding peptide of the invention.
- the mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
- the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic' s structure and/or inhibitory or binding activity.
- routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered.
- a mimetic composition is within the scope of the invention if it is capable of binding to a PDZ domain and or inhibiting a PL-PDZ interaction.
- Polypeptide mimetic compositions can contain any combination of nonnatural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
- a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
- DCC dicyclohexylcarbodiimide
- DIC diisopropylcarbodiimide
- a polypeptide can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues.
- Nonnatural residues are well described in the scientific and patent literature; a few exemplary nonnatural compositions useful as mimetics of natural amino acid residues and guidelines are described below.
- Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L- naphylalanine; D- or L- phenylglycine; D- or L-2 thieneylalanine; D- or L-l, -2, 3-, or 4- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridmyl)-alanine; D- or L-(3-pyridinyl)- alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D- (trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-fluorophenylala_nine; D- or L-p-biphenylphenylalanine; K- or L-p-methoxybiphenylphen
- Aromatic rings of a nonnatural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
- Mimetics of acidic amino acids can be generated by substitution by, e.g., non- carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine.
- Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
- Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids omithine, citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.
- Nitrile derivative e.g., containing the CN-moiety in place of COOH
- Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues.
- Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2- cyclohexanedione, or ninhydrin, preferably under alkaline conditions.
- Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
- Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives.
- alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines
- Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha- bromo-beta-(5-imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro- 2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-l,3-diazole.
- cysteinyl residues e.g., bromo-trifluoroacetone, alpha- bromo-beta-(5-imidozoyl) propionic acid
- chloroacetyl phosphate N-alkylmaleimides
- 3-nitro- 2-pyridyl disulfide methyl 2-pyridyl disulfide
- Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-ammo-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, truiitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate.
- imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, truiitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate.
- Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide.
- Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4- hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline.
- Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para- bromophenacyl bromide.
- mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.
- a component of a natural polypeptide e.g., a PL polypeptide or PDZ polypetide
- an amino acid or peptidomimetic residue
- any amino acid naturally occurring in the L-configuration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, generally referred to as the D- amino acid, but which can additionally be referred to as the R- or S- form.
- the mimetics of the invention can also include compositions that contain a structural mimetic residue, particularly a residue that induces or mimics secondary structures, such as a beta turn, beta sheet, alpha helix structures, gamma turns, and the like.
- a structural mimetic residue particularly a residue that induces or mimics secondary structures, such as a beta turn, beta sheet, alpha helix structures, gamma turns, and the like.
- substitution of natural amino acid residues with D-amino acids; N-alpha-methyl amino acids; C-alpha-methyl amino acids; or dehydroamino acids within a peptide can induce or stabilize beta turns, gamma turns, beta sheets or alpha helix conformations.
- Beta turn mimetic structures have been described, e.g., by Nagai (1985) Tet. Lett.26:647-650; Feigl (1986) J. Amer. Chem. Soc.
- Beta sheet mimetic structures have been described, e.g., by Smith (1992) J. Amer. Chem. Soc. 114:10672-10674.
- a type VI beta turn induced by a cis amide surrogate, 1,5-disubstituted tetrazol is described by Beusen (1995) Biopolymers 36:181-200.
- Incorporation of achiral omega-amino acid residues to generate polymethylene units as a substitution for amide bonds is described by Banerjee
- peptide variants and “conservative amino acid substitutions” refer to peptides that differ from a reference peptide (e.g., a peptide having the sequence of the carboxy-terminus of a specified PL protein) by substitution of an amino acid residue having similar properties (based on size, polarity, hydrophobicity, and the like).
- a reference peptide e.g., a peptide having the sequence of the carboxy-terminus of a specified PL protein
- substitution of an amino acid residue having similar properties based on size, polarity, hydrophobicity, and the like.
- amino acids may be generally categorized into three main classes: hydrophilic amino acids, hydrophobic amino acids and cysteine-like amino acids, depending primarily on the characteristics of the amino acid side chain. These main classes may be further divided into subclasses.
- Hydrophilic amino acids include amino acids having acidic, basic or polar side chains and hydrophobic amino acids include amino acids having aromatic or apolar side chains.
- Apolar amino acids may be further subdivided to include, among others, aliphatic amino acids.
- the definitions of the classes of amino acids as used herein are as follows: "Hydrophobic Amino Acid” refers to an amino acid having a side chain that is uncharged at physiological pH and that is repelled by aqueous solution. Examples of genetically encoded hydrophobic amino acids include He, Leu and Val. Examples of non- genetically encoded hydrophobic amino acids include t-BuA.
- Aromatic Amino Acid refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated ⁇ -electron system (aromatic group).
- aromatic group may be further substituted with groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfanyl, nitro and amino groups, as well as others.
- genetically encoded aromatic amino acids include Phe, Tyr and Trp.
- aromatic amino acids include phenylglycine, 2-naphthylalanine, ⁇ -2-thienylalanine, 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, 4-chloro-phenylalanine, 2-fluorophenyl-alanine, 3- fluorophenylalanine and 4-fluorophenylalanine.
- Apolar Amino Acid refers to a hydrophobic amino acid having a side chain that is generally uncharged at physiological pH and that is not polar. Examples of genetically encoded apolar amino acids include Gly, Pro and Met. Examples of non-encoded apolar amino acids include Cha.
- Aliphatic Amino Acid refers to an apolar amino acid having a saturated or unsaturated straight chain, branched or cyclic hydrocarbon side chain.
- Examples of genetically encoded aliphatic amino acids include Ala, Leu, Val and He.
- Examples of non-encoded aliphatic amino acids include Nle.
- Hydrophilic Amino Acid refers to an amino acid having a side chain that is attracted by aqueous solution.
- examples of genetically encoded hydrophilic amino acids include Ser and Lys.
- examples of non-encoded hydrophilic amino acids include Cit and hCys.
- Acidic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Examples of genetically encoded acidic amino acids include Asp and Glu. "Basic Amino Acid” refers to a hydrophilic amino acid having a side chain pK value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Examples of genetically encoded basic amino acids include Arg, Lys and His. Examples of non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3-diaminopropionic acid, 2,4- diaminobutyric acid and homoarginine.
- Poly Amino Acid refers to a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has a bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.
- genetically encoded polar amino acids include Asx and Glx.
- non-genetically encoded polar amino acids include citrulline, N-acetyl lysine and methionine sulfoxide.
- cyste-Like Amino Acid refers to an amino acid having a side chain capable of forming a covalent linkage with a side chain of another amino acid residue, such as a disulfide linkage.
- cysteine-like amino acids generally have a side chain containing at least one thiol (SH) group.
- examples of genetically encoded cysteine-like amino acids include Cys.
- examples of non-genetically encoded cysteine-like amino acids include homocysteine and penicillamine.
- the above classification are not absolute - several amino acids exhibit more than one characteristic property, and can therefore be included in more than one category.
- tyrosine has both an aromatic ring and a polar hydroxyl group.
- tyrosine has dual properties and can be included in both the aromatic and polar categories.
- cysteine in addition to being able to form disulfide linkages, cysteine also has apolar character.
- cysteine can be used to confer hydrophobicity to a peptide.
- Certain commonly encountered amino acids which are not genetically encoded of which the peptides and peptide analogues of the invention may be composed include, but are not limited to, ⁇ -alanine (b-Ala) and other omega-amino acids such as 3-aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; ⁇ -aminoisobutyric acid (Aib); ⁇ -aminohexanoic acid (Aha); ⁇ -aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (
- TABLE 1 is for illustrative purposes only and does not purport to be an exhaustive list of amino acid residues which may comprise the peptides and peptide analogues described herein.
- Other amino acid residues which are useful for making the peptides and peptide analogues described herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited therein.
- Amino acids not specifically mentioned herein can be conveniently classified into the above-described categories on the basis of known behavior and/or their characteristic chemical and/or physical properties as compared with amino acids specifically identified.
- a "detectable label” has the ordinary meaning in the art and refers to an atom (e.g., radionuclide), molecule (e.g., fluorescein), or complex, that is or can be used to detect (e.g., due to a physical or chemical property), indicate the presence of a molecule or to enable binding of another molecule to which it is covalently bound or otherwise associated.
- label also refers to covalently bound or otherwise associated molecules (e.g., a biomolecule such as an enzyme) that act on a substrate to produce a detectable atom, molecule or complex.
- Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
- Labels useful in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, enhanced green fluorescent protein, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes ( e.g., hydrolases, particularly phosphatases such as alkaline phosphatase, esterases and glycosidases, or oxidoreductases, particularly peroxidases such as horse radish peroxidase, and others commonly used in ELISAs), substrates, cofactors, inhibitors, cherm uminescent groups, chromogenic agents, and colorimetric labels such as colloidal gold
- Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
- Means of detecting such labels are well known to those of skill in the art.
- radiolabels and chemiluminescent labels may be detected using photographic film or scintillation counters
- fluorescent markers may be detected using a photodetector to detect emitted light (e.g., as in fluorescence-activated cell sorting).
- Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
- a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
- the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. Non-radioactive labels are often attached by indirect means.
- a ligand molecule e.g., biotin
- an anti-ligand e.g., streptavidin
- a signal generating system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
- ligands and anti-ligands can be used.
- a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands.
- any haptenic or antigenic compound can be used in combination with an antibody.
- the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
- Means of detecting labels are well known to those of skill in the art.
- the label is a radioactive label
- means for detection include a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging.
- the label is a fluorescent label
- it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence.
- the fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
- CCDs charge coupled devices
- enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
- simple colorimetric labels may be detected by observing the color associated with the label. It will be appreciated that when pairs of fluorophores are used in an assay, it is often preferred that they have distinct emission patterns (wavelengths) so that they can be easily distinguished.
- the term "substantially identical" in the context of comparing amino acid sequences means that the sequences have at least about 70%, at least about 80%, or at least about 90% amino acid residue identity when compared and aligned for maximum correspondence.
- An algorithm that is suitable for determining percent sequence identity and sequence similarity is the FASTA algorithm, which is described in Pearson, W.R. & Lipman, D.J., 1988, Proc. N ⁇ tl Ac ⁇ d. Sci. U.S.A. 85: 2444. See also W. R. Pearson, 1996, Methods Enzymol 266: 227-258.
- hematopoietic cells include leukocytes including lymphocytes (T cells, B cells and NK cells), monocytes, and granulocytes (i.e., neutrophils, basophils and eosinophils), macrophages, dendritic cells, megakaryocytes, reticulocytes, erythrocytes, and CD34 + stem cells.
- test compound or “test agent” are used interchangably and refer to a candidate agent that may have enhancer/agonist, or inhibitor/antagonist activity, e.g., inhibiting or enhancing an interaction such as PDZ-PL binding.
- the candidate agents or test compounds may be any of a large variety of compounds, both naturally occurring and synthetic, organic and inorganic, and including polymers (e.g., oligopeptides, polypeptides, oligonucleotides, and polynucleotides), small molecules, antibodies (as broadly defined herein), sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (e.g., peptide mimetics, nucleic acid analogs, and the like), and numerous other compounds.
- test agents are prepared from diversity libraries, such as random or combinatorial peptide or non-peptide libraries.
- libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro trahslation-based libraries.
- chemically synthesized libraries are described in Fodor et al., 1991 , Science 251 :767-773 ; Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et al., 1994, J. Medicinal Chemistry 37(9): 1233-1251; Ohl eyer et al., 1993, Proc. Natl. Acad Sci.
- phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R.B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; andPCT PublicationNo. WO 94/18318 dated August 18, 1994.
- In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated April 18, 1991; and Mattheakis et al., 1994, Proc. Natl.
- a benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91 :4708-4712) can be adapted for use.
- Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad Sci. USA 89:9367-9371) can also be used.
- Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).
- binding refers to binding between two molecules, for example, a ligand and a receptor, characterized by the ability of a molecule (ligand) to associate with another specific molecule (receptor) even in the presence of many other diverse molecules, i.e., to show preferential binding of one molecule for another in a heterogeneous mixture of molecules. Specific binding of a ligand to a receptor is also evidenced by reduced binding of a detectably labeled ligand to the receptor in the presence of excess unlabeled ligand (i.e., a binding competition assay).
- a "plurality" of PDZ proteins has its usual meaning.
- the plurality is at least 5, and often at least 25, at least 40, or at least 60 different PDZ proteins.
- the plurality is selected from the list of PDZ polypeptides listed in Table 2 or Table 7.
- the plurality of different PDZ proteins are from (i.e., expressed in) a particular specified tissue or a particular class or type of cell.
- the plurality of different PDZ proteins represents a substantial fraction (e.g., typically at least 50%, more often at least 80%) of all of the PDZ proteins known to be, or suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZ proteins known to be present in lymphocytes or hematopoetic cells.
- the plurality is at least 50%, usually at least 80%, at least 90% or all of the PDZ proteins disclosed herein as being expressed in hematopoietic cells (see Tables 2 and 6).
- the plurality includes at least 1, often at least 2, sometimes at least 5 or at least 10 and sometimes all of the following PDZ proteins: BAI I associated protein, Connector enhancer, DLG5 (pdlg), DVL3, GTPase, Guanin-exchange factor 1, PDZ domain containing prot, KIAA147, KIAA0300, K1AA0380, KIAA0440, KIAA0545, KIAA0807, KIAA0858, KIAA0902, novel serine protease, PDZK1, PICK8, PTN-3, RPIP8, serine protease, 26s subunit p27, hSYNTENIN, TAXI-IP, TAX2-like protein, wwp3, XI 1 prot.
- PDZ proteins BAI I associated protein, Connector enhancer, DLG5 (pdlg), DVL3, GTPase, Guanin-exchange factor 1, PDZ domain containing prot, KIAA147
- a "plurality" may refer to at least 5, at least 10, and often at least 25 PLs such as those specifcally listed herein, or to the classes and percentages set forth supra for PDZ domains.
- the present inventors have discovered that interactions between PDZ proteins and PL proteins play an important and extensive role in the biological function of hematopoietic cells and other cells involved in the immune response.
- PDZ-PL interactions were known in the nervous system (i.e., in neurons), their universal importance in hematopoietic cell function, especially in function of T cells and B cells, and their fundamental role in modulation of the immune response has not been recognized.
- cell adhesion molecules that mediate cellrcell interaction in the hematopoietic system are PDZ-binding proteins (PL proteins) and bind to PDZ proteins.
- biological function in the context of a cell, refers to a detectable biological activity normally carried out by the cell, e.g., a phenotypic change such as proliferation, cell activation (e.g., T cell activation, B cell activation, T-B cell conjugate formation), cytokine release, degranulation, tyrosine phosphorylation, ion (e.g., calcium) flux, metabolic activity, apoptosis, changes in gene expression, maintenance of cell structure, cell migration, adherance to a substrate, signal transduction, cell-cell interactions, and others described herein or known in the art.
- a phenotypic change such as proliferation, cell activation (e.g., T cell activation, B cell activation, T-B cell conjugate formation), cytokine release, degranulation, tyrosine phosphorylation, ion (e.g., calcium) flux, metabolic activity, apoptosis, changes in gene expression, maintenance of cell structure, cell migration, adher
- the present invention relates to peptides, peptide analogues or mimetics, pharmaceutical compositions, and methods of using such compositions to regulate the biological activities of hematopoietic cells, e.g. T cells and B cells, or other cells (e.g., endothelial cells) that necessary for immune function.
- the invention further relates to methods of using the compositions to modulate hematopoietic cell activation and immune function, as well as assays for such inhibitors.
- TABLE 2 summarizes an extensive analysis of protein interactions in T cells and B cells.
- PDZ proteins the vast majority of which were not previously known to be expressed in immune system cells, are listed in the top row of TABLE 2.
- the first column of the table lists PL proteins. Positions in the matrix denoted by the letter “A,” “G,” “G ⁇ ” or “G”” indicate that an interaction between the PDZ protein and the PL has been detected in novel binding assays (described in detail infra). A blank cell indicates that no interaction was detected using the assays of the invention.
- the PDZ proteins listed in TABLE 2 are naturally occurring proteins containing a PDZ domain.
- the present invention is particularly directed to the detection and modulation of interactions between PDZ proteins and PL proteins in hematopoietic cells.
- Exemplary PL proteins are listed in TABLE 2. Notably, as discussed infra, many of these PL proteins have not previously been recognized as such in any cell system.
- a variety of PL protein classes are known, and the PL proteins described herein can be characterized as (1) "PL adhesion proteins” (2) "PL ion channel proteins” (3) "PL adaptor proteins” (4) "PL intracellular proteins” and (5) "PL cytokine receptor proteins.”
- an adhesion protein is a cell surface protein involved in cell-cell interaction by direct contact with cell surface molecules (e.g., transmembrane proteins or surface proteins) on a different cell.
- cell surface molecules e.g., transmembrane proteins or surface proteins
- the PL adhesion protein localizes at the interface of the two cells and directly contacts a cell surface molecule on the second cell.
- a cell-cell interface is a region where the plasma membranes of two different cells are in close (generally ⁇ 10 nm, often about 1 nm) apposition.
- direct molecular contact means interaction of molecules at distances where Nan der Walls forces are significant, generally less than about 1 nm.
- Exemplary PL adhesion proteins include CD6; CD49E (alpha-4); CD49F (a form, alpha ⁇ ); CD138 (syndecan); CLASP-1; CLASP-4; NCAM1; CLASP-2; D ⁇ AM-1; CD83; CD44 (long form); CD97; (CD55L); CD3 ⁇ ; DOCK2; CD34; and FceRIb.
- the PL proteins of the invention are PL adhesion proteins.
- the invention provides methods and reagents, as detailed herein, for inhibiting interactions between PL adhesion proteins and PDZ proteins to modulate an immune response.
- the inhibition or modulation occurs in a hematopoietic cell.
- the inhibition or modulation occurs in an endothelial cell. In a related embodiment, the inhibition or modulation occurs in. an endothelial cell. In a related embodiment, the inhibition or modulation occurs in an epithelial cells, keratinocytes, hepatocytes, cardiac myocytes.
- an ion channel protein means a transmembrane protein that itself catalyzes the passage of an ion from aqueous solution on one side of a lipid bilayer membrane to aqueous solution on the other side (e.g., by forming a small pore in the membrane).
- PL ion channel proteins is Kvl .3.
- the PL proteins of the invention are PL ion channel proteins.
- the invention provides methods and reagents, as detailed herein, for inhibiting interactions between PL ion channel proteins and PDZ proteins to modulate an immune response.
- the inhibition or modulation occurs in a hematopoeitic cell.
- the inhibition or modulation occurs in an endothelial cell.
- an intercellular (i.e., cytosolic) protein has the normal meaning in the art and refers to a protein that is not membrane bound, e.g., has no transmembrane domain.
- the PL proteins of the invention are PL intercellular proteins.
- Exemplary PL intercellular proteins include Glycophorin C and LPAP.
- the invention provides methods and reagents, as detailed herein, for inhibiting interactions between PL cytoplasmic proteins and PDZ proteins to modulate an immune response.
- the inhibition or modulation occurs in a hematopoeitic cell.
- the inhibition or modulation occurs in an endothelial cell.
- a cytokine receptor has the normal meaning in the art and refers to a membrane protein with an extracellular domain that specifically binds a cytokine.
- exemplary PL cytokine receptor proteins include CDW125 (TL5R), CDW128A (EL8RA), and BRL-1.
- the PL proteins of the invention are PL cytokine proteins.
- the invention provides methods and reagents, as detailed herein, for inhibiting interactions between PL cytokine proteins and PDZ proteins to modulate an immune response.
- the inhibition or modulation occurs in a hematopoeitic cell.
- the inhibition or modulation occurs in an endothelial cell.
- an adaptor protein means a molecule (e.g., protein) that contributes to the formation of a multimolecular complex by binding two or more other biomolecuics.
- the binding of the two or more other molecules by the adaptor molecule/protein generally involves direct molecular contact between the adaptor protein and each of the two or more other moiccules.
- One exemplary PL adaptor protein is LPAP.
- the PL proteins of the invention are PL adaptor proteins.
- the invention provides methods and reagents, as detailed herein, for inhibiting interactions between PL adaptor proteins and PDZ proteins to modulate an immune response. In an embodiment, the inhibition or modulation occurs in a hematopoeitic cell.
- the inhibition or modulation occurs in an endothelial cell.
- the PL proteins of the invention are characterized by specific C-terminal (i.e., PL domain) amino acid sequences or amino acid motifs, as described elsewhere in this disclosure.
- the PL proteins of the invention bind a PDZ protein expressed in T lymphocytes, B lymphocytes, or both T and B lymphocytes.
- the PL protein binds a PDZ protein expressed in endothelial cells.
- the PL proteins and/or the PDZ protein to which it binds are not expressed in the nervous system (e.g., neurons).
- the PL protein of the invention binds only one PDZ protein listed in TABLE 2. In other embodiments, the PL protein binds 1 to 3, 3 to 5, or more than 5 different PDZ proteins listed in TABLE 2.
- the PL protein is expressed or up- regulated upon cell activation (e.g., in activated B lymphocytes, T lymphocytes) or upon entry into mitosis (e.g., up-regulation in rapidly proliferating cell populations).
- the PL protein is (i) a protein that mediates immune cell (e.g., hematopoietic cell) activation or migration, (ii) a protein that does not mediate apoptosis in a cell type, (iii) a protein that is other than a G-protein coupled seven transmembrane helix receptor, (iv) a protein that is G-protein coupled seven transmembrane helix receptor but not a cytokine receptor, or (v) a protein that is not a G-protein coupled seven transmembrane helix receptor and is a cytokine receptor.
- a protein that mediates immune cell e.g., hematopoietic cell activation or migration
- a protein that does not mediate apoptosis in a cell type e.g., hematopoietic cell
- a protein that is other than a G-protein coupled seven transmembrane helix receptor e.g.,
- PDZ proteins are expressed in immune system cells, and play a fundamental biological role in modulation of the immune response.
- PDZ proteins DLG1 and TIAM-1 have been previously. described to be in T cells.
- the present inventors discovered, using a BLAST search of the Human EST database and the experiments described infra, that several additional PDZ proteins are present in hematopoietic cells including MPP1, P-DLG, NELI-1, PSD95, syntenin in T cells and CASK, DLG1, DLG2, ZIP KINASE, syntrophin 2, P-dlg, PSD95, and syntenin in B cells.
- RNA was prepared using the "trizol" RNA preparation kit (GIBCO-BRL; Cat. # 15596-018) according to the manufacturer's recommendations. Briefly, 1-5 x 10 7 lymphoblasts were harvested by centrifugation at 200 x g for 10 minutes at 20 ° C. Cells were resuspended in 100 ⁇ l PBS buffer and 1 ml of TRIZOL reagent was added per 5 x 10 6 cells.
- the cells resuspension was mixed and after 5 minutes incubation at room temperature (RT), chloroform was added at 0.2 ml per ml TRIZOL. The resuspension was vigorously shaken and incubated for 3 more minutes at RT. Samples were then centrifuged at 12000 x g for 15 minutes at 4 ° C, the aqueous phase was recovered and RNA was precipitated with 2-propanol.
- RNA concentration and purity were determined by the measurement of 260/280 nm light absorption by the nucleic acid.
- the SUPERSCRIPT H reverse transcriptase cDNA kit (GBCO-BRL; Cat. # 18064-014) was used. RNA input per 200 ⁇ l cDNA reaction sample was 10 ⁇ g.
- PCR was conducted using primers designed to amplify specifically PDZ domain-containing regions of PDZ proteins of interest. Oligonucleotide primers were designed to amplify one or more PDZ-encoding domains.
- the DNA sequences encoding the various PDZ domains of interest were identified by inspection (i.e., conceptual translation of the PDZ protein cDNA sequences obtained from GenBank, followed by alignment with the PDZ domain amino acid sequence). TABLE 3 shows the PCR primers, the PDZ-encoded domains amplified, and the GenBank accession number of the PDZ-domain containing proteins.
- the PCR primers included endonuclease restriction sequences at then- ends to allow ligation with pGEX-3X cloning vector (Pharmacia, GenBank XXII 3852 ) in frame with glutathione-S transferase (GST).
- cDNA sequences representing the same gene have several database entries under different accession numbers and names. Accession numbers shown correspond to the gene name used in this description, and numbering of nucleotides and amino acids correlates to the Genbank entry versions specified by the given accession number. Amin ⁇ acid sequences shown coirespond to the cloned DNA portions of PDZ domain containing genes. As is apparent from the primer sequences, in some constructs, the first N-terminal and / or last C-terminal amino acid corresponds to a linker amino acid introduced by the cloning process but is not represented at that position in the corresponding gene. PCR primers were designed such (hat restriction nuclease recognition sites were generated at the ends of the RT-PCR generated fragments. Therefore, 5' primer sequences do not entirely match with the conesponding cDNA sequences.
- Syntenin Syntenin GI Eco Rl gene 3342559 R ⁇ IKQGIREVILCKDQDGKIGLRLKSID 5'- 5'-
- Enhancer connector GI ECO Rl gene enhancer of 3930780 PDZ domain 1 (of 1) 5'- KSR-like AGGGGATCCTGG GGSAATTCCGGTA protein CNK1 LEQKAVLEQVQLDSPLGLEIHTTSNCQH AACAGAAGGCCG TCGGGATCTTCCT
- sequence analysis of the PDZ clones revealed differences to the DNA and/or protein sequence as published in the databases, summarized in TABLE 3A.
- GenBank files (for accession no.; see Table 3).
- A' and G Two complementary assays, termed “A' and "G,” were developed to detect binding between a PDZ-domain polypeptide and candidate PDZ ligand. In each of the two different assays, binding is detected between a peptide having a sequence conesponding to the
- C-te ⁇ ninus of a protein anticipated to bind to one or more PDZ domains i.e. a candidate PL peptide
- a PDZ-domain polypeptide typically a fusion protein containing a PDZ domain
- the candidate PL peptide is immobilized and binding of a soluble PDZ- domain polypeptide to the immobilized peptide is detected (the "A”' assay is named for the fact that in one embodiment an avidin surface is used to immobilize the peptide).
- the PDZ-domain polypeptide is immobilized and binding of a soluble PL peptide is detected (The “G” assay is named for the fact that in one embodiment a GST-binding surface is used to immobilize the PDZ-domain polypeptide).
- Preferred embodiments of these assays are described in detail infra. However, it will be appreciated by ordinarily skilled practitioners that these assays can be modified in numerous ways while remaining useful for the purposes of the present invention.
- GST-PDZ domain fusion proteins were prepared for use in the assays of the invention.
- PCR products containing PDZ encoding domains (as described in ⁇ 6.1 s ⁇ ra) were subcloned into an expression vector to permit expression of fusion proteins containing a PDZ domain and a heterologous domain (i.e., a glutathione-S transferase sequence, "GST").
- PCR products i.e., DNA fragments
- PDZ domain encoding DNA was extracted from agarose gels using the "sephaglas" gel extraction system (Pharmacia) according to the manufacturer' s recommendations.
- PCR primers were designed to include endonuclease restriction sites to facilitate ligation of PCR fragments into a GST gene fusion vector (pGEX-3X; Pharmacia, GenBank accession no. XXU13852) in-frame with the glutathione-S transferase coding sequence.
- This vector contains a IPTG inducible lacZ promoter.
- the pGEX-3X vector was linearized using Bam HI and Eco Rl or, in some cases, Eco Rl or Sma I, as shown in TABLE 3, and dephosphorylated. For most cloning approaches, double digestion with Bam HI and Eco Rl was performed, so that the ends of the PCR fragments to clone were Bam HI and Eco Rl.
- restriction endonuclease combinations used were Bgl II and Eco Rl, Bam HI and Mfe I, or Eco Rl only, Sma I only, or BamHl only (see TABLE 3).
- the DNA portion cloned represents the PDZ domains and the cDNA portion located between individual domains. Precise locations of cloned fragments used in the assays are indicated in TABLE 3. DNA linker sequences between the GST portion and the PDZ domain containing DNA portion vary slightly, dependent on which of the above described cloning sites and approaches were used.
- the amino acid sequence of the GST-PDZ fusion protein varies in the linker region between GST and PDZ domain.
- Protein linkers sequences corresponding to different cloning sites/approaches are shown below. Linker sequences (vector DNA encoded) are bold, PDZ domain containing gene derived sequences are in italics.
- the PDZ-encoding PCR fragment and linearized pG ⁇ X-3X vector were ethanol precipitated and resuspended in 10 ul standard ligation buffer. Ligation was performed for 4-10 hours at 7°C using T4 DNA ligase. It will be understood that some of the resulting constructs include very short linker sequences and that, when multiple PDZ domains were cloned, the constructs included some DNA located between individual PDZ domains.
- the ligation products were transformed in DH5 ⁇ or BL-21 E.coli bacteria strains. Colonies were screened for presence and identity of the cloned PDZ domain containing DNA as well as for correct fusion with the glutathione S-transferase encoding DNA portion by PCR and by sequence analysis. Positive clones were tested in a small scale assay for expression of the GST/PDZ domain fusion protein and, if expressing, these clones were subsequently grown up for large scale preparations of GST/PDZ fusion protein.
- GST-PDZ domain fusion protein was overexpressed following addition of IP TG to the culture medium and purified.
- Detailed procedure of small scale and large scale fusion protein expression and purification are described in "GST Gene Fusion System” (second edition, revision 2; published by Pharmacia).
- a small culture 3-5mls
- a bacterial strain DH5 ⁇ , BL21 or JM109
- the fusion protein construct was grown overnight in LB-media at 37°C with the appropriate antibiotic selection (lOOug/ml ampicillin; a.k.a. LB- amp).
- the overnight culture was poured into a fresh preparation of LB-amp (typically 250- 500mls) and grown until the optical density (OD) of the culture was between 0.5 and 0.9 (approximately 2.5 hours).
- IPTG isopropyl ⁇ -D-thiogalactopyranoside
- IPTG isopropyl ⁇ -D-thiogalactopyranoside
- Bacteria were collect by centrifugation (4500 g) and resuspended in Buffer A- (50mM Tris, pH 8.0, 50mM dextrose, ImM EDTA, 200uM phenylmethylsulfonylfluoride).
- Buffer A+ Buffer A-, 4mg ml lysozyme
- Buffer B Buffer B
- NP40 a.k.a. IGEPAL CA-630
- 200uM phenylmethylsulfonylfluoride was added and incubated for an additional 20 min.
- the bacterial cell lysate was centrifuged (x20,000g), and supernatant was added to glutathione Sepharose 4B (Pharmacia, cat no.
- PDZ domains are known to be bound by the C-terminal residues of PDZ-binding proteins.
- cell surface receptor proteins were identified and peptides having the sequence corresponding to the C-terminus of each protein were synthesized. TABLE 4 lists these proteins, and provides corresponding C- terminal sequences and GenBank accession numbers. "Clasp 1" is described in WO 00/20434 (published 13 April 2000).
- Synthetic peptides of defined sequence can be synthesized by any standard resin-based method (see, e.g., U. S. Pat. No.4,108,846; see also, Caruthers et al., 1980, Nucleic Acids Res. Symp. Ser., 215-223; Horn et al., 1980, Nucleic Acids Res. Symp. Ser., 225-232; Roberge, et al., 1995, Science 269:202).
- the peptides used in the assays described herein were prepared by the FMOC (see, e.g., Guy and Fields, 1997, Meth.
- peptides were labeled with biotin at the ain o-terminus by reaction with a four-fold excess of biotin methyl ester in dimethylsulfoxide with a catalytic amount of base.
- the peptides were cleaved from the resin using a halide containing acid (e.g. trifluoroacetic acid) in the presence of appropriate antioxidants (e.g. ethanedithiol) and excess solvent lyophilized.
- peptides can be redissolved and purified by reverse phase high performance liquid chromatography (HPLC),
- HPLC solvent system involves a Nydac C-18 semi-preparative column running at 5 mL per minute with increasing quantities of acetonitrile plus 0.1% trifluoroacetic acid in a base solvent of water plus 0.1% trifluoroacetic acid.
- HPLC purification the identities of the peptides are confirmed by MALDI cation-mode mass spectrometry.
- exemplary biotinylated peptides are provided in TABLE 4.
- one binding partner of a PDZ-PL pair is imrnobilized, and the ability of the second binding partner to bind is determined.
- these assays can be used to identify yet more novel PDZ-PL interactions in hematopoietic cells.
- they can be used to identify antagonists of PDZ-PL interactions (see infra).
- fusion protein are used in the assays and devices of the invention. Methods for constructing and expressing fusion proteins are well known. Fusion proteins generally are described in Ausubel et al., supra, Kroll et al., 1993, D ⁇ A Cell. Biol. 12:441, and Tmai et al., 1997, Cell 91:521-30. Usually, the fusion protein includes a domain to facilitate immobilization of the protein to a solid substrate ("an immobilization domain").
- the immobilization domain includes an epitope tag (i.e., a sequence recognized by a antibody, typically a monoclonal antibody) such as polyhistidine (Bush et al, 1991, J. Biol Chem 266:13811-14), SEAP (Berger et al, 1988, Gene 66:1-10), or Ml and M2 flag (see, e.g, U.S. Pat. Nos.5,011,912; 4,851,341; 4,703,004; 4,782,137).
- the immobilization domain is a GST coding region.
- the protein in addition to the PDZ-domain and the particular residues bound by an immobilized antibody, protein A, or otherwise contacted with the surface, the protein (e.g., fusion protein), will contain additional residues.
- these are residues naturally associated with the PDZ-domain (i.e., in a particular PDZ-protein) but they may include residues of synthetic (e.g., poIy(alanine)) or heterologous origin (e.g., spacers of, e.g., between 10 and 300 residues).
- PDZ domain-containing polypeptide used in the methods of the invention are typically made by (1) constructing a vector (e.g., plasmid, phage or phagemid) comprising a polynucleotide sequence encoding the desired polypeptide, (2) introducing the vector into an suitable expression system (e.g., a prokaryotic, insec mammalian, or cell free expression system), (3) expressing the fusion protein and (4) optionally purifying the fusion protein.
- a vector e.g., plasmid, phage or phagemid
- an suitable expression system e.g., a prokaryotic, insec mammalian, or cell free expression system
- expression of the protein comprises inserting the coding sequence into an appropriate expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence required for the expression system employed, e.g., control elements including enhancers, promoters, transcription terminators, origins of replication, a suitable initiation codon (e.g., methionine), open reading frame, and translational regulatory signals (e.g., a ribosome binding site, a termination codon and a polyadenylation sequence.
- control elements including enhancers, promoters, transcription terminators, origins of replication, a suitable initiation codon (e.g., methionine), open reading frame, and translational regulatory signals (e.g., a ribosome binding site, a termination codon and a polyadenylation sequence.
- a suitable initiation codon e.g., methionine
- open reading frame e.g., open reading frame
- translational regulatory signals e.g
- the coding sequence of the fusion protein includes a PDZ domain and an immobilization domain as described elsewhere herein.
- Polynucleotides encoding the amino acid sequence for each domain can be obtained in a variety of ways known in the art; typically the polynucleotides are obtained by PCR amplification of cloned plasmids, cDNA libraries, and cDNA generated by reverse transcription of RNA, using primers designed based on sequences determined by the practitioner or, more often, publicly available (e.g., through GenBank).
- the primers include linker regions (e.g., sequences including restriction sites) to facilitate cloning and manipulation in production of the fusion construct.
- the polynucleotides corresponding to the PDZ and immobilization regions are joined, in-frame to produce the fusion protein- encoding sequence.
- the fusion proteins of the invention may be expressed as secreted proteins (e.g., by including the signal sequence encoding DNA in the fusion gene; see, e.g., Lui et al, 1993, PNAS USA, 90:8957-61) or as nonsecreted proteins.
- the PDZ-containing proteins are immobilized on a solid surface.
- the substrate to which the polypeptide is bound may in any of a variety of forms, e.g., a microtiter dish, a test tube, a dipstick, a microcentrifuge tube, a bead, a spinnable disk, and the like.
- Suitable materials include glass, plastic (e.g., polyethylene, PNC, polypropylene, polystyrene, and the like), protein, paper, carbohydrate, lipip monolayer or supported lipid bilayer, and other solid supports.
- Other materials that may be employed include ceramics, metals, metalloids, semiconductive materials, cements and the like.
- the fusion proteins are organized as an array.
- array refers to an ordered arrangement of immobilized fusion proteins, in which particular different fusion proteins (i.e., having different PDZ domains) are located at different predetermined sites on the substrate. Because the location of particular fusion proteins on the array is known, binding at that location can be correlated with binding to the PDZ domain situated at that location. Immobilization of fusion proteins on beads (individually or in groups) is another particularly useful approach, h one embodiment, individual fusion proteins are immobilized on beads. In one embodiment, mixtures of distinguishable beads are used.
- Distinguishable beads are beads that can be separated from each other on the basis of a property such as size, magnetic property, color (e.g., using FACS) or affinity tag (e.g., a bead coated with protein A can be separated from a bead not coated with protein A by using IgG affinity methods). Binding to particular PDZ domain may be determined; similarly, the effect of test compounds (i.e., agonists and antagonists of binding) may be determined.
- a property such as size, magnetic property, color (e.g., using FACS) or affinity tag (e.g., a bead coated with protein A can be separated from a bead not coated with protein A by using IgG affinity methods).
- Binding to particular PDZ domain may be determined; similarly, the effect of test compounds (i.e., agonists and antagonists of binding) may be determined.
- Methods for immobilizing proteins are known, and include covalent and non- covalent methods.
- One suitable immobilization method is antibody-mediated immobilization. According to this method, an antibody specific for the sequence of an "immobilization domain" of the PDZ-domain containing protein is itself immobilized on the substrate (e.g., by adsorption).
- One advantage of this approach is that a single antibody may be adhered to the substrate and used for immobilization of a number of polypeptides (sharing the same immobilization domain).
- an immobilization domain consisting of poly-histidine (Bush et al, 1991, J Biol Chem 266:13811-14) can be bound by an anti-histidine monoclonal antibody (R&D Systems, Minneapolis, M ⁇ ); an immobilization domain consisting of secreted alkaline phosphatase (“SEAP”) (Berger et al, 1988, Gene 66:1-10) can be bound by anti-SEAP (Sigma Chemical Company, St Louis, MO); an immobilization domain consisting of a FLAG epitope can be bound by anti-FLAG.
- SEAP secreted alkaline phosphatase
- ligand-antiligand immobilization methods are also suitable (e.g., an immobilization domain consisting of protein A sequences (Harlow and Lane, 1988, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory; Sigma Chemical Co., St. Louis, MO) can be bound by IgG; and an immobilization domain consisting of sfrepavidin can be bound by biotin (Harlow & Lane, supra; Sigma Chemical Co., St. Louis, MO).
- the immobilization domain is a GST moiety, as described herein.
- glass and plastic are especially useful substrates.
- the substrates may be printed with a hydrophobic (e.g., Teflon) mask to form wells.
- Preprinted glass slides with 3, 10 and 21 wells per 14.5 cm 2 slide "working area" are available from, e.g., SPI Supplies, West Chester, PA; also see U.S. Pat No. 4,011,350).
- a large format (12.4 cm x 8.3 cm) glass slide is printed in a 96 well format is used; this format facilitates the use of automated liquid handling equipment and utilization of 96 well format plate readers of various types (fluorescent, colorimetric, scintillation).
- higher densities may be used (e.g., more than 10 or 100 polypeptides per cm 2 ). See, e.g., MacBeath et al, 2000, Science 289:1760-63.
- antibodies are bound to substrates (e.g., glass substrates) by adsorption.
- S ⁇ itable adsorption conditions are well known in the art and include incubation of 0.5-50ug/ml (e.g., 10 ug/ml) mAb in buffer (e.g., PBS, or 50 to 300 mM Tris, MOPS, HEPES, PIPES, acetate buffers, pHs 6.5 to 8, at 4°C) to 37°C and from lhr to more than 24 hours.
- buffer e.g., PBS, or 50 to 300 mM Tris, MOPS, HEPES, PIPES, acetate buffers, pHs 6.5 to 8, at 4°C
- Proteins may be covalently bound or noncovalently attached through nonspecific bonding. If covalent bonding between a the fusion protein and the surface is desired, the surface will usually be polyfunctional or be capable of being polyfunctionalized.
- Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like. The manner of linking a wide variety of compounds to various surfaces is well known and is amply illustrated in the literature.
- the invention provides an assay in which biotinylated candidate PL peptides are immobilized on an avidin coated surface. The binding of PDZ- domain fusion protein to this surface is then measured.
- the PDZ- domain fusion protein is a GST PDZ fusion protein and the assay is carried out as follows:
- Avidin is bound to a surface, e.g. a protein binding surface.
- avidin is bound to a polystyrene 96 well plate (e.g., Nunc Polysorb (cat #475094) by addition of 100 uL per well of 20 ug/mL of avidin (Pierce) in phosphate buffered saline without calcium and magnesium, pH 7.4 ("PBS", GibcoBRL) at 4°C for 12 hours.
- PBS phosphate buffered saline without calcium and magnesium, pH 7.4
- the plate is then treated to block nonspecific interactions by addition of 200 uL per well of PBS containing 2 g per 100 mL protease-free bovine serum albumin ("PBS/BSA”) for 2 hours at 4°C.
- PBS/BSA protease-free bovine serum albumin
- Biotinylated PL peptides (or candidate PL peptides, e.g. see TABLE 4) are immobilized on the surface of wells of the plate by addition of 50 uL per well of 0.4 uM peptide in PBS BSA for 30 minutes at 4°C.
- each different peptide is added to at least eight different wells so that multiple measurements (e.g. duplicates and also measurements using different (3ST/PDZ-domain fusion proteins and a GST alone negative control) can be made, and also additional negative control wells are prepared in which no peptide is immobilized.
- the plate is washed 3 times with PBS.
- GST/PDZ-domain fusion protein (prepared as described supra) is allowed to react with the surface by addition of 50 uL per well of a solution containing 5 ug/mL GST PDZ-domain fusion protein in PBS BSA for 2 hours at 4°C.
- GST alone i.e. not a fusion protein
- specified wells generally at least 2 wells (i.e. duplicate measurements) for each immobilized peptide.
- the plate is washed 3 times with PBS to remove unbound fusion protein.
- the binding of the GST/PDZ-domain fusion protein to the avidin- biotinylated peptide surface can be detected using a variety of methods, and detectors known in the art.
- 50 uL per well of an anti-GST antibody in PBS/BSA (e.g. 2.5 ug/mL of polyclonal goat-anti-GST antibody, Pierce) is added to the plate and allowed to react for 20 minutes at 4°C.
- the plate is washed 3 times with PBS and a second, delectably labeled antibody is added.
- 50 uL per well of 2.5 ug/mL of horseradish peroxidase (HRP)-conjugated polyclonal rabbit anti-goat inununoglobulin antibody is added to the plate and allowed to react for 20 minutes at 4°C.
- HRP horseradish peroxidase
- the plate is washed 5 times with 50 mM Tris pH 8.0 containing 0.2% Tween 20, and developed by addition of 100 uL per well of HRP-substrate solution (TMB, Dako) for 20 minutes at room temperature (RT).
- TMB HRP-substrate solution
- RT room temperature
- the reaction of the HRP and its substrate is terminated by the addition of 100 uL per well of IM sulfuric acid and the optical density (O.D.) of each well of the plate is read at 450 nm.
- Specific binding of a PL peptide and a PDZ-domain polypeptide is detected by comparing the signal from the well(s) in which the PL peptide and PDZ domain polypeptide are combined with the background signal(s).
- the background signal is the signal found in the negative controls.
- a specific or selective reaction will be at least twice background signal, more typically more than 5 times background, and most typically 10 or more times the background signal.
- a statistically significant reaction will involve multiple measurements of the reaction with the signal and the background differing by at least two standard errors, more typically four standard errors, and most typically six or more standard errors.
- a statistical test e.g.
- GST/PDZ-domain fusion protein to an avidin surface not exposed to (i.e. not covered with) the PL peptide is one suitable negative control (sometimes referred to as "B").
- the signal from binding of GST polypeptide alone (i.e. not a fusion protein) to an avidin-coated surface that has been exposed to (i.e. covered with) the PL peptide is a second suitable negative control (sometimes referred to as "B2"). Because all measurements are done in multiples (i.e. at least dupUcate) the arithmetic mean (or, equivalently, average) of several measurements is used in determining the binding, and the standard error of the mean is used in determining the probable error in the measurement of the binding.
- the standard error of the mean of N measurements equals the square root of the following: the sum of the squares of the difference between each measurement and the mean, divided by the product of (N) and (N-l).
- specific binding of the PDZ protein to the plate-bound PL peptide is determined by comparing the mean signal ("mean S") and standard error of the signal ("SE") for a particular PL-PDZ combination with the mean Bl and/or mean B2.
- mean S mean signal
- SE standard error of the signal
- the invention provides an assay in which a GST/PDZ fusion protein is immobilized on a surface ("G" assay). The binding of labeled PL peptide (as listed in TABLE 4) to this surface is then measured.
- the assay is carried out as follows:
- a PDZ-domain polypeptide is bound to a surface, e.g. a protein binding surface.
- a GST/PDZ fusion protein containing one or more PDZ domains is bound to a polystyrene 96-well plate.
- the GST/PDZ fusion protein can be bound to the plate by any of a variety of standard methods known to one of skill in the art, although some care must be taken that the process of binding the fusion protein to the plate does not alter the ligand-binding properties of the PDZ domain.
- the GST/PDZ fusion protein is bound via an anti-GST antibody that is coated onto the 96-well plate. Adequate binding to the plate can be achieved when: a.
- Biotinylated PL peptides (or candidate PL peptides, e.g. as shown in TABLE 4) are allowed to react with the surface by addition of 50 uL per well of 20 uM solution of the biotinylated peptide in PBS/BSA for 10 minutes at 4°C, followed by an additional 20 minute incubation at 25°C. The plate is washed 3 times with ice cold PBS.
- the binding of the biotinylated peptide to the GST/PDZ fusion protein surface can be detected using a variety of methods and detectors known to one of skill in the art.
- 100 uL per well of 0.5 ug/mL streptavidin-horse radish peroxidase (HRP) conjugate dissolved in BSA/PBS is added and allowed to react for 20 minutes at 4°C.
- the plate is then washed 5 times with 50 mM Tris pH 8.0 containing 0.2% Tween 20, and developed by addition of 100 uL per well of HRP-substrate solution (TMB, Dako) for 20 minutes at room temperature (RT).
- TMB HRP-substrate solution
- RT room temperature
- the reaction of the HRP and its substrate is terminated by addition of 100 uL per well of I M sulfuric acid, and the optical density (O.D.) of each well of the plate is read at 450 urn.
- Specific binding of a PL peptide and a PDZ domain polypeptide is determined by comparing the signal from the well(s) in which the PL peptide and PDZ domain polypeptide are combined, with the background signal(s).
- the background signal is the signal found in the negative contrbl(s).
- a specific or selective reaction will be at least twice background signal, more typically more than 5 times background, and most typically 10 or more times the background signal.
- a statistically significant reaction will involve multiple measurements of the reaction with the signal and the background differing by at least two standard errors, more typically four standard errors, and most typically six or more standard errors.
- a statistical test e.g.
- the signal from binding of a given PL peptide to immobilized (surface bound) GST polypeptide alone is one suitable negative control (sometimes referred to as "B 1"). Because all measurement are done in multiples (i.e. at least duplicate) the arithmetic mean (or, equivalently, average.) of several measurements is used in determining the binding, and the standard error of the mean is used in determining the probable error in the measurement of the binding.
- the standard error of the mean of N measurements equals the square root of the following: the sum of the squares of the difference between each measurement and the mean, divided by the product of (N) and (N-l).
- specific binding of the PDZ protein to the platebound peptide is determined by comparing the mean signal ("mean S") and standard error of the signal ("SE”) for a particular PL-PDZ combination with the mean Bl. In experiments summarized in TABLE 2, binding was determined to be specific (denoted
- the modified assays use lesser quantities of labeled PL peptide and have slightly different biochemical requirements for detection of PDZ-ligand binding compared to the specific assay conditions described supra.
- the assay conditions described in this section are referred to as the "G' assay” and the “G” assay,” with the specific conditions described in ⁇ 6.2.3.2 being referred to as the "G° assay.”
- the "G' assay” is identical to the "G° assay” except at step (2) the peptide concentration is 10 uM instead of 20 uM. This results in slightly lower sensitivity for detection of interactions with low affinity and or rapid dissociation rate. Correspondingly, it slightly increases the certainty that detected interactions are of sufficient affinity and half-life to be of biological importance and useful therapeutic targets.
- the “G” assay” is identical to the “G° assay” except that at step (2) the peptide concentration is 1 uM instead of 20 uM and the incubation is performed for 60 minutes at 25 °C (rather than, e.g., 10 minutes at 4°C followed by 20 minutes at 25°C). This results in lower sensitivity for interactions of low affinity, rapid dissociation rate, and/or affinity that is less at 25°C than at 4°C. Interactions will have lower affinity at 25°C than at 4°C if (as we have found to be generally true for PDZ-ligand binding) the reaction entropy is negative (i.e. the entropy of the products is less than the entropy of the reactants).
- the PDZ-PL binding signal may be similar in the "G” assay” and the “G° assay” for interactions of slow association and dissociation rate, as the PDZ-PL complex will accumulate during the longer incubation of the "G” assay.”
- comparison of results of the "G” assay” and the “G° assay” can be used to estimate the relative entropies, enthalpies, and kinetics of different PDZ-PL interactions.
- thermodynamics and kinetics of PDZ-PL interactions can be used in the design of efficient inhibitors of the interactions.
- a small molecule inhibitor based onthe chemical structure of a PL that dissociates slowly from a given PDZ domain may itself dissociate slowly and thus be of high affinity.
- step (2) of the "G assay” variation of the temperature and duration of step (2) of the "G assay” can be used to provide insight into the kinetics and thermodynamics of the PDZ-ligand binding reaction and into design of inhibitors of the reaction.
- the PDZ-PL detection assays can employ a variety of surfaces to bind the PL and PDZ-containing proteins.
- a surface can be an "assay plate" which is formed from a material (e.g. polystyrene) which optimizes adherence of either the PL protein or PDZ- containing protein thereto.
- the individual wells of the assay plate will have a high surface area to volume ratio and therefore a suitable shape is a flat bottom well (where the proteins of the assays are adherent).
- Other surfaces include, but are not limited to, polystyrene or glass beads, polystyrene or glass slides, and alike.
- the assay plate can be a "microtiter" plate.
- microtiter plate when used herein refers to a multiwell assay plate, e.g., having between about 30 to 200 individual wells, usually 96 wells. Alternatively, high density arrays can be used. Often, the individual wells of the microtiter plate will hold a maximum volume of about 250 ul.
- the assay plate is a 96 well polystyrene plate (such as that sold by Becton Dickinson Labware, Lincoln Park, N.J.), which allows for automation and high throughput screening. Other surfaces include polystyrene microtiter ELISA plates such as that sold by Nunc Maxisorp, Inter Med, Denmark. Often, about 50 ul to 300 ul, more preferably 100 ul to 200 ul, of an aqueous sample comprising buffers suspended therein will be added to each well of the assay plate.
- the detectable labels of the invention can be any detectable compound or composition which is conjugated directly or indirectly with a molecule (such as described above).
- the label can be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can catalyze a chemical alteration of a substrate compound or composition which is detectable.
- the preferred label is an enzymatic one which catalyzes a color change of a non-radioactive color reagent.
- the label is indirectly conjugated with the antibody.
- One of skill is aware of various techniques for indirect conjugation.
- the antibody can be conjugated with biotin and any of the categories of labels mentioned above can be conjugated with avidin, or vice versa (see also "A” and “G” assay above).
- Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
- the antibody is conjugated with a small hapten (e.g. digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g. anti-digoxin antibody).
- a small hapten e.g. digoxin
- an anti-hapten antibody e.g. anti-digoxin antibody
- washing is meant exposing the solid phase to an aqueous solution (usually a buffer or cell culture media) in such a way that unbound material (e.g., non-adhering cells, non-adhering capture agent, unbound ligand, receptor, receptor construct, cell lysate, or HRP antibody) is removed therefrom.
- a detergent e.g., Triton X
- the aqueous washing solution is decanted from the wells of the assay plate following washing. Conveniently, washing can be achieved using an automated washing device. Sometimes, several washing steps (e.g., between about 1 to 10 washing steps) can be required.
- blocking buffer refers to an aqueous, pH buffered solution containing at least one blocking compound which is able to bind to exposed surfaces of the substrate which are not coated with a PL or PDZ- containing protein.
- the blocking compound is normally a protein such as bovine serum albumin (BSA), gelatin, casein or milk powder and does not cross-react with any of the reagents in the assay.
- BSA bovine serum albumin
- the block buffer is generally provided at a pH between about 7 to 7.5 and suitable buffering agents include phosphate and TRIS.
- suitable buffering agents include phosphate and TRIS.
- enzyme-substrate combinations include, for example:
- HRPO Horseradish peroxidase
- AP alkaline phosphatase
- para-Nitrophenyl phosphate as chromogenic substrate.
- ⁇ -D-galactosidase ( ⁇ D-Gal) with a chromogenic substrate (e.g. p- nitrophenyl- ⁇ -D-galactosidase) or fluorogenic substrate 4-methylumbellife ⁇ yl- ⁇ -D- galactosidase.
- a chromogenic substrate e.g. p- nitrophenyl- ⁇ -D-galactosidase
- fluorogenic substrate 4-methylumbellife ⁇ yl- ⁇ -D- galactosidase 4-methylumbellife ⁇ yl- ⁇ -D- galactosidase.
- TABLE 2 shows the results of assays in which, specific binding was detected using the "A” and “G” assays described herein.
- the top row of the table specifies the source of the PDZ domain used in the GST-PDZ fusion proteins (see TABLE 3).
- the first column lists the cell surface proteins from which C-terminal peptide sequences were derived and the second column ("code") identifies the peptide used in the assay (see TABLE 4).
- code identifies the peptide used in the assay (see TABLE 4).
- the third column, “Seq” provides the sequence of the four (4) C-terminal residues of the cell surface protein and peptide.
- A indicates specific binding as detected in the "A” assay.
- “G” indicates specific binding as shown in the "G” assay.
- a blank indicates that no specific binding was detected using the "A” or “G” assays.
- An asterisk (*) indicates that a pairwise interaction between the PDZ protein and the
- TABLE 2 shows the results of assays (referred to as "PRISM MATRIX”) to detect binding between PDZ proteins and candidate PL peptides.
- PRISM MATRIX results of assays
- a number of specific PDZ-PL interactions are identified by the MATRIX and key amino acids and positions important in PDZ binding ("PL motifs") are deduced from these results.
- the assay can further aid in the rapid discovery and characterization of novel PL proteins and PL motifs to help in rational drug design and synthesis of PL-PDZ interaction inhibitors.
- PL motifs important in PDZ binding e.g., the C-terminal motifs S/T-X-V/I L (for DLG1) and Y/F-Y/F-I/L/F for MPP1 (see, Doyle et al., 1996, Cell 85, 1067; Songyang et al., 1997, Science 275, 73).
- the reported motifs are not sufficiently specific (i.e. a large number of proteins meet these criteria yet are not necessarily actual PDZ ligands) and cover only a small number of PDZ proteins (approximately 10).
- the PRISM MATRIX can be used to determine ligand specificity and to deduce ligand binding motifs for any PDZ protein because it can precisely determine sequences of amino acids that do or do not result in specific PDZ binding.
- the assay has revealed a significant of new PDZ domain binding motifs (i.e.
- PL motifs C-terminal sequence of CD6, ISAA (SEQ ID NO: 14); C- terminal sequence of CD49E, TSDA (SEQ ID NO: 24); C- terminal sequence of CD49F, TSDA (SEQ ID NO: 24); C-terminal sequence of Clasp-1, SAEV (SEQ ID NO: 175); C- terminal sequence of CLASP-4, YAEV (SEQ ID NO: 192); C- terminal sequence of CD44, KIGV (SEQ ID NO: 104); C- terminal sequence of Fas Ligand, LYKL (SEQ ID NO: _ ; C-terminal sequence of EL5R, DSVF (SEQ ID NO: 94); C- terminal sequence of BLR-1, LTTF (SEQ ID NO: 217).
- novel PL sequences allow the definition of novel PL motifs (See TABLE 5A, infra).
- the specificity with which these novel motifs are defined is enhanced by the fact that the MATRIX reports both positive results (i.e. PDZ-PL) combinations that result in specific binding interactions) and negative results (i.e. PDZ-PL combinations that do not result in specific binding),
- the C-terminal sequence of CD6, SAA and the C-terminal sequence of CD49E, SDA bind to the PDZ-domain polypeptide 41.8 while the related C-terminal sequence of CD166, TEA and C- terminal sequence of CD148, YIA do not.
- X* is any non-aromatic amino acid (any residue other than Y, F or W).
- the invention provides a method for identifying a PDZ-domain binding protein
- PDZ ligand or PL that binds to a specified PDZ protein.
- a plurality of putative PL peptides e.g., peptides or polypeptides that include at or near their carboxy terminus a sequence of the C-terminus of a naturally occurring protein known or suspected of being a PL protein, i.e., binding to at least one PDZ domain
- Binding assays typically the A and G assays as described elsewhere herein, are carried out to identify peptides that do and do not bind the specified PDZ protein (e.g., by detecting binding to a PDZ domain sequence from the specified PDZ protein).
- PLs that bind the specified PDZ are identified, and typically at least a plurality (e.g., in this context, at least 10, more often at least 20, and typically at least 40 or more) PLs that dp not bind the PDZ are identified.
- sequences of the binding and nonbinding peptides are compared, for example as described infra, and a motif(s) characteristic of peptides that bind the PDZ domain sequence and not characteristic of peptides that do not bind the PDZ domain sequence is determined.
- known proteins are examined to identify sequences (typically at or near the c-terminus, e.g., within 1, 2 or 3 residues of the c-terminus) that match the motif identified.
- the search parameters may include other characteristics of the protein sequences being searched, such as an expression property (e.g., expression in a particular cell type, e.g., lymphocytes, or disease state), a functional property (e.g., receptor activity), and/or a structural property (e.g., similarity to a reference sequence).
- an expression property e.g., expression in a particular cell type, e.g., lymphocytes, or disease state
- a functional property e.g., receptor activity
- a structural property e.g., similarity to a reference sequence.
- this identification is carried out, at least in part, by a computer- implemented search of a database such as GenBank for proteins having the specified motif, although comparison can be made manually, particularly when the search is limited to a specific class of putative PLs.
- the assay also includes the further step of characterizing or confirming the binding properties of the identified PL(s) and PDZ, typically by carrying out in vivo or in vitro binding assays described herein (e.g., the G assay) or known in the art (e.g., precipitation assays).
- a PRISM MATRIX i.e., the representation of PL-PDZ binding interactions, e.g., interactions occuring in lymphocytes, e.g., as shown in TABLE 2 is used to identify C-terminal peptide sequence motifs characteristic of PLs (i.e., sequences that mediate binding to a particular PDZ domain-containing protein).
- the MATRIX is specifically arranged to facilitate identification of these motifs.
- the PL ligands in the MATRIX shown in TABLE 2 are ordered on the basis of C-terminal amino acid similarity, with weight given to residues reported to be important in PDZ binding (Doyle et al., 1996, Cell 85, 1067).
- peptides are first ordered based on the most C-terminal residues (zero position) in the following amino acid order: G, A, C, S, T, N, Q, D, E, H, K, R, V, I, L, M, P, F, Y, W.
- the same raking scheme was then applied to the next most important residue for peptide binding, the -2 position, followed by the -1 position and the -3 position.
- the PDZ domains of each of the GST-PDZ fusion proteins in the MATRIX are also ordered based on amino acid sequence similarity, in this case based on multiple sequence alignment using the CLUSTAL software package.
- the GST-PDZ fusions can also be arranged to give additional weight in alignment to residues known in the art to be important for ligand binding.
- amino acids constitute the motif at the C-terminus.
- the C-terminal amino acids from ligand CLASP-1 to ligand CD34 range from V to L (including positive interactions with ligands terminating in I, the amino acid separating V and L in the amino acid order supra).
- the amino acid separating V and L in the amino acid order supra is V/I/L.
- a preliminary motif at the -2 position identify a subdivision of the overall region, such that the subdivision contains several interactions and is surrounded by areas of no or few interactions.
- the amino acid(s) present at the -2 position in the subdivision For example, in the case of DLG1, one subdivision of many interactions is from the ligand CDW128A to the ligand DOCK2.
- the amino acid(s) present at the -2 position in this subdivision are S and T (adjacent in the amino acid ordering), defining the — 2 position motif S/T.
- the set of preferred versus disfavored amino acids at the -1 position, identified in step 6 supra, is examined. If these amino acids fall into logical structural categories, e.g. the preferred amino acids are all hydrophilic while the disfavored amino acids are all hydrophobic, this confirms a -1 position motif.
- preferred amino acids include R, E, and G, and disfavored include R and E. Since these sets overlap, there appears to be no -1 position motif.
- DLGl, PSD95, NeDLG, 41 kd, W P3) bind a sufficient number of ligands in the current MATRIX for the above algorithm to be practical.
- motifs are D/E/K/R (all charged amino acids) or X (any amino acid) (as the set ⁇ Y, V, K, E, E ⁇ does not have a clear structural basis). 4. Revise the motifs at each position based on ligands that failed to bind to this PDZ. To do this, assemble a net motif using the least restrictive proposed motif at each position. Next determine how many ligands fitting this least restrictive overall motif fail to bind to this PDZ. Then see if the more restrictive proposed motif at each position would succeed in preventing these ligands from being predicted to bind. If so, the more restrictive motif is adopted.
- the least restrictive net motif (based on the above) is X - A S/T/Y - F/Y/D/E/K R - V/I/L.
- Fifteen different ligands in TABLE 2 (ten of which do not bind AF6) are included by this motif. Restricting the motif to F/Y at the -1 position eliminates all of the false predictions, while eliminating a minority (two of five) of the accurate predictions. As noted above, this also results in a more logical structural basis for the motif.
- X - A/S/T/Y - F/Y - V/I/L appears to be a promising motif. 5. Check whether the promising motif includes accurately predicts all of the "strong" interactions.
- PDZ domain-containing proteins in the MATRIX that bind to only one or two ligands provide a special challenge for defining PL motifs.
- the combination of a PDZ domain binding to even a single ligand combined with the failure of that PDZ to bind related ligands allows prediction of a motif specific to that PDZ.
- the following process can be applied to define a PDZ-specific ligand motif based on only a single ligand that binds to that PDZ:
- a preliminary guess at the C-terminal motif be X - XI - X - X2, where XI and X2 refer to amino acids chemically similar or identical to the amino acid found at the -2 position and zero positions respectively of the ligand that is known to bind (X is any amino acid).
- X is any amino acid.
- the C-terminal sequence known to bind is that of CD105, SSMA, therefore the preliminary motif is X - A/S/T/Y - X - A/S/V.
- K545 this means excluding the ligand CD49F (C-terminus TSDA) which is most easily accomplished by placing a limit on the -1 position (the zero and -2 positions are identical to SSMA, and the -3 position is very similar).
- a logical limit on the -1 position is to hydrophobic amino acids.
- one sufficient motif i.e. that only predicts the interaction which occurs is X -A/S/T/Y-M- A/S/V.
- ligands not already in the MATRIX that bind to a specific PDZ protein.
- Such searches can be performed using publicly available software such as BLAST (available at www.ncbi.nlm.nih.gov'). Knowledge of these other ligands is of practical value in several respects:
- Table 6 shows examplary PL motifs derived from the PRISM MATRIX according to the invention.
- the "A” and “G” assays of the invention can be used to determine the "apparent affinity" of binding of a PDZ ligand peptide to a PDZ-domain polypeptide.
- Apparent affinity is determined based on the concentration of one molecule required to saturate the binding of a second molecule (e.g., the binding of a ligand to a receptor).
- Two particularly useful approaches for quantitation of apparent affinity of PDZ-ligand binding are provided infra.
- a GST/PDZ fusion protein, as well as GST alone as a negative control, are bound to a surface (e.g., a 96-well plate) and the surface blocked and washed as described supra for the "G" assay.
- a surface e.g., a 96-well plate
- a solution of biotinylated PL peptide (e.g. as shown in TABLE 4) is added to the surface in increasing concentrations in PBS/BSA (e.g. at 0.1 uM, 0.33 uM, 1 uM, 3.3 uM, 10 uM, 33 uM, and 100 uM).
- the PL peptide is allowed to react with the bound GST/PDZ fusion protein (as well as the GST alone negative control) for 10 minutes at 4°C followed by 20 minutes at 25°C. The plate is washed 3 times with ice cold PBS to remove unbound labeled peptide.
- the net binding signal is determined by subtracting the binding of the peptide to GST alone from the binding of the peptide to the GST/PDZ fusion protein. The net binding signal is then plotted as a function of ligand concentration and the plot is fit (e.g.
- FIGURE 2 shows varying concentrations of biotinylated CLASP-2 (FIG.2A) or Fas (FIG.2B). C-terminal peptides reacted with immobilized (plate bound) GST polypeptide or GST/PDZ fusion proteins (GST/DLG1, GST/NeDLG, and GDT/PSD95) in duplicate.
- the signals were normalized, plotted and fit to a saturation binding curve, yielding an apparent affinity of 21 uM for DLGl -CLASP-2 interaction, 7.5 uM for NeDLG-CLASP-2 interaction, 45 uM for PSD95-CLASP-2 interaction, and 54 uM for DLGl -Fas interaction, 54 uM for NeDLG-Fas interaction, and 85 uM for PSD95-Fas interaction.
- Approach 2 (1) A fixed concentration of a PDZ-domain polypeptide and increasing concentrations of a labeled PL peptide (labeled with, for example, biotin or fluorescein, see TABLE 4 for representative peptide amino acid sequences) are mixed together in solution and allowed to react.
- preferred peptide concentrations are 0.1 uM, 1 uM, 10 uM, 100 uM, 1 mM.
- appropriate reaction times can range from 10 minutes to 2 days at temperatures ranging from 4°C to 37°C.
- the identical reaction can also be carried out using a non-PDZ domain-containing protein as a control (e.g., if the PDZ-domain polypeptide is fusion protein, the fusion partner can be used).
- (2) PDZ-ligand complexes can be separated from unbound labeled peptide using a variety of methods known in the art.
- the complexes can be separated using higb performance size-exclusion chromatography (HPSEC, gel filtration) (Rabinowitz et al., 1998, Immunity 9:699), affinity chromatography(e.g. using glutathione Sepharose beads), and affinity absorption (e.g., by binding to an anti-GST-coated plate as described supra).
- higb performance size-exclusion chromatography HPSEC, gel filtration
- affinity chromatography e.g. using glutathione Sepharose beads
- affinity absorption e.g., by binding to an anti-GST-coated plate as described supra.
- the PDZ-ligand complex is detected based on presence of the label on the peptide ligand using a variety of methods and detectors known to one of skill in the art For example, if the label is fluorescein and the separation is achieved using HPSEC, an in-line fluorescence detector can be used. The binding can also be detected as described supra for the G assay.
- the PDZ-ligand binding signal is plotted as a function of ligand concentration and the plot is fit. (e.g., by using the Kaleidagraph software package curve fitting algorithm) to the following equation, where "Signal ⁇ igand]” is the binding signal at PL peptide concentration "[ligand],” “Kd” is the apparent affinity of the binding event, and "Saturation Binding” is a constant determined by the curve fitting algorithm to optimize the fit to the experimental data:
- the invention provides a method of determining the apparent affinity of binding between a PDZ domain and a ligand by immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with a plurality of different concentrations of the ligand, determining the amount of binding of the ligand to the immobilized polypeptide at each of the concentrations of ligand, and calculating the apparent affinity of the binding based on that data.
- the polypeptide comprising the PDZ domain and a non-PDZ domain is a fusion protein.
- the e.g., fusion protein is GST-PDZ fusion protein
- other polypeptides can also be used (e.g., a fusion protein including a PDZ domain and any of a variety of epitope tags, biotinylation signals and the like) so long as the polypeptide can be immobilized In an orientation that does not abolish the ligand binding properties of the PDZ domain, e.g, by tethering the polypeptide to the surface via the non-PDZ domain via an anti-domain antibody and leaving the PDZ domain as the free end. It was discovered, for example, reacting a PDZ- GST fusion polypeptide directly to a plastic plate provided suboptimal results.
- binding affinity itself can be determined using any suitable equation (e.g., as shown supra; also see Cantor and Schimmel (1980) BIOPHYSICAL CHEMISTRY WH Freeman & Co., San Francisco) or software.
- the polypeptide is immobilized by binding the polypeptide to an immobilized immunoglobulin that binds the non-PDZ domain (e.g., an anti- GST antibody when a GST-PDZ fusion polypeptide is used).
- the step of contacting the ligand and PDZ-domain polypeptide is carried out under the conditions provided supra in the description of the "G" assay. It will be appreciated that binding assays are conveniently carried out in multiwell plates (e.g., 24-well, 96-well plates, or 384 well plates).
- the present method has considerable advantages over other methods for measuring binding affinities PDZ-PL affinities, which typically involve contacting varying concentrations of a GST-PDZ fusion protein to a ligand-coated surface.
- some previously described methods for determining affinity e.g., using immobilized ligand and GST-PDZ protein in solution
- an estimate of the relative strength of binding of different PDZ-PL pairs can be made based on the absolute magnitude of the signals observed in the "G assay.” This estimate will reflect several factors, including biologically relevant aspects of the interaction, including the affinity and the dissociation rate. For comparisons of different ligands binding to a given PDZ domain-containing protein, differences in absolute binding signal likely relate primarily to the affinity and/or dissociation rate of the interactions of interest.
- the assays described supra and other assays can also be used to identify the binding of other molecules (e.g., peptide mimetics, small molecules, and the like) to PDZ domain sequences.
- other molecules e.g., peptide mimetics, small molecules, and the like
- combinatorial and other libraries of compounds can be screened, e.g., for molecules that specifically bind to PDZ domains in hematopoietic cells. Screening of libraries can be accompUshed by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol.
- screening can be carried out by contacting the library members with a hematopoietic cell PDZ-domain polypeptide immobilized on a solid support (e.g. as described supra in the "G” assay) and harvesting those library members that bind to the protein.
- a solid support e.g. as described supra in the "G” assay
- panning techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited hereinabove.
- the two-hybrid system for selecting interacting proteins in yeast can be used to identify molecules that specifically bind to a PDZ domain- containing protein. Furthermore, the identified molecules are further tested for their ability to inhibit transmembrane receptor interactions with a PDZ domain.
- antagonists of an interaction between a PDZ protein and a PL protein are identified.
- a modification of the "A” assay described supra is used to identify antagonists.
- a modification of the "G” assay described supra is used to identify antagonists.
- screening assays are used to detect molecules that specifically bind to PDZ domains in hematopoietic cells.
- Such molecules are useful as agonists or antagonists of PDZ-protein-mediated cell function (e.g., cell activation, e.g., T cell activation, vesicle transport, cytokine release, growth factors, transcriptional changes, cytoskeletin rearrangement, cell movement, chemotaxis, and the like).
- cell activation e.g., T cell activation, vesicle transport, cytokine release, growth factors, transcriptional changes, cytoskeletin rearrangement, cell movement, chemotaxis, and the like.
- such assays are performed to screen for leukocyte activation inhibitors for drug development.
- the invention thus provides assays to detect molecules that specifically bind to PDZ domain- containing proteins in hematopoietic cells.
- recombinant cells expressing PDZ domain-encoding nucleic acids can be used to produce PDZ domains in these assays and to screen for molecules that bind to the domains.
- Molecules are contacted with the PDZ domain (or fragment thereof) under conditions conducive to binding, and then molecules that specifically bind to such domains are identified. Methods that can be used to carry out the foregoing are commonly known in the art.
- antagonists are identified by conducting the A or G assays in the presence and absence of a known or candidate antagonist. When decreased binding is observed in the presence of a compound, that compound is identified as an antagonist Increased binding in the presence of a compound signifies that the compound is an agonist
- a test compound in one assay, can be identified as an inhibitor (antagonist) of binding between a PDZ protein and a PL protein by contacting a PDZ domain polypeptide and a PL peptide in the presence and absence of the test compound, under conditions in which they would (but for the presence of the test compound) form a complex, and detecting the formation of the complex in the presence and absence of the test compound.
- the PL peptide comprises an amino acid sequence substantially identical to the C-terminal sequence of a PL protein (e.g., CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, NCAM1, Clasp-2, CD95, D ⁇ AM-1, CD83, CD44, CD4, CD97, Neurexin, CD3n, DOCK2, CD34, FceRIb, or FasLigand).
- a PL protein e.g., CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, NCAM1, Clasp-2, CD95, D ⁇ AM-1, CD83, CD44, CD4, CD97, Neurexin, CD3n, DOCK2, CD34, FceRIb, or FasLigand.
- the "G” assay is used in the presence or absence of an candidate inhibitor.
- the "A” assay is used in the presense or absence of a candiate inhibitor.
- one or more PDZ domain- containing GST-fusion proteins are bound to the surface of wells of a 96-well plate as described supra (with appropriate controls including nonfusion GST protein). All fusion proteins are bound in multiple wells so that appropriate controls and statistical analysis can be done.
- a test compound in BSA PBS (typically at multiple different concentrations) is added to wells.
- a detectably labeled (e.g., biotinylated) peptide known to bind to the relevant PDZ domain is added in each of the wells at a final concentration of, e.g., between about 2 uM and about 40 uM, typically 5 uM, 15 uM, or 25 uM.
- This mixture is then allowed to react with the PDZ fusion protein bound to the surface for 10 minutes at 4°C followed by 20 minutes at 25°C. The surface is washed free of unbound peptide three times with ice cold PBS and the amount of binding of the peptide in the presence and absence of the test compound is determined.
- the level of binding is measured for each set of replica wells (e.g. duplicates) by subtracting the mean GST alone background from the mean of the raw measurement of peptide binding in these wells.
- the A assay is carried out in the presence or absence of an test candidate to identify inhibitors of PL-PDZ interactions.
- a test compound is determined to be a specific inhibitor of the binding of the PDZ domain (P) and a PL (L) sequence when, at a test compound concentration of less than or equal to 1 mM (e.g., less than or equal to: 500 uM, 100 uM, 10 uM, 1 uM, 100 nM or 1 nM) the binding of P to L in the presence of the test compound less than about 50% of the binding in the absence of the test compound, (in various embodiments, less than about 25%, less than about 10%, or less than about 1%).
- the net signal of binding of P to L in the presence of the test compound plus six (6) times the standard error of the signal in the presence of the test compound is less than the binding signal in the absence of the test compound.
- assays for an inhibitor are carried out using a single PDZ protein-PL protein pair (e.g., a PDZ domain fusion protein and a PL peptide).
- the assays are carried out using a plurality of pairs, such as a plurality of different pairs listed in TABLE 2.
- antagonists can be identified by carrying out a series of assays using a candidate inhibitor and different PL-PDZ pairs (e.g., as shown in the matrix of TABLE 2) and comparing the results of the assays. All such pairwise combinations are contemplated by the invention (e.g., test compound inhibits binding of PLi to PDZi to a greater degree than it inhibits binding of PLi to PDZ 2 or PL 2 to PDZ 2 ).
- test compound inhibits binding of PLi to PDZi to a greater degree than it inhibits binding of PLi to PDZ 2 or PL 2 to PDZ 2 ).
- inhibitors with different specificities can readily be designed.
- the Ki (“potency") of an inhibitor of a PDZ-PL interaction can be determined.
- Ki is a measure of the concentration of an inhibitor required to have a biological effect
- administration of an inhibitor of a PDZ-PL interaction in an amount sufficient to result in an intracellular inhibitor concentration of at least between about 1 and about 100 Ki is expected to inhibit the biological response mediated by the target PDZ-PL interaction.
- the Kd measurement of PDZ-PL binding as determined using the methods supra is used in determining Ki
- the invention provides a method of determining the potency
- the polypeptide is immobilized by binding the polypeptide to an immobilized immunoglobulin that binds the non-PDZ domain.
- This method which is based on the "G” assay described supra, is particularly suited for high-throughput analysis of the Kli for inhibitors of PDZ-ligand interactions. Further, using this method, the inhibition of the PDZ-ligand interaction itself is measured, without distortion of measurements by avidity effects.
- the concentration of ligand and concentrations of inhibitor are selected to allow meaningful detection of inhibition.
- the concentration of the ligand whose binding is to be blocked is close to or less than its binding affinity (e.g., preferably less than the 5x Kd of the interaction, more preferably less than 2x Kd, most preferably less than lx Kd).
- the ligand is typically present at a concentration of less than 2 Kd (e.g., between about 0.01 Kd and about 2 Kd) and the concentrations of the test inhibitor typically range from 1 nM to 100 uM (e.g. a 4-fold dilution series with highest concentration 10 uM or 1 mM).
- the Kd is determined using the assay disclosed supra.
- Tj is expressed as a molar concentration.
- an enhancer (sometimes referred to as, augmentor or agonist) of binding between a PDZ domain and a ligand is identified by immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with the ligand in the presence of a test agent and determining the amount of ligand bound, and comparing the amount of ligand bound in the presence of the test agent with the amount of ligand bound by the polypeptide in the absence of the test agent.
- At least two-fold (often at least 5-fold) greater binding in the presence of the test agent compared to the absence of the test agent indicates that the test agent is an agent that enhances the binding of the PDZ domain to the ligand.
- agents that enhance PDZ-ligand interactions are useful for disruption (dysregulation) of biological events requiring normal PDZ-ligand function (e.g., cancer cell division and metastasis, and activation and migration of immune cells).
- the invention also provides methods for determining the "potency" or "K enhancer " of an enhancer of a PDZ- ligand interaction.
- the K enhancer of an enhancer of a PDZ-PL interaction can be determined, e.g., using the Kd of PDZ- PL binding as determined using the methods described supra.
- Kcnhancer is a measure of the concentration of an enhancer expected to have a biological effect.
- administering in an amount sufficient to result in an intracellular inhibitor concentration of at least between about 0.1 and about 100 Kenhancer (e.g., between about 0.5 and about 50 Kenhanc er ) is expected to disrupt the biological response mediated by the target PDZ-PL interaction.
- Kenhancer e.g., between about 0.5 and about 50 Kenhanc er
- the invention provides a method of detern ⁇ iing the potency (K e n h a nc er) of an enhancer or suspected enhancer of binding between a PDZ domain and a ligand by immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with a plurality of different mixtures of the ligand and enhancer, wherein the different mixtures comprise a fixed amount of ligand, at least a portion of which is detectably labeled, and different concentrations of the enhancer, determining the amount of ligand bound at the different concentrations of enhancer, and calculating the potency (Kenhancer) of the enhancer from the binding based on the amount of ligand bound in the presence of different concentrations of the enhancer.
- the concentration of ligand and concentrations of enhancer are selected to allow meaningful detection of enhanced binding.
- the ligand is typically present at a concentration of between about 0.01 Kd and about 0.5 Kd and the concentrations of the test agent/enhancer typically range from 1 nM to 1 mM (e.g. a 4-fold dilution series with highest concentration 10 uM or 1 mM).
- the Kd is determined using the assay disclosed supra.
- the potency of the binding can be determined by a variety of standard methods based on the amount of ligand bound in the presence of different concentrations of the enhancer or augmentor. For example, a plot of labeled ligand binding versus enhancer concentration can be fit to the equation:
- the present invention provides powerful methods for analysis of PDZ-ligand interactions, including high-throughput methods such as the "G" assay and affinity assays described supra.
- the affinity is determined for a particular ligand and a pluraUty of PDZ proteins.
- the plurality is at least 5, and often at least 25, or at least 40 different PDZ proteins.
- the plurality of different PDZ proteins are from a particular tissue (e.g., central nervous system, spleen, cardiac muscle, kidney) or a particular class or type of cell, (e.g., a hematopoietic ceU, a lymphocyte, a neuron) and the like.
- the pluraUty of different PDZ proteins represents a substantial fraction (e.g., typicaUy a majority, more often at least 80%) of all of the PDZ protems known to be, or suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZ proteins known to be present in lymphocytes.
- the pluraUty is at least 50%, usuaUy at least 80%, at least 90% or all of the PDZ proteins disclosed herein as being expressed in hematopoietic cells (see Table 7).
- the binding of a ligand to the plurality of PDZ proteins is determined. Using this method, it is possible to identify a particular PDZ domain bound with particular specificity by the ligand.
- the binding may be designated as "specific” if the affinity of the ligand to the particular PDZ domain is at least 2-fold that of the binding to other PDZ domains in the plurality (e.g., present in that cell type).
- the binding is deemed "very specific” if the affinity is at least 10-fold higher than to any other PDZ in the plurality or, alternatively, at least 10-fold higher than to at least 90%, more often 95% of the other PDZs in a defined plurality.
- the binding is deemed “exceedingly specific” if it is at least 100-fold higher.
- a ligand cound bind to 2 different PDZs with an affinity of 1 uM and to no other PDZs out of a set 40 with an affinty of less than 100 uM. This would constitute specific binding to those 2 PDZs.
- Similar measures of specif ⁇ ty are used to describe binding of a PDZ to a plurality of PLs. It will be recognized that high specificity PDZ-PL interactions represent potentially more valuable targets for achieving a desired biological effect. The ability of an inhibitor or enhancer to act with high specificity is often desirable.
- the invention provides a method of identifying a high specificity interaction between a particular PDZ domain and a ligand known or suspected of binding at least one PDZ domain, by providing a plurality of different immobilized polypeptides, each of said polypeptides comprising a PDZ domain and a non-PDZ domain; determining the affinity of the Ugand for each of said polypeptides, and comparing the affinity of binding of the Ugand to each of said polypeptides, wherein an interaction between the ligand and a particular PDZ domain is deemed to have high specificity when the ligand binds an immobilized polypeptide comprising the particular PDZ domain with at least 2-fold higher affinity than to immobilized polypeptides not comprising the particular PDZ domain.
- the affinity of binding of a specific PDZ domain to a pluraUty of ligands (or suspected ligands) is determined.
- the invention provides a method of identifying a high specificity interaction between a PDZ domain and a particular ligand known or suspected of binding at least one PDZ domain, by providing an immobilized polypeptide comprising the PDZ domain and a non-PDZ domain; determining the affinity of each of a plurality of ligands for the polypeptide, and comparing the affinity of binding of each of the ligands to the polypeptide, wherein an interaction between a particular ligand and the PDZ domain is deemed to have high specificity when the ligand binds an immobilized polypeptide comprising the PDZ domain with at least 2-fold higher affinity than other ligands tested.
- the binding may be designated as "specific” if the affinity of the PDZ to the particular PL is at least 2-fold that of the binding to other PLs in the plurality (e.g., present in that cell type).
- the binding is deemed “very specific” if the affinity is at least 10-fold higher than to any other PL in the plurality or, alternatively, at least 10-fold higher than to at least 90%, more often 95% of the other PLs in a defined plurality.
- the binding is deemed “exceedingly specific” if it is at least 100-fold higher.
- the plurality is at least 5 different ligands, more often at lease 10.
- the pluraUty of ligands comprises at least 1, typically at least 2, more often at least 5, and sometimes at least 10 Ugands selected from CD105, NCAMl, CD95, Spectrin beta, KN1.3, D ⁇ AM1, Neuroligin 3, TAX, CD44 (long form), CD38, CD3n, LPAP, CD46 (form 1), CDwl28B (IL-8 receptor B), DOCK2, PAG, CD34, BLR-1 (or a polypeptide comprising a C-terminal sequence (e.g., at least about 3, 4, 6, 8 or 10 residues) from such a ligand).
- Ugands selected from CD105, NCAMl, CD95, Spectrin beta, KN1.3, D ⁇ AM1, Neuroligin 3, TAX, CD44 (long form), CD38, CD3n, LPAP, CD46 (form 1), CDwl28B (IL-8 receptor B), DOCK2, PAG, CD34, BLR-1 (or a polypeptide comprising a C
- One discovery of the present inventors relates to the important and extensive roles played by interactions between PDZ proteins and PL proteins, particularly in the biological function of hematopoietic cells and other cells involved in the immune response. Further, it has been discovered that valuable information can be ascertained by analysis (e.g., simultaneous analysis) of a large number of PDZ-PL interactions. In a preferred embodiment the analysis encompasses all of the PDZ proteins expressed in a particular tissue (e.g., spleen) or type or class of cell (e.g., hematopoietic ceU, neuron, lymphocyte, B ceU, T ceU and the like).
- tissue e.g., spleen
- type or class of cell e.g., hematopoietic ceU, neuron, lymphocyte, B ceU, T ceU and the like.
- the analysis encompasses at least about 5, or at least about 10, or at least about 12, or at least about 15 and often at least 50 different polypeptides, up to about 60, about 80, about 100, about 150, about 200, or even more different polypeptides; or a substantial fraction (e.g., typically a majority, more often at least 80%) of all of the PDZ proteins known to be, or suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZ proteins known to be present in lymphocytes.
- the plurality is at least 50%, usually at least 80%, at least 90% or all of the PDZ proteins disclosed herein as being expressed in hematopoietic cells (see Table 7).
- the arrays and methods of the invention are directed to analyis of PDZ and PL interactions, and involve selection of such proteins for analysis. While the devices and methods of the invention may include or involve a small number of control polypeptides, they typically do not include significant numbers of proteins or fusion proteins that do not include either PDZ or PL domains (e.g., typically, at least about 90% of the arrayed or immobilized polypeptides in a method or device of the invention is a PDZ or PL sequence protein, more often at least about 95%, or at least about 99%).
- the array includes at least one, preferably at least 1, more often at least 5 or at least 10 and sometimes all of the following PDZ proteins present in lymphocytes: BAI I associated prot., Connector enhancer, DLG5 (pdlg), DNL3, GTPase, Guanin-exchange factor 1, PDZ domain containing prot, KIAA147, KIAA0300, KIAA0380, KIAA0440, KIAA0545, KIAA0807, KIAA0858, KIAA0902, novel serine protease, PDZK1 , PICK8, PT ⁇ -3, RPIP8, serine protease, 26s subunit p27, hSYNTENIN, TAXI-IP, TAX2-like protein, wwp3, XI 1 prot. beta, ZOl .
- simultaneous analysis facilitates, for example, the direct comparison of the effect of an agent (e.g., an potential interaction inhibitor) on the interactions between a substantial portion of PDZs and/or PLs in a tissue or ceU.
- an agent e.g., an potential interaction inhibitor
- the invention provides an array of immobilized polypeptide comprising the PDZ domain and a non-PDZ domain on a surface.
- the array comprises at least about 5, or at least about 10, or at least about 12, or at least about 15 and often at least 50 different polypeptides.
- the different PDZ proteins are from a particular tissue (e.g., central nervous system, speen, cardiac muscle, kidney) or a particular class or type of cell, (e.g., a hematopoietic ceU, a lymphocyte, a neuron) and the like.
- the pluraUty of different PDZ proteins represents a substantial fraction (e.g., typically a majority, more often at least 80%) of all of the PDZ proteins known to be, or suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZ proteins known to be present in lymphocytes.
- the pluraUty is at least 50%, usually at least 80%, at least 90% or all of the PDZ proteins disclosed herein as being expressed in hematopoietic ceUs (see Table 7) e.g.; all of the PDZ proteins known to be present in lymphocytes.
- the plurality is at least 50%, usuaUy at least 80%, at least 90% or all of the PDZ proteins disclosed herein as being expressed in hematopoietic ceUs (see Table 7).
- the array includes at least one, preferably at least 1, typically at least 5 and sometimes all of the following PDZ proteins present in lymphocytes: BAI I associated prot., Connector enhancer, DLG5 (pdlg), DNL3, GTPase, Guanin-exchange factor 1, PDZ domain containing prot., KIAA147, KIAA0300, KIAA0380, KIAA0440, KIAA0545, KIAA0807, KIAA0858, KIAA0902, novel serine protease, PDZK1, PICK8, PT ⁇ -3, RPIP8, serine protease, 26s subunit p27, hSYNTENIN, TAXI-IP, TAX2-like protein, wwp3, XI 1 prot.
- BAI I associated prot. Connector enhancer
- DLG5 pdlg
- DNL3, GTPase Guanin-exchange factor 1
- PDZ domain containing prot.
- array refers to an ordered series of of immobilized polypeptides in which the identity of each polypeptide is associated with its location.
- the plurality of polypeptides are arrayed in a "common” area such that they can be simultaneously exposed to a solution (e.g., containing a ligand or test agent).
- a solution e.g., containing a ligand or test agent.
- the pluraUty of polypeptides can be on a sUde, plate or similar surface, which may be plastic, glass, metal, silica, beads or other surface to which proteins can be immobilized.
- the different immobilized polypeptides are situated in separate areas, such as different wells of multi-well plate (e.g., a 24-well plate, a 96-well plate, a 384 weU plate, and the like). It wiU be recognized that a similar advantage can be obtained by using multiple arrays in tandem.
- multi-well plate e.g., a 24-well plate, a 96-well plate, a 384 weU plate, and the like.
- the invention provides a method for dete ⁇ nining if a test compound inhibits any PDZ-ligand interaction in large set of PDZ-ligand interaction (e.g., some or all of the PDZ-Ugands interactions described in Table 2; a majority of the PDZ-ligands identified in a particular cell or tissue as described supra (e.g., lymphocytes) and the like.
- the PDZ domains of interest are expressed as GST-PDZ fusion proteins and immobilized as described herein. For each PDZ domain, a labeled Ugand that binds to the domain with a known affinity is identified as described herein.
- PDZ-PL interactions occur in cells of the hematopoietic system.
- modulator e.g., inhibitor
- an agent that inhibits all PDZ-PL interactions in a cell e.g., a lymphocyte
- an agent that inhibits only one, or a small number, of specific PDZ-PL interactions e.g., a lymphocyte
- the profile of PDZ interactions inhibited by a particular agent is referred to as the "inhibition profile" for the agent, and is described in detail below.
- the profile of PDZ interactions enhanced by a particular agent is referred to as the "enhancement profile" for the agent. It will be readily apparent to one of skill guided by the description of the inhibition profile how to determine the enhancement profile for an agent.
- the present invention provides methods for determining the PDZ interaction (inhibition/enhancement) profile of an agent in a single assay.
- the invention provides a method for determining the PDZ-PL inhibition profile of a compound by providing (i) a plurality of different immobilized polypeptides, each of said polypeptides comprising a PDZ domain and a non-PDZ domain and (U) a plurality of conesponding ligands, wherein each ligand binds at least one PDZ domain in (i), then contacting each of said immobUized polypeptides in (i) with a conesponding Ugand in (ii) in the presence and absence of a test compound, and determining for each polypeptide- ligand pair whether the test compound inhibits binding between the immobilized polypeptide and the conesponding ligand.
- the pluraUty is at least 5, and often at least 25, or at least 40 different PDZ proteins.
- the plurality of different ligands and the plurality of different PDZ proteins are from the same tissue or a particular class or type of cell, e.g., a hematopoietic cell, a lymphocyte, a neuron and the like.
- the plurality of different PDZs represents a substantial fraction (e.g;, at least 80%) of all of the PDZs known to be, or suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZs known to be present in lymphocytes (for example, at least 80%, at least 90% or aU of the PDZs disclosed herein as being expressed in hematopoietic cells).
- the inhibition profile is determined as follows: A pluraUty (e.g., all known) PDZ domains expressed in a cell (e.g., lymphocytes) are expressed as GST- fusion proteins and immobiUzed without altering their ligand binding properties as described supra. For each PDZ domain, a labeled ligand that binds to this domain with a known affinity is identified. If the set of PDZ domains expressed in lymphocytes is denoted by ⁇ PI ...Pn ⁇ , any given PDZ domain Pi binds a (labeled) ligand Li with affinity K ⁇ i.
- a pluraUty e.g., all known
- PDZ domains expressed in a cell e.g., lymphocytes
- a labeled ligand that binds to this domain with a known affinity is identified. If the set of PDZ domains expressed in lymphocytes is denoted by ⁇ PI ...Pn ⁇ , any given PDZ domain
- the "G” assay (supra) can be performed as foUows in 96- well plates with rows A-H and columns 1-12.
- Column 1 is coated with PI and washed.
- the conesponding ligand LI is added to each washed coated well of column 1 at a concentration 0.5 dl with (rows B, D, F, H) or without (rows A, C, E, F) between about 1 and about 1000 uM) of test compound X.
- Column 2 is coated with P2, and L2 (at a concentration 0.5 _ d 2) is added with or without inhibitor X. Additional PDZ domains and ligands are similarly tested.
- Compound X is considered to inhibit the binding of Li to Pi if the average signal in the weUs of column i containing X is less than half the signal in the equivalent weUs of the column lacking X.
- the test compound X is a mixture of compounds, such as the product of a combinatorial chemistry synthesis as described supra.
- the test compound is known to have a desired biological effect, and the assay is used to determine the mechanism of action (i.e., if the biological effect is due to modulating a PDZ-PL interaction).
- an agent that modulates only one, or a few PDZ-PL interactions, in a panel is a more specific modulator than an agent that modulate many or most interactions.
- a panel e.g., a panel of all known PDZs lymphocytes, a panel of at least 10, at least 20 or at least 50 PDZ domains
- an agent that modulates less than 20% of PDZ domains in a panel is deemed a "specific" inhibitor, less than 6% a "very specific” inhibitor, and a single PDZ domain a "maximally specific" inhibitor.
- compound X may be a composition containing mixture of compounds (e.g., generated using combinatorial chemistry methods) rather than a single compound.
- the assay above is performed using varying concentrations of the test compound X, rather than fixed concentration. This allows determination of the Ki of the X for each PDZ as described above.
- a mixture of different labeled ligands is created that such that for every PDZ at least one of the ligands in the mixture binds to this PDZ sufficiently to detect the binding in the "G" assay.
- This mixture is then used for every PDZ domain.
- compound X is known to have a desired biological effect, but the chemical mechanism by which it has that effect is unknown. The assays of the invention can then be used to determine if compound X has its effect by binding to a PDZ domain.
- PDZ-domain containing proteins are classified in to groups based on their biological function, e.g. into those that regulate chemotaxis versus those that regulate transcription.
- An optimal inhibitor of a particular function e.g., including but not limited to an anti-chemotactic agent, an anti-T cell activation agent, cell-cycle control, vesicle transport, apoptosis, etc.
- wiU inhibit multiple PDZ-ligand interactions involved in the function (e.g., chemotaxis, activation) but few other interactions.
- the assay is used in one embodiment in screening and design of a drug that specifically blocks a particular function.
- an agent designed to block chemotaxis might be identified because, at a given concentration, the agent inhibits 2 or more PDZs involved in chemotaxis but fewer than 3 other PDZs, or that inhibits PDZs involved in chemotaxis with a Ki > 10-fold better than for other PDZs.
- the invention provides a method for identifying an agent that inhibits a first selected PDZ-PL interaction or plurality of interactions but does not inhibit a second selected PDZ-PL interaction or plurality of interactions.
- the two (or more) sets of interactions can be selected on the basis of the known biological function of the PDZ proteins, the tissue specificity of the PDZ proteins, or any other criteria.
- the assay can be used to determine effective doses (i.e., drug concentrations) that result in desired biological effects while avoiding undesirable effects.
- the invention provides a method for dete ⁇ nining likely side effects of a therapeutic that inhibits PDZ-ligand interactions.
- the method entails identifying those target tissues, organs or ceU types that express PDZ proteins and Ugands that are disrupted by a specified inhibitor. If, at a therapeutic dosage, a drug intended to have an effect in one organ system (e.g., hematopoietic system) disrupts PDZ-PL interactions in a different system (e.g., CNS) it can be predicted that the drug will have effects ("side effects") on the second system. It will be apparent that the information obtained from this assay wiU be useful in the rational design and selection of drugs that do not have the side-effect.
- a comprehensive PDZ protein set is obtained.
- a "perfectly comprehensive" PDZ protein set is defined as the set of all PDZ proteins expressed in the subject animal (e.g., humans).
- a comprehensive set may be obtained by analysis of, for example, the human genome sequence.
- a "perfectly comprehensive" set is not required and any reasonably large set of PDZ domain proteins (e.g., the set of all known PDZ proteins; or the set listed in Table 7) will provide valuable information.
- the method involves some of all of the following steps: a) For each PDZ protein, determine the tissues in which it is highly expressed. This can be done experimentally although the information generally will be available in the scientific literature; b) For each PDZ protein (or as many as possible), identify the cognate PL(s) bound by the PDZ protein; c) Determine the Ki at which the test agent inhibits each PDZ-PL interaction, using the methods described supra; d) From this information it is possible to calculate the pattern of PDZ-PL interactions disrupted at various concentrations of the test agent
- Additional steps can also be carried out, including dete ⁇ nining whether a specified tissue or cell type is exposed to an agent following a particular route of administration. This can be determined using basis pharmacokinetic methods and principles.
- the PDZ binding moieties and PDZ protein -PL protein binding antagonists of the invention are used to modulate biological activities or functions of ceUs (e.g., hematopoietic cells, such as T cells and B cells and the like), endothelial cells, and other immune system ceUs, as described herein, and for treatment of diseases and conditions in human and nonhuman animals (e.g., experimental models). Exemplary biological acitivities are listed supra.
- the compounds of the invention are useful for treating (ameUorating symptoms of) a variety of diseases and conditions, including diseases characterized by inflammatory and humoral immune responses, e.g., inflammation, allergy (e.g., systemic anaphylaxis, hypersensitivity responses, drug allergies, insect sting allergies; inflammatory bowel diseases, ulcerative colitis, Ueitis and enteritis; psoriasis and inflammatory dermatoses, scleroderma; respiratory allergic diseases such as asthma, allergic rhinitis, hypersensitivity lung diseases, and the like vasculitis, rh incompatibility, transfusion reactions, drug sensitivities, PIH, atopic dermatitis, eczema, rhinnitis; autoimmune diseases, such as arthritis (rheumatoid and psoriatic), multiple sclerosis, systemic lupus erythematosus, insulin-dependent diabetes,
- inflammatory and humoral immune responses e.g., inflammation, allergy (e
- MCTD MCTD
- IDDM Hashimoto thyroiditis
- Goodpasture syndrome psoriasis and the like
- osteoarthritis polyarthritis
- graft rejection e.g., allograft rejection, e.g., renal allograft rejection, graft- vs-host disease, transplantation rejection (cardiac, kidney, lung, liver, small bowel, cornea, pancreas, cadaver, autologous, bone manow, xenotransplantation)
- atherosclerosis angiogenesis-dependent disorders
- cancers e.g., melanomas and breast cancer, prostrate cancer, leukemias, lymphomas, metastatic disease
- infectious diseases e.g., viral infection, such as MN, measles, parainfluenza, virus-mediated cell fusion,
- ischemia e.g., post-myocardial infarction complications, joint injury, kidney, scleroderma.
- PL proteins and PDZ proteins listed in TABLE 2 are well characterized, and one of skill, guided by this disclosure (including the discovery of the interactions between PL proteins and PDZ proteins described herein), will recognize many uses for modulators (e.g., enhancers or inhibitors) of PDZ-PL interactions such as those described in TABLE 2.
- modulators e.g., enhancers or inhibitors
- PDZ-PL interactions such as those described in TABLE 2.
- CD6 binds PDZ protein 41.8.
- CD6 is expressed on thymocytes, T cells, and B ceU chronic lymphocytic leukemias.
- CD6 plays a role in T cell co- stimulation and CD6 negative T cells are less autoreactive than CD6 positive T cells.
- Inhibition of CD6 and CD6/41.8 interactions is predicted to reduce the symptoms of graft-versus-host disease (GNHD) or psoriasis.
- GNHD graft-versus-host disease
- GNHD graft-versus-host disease
- CD6-PDZ interaction inhibitors wiU improve overall survival of transplantation patients (e.g., leukemia patients).
- CD49e binds to the PDZ-domain-containing protein41.8kD.
- CD49e is a 110 kD transmembrane membrane protein of the integrin alpha family (integrin alpha 5). Paired with the integrin beta- 1 subunit it forms NLA-5.
- NLA-5 is expressed predominantly on hematopoietic and lymphoid lineage cells including monocytes, basophils, T cells, and activated B cells.
- NLA-5 is the receptor for the ubiquitously-expressed adhesion molecule fibronectin. Tissue injury such as myocardial infarction releases soluble fragments of fibronectin.
- Binding of these soluble fragments to NLA-5 results in chemotaxis of immune cells including monocytes to the source of fibronectin, as well as down-modulation of NLA ⁇ 5 expression on these ceUs.
- Such ligand- induced down-modulation is a common and required feature of chemotaxic receptors.
- the 41.8/CD49e interaction is believed to be necessary for proper membrane distribution of CD49e and/or recycling of CD49e such that when it is disrupted, the migration and adherence to fibronectin -containing surfaces is similarly disrupted, resulting in an inabiUty of immune system cells to effectively migrate toward a fibronectin source and adhere to fibronectin-containing surfaces.
- Such disruption would therefore result in desirable reduced inflammatory processes, including reduced post-myocardial infarction inflammation.
- Other diseases to be treated include but are not Umited to joint inflamation, psoriasis, contact aUergy, Crohn's Disease, inflammatory bowel disease, eczema, atopic dermatitis.
- CD49F (VLA-6 ⁇ subunit) As shown supra, CD49F binds PDZ protein 41.8.
- CD49f is known as an integrin subunit that pairs either with the ⁇ l integrin subunit (CD29), forming VLA-6, or with CD 104 ( ⁇ 4 integrin subunit).
- the integrin supergene family consists of a number of cell surface ⁇ heterodimers important for many different physiologic processes, including embryogenesis, thrombosis, wound healing, tumorigenesis and immune responses. Each ⁇ chain can pair with various ⁇ chains.
- Both NLA-6 and CD49-&OD104 are widely expressed on epithelia in non-lymphoid tissues.
- VLA-6 is also expressed on platelets, monocytes, thymocytes and T lymphocytes, with an increased expression on activated and resting memory T cells. Inhibition of interactions between NLA-6 and 41.8 has anumber of therapeutic functions such as the prevention and treatment of metastatic cancers, and treatment of overactive immunity.
- NLA-6 is associated with invasivion of prostrate carcinoma and plays a role in the metastasis of breast cancer. Blockage of VLA-6 function combined with conventional treatment for prostrate cancer, would be a more effective treatment by preventing metastatic disease (see, Cress et al., 1995, Cancer Metastasis R). Blockage of CD49f through PDZ interaction may also treat Rh incompatibility by blunting memory response or in the treatment of keloids.
- CD138 (syndecan-1)
- CD 138 is a transmembrane proteoglycan receptor with the extracellular domain functioning as a ligand binding domain for various extracellular matrix components and the intracellular portion functioning to alter cytoskeleton and transduce intraceUular signals.
- CDi38 also binds FGF2 and may be a co-receptor for FGF receptor (Yayon et al., 1991).
- CD138 interacts with 41.8kD protein and TIAM1.
- the c- terminus of CD138 has also been reported to bind the PDZ domains of syntenin and human CASK (Cohen et al., 1998, J Cell. Biol. 142:129-138; Grootjans et al., 1997, PNAS 94: 13683- 13688; Hsueh et al., 1998, J. Cell Biol.
- CD138 is expressed in pre-B cells, immature B cells, plasma cells, neural cells, the basolateral surface of epithelial cells, embryonic mesenchymal ceUs, vascular smooth muscle ceUs, endothelial ceUs and neural ceUs but not mature circulating B cells.
- the interaction between CD138 and the PDZ domains of the 41.8kD protein and TIAM1 proteins is beUeved to be necessary for the proper distribution of CD138 on the cell surface. Disruption of the interaction by administration of an effective amount of an antagonist is expected to interfere with the migration and adherence of cells to the extracellular matrix, resulting in reduced inflammatory and humoral immune responses.
- Inhibition of CD138 may be used to treat without limitation diseases such as post-myocardial infarction inflamatory damage, joint injury, rheumatoid arthritis, vasculitis, drug reaction, scleraderma, SLE, Hashimoto thyroiditis, Goodpasture's syndrome, juvenile insulin-dependent diabetes, psoriasis.
- diseases such as post-myocardial infarction inflamatory damage, joint injury, rheumatoid arthritis, vasculitis, drug reaction, scleraderma, SLE, Hashimoto thyroiditis, Goodpasture's syndrome, juvenile insulin-dependent diabetes, psoriasis.
- CD98 interacts with MPP2.
- CD98 is expressed at high levels on monocytes and at low levels on T cells, B cells, splenocytes, NK cells, and granulocytes.
- CD98 plays roles in adhesion, fusion and is a L-type amino acid transporter.
- CD98 is also involved in virus-mediated cell fusion (e.g. paramyoviruses: parainfluenza virus type 2, Newcastle disease virus, and rubulaviruses) and antagonism of CD98 function is expected to treat )viral infections and limit viral spread.
- CD98 inhibitors can be an antiviral agent for, but not limited to paramyovirus, parainfluenza, Newcastle disease and rubula. Other roles include treatment for acute leukemias.
- CLASP-1 interacts with DLGl, PSD95, and NeDLG.
- CLASP- 1 is a member of a superfamily of immune-cell associated proteins with similar motifs (see
- CLASP-1 functions in the maintenance of the immune synapse.
- the CLASP-1 transcript is present in lymphoid organs and neural tissue, and the protein is expressed by T and B lymphocytes and macrophages in the MOMA-i subregion of the marginal zone of the spleen, an area known to be important in T:B cell interaction.
- CLASP-1 staining of individual T and B cells exhibits a preactivation structural polarity, being organized as a "ball” or “cap” structure in B cells, and forming a "ring", "ball” or “cap” structure in T cells. The placement of these strtictures is adjacent to the microtubule-organizing center (“MTOC").
- MTOC microtubule-organizing center
- T-B ceU conjugates that are fully committed to differentiation.
- Antibodies to the extracellular domain of CLASP-1 also block T-B ceU conjugate formation and T cell activation.
- Antagonism of CLASP-1 function is expected to interfere with immune responses (e.g., T and B cell activation), signal transduction, cell-cell interactions, and membrane organization.
- Diseases to be treatment by CLASP-1 agonists/antagonists include, but is not limited to, rheumaotoid arthritis, juvenile diabetes, organ rejection, graft-versus-host disease, scleroderma, multiple sclerosis.
- CLASP-4 interacts with DLGl, PSD95, NeDLG, LDP, AF6, 41.8, and MINT1.
- CLASP-4 is a member of a superfamily of immune-ceU associated proteins with similar motifs (see copending U.S Pat. Application 60/196,527 filed April 11, 2000).
- the CLASP-4 protein is expressed primarily in peripheral blood lymphocytes. Inhibition of the interaction of CLASP-4 and PDZ domains will interfere with immune responses (e.g., T and B cell activation), signal transduction, cell-cell interactions, and membrane organization.
- rheumaotoid arthritis include, but is not limited to, rheumaotoid arthritis, juvenile diabetes, organ rejection, graft-versus-host disease, scleroderma, multiple sclerosis, acute leukemias, leukemic blast crisis, post-infarction inflamation (cardiac, etc.), atherosclerosis.
- VCAM-1 The vascular cell adhesion molecule- 1 (VCAM-1, CD 106) is predominantly expressed by vascular endothelium (i.e., endothelial cells) but has been detected in macrophages, dendritic cells, bone manow-derived cells, fibroblasts, cortical thymic epitheUal cells, vascular smooth muscle cells, myoblasts and myotubes.
- VCAM-1 mediates adhesion through interacting with an integrin ligand, VLA-4, which is expressed by lymphocytes, monocytes and eosinophils.
- VCAM-1 and VLA-4 The interaction between VCAM-1 and VLA-4 is important for activation, flattening and extravasation of VLA-4 expressing cells when the endotheUum itself has become activated due to inflammation or injury (Salomon et al., 1997, Blood 89:2461- 2471; St-Piene et al., 1996, Eur. J. Immunol. 26:2050-2055; Bell et al., 1995, Int. Immunol. 7:1861-1871).
- VCAM-1 is a ligand for the PDZ domains of MPP1, DLGl, NeDLGl, LDP, 41.8 protein, TIAM1, K807, WWP3 and K303. These interactions are believed to mediate the function of endothelial cell interactions with integrin expressing leukocytes. When the PDZ-PL interactions are disrupted, the adherence of leukocytes to the endotheUum will similarly be disrupted, resulting in, e.g., reduction of inflammation.
- VCAM-1 binding to PDZ proteins is useful for reducing abnormal VCAM-1 inflammatory responses and associated pathologies such as (but not limited to) renal allograft rejection, insulin-dependent diabetes, rheumatoid arthritis, post-myocardial infarction complications and systemic lupus erythematosus (Pasloske et al., 1994, Ann Rev Med 45:283; Ockenhouse et al., 1992, J. Exp. Med. 176:1183; Solezk et al., 1997, Kidney Int. 51:1476; Tedla, et al., 1999, Clin. Exp. Immunol.
- CLASP-2 interacts with PSD-95, NeDLG, and DLGl.
- CLASP-2 is a member of a superfamily of immune-cell associated proteins with similar motifs
- CLASP-2 transcript is present most strongly in placenta followed by lung, kidney and heart and the protein is expressed in T and B cells, and kidney epithelial ceUs.
- CLASP-2 and PDZ domains will interfere with CLASP-2 function resulting in interference with T and B cell function (e.g., T and B cell activation), signal transduction, cell-cell interactions, and membrane organization.
- T and B cell function e.g., T and B cell activation
- blocking CLASP-2 function or expression can selectively block immune responses in the heart (for example, to selectively stop immune response in the heart compartment e.g., following cardiac transplant rejection or post-MI inflammation, without compromising immunity elsewhere).
- Other diseases to be treatment by CLASP-1 agonists/antagonists include, but is not limited to, rheumaotoid arthritis, juvenile diabetes, organ rejection, graft-versus-host disease, scleroderma, multiple sclerosis.
- CD95 Fas Apo-1
- Fas ligand Fas ligand
- Agents that modulate (e.g., inhibit) the interaction of CD95 and PDZ domains are useful for treatment of diseases, e.g., organ transplantation, graft-versus-host disease, Crohn's Disease, Ulcerative colitis, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, multiple sclerosis, scleroderma, mixed connective tissue disease, leukemia and other malignancies.
- diseases e.g., organ transplantation, graft-versus-host disease, Crohn's Disease, Ulcerative colitis, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, multiple sclerosis, scleroderma, mixed connective tissue disease, leukemia and other malignancies.
- Kvl.3 binds DLGl, PSD95, NeDLG, LIMK, 41.8, RGS12, DVL1, and MINT1.
- Kvl.3 is a Shaker-related channel protein that is involved in modulating the membrane potential of T lymphocytes (Lewis and Cahalan, 1995, Ann. Rev. Immunol. 13:623). Inhibition ofthe Kvl.3 channel chronically depolarizes the T ceU membrane, reduces calcium entry via calcium-activated release calcium channels in the plasma membrane, and consequently inhibits the calcium-signaling pathway essential for lymphocyte activation.
- Hanada et al. reported that Kvl.3 is associated with DLGl and PSD95 in Jurkat T cells (J. Biol. Chem.
- Kvl.3-PDZ protein agonist/antagonists will disrupt T cell signaling and can be a useful therapeutic drug to treat but not limited to, organ transplantation, graft-versus-host disease, Crohn's Disease, Ulcerative colitis, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, multiple sclerosis, scleroderma, mixed connective tissue disease.
- DNAM-1 binds several PDZ proteins, including MPP1, MPP2, DLGl, PSD95, NeDLG, LIM, AF6, 41.8, RGS12 and WWP3.
- DNAM-1 is associated with Fyn constitutively but required the presence of pervanadate (a tyrosine phosphatase inhibitor) (Shibuya, et al., 1999, Immunity. 1 :615-623).
- pervanadate a tyrosine phosphatase inhibitor
- DNAM-1 is phosphorylated at Ser329 (Shibuya, et al., 1999, Immunity 1:1671-75) and associates with LFA-1.
- Fyn becomes associated with DNAM-1 independent of pervanadate. Fyn phosphorylates DNAM-1 at Tyr322, but does not require Tyr322 to continue binding to DNAM-1.
- DNAM-1 itself does not have a SH3 binding domain but has a Src phosphorylation site at Tyr322, an adaptor molecule must be present to bridge DNAM-1 and Fyn.
- DLGl has been described in the literature to be present in T cells (Hanada, et al. 1997, J. Biol. Chem.272:26899), but does not bind to Fyn.
- PSD95 does not have a SH3 site, several of the other PDZ proteins do have SH3 binding domains including but not limited to NeDLG, RGS12, WWP3 and MPP2, and can fulfill this function.
- the adaptor PDZ list supra describes binding to DNAM-1 through the PDZ domain.
- Tyrl39 is a candidate phosphorylation site to control association of Fyn to DNAM-1 and the adaptor PDZ.
- DNAM-1 may complex with beta-catenin, actin and cadherin (Dobrosotskaya and James, 2000, Biochem. Biophys. Res. Commun.270:903-909).
- inhibition of PDZ association with DNAM-1 using the reagents of the invention wiU inhibit Fyn association with DNAM-1 and the subsequent Tyr322 phosphorylation and activation of cytotoxic T cells.
- Disease that can be treated include but are not limited to Crohn's Disease, multiple sclerosis, ulcerative colitis, inflammatory bowel disease, graft-versus-host, juvenile diabetes, and Hashimoto's Disease.
- CD83 is a transmembrane glycoprotein, expressed predominantly on activated dendritic cells (DCs), Langerhans cells in the skin, with some weak expression detected or activated peripheral lymphocytes, and interdigitating reticulum ceUs within the T cell zones o. lymphoid organs (Zhou and Tedder, 1995, J. Immunol. 154:3821-3835; Zhou et al., 1992, j
- CD83 is up-regulated de novo upon activation of an immature DC; and is the major discriminating marker and characteristic for activated, mature DC (Czerniecki et al., 1997, J. Immunol. 159:3823-3837). DCs function as antigen presenting eel
- CD83 binds to the PDZ domai of DLGl, PSD95, and NeDLG. These interactions between CD83 and PDZ domains, a between CD83 and DLGl, PSD95, and NeDLG are believed to be important for pro] distribution and recycling of CD83. Disruption of CD83 and PDZ proteins with agonists « • antagonist can be used to treat, but not limited to, psoriasis, cancers, allergies, autoimnr diseases such as multiple sclerosis, system lupus erythematosis.
- CD44 phagocytic glycoprotein 1 , lymphocyte homing receptor, p85 and HCAM
- CD44 is single pass transmembrane protein that has several different isofi due to alternative splicing. It has a broad pattern of expression being detected on hematopoietic and non-hematopoietic ceU types including epitheUal, endotheUal, mesenchymal and neuronal cells.
- CD44H is a major isoform that is expressed in lymphoid, myeloid and erythroid cells (reviewed in Barclay et al., 1997, The Leukocyte Antigen Facts Book, 2ed, Academic Press).
- CD44 is a receptor for hyaluronate (HA), which is a constituent of the extracellular matrix (ECM).
- CD44 functions as an adhesion molecule on the surface of leukocytes and erythrocytes that binds HA polymers in the ECM, and it can also act as a signaling receptor when HA becomes soluble during inflammatory reactions or tissue damage.
- the cytoplasmic region of CD44 has been shown to bind or be associated with the actin cytoskeleton through interactions with spectrin and members of the ERM (ezrin, radixin, and meosin) family (reviewed in Lesley et al., 1993; Bajorath, 2000, Proteins 39:103- 111).
- CD44 is associated with the non-receptor tyrosine kinase p56Lck (Taher et al., 1996, J. Biol. Chem. 271:2863-2867. CD44 has been shown to be a co-stimulatory molecule with CD3/TCR engagement to activate T cells (reviewed in Aruffo, 1996, J. Clin. Invest. 98:2191-2192). As described supra by experiments reported herein, the C-terminus of CD44 is a ligand for the PDZ domain contained in MPP1, prIL-16 and MINT1.
- CD44 with PDZ domains, and between CD44 with MPP1, prIL-16 and MINT1 function in maintenance of leukocyte structure and in leukocyte signaling.
- CD44- PDZ interaction when a CD44- PDZ interaction is disrupted, CD44 will fail to transduce proper intracellular signals, and maintain proper distribution of CD44 on the surface, which wiU prevent adhesion of leukocytes to the endothelium during inflammation and tissue damage.
- Administration of agonists/antagonists of this interaction will thus result in, but not limited to, reduced inflammatory responses during tissue ischemia and cell lysis (e.g., rhabdomyosis), vascular insufficiencies (e.g. frostbite), psoriasis, eczema, graft-versus-host disease, granuloma annulare, scleroderma.
- CD97 binds the PDZ domains of DLGl and 41.8.
- CD97 is a 79.7 kD seven-span transmembrane protein expressed on granulocytes and monocytes and at low levels on resting T cells and B cells. Upon T or B cell activation expression levels of
- CD97 When expressed on COS cells, CD97 confers adhesion to lymphocytes and to erythrocytes.
- the interaction of CD97 with DLGl and the 41.8 kd protein can be altered to interfere with proper membrane distribution of CD97 and/or recycling of CD97. Such modulation will affect CD97 dependent adherence of cells with therapeutic benefit.
- agonists and antagonists of CD97-PDZ protein interaction can be used to treat rheumatoid arthritis, osteoarthritis, Crohn's Disease, Ulcerative colitis, psoriasis.
- Glycophorin C As is shown supra, the c-terminus of Glycophorin C (GC) interacts with the
- Glycophorin C is an integral membrane protein expressed in erythroid cells, thymus, stomach, breast adult and fetal liver, monocytes, T and B cells (Le Van Kim et al., 1989, J. Biol Chem. 264:20407-20414) and is known for its role in human erythrocytes where it interacts with MPPl and protein 4.1 to regulate the shape, integrity and mechanical stability of red cells (Marfatia et al., 1997, J. Biol. Chem. 272:24191-24197; Reid et al., 1987, Blood 69:1068-1012.).
- Glycophorin C and PDZ proteins DLG, PSD95, NeDLG, MMP2, AF6, 41.8, and MINT1 are beUeved necessary for maintenance of the physical integrity of cells in which they are expressed. Modulation of GC-PDZ interactions will alter with the function of these and can be utilized to treat, but not limited to, polycythemia vera, spherocytosis.
- CDwl28A (IL8RA) As is described supra, CDW128A binds to the PDZ domains of DLGl and
- IL-8 receptor There are two forms for the IL-8 receptor, IL-8RA (CDwl28A) and IL-8RB (CDwl28B) both of which are members of the G protein-coupled receptor superfamily and chemokine receptor branch of rhodopsin family.
- CDwl28A and CDwl28B both bind IL-8 with the same affinity but only CDwl28B, binds three other IL-8-related CXC chemokines: melanoma growth-stimulating activity (GRO MGSA), neutrophil-activating peptide 2 (NAP-2) and ENA-78. See, e.g., Ahuja and Murphy, 1996, JBiol Chem 271:20545-50.
- CDwl28A is expressed on all granulocytes, a subset of T cells, monocytes, endothelial ceUs, keratinocytes, erythrocytes, and melanoma cells.
- IL-8 induces chemotaxis of neutrophils, basophils, and T lymphocytes and increases neutrophil and monocyte adhesion to endothelial cells.
- the binding of IL-8 to IL8RA induces a transient increase in intracellular calcium levels, activation of phospholipase D, a respiratory burst of neutrophils and chemotaxis. This pro-inflammatory response is effective in normal immune responses.
- Inhibitors of CDwl28A are useful for treatment of psoriasis, rheumatoid arthritis, polyarthritis, and for control of angiogenesis-dependent disorders such as melanomas and breast cancer.
- CD3-eta( ⁇ ) CD3- ⁇ is a splice variant of CD3 zeta and a component of the CD3/TCR complex, which is required for antigen recognition, signal transduction and activation of T cells (Weiss and Liftman, 1994, Cell 76:263-274.). See, Barclay et al., 1997, The Leucocyte antigen facts book, 2nd Ed, Academic Press. As shown by experiments reported herein, the C-terminal region of CD3- ⁇ is a ligand for the PDZ domains of MINTl, 41.8 protein, DLGl, and PSD95.
- CD3- ⁇ with PDZ domains MINTl, 41.8 protein, DLGl, K807, TIP, and PSD95 are believed to be important activation of T cells, which is required for all cellular immune responses. Modulation of this interaction by agonists and antogonists can be used to treat but is not limited to, acute and chronic allograft rejection, multiple sclerosis, graft-versus- host disease, rheumatoid arthritis.
- LPAP is a transmembrane protein expressed on resting T-and B-cells. LPAP has been shown to bind to CD45, a protein that is part of the T-cell receptor complex and has been found to co-localize with CD4, CD2 and Thy-1. LPAP has also been co-immune precipitated with p56(lck) and ZAP-70. The actual function of LPAP is unknown, but data obtained from LPAP deficient mice and Jurkat cell lines suggest that LPAP is an assembly molecule important for the organization of a functional CD45 complex.
- LPAP binds to DLG-1, MINT-1, KIAA0807 and TIP-1 (sometimes "TAX Interacting Protein 1" or “TAX Interacting Protein” or “TAX H ”)- Notably, DLG-1, MINT-1, KIAA0807 and TIP-1 are expressed in T-ceUs. It has been shown that DLG-1 coprecipitates with p56(lck) in T-cells (Hanada et al., 1997, J. Biol C ⁇ e/M.272(43):26899-26904).
- the assay described herein also demonstrates that DLG-1 binds to CD95 and KV1.3, MINT-1 binds to KV1.3, TIP-1 binds to CD95, KV1.3, CD3 ⁇ and HTLV- 1 TAX oncoprotein. All these molecules are involved in signaling by the TCR or in the regulation of ceU death by apoptosis.
- LPAP is believed to function in organizing the signaling of CD45 in T-cells by recruiting p56(LCD) as a substrate for CD45. Blocking the function of CD45 has been shown to severely impair the T-cell response.
- the human PDZ domain containing protein KIAA0807 shows a high degree of similarity to the mouse protein MAST205, a serine/threonine protein kinase, and computer based protein domain homology se&rch predicts protein tyrosine kinase activity for KIAA0807. Binding of KIAA0807 to LPAP appears to be involved in, and possibly crucial for, recruiting KIAA0807 encoded protein into the CD45 complex. Further, protein kinase activity of the KIAA0807 protein may add specific kinase function to the CD45 complex and might tune phosphatase activity of CD45. Thus, inhibition of the PDZ domain-PDZ ligand mediated interaction between LPAP and KIAA0807 can be used to alter (e.g., diminish or abolish) immune response.
- TTP-1 binds to LPAP and to CD95.
- CDp5 has been shown to be a pivotal component of programmed cell death (apoptosis).
- Binding of TEP-1 to CD95 might be regulated by changes in phosphorylation of either binding partner, and LPAP mediated recruitment of TIP-1 into the CD45 (phosphatase / kinase)- complex might ensure proximity of phosphatase / kinase activity when TEP-1 engages in CD95 binding.
- TIP-1 / LPAP binding might compete for TTP-1 / CD95 binding, thus being involved in switching from T-cell proliferation to apoptosis.
- LPAP null mice have shown impaired lymphocyte responses to antigen receptor (Motoya, et al., 1999, J. Biol. Chem.274(3):1407-1414) and have suggested that LPAP plays a role in regulation of lymphocyte expansion in particular lymphatic organs (Ding, et al., 1999, Eur. J. Immunol. 29(12):3956-3961). Therefore, inhibiting the interaction between LPAP and PDZ proteins is expected to alter the CD45-mediated path from the rest of the immune response and to change the pattern of lymphocyte distribution.
- Agonists and antagonist of PL - PDZ binding can be used to treat a variety of disease, including immune disorders such as (but not limited to) rheumatoid arthritis, transplant rejection, multiple sclerosis, scleroderma, graft-versus host disease.
- CD46 Complement membrane cofactor protein (MCP)
- CD46 is a membrane protein expressed on all nucleated cells, but not on erythrocytes. CD46 is a member of the regulator of complement activation protein family. Its primary function is the protection of cells from complement attack by inactivating membrane deposited C3b/C4b complement (Liszewski, et al., 1999, Adv. Immunol. 61 :201-283). CD46 exists in more than 8 isoforms that are generated by differential splicing, with molecular weights ranging from 45 to 70 kD. In addition to the above function, CD46 also serves as the receptor for the measles virus and for other pathogenic microorganisms (e.g.
- CD46 also appears to be over-expressed on certain tumors (Jurianz et al., 1999, Mol Immunol 36:929-939) thus rendering tumor ceUs insensitive to the action of complement. See, Barclay et al., (1997) The Leucocyte antigen facts book, 2 nd ed, Academic Press.
- CD46 binds DLGl, PSD95 and Ne-DLG. This interaction is believed to be necessary for proper membrane distribution of CD46 and/or recycling of CD46. Alteration of the CD46-PDZ interaction can reduce the ability of measles virus and other pathogens to enter cells, renders CD46-expressing tumors susceptible to attack by complement.
- the administration of CD46-PDZ interaction agonists and antagonists is useful for the treatment of , but not limited to, cancers and viral infectious diseases.
- CDwl28B As is described swpr ⁇ , CDwl28B binds to the PDZ domains of DLGl, NeDLG,
- CDwl28A and CDwl28B both binds IL-8 with equal affinity but only CDwl28B, also binds three other IL-8-related CXC chemokines: melanoma growth-stimulating activit] (GRO/MGSA), neutrophil-activating peptide 2 (NAP-2) and ENA-78. See, e.g., Ahuja, SK an ⁇ Murphy, PM. 1996. JBiol Chem 271:20545-50.
- CDwl28B is expressed on all granulocytes, a subset of T ceUs, monocyte, endothelial cells, keratinocytes, erythrocytes, and melanoma cells.
- IL-8 induces chemotaxis * neutrophils, basophils, and T lymphocytes but diminished relative to IL8RA and increaa neutrophil and monocyte adhesion to endothelial ceUs.
- the binding of IL-8 A to its recept induces a transient increase in intracellular calcium levelsand granule release but does r induce activation of phospholipase D or a respiratory burst in neutrophils. This pro- inflammatory response is effective in normal immune responses.
- Inhibitors of CDwl 28B are useful for treatment of psoriasis, rheumatoid arthritis, polyarthritis, and for control of angiogenesis-dependent disorders such as melanomas and breast cancer.
- the DOCK family is a group of transmembrane proteins that interact with the cytoskeleton to affect cell shape, and maintain structural integrity of functional subdomains within a ceU.
- Members of this new family include Drosophila myoblast city (mbc), DOCKl 8( (DOCKl), DOCK2, DOCK3, CED5, KIAA0209 and CLASP.
- the prototypical molecule DOCKl or DOCKl 80 is the human homologue of the C. elegans gene, CED5, which j involved in the engulfinent and phagocytosis by macrophages of cells undergoing apoptos: (apoptotic cells).
- DOCK2 is most closely related to DOCK DOCK2 expression appears to be confined to cells of leukocytic origin.
- DOCK2 is found peripheral blood lymphocytes and can convert a flatten cell into a rounded morphology up transfection (Nagase, et al., 1996, DNA Res 3:321-29;, Nishihara, 1999, Hokkaido Igaku Zas 74:157).
- DOCK2 mRNA is highly expressed in peripheral blood lymphocytes, with expression in thymus and spleen and very weak expression in colon and small intestir -[mmunohistochemistry with antibodies directed against DOCK2 detect expression of DOC in macrophages in the mterstitium and alveoU.
- DOCK2 protein is also detected in lymphoc of the lymph node, and macrophage and lymphocytes in tonsillar tissue (Nishihara et al. If Bioch. Biophys. Acta 1452:179).
- DOCK2 like DOCKl, has been shown to bind Rac GTPase, which could account for it's ability to round-up flat NRK (normal rat kidney) (Nishihara, 1999, Hokkaido Igaku Zasshi 74:157).
- DOCK2 is a PL and binds to the PDZ-containing pre
- KIAA807 DLGl, PSD95, NeDLG, Syntrophin l (Synt ⁇ l) and KIAA0561.
- KIAA807 contains a kinase domain suggesting that the interaction be* KIAA807 and DOCK may have a role in signal transduction.
- the function of KIAAO unknown at this time.
- DLGl, PSD95, NeDLG and Syntrophin ⁇ l have roles in cell sig and structure (Gomperts, 1996, CeU 84:659; Shen and Wyszynski, 1997, Bioessays 1 Sheng and Kim, 1996, Curr. Opin. Neurobiol. 6:602; Leu et al., 1994, Proc.
- Modulation of DOCK2-PDZ interactions by agonists and antagonists can be used to treat diseases such as, but not limited to, acute lymphoytic leukemias, leukemic blast crisis, post-myocardial infarction inflammation, and post-traumatic inflammation.
- CD34 binds DLGl, PSD95, K807, and NeDLG.
- CD34 is expressed on a smaU subpopulation of bone manow ceUs which includes hematopoietic stem cells.
- CD34 is also present on bone manow stomal cells and on endothelial ceUs.
- the selectins CD62L (L-selectin) and CD62E (E-selectin) bind CD34.
- CD34 mediates attachment and rolling of leukocytes.
- the hematopoietic stem cell properties of CD34 include myeloid differentiation of stem cells. Modulation of the CD34-PDZ interaction with agonists and/or antagonists can be used to treat but is not limited to, myelodysplasia, leukemias, post-traumatic inflammation, post-myocardial infarction inflammation.
- the high affinity receptor for human IgE is composed of an ⁇ , ⁇ , and disulfide-linked ⁇ homodimer.
- the ⁇ -chain binds the Fc portion of IgE, whereas the ⁇ -chain serves to amptify signals that are transduced through the ⁇ -chain homodimer.
- the Fc ⁇ R ⁇ l is a PDZ ligand and is a member of the CD20/ Fc ⁇ R ⁇ l receptor family. As is shown supra, Fc ⁇ R ⁇ l binds MINTl.
- Fc ⁇ RI on basophils and mast cells plays a central role in the initiation of allergic responses. Signaling through the Fc ⁇ RI begins by crosslinking of a multivalent allergen bound to IgE. The result is vesicular degranulation, release of histamine, leukotrienes and pro-inflammatory cytokines (IL-6 and TNF ⁇ ), factors responsible for the symptoms of immediate hypersensitivity.
- IL-6 and TNF ⁇ pro-inflammatory cytokines
- Alteration of signaling by targeting PL/PDZ interaction with agonists and antagonists can be used to treat, but is not limited to, asthma, atopic dermatitis, eczema, drug reaction, mastocytosis, urticaria, eosinophilia myalgia syndrome (Turner, H., et. al., 1999, Nature 402 SUPP:B24).
- CD95 Fas/Apo-1
- FasL Fas ligand
- CD95 and Fas ligand are a receptor-ligand pair critically involved in lymphocyte homeostasis and peripheral tolerance. Binding of Fas by its ligand results in apoptotic cell death, an important major mechanism for safe clearance of unwanted cell during resolution of the acute inflammatory response. FasL is mainly restricted to activated T lymphocytes and is rapidly induced. Fas ligand is frequently up-regulated in breast cancer, as compared with normal breast epithelial cells and benign breast disease. As is shown supra, FasL binds the KIAA0561 PDZ domain.
- the PDZ-PL modifiers are useful for treatment of, but not limited to, tumors, e.g., tumors unresponsive to conventional chemotherapy.
- CDW125 binds PTN-4 and RGS12.
- CDW125 is an IL-5 receptor expressed on eosinophils and basophils.
- IL5 promotes growth and differentiation of eosinophil precursors and actives mature eosinophils (Takatsu et al 1994, Adv. Immunol. 57:145-190).
- the secreted form of CDwl25 has antagonistic properties and is able to inhibit IL-5-induced eosinophU proliferation and differentiation.
- Modulation of CDW125 binding to PDZ domains may be used to treat, but is not limited to, asthma, atopic dermatitis, eczema, drug reaction, urticaria, mastocytosis, eosinophilia.
- BLR-1 Burkitt's lymphoma receptor-1
- BLR-1 is a transmembrane receptor detected primarily on B cells, and shown to be upregulated in stimulated T ceUs (Dobner et al., 1992, Eur J. Immunol. 22:2795-2799.; Flynn et al., 1998, J. Exp. Med 188:297-304). BLR-1 functions in chemotaxis of B and T ceUs into follicles of secondary lymphoid organs (e.g. spleen) for proper development and selection toward antigens (Forster et al., 1996, Cell 87:1037-1047. Its ligand is B-lymphocyte chemoattractant (BLC), which is strongly expressed in the follicles of Peyer's patches, spleen
- MAP17, cMOAT, CFTR, and scavenger receptor type B are four known examples of membrane bound and clustered proteins that engage via PDZ / PL mediated interactions with PDZ domains of PDZKl .
- MAP 17 is expressed in normal kidney epitheUum, but it is strongly upregulated in human kidney, colon, lung and breast carcinomas (Kocher, et al., Lab Invest., 1998, 78:117-125).
- cMOAT canalicular multispecific anionic transporter
- CFTR functions as a chloride channel which causes cystic fibrosis when mutated. CFTR binds to PDZKl PDZ domains 2-4. The clustering of several CFTR molecules mediated by PDZK-1 has been shown to potentiate CFTR function (Wang et al., 2000, Cell, 103:169-179). Scavenger receptor type B is expressed in Uver cells and is involved in the uptake of cholesterol from high density lipoproteins. The interaction between scavenger receptor type B and PDZK-1 seem to be associated with the regulation of atherogenesis (Ikemoto et al., 2000, Proc. Natl. Acad Sci. 97:6538-6543).
- PDZKl PDZ domain 1 might interact with the PL of another PDZKl molecule in a head-to-tail fashion (inter-molecular), thus enabeling the local formation of stable high density homo- or heteromeric complexes.
- the equilibrium between intra-molecular and inter-molecular PDZKl PL/PDZ binding might depend on the local PDZKl concentration, whereas phosphorylation changes at the PDZKl PL and / or the BLR-PL might function as an on / off switch for either interaction.
- BLR-1 PL phosphorylation might be regulated by KIAA0807 and KIAA0561 proteins when interacting with BLR-1.
- KIAA0807 and KIAA0561 encode human PDZ domain containing proteins that are highly similar to the mouse protein MAST205, a serine/threonin protein kinase, and computer based protein domain homology search predicts protein thyrosine kinase activity for both, KIAA0807 and KIAA0561.
- PL / PDZ mediated binding of BLR-1 to KIAA0807 and to KIAA0561 proteins as detected by the herein described assay might change the phosphorylation status of BLR-1 and thus regulate its activity and binding properties; in addition, the BLR-1 / KIAA0807 and the BLR-1/KIAA0561 interactions might associate protein kinase activity with BLR-1 which, in turn, might modulate the phosphorylation status of PDZKl or any other of its PDZ domain containing interacting proteins in the course of (BLR-1) transport to and / or clustering in the cell membrane.
- the phosphorylation status of either binding partner might constitute the switch between engagement into and interruption of a PL/PDZ mediated protein- protein interaction.
- BLR-1 binds to the PDZ domain containing proteins PDZKl, MINTl, KIAA0807 or KIAA0561.
- BLR-1 normal function of the receptor is required for physiological B lymphocyte chemotaxis and thus for B lymphocyte function. This function of the receptor is disrupted by small molecule therapeutics that disrupt BLR-1/PDZ binding (see, e.g., Example 7, infra).
- lymphoid cells expressing BLR-1 When this interaction is disrupted, the chemotactic abiUties of lymphoid cells expressing BLR-1 is similarly disrupted. Such a disruption results in a reduced immune response, interference with the ability of lymphocytes to properly circulate and develop responses to antigen.
- small molecule therapeutics are thus immunosupressive agents that specifically target B lymphocytes. These therapeutics are of use in the treatment of autoimmune disorders involving overactivity of B lymphocytes, such as rheumatoid arthritis, systemic lupus erythematosis, and pemphigus vulgaria.
- Agonists and antagonists of the BLR-PDZ interaction are used to treat immune system diseases including, but is not limited to, rheumatoid arthritis, transplant rejection, multiple sclerosis, scleroderma, graft-versus host disease, systemic lupus erythematosus, scleroderma and other autoimmune diseases.
- CD4 is a co-receptor with the T cell receptor (TCR) involved in antigen recognition. Both CD4 and TCR belong to the immunoglobulin supergene family. T cell activation is enhanced by increasing the avidity of T cells for effector and target cells.
- the cytoplasmic domain is involved in signal transduction and association with the tyrosine kinase p56 lck .
- CD4 is expressed on most thymocytes, two-thirds of peripheral blood T lymphocytes, monocytes and macrophages.
- HTV-1 Human immunodeficiency virus type-1 infects cells by membrane fusion mediated by its envelope glycoproteins (gpl20-gp41) and is triggered by the interaction of CD4 and a chemokine co-receptor, CCR5 or CXCR4.
- Modulation of CD4-PDZ inhibitors with agonists and antagonists can be used to treat, but is not limited to, HIV infection immediately after exposure to HTV, rheumatoid arthritis, multiple sclerosis, scleroderma, systemic lupus erythematosis, psoriasis.
- PAG is a recently identified, transmembrane adaptor phosphoprotein. It is expressed in hematopoietic cells including peripheral blood lymphocytes, monocytes and neutrophils, and is a substrate for kinases including the Lck and Fyn (Brdicka et al.2000 J. Exp. Med 191:1592-1604).
- kinases including the Lck and Fyn (Brdicka et al.2000 J. Exp. Med 191:1592-1604).
- GEMs glycosphingolipid-enriched microdomains
- GEMs are 70nm detergent resistant membrane islands found within the bulk plasma membrane of a cell. Each GEM has a concentration of Upids with higher saturated fatty acid side-chains, which favors their association. Importantly, GEMs also contain vital signal transduction molecules including kinases (e.g. Lck, Fyn, LAT, and PI-3 '-kinase), adaptor proteins (LAT) and G-proteins. In hematopoietic ceUs GEMs are a required, functional component of immune cell activation since disruption of GEMs attenuates their activation. Additionally, GEMs functionally interact with the lymphocyte cytoskeleton, which is necessary for microtubule organization and proper lymphocyte activation (Xavier and Seed, 1999, Curr. Op. in Immunol. 11 :265-269; Xavier et al., 1998, Immunity 8:723-732; Montixi et al., 1998, EMBO J. 17:5334- 5348).
- PAG is an important mediator of ceUular signal transduction, and is able to recruit other signaling components, namely KIAA807, to GEMs and regulate signal transduction.
- PAG has been shown to be a negative regulator of T-cell activation (Brdicka et al., 2000, J. Exp. Med. 191 : 1592-1604), and the kinase activity of KIAA807 may transduce the negative regulatory effects of PAG. Such an association provides a means to modulate immune function.
- enhancement of the PAG:KIAA807 interaction may reduce an overactive immune system in autoimmune disease states during transplantation rejection, rheumatoid arthritis, systemic lupus erythematosus (SLE) and multiple sclerosis. In contrast disruption of this interaction may stimulate the activation of lymphocytes, which could help patients with immune deficiencies such as HIV- induced AIDS.
- SLE systemic lupus erythematosus
- interactions between PDZ proteins and PL proteins in cells may be disrupted or inhibited by the administration of inhibitors or antagonists.
- Inhibitors can be identified using screening assays described herein.
- the motifs disclosed herein are used to design inhibitors.
- the antagonists of the invention have a structure (e.g., peptide sequence) based on the C-terminal residues of PL-domain proteins Usted in TABLE 2.
- the antagonists of the invention have a structure (e.g., peptide sequence) based on a PL motif disclosed herein.
- the PDZ/PL antagonists and antagonists of the invention may be any of a large variety of compounds, both naturally occurring and synthetic, organic and inorganic, and including polymers (e.g., oUgopeptides, polypeptides, oUgonucleotides, and polynucleotides), small molecules, antibodies, sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturaUy occurring structures (e.g., peptide mimetics, nucleic acid analogs, and the like), and numerous other compounds.
- polymers e.g., oUgopeptides, polypeptides, oUgonucleotides, and polynucleotides
- small molecules antibodies, sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturaUy occurring structures (e.g., peptide mimetics, nucleic acid analogs, and the like)
- analogs of naturaUy occurring structures e
- the peptides and peptide mimetics or analogues of the invention contain an amino acid sequence that binds a PDZ domain in hematopoietic cells such as T cells and B ceUs, or otherwise inhibits the association of PL proteins and PDZ proteins.
- the antagonists comprise a peptide that has a sequence corresponding to the carboxy-terminal sequence of a PL protein Usted in TABLE 2, e.g., a peptide Usted TABLE 4.
- the peptide comprises at least the C-terminal two (3), three (3) or four (4) residues of the PL protein, and often the inhibitory peptide comprises more than four residues (e.g., at least five, six, seven, eight, nine, ten, twelve or fifteen residues) from the PL protein C- te ⁇ ninus.
- the C-terminal domains of specific surface receptors expressed by hematopoietic system and endotheUal cells may themselves be used as inhibitors, and may be used as the basis for rational design of non-peptide inhibitors. See Section 6.6, infra.
- the inhibitor is a peptide, e.g., having a sequence of a PL C-terminal protein sequence. See, e.g. Section 6.5.1, infra
- the antagonist is a fusion protein comprising such a sequence. Fusion proteins containing a transmembrane transporter amino acid sequence are particularly useful. See, e.g. Section 6.9, infra.
- the inhibitor is conserved variant of the PL C-terminal protein sequence having inhibitory activity. See, e.g. Section 6.5.2, infra.
- the antagonist is a peptide mimetic of a PL C-terminal sequence. See, e.g. Section 6.5.3, infra.
- the inhibitor is a small molecule (i.e., having a molecular weight less than 1 kD). See, e.g. Section 6.5.4, infra.
- the antagonists comprise a peptide that has a sequence of a PL protein carboxy-terminus listed in TABLE 2.
- the peptide comprises at least the C- terminal two (2) residues of the PL protein, and typically, the inhibitory peptide comprises more than two residues (e.g, at least three, four, five, six, seven, eight, nine, ten, twelve or fifteen residues) from the PL protein C-te ⁇ hinus.
- the peptide may be any of a variety of lenghts (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, or at least 20 residues) and may contain additional residues not from the PL protein.
- the residues shared by the inhibitory peptide with the PL protein are found at the C-terminus of the peptide.
- the sequence is internal.
- the inhibitory peptide comprises residues from a PL sequence that is near, but not at the c-terminus of a PL protein (see, Gee et al., 1998, J Biological Chem. 273:21980-87).
- the PL protein carboxy-terminus sequence is referred to as the "core
- the "core PDZ motif sequence" of a hematopoietic ceU surface receptor at its C-terminus contains the last four amino acids, this sequence may be used to target PDZ domains in hematopoietic cells.
- the four amino acid core of a PDZ motif sequence may contain additional amino acids at its amino terminus to further increase its binding affinity and or stability.
- the PDZ motif sequence peptide can be from four amino acids up to 15 amino acids. It is prefened that the length of the sequence to be 6-10 amino acids. More preferably, the PDZ motif sequence contains 8 amino acids.
- Additional amino acids at the amino terminal end of the core sequence may be derived from the natural sequence in each hematopoietic cell surface receptor or a synthetic linker. The additional amino acids may also be conservatively substituted. When the third residue from the C-terminus is S, T or Y, this residue may be phosphorylated prior to the use of the peptide.
- the peptide and nonpeptide inhibitors of the are small, e.g., fewer than ten amino acid residues in length if a peptide.
- a limited number of ligand amino acids directly contact the PDZ domain (generally less than eight) (Kozlov et al., 2000, Biochemistry 39, 2572; Doyle et al., 1996, Cell 85, 1067) and that peptides as short as the C-terminal three amino acids often retain similar binding properties to longer (> 15) amino acids peptides (Yanagisawa et al., 1997, J. Biol. Chem.272, 8539).
- FIGURES 3A-H show the use of peptides to inhibit PL-PDZ interactions using the G assay described supra.
- the inhibiton assays were carried out using GST fusion proteins containing PDZ domains from DLGl or PSD95 (see supra and TABLE 3). Binding of biotinylated PL peptides for Clasp 2, CD46, Fas, or KVl.3 (as listed in TABLE 4) was determined in the presence of various competitor peptides (at a concentration of 100 uM) or in the absence of a competitor (equalized as 100% binding).
- the competitor peptides were 8-mers peptides having the sequence of C-terrninus of Clasp 2 (MTSSSSW, SEQ ID NO: 191), CD46 (REVKFTSL, SEQ ID NO: 113), or Fas (RNEIQSLV, SEQ ID NO: 48), a unlabeled 19-mer having the sequence of c-terminus of KVl.3 (i.e., non-biotinylated AA33L as listed in TABLE 3), or a peptide having the sequence of residues 64-76 of hemoglobin (Vidal et al., 1999, J. Immunol. 163, 4811), i.e., an unrelated competitor.
- FIGURES 3C-F show similar assays using shorter peptides to inhibit (e.g., a 3-mer and a 5-mer).
- Figures 3C-E show binding of biotinylated PL peptides for Clasp 2, CD46, Fas, or KVl.3, at the indicated concentration (as listed i_u TABLE 3) to GST fusion proteins containing PDZ domains from NeDLG, DLGl, or PSD95 in the absence or presence of 1 mM 3-mer peptide having the sequence of the C-terminus of Clasp 2 (SW).
- FIGURE 3F shows the effect on binding of a 5-mer CD49E peptide (ATSDA, SEQ ID NO: 25) to GST fusion proteins containing a PDZ domain from 41.8Kd
- the variants have the same or a different abiUty to bind a PDZ domain as the parent peptide.
- amino acid substitutions are conservative, i.e., the amino acid residues are replaced with other amino acid residues having physical and/or chemical properties similar to the residues they are replacing.
- conservative amino acid substitutions are those wherein an amino acid is replaced with another amino acid encompassed within the same designated class.
- peptide mimetics can be prepared using routine methods, and the inhibitory activity of the mimetics can be confirmed using the assays of the invention.
- the antagonist is a peptide mimetic of a PL C-terminal sequence.
- the skilled artisan wiU recognize that individual synthetic residues and polypeptides incorporating mimetics can be synthesized using a variety of procedures and methodologies, which are well described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY.
- Polypeptides incorporating mimetics can also be made using solid phase synthetic procedures, as described, e.g., by Di Marchi, et al., U.S. Pat. No. 5,422,426.
- Mimetics of the invention can also be synthesized using combinatorial methodologies.
- Various techniques for generation of peptide and peptidomimetic libraries are well known, and include, e.g., multipin, tea bag, and split-couple-mix techniques; see, e.g., al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Cun. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers.3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234.
- the inhibitor is a small molecule (i.e., having a molecular weight less than 1 kD).
- Methods for screening small molecules are weU known in the art and include those described supra at Section 6.4.
- the following sections describe specific surface receptors expressed by different cell types in the hematopoietic and immune response system.
- the C-termini of these receptors are used as inhibitors, or serve as the basis for designing PDZ motif sequence peptides, variants, fusion proteins, peptidomimetics, and small molecules for use in inhibiting PDZ-PL interactions.
- the peptides are tested in an assay of the invention for inhibitory or modulatory activity (also see, TABLE 4, and discussion supra).
- a number of surface receptors expressed by T cells contain a PDZ motif sequence (PL sequence). These molecules include CD3 ⁇ , CD4, CD6, CD38, CD49e, CD49f, CD53, CD83, CD90, CD95, CD97, CD98, CDwl37 (41BB), CD166, CDwl28 (D 8 R), DNAM-1, Fas ligand (FasL) and LPAP (Barclay et al., 1997, The Leucocyte Antigen Facts Book, second edition, Academic Press), CLASP-1, CLASP-2, CLASP-4, KVl .3, BLR-1 (CXCR5), PAG and DOCK2.
- PL sequence PDZ motif sequence
- the C-terminal core sequence of CD3 is SSQL (SEQ 3D NO:4).
- QL SEQ ID NO:
- SQL SQL
- SSSQL SEQ ID NO:5
- SSSSQL SEQ ID NO:6
- PSSSSQL SEQ ID NO:7
- PPSSSSQL SEQ ID NO:8
- the C-terminal core sequence of CD4 is CSPI (SEQ ID NO:9).
- PI SEQ ID NO:
- SPI SEQ ID NO:
- TCSPI SEQ ID NO: 10
- KTCSPI SEQ ID NO: 11
- QKTCSPI SEQ ID NO: 12
- FQKTCSPI FQKTCSPI
- SEQ ID NO: 17 may also be used to target a PDZ domain- containing protein in T cells.
- the C-terminal core sequence of CD38 is TSEI (SEQ ID NO:19).
- El SEQ ID NO:
- SEI SEQ ID NO:
- CTSEI SEQ ID NO:20
- SCTSEI SEQ ID NO:21
- SSCTSEI SEQ ID NO:
- DSSCTSEI SEQ ID NO:23
- DSSCTSEI SEQ ID NO:23
- the C-terminal core sequence of CD49e is TSDA (SEQ ID NO:24).
- DA (SEQ ID NO:24).
- KPPATSDA (SEQ ID NO:28) may also be used to target a PDZ domain- containing protein in T cells.
- the C-terminal core sequence of CD49f is TSDA (SEQ ID NO:29).
- DA Naturally-occuring residues are added or removed from the core sequence
- SDA SEQ ID NO:
- LTSDA SEQ ID NO:30
- RLTSDA SEQ ID NO:31
- SEQ ID NO:32 may also be used to target a PDZ domain- containing protein in T ceUs.
- the C-te ⁇ ninal core sequence of CD53 is TIGL (SEQ ID NO:34).
- GL SEQ ID NO:34
- KTSQTIGL (SEQ ID NO:38) may also be used to target a PDZ domain- containing protein in T cells.
- the C-te ⁇ ninal core sequence of CD83 is TELV (SEQ. ID. NO: 177).
- LV (SEQ ID NO: 177).
- ELV SEQ ID NO:
- KTELV SEQ. ID. NO: 178
- HKTELV SEQ. ID. NO: 179
- PHKTELV SEQ. ID. NO: 180
- TPHKTELV SEQ. ID. NO: 181
- the C-terminal core sequence of CD90 is FMSL (SEQ ID NO:39).
- SL SEQ ID NO:39.
- LV (SEQ ID NO:44).
- SLV SEQ ID NO:
- IQSLV SEQ ID NO:45
- EIQSLV SEQ ID NO:46
- NEIQSLV SEQ ID NO:
- RNEIQSLV SEQ ID NO:48
- T ceUs T ceUs
- the C-te ⁇ ninal core sequence of CD97 is ESGI (SEQ ID NO:49).
- GI SEQ ID NO:49.
- SGI SEQ ID NO:
- SESGI SEQ ID NO:50
- ASESGI SEQ ID NO:51
- RASESGI SEQ ID NO:
- LRASESGI SEQ ID NO:53
- T ceUs T ceUs
- the C-terminal core sequence of CD98 is PYAA (SEQ ID NO:54).
- AA SEQ ID NO:54
- SEQ ID NO:57 may also be used to target a PDZ domain- containing protein in T cells.
- the C-terminal core sequence ofCDwl37 is GCEL (SEQ ID NO:59).
- EL SEQ ID NO:59.
- SEQ ID NO:62 may also be used to target a PDZ domain- containing protein in T cells.
- the C-terminal core, sequence of CD 166 is KTEA (SEQ ID NO:64).
- EA SEQ ID NO:
- TEA SEQ ID NO:
- HKTEA SEQ ID NO:65
- NHKTEA SEQ ID NO:66
- SEQ ID NO:67 may also be used to target a PDZ domain-containing protein in T cells.
- ENNHKTEA SEQ ID NO:68
- the C-terminal core sequence of CDwl28 is SSNL (SEQ ID NO:69).
- NL SEQ ID NO:69.
- SNL SEQ ID NO:
- VSSNL SEQ ID NO:70
- NVSSNL SEQ ID NO:71
- VNVSSNL VNVSSNL
- SEQ ID NO:72 may also be used to target a PDZ domain- containing protein in T cells.
- the C-terminal core sequence of DNAM-1 is KTRV (SEQ ID NO:74).
- RV When naturally-occuring residues are added or removed from the core sequence, RV (SEQ ID NO: 74).
- SEQ ED NO:77 SEQ ED NO:77
- SRRPKTRV SEQ ID NO:78
- LYKL SEQ ID NO:79
- TFFGLYKL (SEQ ED NO:83) may also be used to target a PDZ domain- containing protein in T cells.
- the C-terminal core sequence of LPAP is VTAL (SEQ ID NO:84).
- AL SEQ ED NO:
- TAL SEQ ID NO:
- HVTAL SEQ ID NO:85
- LHVTAL SEQ ID NO:86
- GLHVTAL SEQ K
- QGLHVTAL SEQ ID NO:88
- the C-terminal core sequence of CLASP-1 is SAQV (SEQ. ED. NO: 182).
- QV SEQ ED NO:
- AQV SEQ ID NO:
- SSAQV SEQ. ID. NO: 183
- SSSAQV SEQ. TD. NO: 184
- ISSSAQV SEQ. ED. NO: 185
- SISSSAQV SEQ. ID. NO: 1 6
- the C-terminal core sequence of CLASP-2 is SSW (SEQ. ED. NO: 187).
- W SEQ ED NO:
- SW SEQ ID NO:
- SSSW SEQ. ED. NO: 188
- SSSSW SEQ. ED. NO: 189
- TSSSSW SEQ. ED. NO: 190
- MTSSSSW SEQ. ID. NO: 191
- the C-terminal core sequence of CLASP-4 is YAEV (SEQ. ID. NO: 192).
- EV SEQ ID NO:
- AEV SEQ ID NO:
- RYAEV SEQ. ID. NO: 193
- PRYAEV SEQ. TD. NO: 194
- SPRYAEV SEQ. ro. NO: 195
- GSPRYAEV SEQ. ID. NO: 196
- the C-te ⁇ ninal core sequence of KVl.3 is FTDV (SEQ. ID. NO: 202).
- FTDV SEQ. ID. NO: 202
- DV naturally-occuring residues are added or removed from the core sequence
- TDV SEQ ro NO:
- IFTDV SEQ. ED. NO: 203
- KIFTDV SEQ. ID, NO: 204
- KKIFTDV (SEQ. ED. NO: 205), and IKKEFTDV (SEQ. ID. NO: 206) may also be used to target a PDZ domain-containing protein in T ceUs.
- the C-te ⁇ ninal core sequence of DOCK2 is STDL (SEQ. ED. NO: 207).
- DL SEQ. ED. NO: 207
- TDL SEQ. ID. NO:
- LSTDL SEQ. ID. NO: 208
- SLSTDL SEQ. ID. NO: 209
- DSLSTDL (SEQ. ID. NO: 210), and PDSLSTDL (SEQ. ID. NO: 211) may also be used to target a PDZ domain-containing protein in T cells.
- the C-terminal core sequence of BLR-1 is LTTF (SEQ ID NO:).
- TF (SEQ ID NO: 1)
- TTF SEQ ID NO:
- SLTTF SEQ ED NO:
- TSLTTF SEQ ID NO:
- ATSLTTF SEQ ID NO:
- NATSLTTF SEQ ED NO:
- SEQ ED NO: NATSLTTF
- the C-te ⁇ ninal core sequence of PAG is ITRL (SEQ ID NO:).
- RL SEQ ED NO:
- TRL TRL
- SEQ ID NO: DITRL (SEQ ID NO:), RDITRL (SEQ ID NO:), GRDITRL (SEQ ID NO:), and QGRDITRL (SEQ ID NO:) may also be used to target a PDZ domain-containing protein in T cells.
- a number of surface receptors expressed by B cells contain a PDZ domain motif sequence. These molecules include, but are not Umited to, CD38, CD53, CD95, CD97, CD98, CDwl37, CD138, CDwl25 (IL5R), DNAM-1, LPAP, Syndecan-2 (Barclay et al., 1997, The Leucocyte Antigen Facts Book, second edition, Academic Press) and BLR- 1.
- the specific motif sequences of CD38, CD53, CD83, CD95, CD97, CD98, CDwl37, DNAM-1, DOCK2, LPAP, BLR-1 (CXCR5), PAG, CLASP-1, CLASP-2 and CLASP-4 have been described in the preceding paragraphs.
- the C-terminal core sequence of CD138 is EFYA (SEQ ID NO:89).
- YA SEQ ID NO:
- FYA SEQ ro NO:
- EEFYA SEQ ED NO:90
- QEEFYA SEQ ED NO:91
- KQEEFYA SEQ ID NO:92
- TKQEEFYA SEQ ID NO:93
- the C-te ⁇ ninal core sequence of CDwl25 is DSVF (SEQ ID NO:94).
- VF SEQ ID NO:
- SVF SEQ ED NO:
- EDSVF SEQ ID NO:95
- LEDSVF SEQ ED NO:96
- TLEDSVF SEQ ID NO:97
- ETLEDSVF SEQ ID NO:98
- the C-terminal core sequence of Syndecan-2 is EFYA (SEQ. ED. NO: 212).
- EFYA SEQ. ED. NO: 212
- FYA SEQ ED NO:
- KEFYA SEQ. ID. NO: 213
- TKEFYA SEQ. ID. NO: 214
- PTKEFYA SEQ. ID. NO: 215
- APTKEFYA SEQ. ID. NO: 216
- the C-te ⁇ ninal core sequence of BLR-1 is LTTF (SEQ. ID. NO: 217).
- TF SEQ ID NO:
- TTF SEQ ED NO:
- SLTTF SEQ. ID. NO: 2148
- TSLTTF SEQ. ED. NO: 219
- ATSLTTF SEQ. ID. NO: 220
- NATSLTTF SEQ. ID. NO: 221
- a number of surface receptors expressed by NK cells contain a PDZ domain motif sequence. These molecules include, but are not limited to CD38, CD56, CD98 and DNAM-1. The specific motif sequences of CD38, CD98 and DNAM-1 have been described in the preceding paragraphs.
- the C-terminal core sequence of CD56 is ESKA (SEQ ID NO:99).
- KA SEQ ID NO:
- SKA SEQ ID NO:
- NESKA SEQ ID NO:100
- ENESKA SEQ ID NO:101
- KENESKA SEQ ID NO:102
- TKENESKA SEQ ID NO:103
- a number of surface receptors expressed by cells of the monocytic lineage contain a PDZ domain motif sequence. These molecules include, but are not limited to CD38, CD44, CD46, CD49e, CD49f, CD53, CD61, CD95, CD97, CD98, CD148, CDwl28, CDwl37, Ly-6, DNAM-1 and Fc ⁇ RI ⁇ .
- the specific motif sequences of CD38, CD49e, CD49f, CD53, CD95, CD97, CD98, CDwl28, CDwl37, DNAM- 1, Galectin 3 (Mac-2), BLR-1 (CXCR5) and Mannose receptor have been described in the preceding paragraphs.
- the C-terminal core sequence of CD44 is KIGV (SEQ ID NO:104).
- KIGV SEQ ID NO:104
- GV SEQ ED NO:
- IGV SEQ ED NO:
- MKIGV SEQ ID NO: 105
- DMKIGV SEQ ID NO:106
- SEQ ID NO:107 and NVDMKIGV (SEQ ID NO:108) may also be used to target a PDZ domain-containing protein in monocytes.
- the C-terminal core sequence of CD46 is FTSL (SEQ ID NO: 109).
- FTSL SEQ ID NO: 109.
- SL SEQ JJD NO:
- TSL SEQ ED NO:
- KFTSL SEQ IDNO:110
- VKFTSL SEQ ID NO:lll
- SEQ ID NO-.112 and REVKFTSL (SEQ ID NO:113) may also be used to target a PDZ domam-containing protein in monocytes.
- the C-terminal core sequence of CD61 is KSLV (SEQ ID NO:l 14).
- LV SEQ ID NO:
- SLV SEQ ED NO:
- LKSLV SEQ ID O:115
- FLKSLV SEQ ED NO: 116
- RFLKSLV SEQ ED NO:117
- GRFLKSLV SEQ ID NO:118
- GYIA GYIA (SEQ ID NO:l 19).
- IA SEQ DD NO:
- YIA SEQ ID NO:
- NGYIA SEQ ED NO:120
- .TNGYIA SEQ ED NO:121
- KTNGYIA SEQ ID NO:122
- GKTNGYIA GKTNGYIA
- LL SEQ ID NO:
- TLL SEQ ⁇
- LQTLL SEQ ro NO:125
- LLQTLL SEQ ID NO:126
- VLLQTLL SEQ K> NO.-127
- SVLLQTLL SEQ ID NO:1278
- DL SEQ ED NO:
- IDL SEQ E NO:
- PPEDL SEQ ID NO: 130
- SPPIDL SEQ ID NO: 131
- MSPPIDL MSPPIDL
- SEQ E SEQ E NO:132
- EMSPPIDL SEQ ID NO:133
- the C-terminal core sequence of Galectin 3 is YTMI (SEQ ID NO:134).
- MI SEQ ID NO:
- TMI SEQ ro NO:
- SYTMI SEQ ID NO:135
- ASYTMI SEQ ID NO:136
- SASYTMI SEQ ID NO:137
- TSASYTM SEQ ED NO:138
- the C-terminal core sequence of mannose receptor is HSVI (SEQ ED NO: 139).
- SVI SEQ.-ED NO:
- EHSVI SEQ ID NO:140
- NEHSVI SEQ ED NO:141
- QNEHSVI (SEQ ro NO: 142) and EQNEHSVI (SEQ ID NO: 143) may also be used to target
- a number of surface receptors expressed by granulocytes contain a PDZ domain motif sequence. These molecules include, but are not limited to CD53, CD95, CD97, CD98, CD148, CDwl25, CDwl28, Fc ⁇ RI ⁇ and G-CSFR. The specific motif sequences of most of these molecules have been described in the preceding paragraphs.
- the C-te ⁇ ninal core sequence of G-CSFR is TSVL (SEQ ED NO:144). When naturally-occuring residues are added or removed from the core sequence, VL (SEQ ID NO;
- SEQ ID NO:147 and LFPITSVL (SEQ ID NO:148) may also be used to target a PDZ domain- containing protein in monocytes.
- endothelial cells While endothelial cells are not hematopoietic cells, they closely interact with the hematopoietic system as they form the lining of blood vessels. As such, endotheUal cells come in contact with the cells of the hematopoietic system. Thus, the ability to regulate endothelial cell function provides for indirect regulation of hematopoietic cells.
- a number of surface receptors expressed by endothelial ceUs contain a PDZ domain motif sequence. These molecules include, but are not limited to CD34, CD46, CD66b, CD66c, CD105, CD106, CD62e (E-selectin) and VCAMl.
- the C-terminal core sequence of CD34 is DTEL (SEQ ⁇ NO: 149). When naturally-occuring residues are added or removed from the core sequence, EL (SEQ ID NO:
- TEL SEQ ED NO:
- ADTEL SEQ ID NO:150
- VADTEL SEQ ID NO:151
- WADTEL WADTEL
- CD66b and CD66c may also be used to target a PDZ domain-containing protein in endothelial cells.
- the C-terminal core sequence of CD66b and CD66c is VALI (SEQ ID NO: 152)
- LI SEQ ID NO: 154
- ALI SEQ ID NO: 155
- ARVALI SEQ EDNO:156
- LARVALI SEQ ID NO-.157
- VLARVALI VLARVALI
- SMA SEQ E NO:
- TSSMA SEQ ED NO:160
- STSSMA SEQ ID NO:161
- CSTSSMA CSTSSMA
- the C-terminal core sequence ofCD106 is KSKV (SEQ DD NO:163).
- KV SEQ ED NO:
- SKV SEQ ED NO:
- QKSKV SEQ ID NO:164
- AQKSKV SEQ ED NO:165
- EAQKSKV SEQ ID NO:166
- VEAQKSKV SEQ ID NO:167
- the C-terminal core sequence of CD62e is SYIL (SEQ ED NO:168).
- IL SEQ ID NO:
- YIL (SEQ ID NO: ), PSYIL (SEQ ID NO:169), KPSYIL (SEQ ID NO:170), QKPSYE (SEQ ⁇ ) NO:171) and YQKPSYIL (SEQ ID NO:172) may also be used to target a PDZ domain- containing protein in endothelial cells.
- KSKV SEQ. ED. NO: 197.
- SKV SEQ ID NO:
- QKSKV SEQ. ID. NO: 198
- AQKSKV SEQ. ID. NO: 199
- EAQKSKV (SEQ. ED. NO: 200), and VEAQKSKV (SEQ. ED. NO: 201) may also be used to target a PDZ domain-containing protein in endothelial cells.
- Fc ⁇ RI ⁇ , CDwl25, CDwl28 and E -8RB are transmembrane receptors expressed by mast ceUs, basophils and eosinophils. These receptors play a role in the activation of these cells to result in degranulation and histamine release in allergic reactions.
- the C-terminal core sequence of Fc ⁇ RI ⁇ is PIDL (SEQ ID NO:129).
- DL SEQ ID NO:
- IDL SEQ ED NO:
- PPIDL SEQ ID NO:244
- SPPEDL SEQ ID NO:245
- MSPPIDL SEQ ID NO:246
- EMSPPIDL SEQ ED NO:247
- the C-terminal core sequence of CDwl25 is DSVF (SEQ ID NO: 248).
- VF SEQ ED NO:
- SVF SEQ ID NO:
- EDSVF SEQ ID NO:249
- LEDSVF SEQ ID NO:250
- SEQ ID NO:251 may also be used to target a PDZ domain-containing protein in mast cells.
- ETLEDSVF SEQ ID NO:252
- the C-terminal core sequence of CDwl28 is SSNL (SEQ ED NO:253).
- NL SEQ ED NO:253
- SEQ ED NO:256 may also be used to target a PDZ domain-containing protein in mast ceUs.
- the C-terminal core sequence of E -8RB is STTL (SEQ ID NO:258).
- TL SEQ ED NO:
- TTL SEQ ID NO:
- TSTTL SEQ ID NO:259
- HTSTTL SEQ ED NO:260
- GHTSTTL GHTSTTL
- the C-terminal core sequence of NMDA is ESDV (SEQ. ED. NO: 223).
- DV SEQ ED NO: 223.
- SDV SEQ K
- IESDV SEQ. ID. NO: 224
- SEESDV SEQ. ID. NO: 225
- PSIESDV SEQ. ID. NO: 2236
- MPSEESDV SEQ. ED. NO: 227) may also be used to target a PDZ domain-containing protein in neuronal ceUs.
- the C-terminal core sequence of neurexin is EYYV (SEQ. ID. NO: 228).
- YV SEQ ID NO:
- YYV SEQ ID NO:
- KEYYV SEQ. ID. NO: 229
- DKEYYV SEQ. ED. NO: 230
- KDKEYYV SEQ. ID. NO: 231
- NKDKEYYV SEQ. ID. NO: 232
- the C-terminal core sequence of Glycophorin C is EYFI (SEQ. ED. NO: 233).
- YFI SEQ ED NO:
- KEYFI SEQ. ro. NO: 234
- RKEYFI SEQ. ED. NO: 235
- SRKEYFI SEQ. ID. NO: 236
- SSRKEYFI SEQ. ID. NO: 237
- the C-terminal core sequence of CD148 is KTIA (SEQ. ID. NO: 238).
- IA SEQ ED NO:
- TIA SEQ ID NO:
- GKTIA SEQ. ED. NO: 239
- FGKTIA SEQ. ED. NO: 240
- TFGKTIA SEQ. ID. NO: 241
- TTFGKTIA SEQ. ID. NO: 242
- VSFV The C-terminal core sequence of beta-spectrin is VSFV (SEQ. ED. NO: ).
- FV SEQ. ID. NO:
- SFV SEQ. ID. NO:
- LVSFV SEQ. ED. NO:
- SLVSFV SEQ. ID. NO:
- QSLVSFV SEQ. ID. NO:
- GQSLVSFV GQSLVSFV
- the peptides of the invention or analogues thereof may be prepared using virtually any art-known technique for the preparation of peptides and peptide analogues.
- the peptides may be prepared in linear form using conventional solution or solid phase peptide syntheses and cleaved from the resin followed by purification procedures (Creighton, 1983, Protein Structures And Molecular Principles, W.H. Freeman and Co., N.Y.). Suitable procedures for synthesizing the peptides described herein are well known in the art.
- the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure and mass spectroscopy).
- analogues and . derivatives of the peptides can be chemically synthesized.
- the linkage between each amino acid of the peptides of the invention may be an amide, a substituted amide or an isostere of amide.
- Nonclassical amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the sequence.
- Non- classical amino acids include, but are not limited to, the D-isomers of the common amino acids, et-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, ⁇ -Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogues in general.
- the amino acid can be D (dextrorotary) or L (levorotary
- the peptide or the relevant portion may also be synthesized using conventional recombinant genetic engineering techniques.
- a polynucleotide sequence encoding a linear form of the peptide is inserted into an appropriate expression vehicle, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
- the expression vehicle is then transfected into a suitable target ceU which wUl express the peptide.
- the expressed peptide is then isolated by procedures well-established in the art.
- microorganisms such as bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an appropriate coding sequence; or animal cell systems.
- microorganisms such as bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recomb
- the expression elements of the expression systems vary in their strength and specificities.
- any of a number of suitable transcription and translation elements may be used in the expression vector.
- inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptip-lac hybrid promoter) and the like may be used;
- promoters such as the baculovirus polyhedron promoter may be used;
- promoters derived from the genome of plant cells e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein
- plant viruses e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV
- the expression of sequences encoding the peptides of the invention may be driven by any of a number of promoters.
- viral promoters such as the 35S R ⁇ A and 19S R ⁇ A promoters of CaMV (Brisson et al, 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsuet ⁇ ./., 1987, EMBO J.6:307-311) may be used; alternatively, plant promoters such as the smaU subunit of RUBISCO (Coruzzi et al, 1984, EMBO J.3:1671-1680; BrogUe et al, 1984, Science 224:838- 843) or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B (Gurley et al, 1986, Mol.
- Autographa c ⁇ lifornica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes.
- the virus grows in Spodopterafrugiperda cells.
- a coding sequence may be cloned into non-essential regions (for example the polyhedron gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedron promoter).
- Successful insertion of a coding sequence wiU result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedron gene).
- a number of viral based expression systems may be utilized.
- a coding sequence may be ligated to an adenovirus transcription translation control complex, e.g., the late promoter and tripartite leader sequence.
- This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) wiU result in a recombinant virus that is viable and capable of expressing peptide in infected hosts, (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad.
- the vaccinia 7.5 K promoter may be used, (see, e.g. , Mackett et al, 1982, Proc. Natl. Acad. Sci. USA 79:7415-7419; Mackett et al, 1984, J. Virol.49:857-864; PanicaU et al, 1982, Proc. Natl. Acad. Sci. USA 79:4927-4931).
- the peptides and peptide analogues of the invention can be purified by art- known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like.
- the actual conditions used to purify a particular peptide or analogue will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those having skill in the art.
- the purified peptides can be identified by assays based on their physical or functional properties, including radioactive labeling followed by gel electrophoresis, radioimmuno-assays, ELISA, bioassays, and the like.
- any antibody which specifically binds the peptides or peptide analogues may be used.
- various host animals including but not limited to rabbits, mice, rats, etc., may be immunized by injection with a peptide.
- the peptide may be attached to a suitable carrier, such as BSA or KLH, by means of a side chain functional group or linkers attached to a side chain functional group.
- adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oU emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.
- BCG Bacilli Calmette-Guerin
- Corynebacterium parvum bacilli Calmette-Guerin
- Monoclonal antibodies to a peptide may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein, 1975, Nature 256:495-497, the human B-cell hybridoma technique, Kosbor et al, 1983, Immunology Today 4:72; Cote et al, 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030 and the EBV-hybridoma technique (Cole et al, 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)).
- fragments include but are not limited to F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and Fab fragments, which can be generated b reducing the disulfide bridges of the F(ab') 2 fragments.
- Fab expression libraries may be constructed (Huse et al, 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the peptide of interest.
- the antibody or antibody fragment specific for the desired peptide can be attached, for example, to agarose, and the antibody-agarose complex is used in immunochromatography to purify peptides of the invention. See, Scopes, 1984, Protein
- the PDZ domain binding and PDZ-PL inhibitory compounds of the present invention are useful in regulating diverse activities of hematopoietic cells (e.g., T cells and B cells) and other cells involved in the immune response.
- hematopoietic cells e.g., T cells and B cells
- the compounds of the invention are used to inhibit leukocyte activation, which is manifested in measurable events including but not limited to, cytokine production, ceU adhesion, expansion of cell numbers, apoptosis and cytotoxicity.
- the compounds of the invention may be used to treat diverse conditions associated with undesirable leukocyte activation, including but not limited to, acute and chronic inflammation, graft-versus-host disease, transplantation rejection, hypersensitivities and autoimmunity such as multiple sclerosis, rheumatoid arthritis, peridontal disease, systemic lupus erythematosis, juvenile diabetes mellitis, non-insulin-dependent diabetes, and aUergies, and other conditions listed herein (see, e.g., Section 6.4, supra).
- the invention also relates to methods of using such compositions in modulating leukocyte activation as measured by, for example, cytotoxicity, cytokine production, cell proliferation, and apoptosis.
- Assays for activation are well known.
- PDZ/PL interaction antagonists can be evaluated in the following: (1) cytotoxic T lymphocytes can be incubated with radioactively labeled target cells and the antigen-specific lysis of these target ceUs detected by the release of radioactivity, (2) helper T lymphocytes can be incubated with antigens and antigen presenting cells and the synthesis and secretion of cytokines measured by standard methods (Windhagen A; et al., 1995, Immunity 2(4): 373-80), (3) antigen presenting cells can be incubated with whole protein antigen and the presentation of that antigen on MHC detected by either T lymphocyte activation assays or biophysical methods (Harding et al., 1989, Proc.
- mast cells can be incubated with reagents that cross-link their Fc-epsilon receptors and Wstamine release measured by enzyme immunoassay (Siraganian, et al., 1983, TIPS 4: 432-437).
- the effect of PDZ/PL interaction antagonists on products of leukocyte activation in either a model organism (e.g., mouse) or a human patient can also be evaluated by various methods that are well known.
- a model organism e.g., mouse
- the production of antibodies in response to vaccination can be readily detected by standard methods cunently used in clinical laboratories, e.g., an ELISA;
- the migration of immune cells to sites of inflammation can be detected by scratching the surface of skin and placing a sterile container to capture the migrating cells over scratch site (Peters et al., 1988, Blood 72: 1310-5);
- the proUferation of peripheral blood mononuclear cells in response to mitogens or mixed lymphocyte reaction can be measured using 3 H-thymidine;
- the phagocytic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PMBCs in wells together with labeled particles (Peters et
- human peripheral blood mononuclear cells PBMC
- human T cell clones e.g., Jurkat E6, ATCC TUB- 152
- EBV-transfo ⁇ ned B cell clones e.g., 9D10, ATCC CRL-8752
- antigen-specific T cell clones or lines can be used to examine PDZ PL interaction antagonists in vitro. Inhibition of activation of these cells or ceU lines can be used for the evaluation of potential PDZ PL interaction antagonists.
- Standard methods by which hematopoietic cells are stimulated to undergo activation characteristic of an immune response are, for example:
- Antigen specific stimulation of immune responses Either pre-immunized or na ⁇ ve mouse splenocytes can be generated by standard procedures. In addition, antigen-specific T ceU clones and hybridomas (e.g., MBP-specific), and numerous B ceU lymphoma ceU fines (e.g., CH27), have been previously characterized and are available for the assays discussed below. Antigen specific splenocytes or B-cells can be mixed with antigen specific T-cells in the presence of antigen to generate an immune response. This can be performed in the presence or absence of PDZ/PL interaction antagonists to assay whether PDZ/PL interaction antagonists modulate the immune response infra.
- MBP-specific antigen-specific T ceU clones and hybridomas
- B ceU lymphoma ceU fines e.g., CH27
- T cell activation A) Non-specific T cell activation.
- the following methods can be used to activate T ceUs in the absence of antigen: 1) cross-Unking T cell receptor (TCR) by addition of antibodies against receptor activation molecules (e.g., TCR, CD3, or CD2) together with antibodies against co-stimulator molecules, for example anti-CD28; 2) activating cell surface receptors in a non-specific fashion using lectins such as concanavalin A (con A) and phytohemagglutinin (PHA); 3) mirnicking cell surface receptor-mediated activation using pharmacological agents that activate protein kinase C (e.g., phorbol esters) and increase cytoplasmic Ca 2+ (e.g., ionomycin).
- TCR cross-Unking T cell receptor
- receptor activation molecules e.g., TCR, CD3, or CD2
- co-stimulator molecules for example anti-CD28
- co-stimulator molecules for example anti-CD28
- Non-specific B cell activation 1) application of antibodies against cell surface molecules such as IgM, CD20, or CD21. 2) Lipopolysaccharide (LPS), phorbol esters, calcium ionophores and ionomycin can also be used to by-pass receptor triggering.
- LPS Lipopolysaccharide
- phorbol esters phorbol esters
- calcium ionophores ionomycin
- ionomycin can also be used to by-pass receptor triggering.
- MLR Mixed lymphocyte reaction
- PBMC to activate lymphocytes by presentation of mismatched tissue antigens, which occurs in all cases except identical twins.
- a standard approach is to generate tetanus toxin-specific T cells from a donor that has recently been boosted with tetanus toxin.
- Major histocompatability complex- (MHC-) matched antigen presenting cells and a source of tetanus toxin are used to maintain antigen specificity of the cell line or T cell clone (Lanzavecchia, A., et al., 1983, Eur. J. Immun. 13: 733-738).
- Tyrosine phosphorylation of early response proteins such as HS 1 , PLC-r, ZAP- 76, and Vav is an early biochemical event following leukocyte activation.
- the tyrosine phosphorylated proteins can be detected by Western blot using antibodies against phosphorylated tyrosine residues. Tyrosine phosphorylation of these early response proteins can be used as a standard assay for leukocyte activation (J. Biol. Chem., 1997, 272(23): 14562- 14570). Any change in the phosphorylation pattern of these or related proteins when immune responses are generated in the presence of potential PDZ PL interaction antagonists is indicative of a potential PDZ PL interaction antagonists.
- B IntraceUular Calcium Flux
- the kinetics of intracellular Ca 2+ concentrations are measured over time after stimulation of cells preloaded with a calcium sensitive dye.
- the indicator dye e.g., Fluor-4 (Molecular Probes)
- the indicator dye exhibits an increase in fluorescence level using flow cytometry, solution fluorometry, and confocal microscopy. Any change in the level or timing of calcium flux when immune responses are generated in the presence of PDZ/PL interaction antagonists is indicative of an inhibition of this response.
- lymphocyte activation marker levels such as CD69, IL-2R, MHC class ⁇ , B7, and TCR are commonly measured with fluorescently labeled antibodies using flow cytometry.
- AU antibodies are commercially available. Any change in the expression levels of lymphocyte activation markers when immune responses are generated in the presence of the PDZ/PL interaction antagonists is indicative of an inhibition of this response.
- MTS [5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3(4- sulfophenyl)tetrazolium, inner salt] is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays (Barltrop, J.A. et al., 1991, Bioorg. & Med. Chem. Lett. 1: 611). 1-5 days after lymphocyte activation, MTS tetrazolium compound, Owen' s reagent is bioreduced by ceUs into a colored formazan product that is soluble in tissue culture media. Color intensity is read at 490 nm minus 650 nm using a microplate reader.
- BrdU-pulsed cells are labeled with an enzyme- conjugated anti-BrdU antibody (Gratzner, H.G., 1982, Science 218: 474-475.).
- a colorimetric, soluble substrate is used to visualize proUferating ceUs that have incorporated BrdU. Reaction is stopped with sulfuric acid and plate is read at 450 nm using a microplate reader. Any statistically significant increase or decrease in color intensity of the PDZ/PL interaction antagonist-treated sample, as compared to control sample (no treatment), suggest an effect of the PDZ/PL interaction antagonist on biological function.
- Apoptosis by Annexin V Programmed ceU death or apoptosis is an early event in a cascade of catabolic reactions leading to cell death. A lose in the integrity of the cell membrane allows for the binding of fiuorescently conjugated phosphatidylserine. Stained cells can be measured by fluorescence microscopy and flow cytometry (Vermes, I., 1995, J. Immunol. Methods. 180: 39-52). In one embodiment any statistically significant increase or decrease in apoptotic cell number of the PDZ/PL interaction antagonist-treated sample, as compared to control sample (no treatment), suggest an effect of the PDZ PL interaction antagonist on biological function. For evaluating apoptosis in situ, assays for evaluating cell death in tissue samples can also be used in vivo studies.
- cytokine production is measured using a standard two-antibody sandwich ELISA protocol as described by the manufacturer. The presence of horseradish peroxidase is detected with 3, 3'5, 5' tertamethyl benziidine (TMB) substrate and the reaction is stopped with sulfuric acid. The absorbency at 450 nm is measured using a microplate reader.
- TMB 3, 3'5, 5' tertamethyl benziidine
- T ceU activation requires the import of nuclear factor of activated T cells (NF- AT) to the nucleus.
- NF-AT nuclear factor of activated T cells
- This translocation of NF-AT can be visualized by immunostaining with anti-NF-AT antibody (Cell 1998, 93: 851-861). Therefore, NF-AT nuclear translocation has been used to assay T cell activation.
- NF-AT/luciferase reporter assays have been used as a standard measurement of T cell activation (MCB 1996, 12: 7151-7160). Any statistically significant increase or decrease in the nuclear translocation of NF-AT brought about by the PDZ PL interaction antagonist-treated sample, as compared to control sample (no treatment), suggest an effect of the PDZ/PL interaction antagonist on biological function.
- the compounds of the invention may be tested in different dosages, formulations and route of administration in a cardiac transplant mouse model to optimize their abiUty to inhibit rejection responses to solid organ transplants (Fulmer et al., 1963, Am. J. Anat. 113:273; Jockusch et al., 1983, Exp. Neurol. 81:749).
- the compounds of the inventions may be used to inhibit PDZ domain interactions with CD3, CD4, CD6 and CDwl37.
- the compounds of the invention may be used to inhibit PDZ domain interactions with CD53 and CD138 in B cells.
- the compounds of the invention may be used to inhibit PDZ domain interactions with Fc ⁇ RI ⁇ , CDwl25 and CDwl28.
- a PDZ motif sequence (PL sequence) of CD95 may be used to induce apoptosis of lymphomas.
- the PDZ-PL antagonists of the invention are introduced into a cell to modulate (i.e., increase or decrease) a biological function or activity of the ceU.
- Many smaU organic molecules readily cross the cell membranes (or can be modified by one of skill using routine methods to increase the ability of compounds to enter cells, e.g., by reducing or eliminating charge, increasing lipophilicity, conjugating the molecule to a moiety targeting a ceU surface receptor such that after interacting with the receptor).
- Methods for introducing larger molecules e.g., peptides and fusion proteins are also well known, including, e.g., injection, liposome-mediated fusion, application of a hydrogel, conjugation to a targeting moiety conjugate endocytozed by the cell, electroporation, and the like).
- the antagonist or agent is a fusion polypeptide or derivatized polypeptide.
- a fusion or derivatized protein may include a targeting moiety that increases the ability of the polypeptide to traverse a cell membrane or causes the polypeptide to be delivered to a specified cell type (e.g., liver cells or tumor cells) preferentially or cell
- a transmembrane transporter sequence is fused to a hematopoietic cell surface receptor carboxyl terminal sequence at its amino-terminus with or without a linker.
- the C-terminus of a PDZ motif sequence (PL sequence) must be free in order to interact with a PDZ domain.
- the transmembrane transporter sequence may be used in whole or in part as long as it is capable of facilitating entry of the peptide into a cell.
- a hematopoietic cell surface receptor C-terminal sequence may be used alone when it is delivered in a manner that allows its entry into cells in the absence of a transmembrane transporter sequence.
- the peptide may be deUvered in a liposome formulation or using a gene therapy approach by deUvering a coding sequence for the PDZ motif alone or as a fusion molecule into a target ceU.
- the compounds of the of the invention may also be administered via Uposomes, which serve to target the conjugates to a particular tissue, such as lymphoid tissue, or targeted selectively to infected cells, as well as increase the half-life of the peptide composition.
- Uposomes serve to target the conjugates to a particular tissue, such as lymphoid tissue, or targeted selectively to infected cells, as well as increase the half-life of the peptide composition.
- Liposomes include emulsions, foams, miceUes, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
- the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
- a molecule which binds to e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
- liposomes fiUed with a desired peptide or conjugate of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the selected inhibitor compositions.
- Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generaUy include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
- the selection of lipids is generaUy guided by consideration of, e.g., liposome size, acid lability and stabiUty of the liposomes in the blood stream.
- a variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos.4,235,871, 4,501,728 and 4,837,028.
- a Ugand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for ceU surface determinants of the desired immune system cells.
- a liposome suspension containing a peptide or conjugate may be administered intravenously, locaUy, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the conjugate being delivered, and the stage of the disease being treated.
- the peptide may be linked to a cell-specific targeting moiety, which include but are not limited to, ligands for diverse leukocyte surface molecules such as growth factors, hormones and cytokines, as well as antibodies or antigen-binding fragments thereof. Since a large number of ceU surface receptors have been identified in leukocytes, Ugands or antibodies specific for these receptors may be used as cell-specific targeting moieties.
- a cell-specific targeting moiety include but are not limited to, ligands for diverse leukocyte surface molecules such as growth factors, hormones and cytokines, as well as antibodies or antigen-binding fragments thereof. Since a large number of ceU surface receptors have been identified in leukocytes, Ugands or antibodies specific for these receptors may be used as cell-specific targeting moieties.
- interleukin-2, B7-1 (CD80), B7-2 (CD86) and CD40 or peptide fragments thereof may be used to specifically target activated T ceUs (The Leucocyte Antigen Facts Book, 1997, Barclay et al. (eds.), Academic Press).
- CD28, CTLA-4 and CD40L or peptide fragments thereof may be used to specifically target B cells.
- Fc domains may be used to target certain Fc receptor-expressing cells such as monocytes.
- Antibodies are the most versatile cell-specific targeting moieties because they can be generated against any cell surface antigen. Monoclonal antibodies have been generated against leukocyte lineage-specific markers such as certain CD antigens. Antibody variable region genes can be readily isolated from hybridoma cells by methods well known in the art. However, since antibodies are assembled between two heavy chains and two light chains, it is prefened that a scFv be used as a cell-specific targeting moiety in the present invention. Such scFv are comprised of V H and V domains linked into a single polypeptide chain by a flexible linker peptide.
- the PDZ motif sequence may be linked to a transmembrane transporter sequence and a cell-specific targeting moiety to produce a tri-fusion molecule. This molecule can bind to a leukocyte surface molecule, passes through the membrane and targets PDZ domains. Alternatively, a PDZ motif sequence (PL sequence) may be linked to a cell- specific targeting moiety that binds to a surface molecule that internalizes the fusion peptide.
- microspheres of artificial polymers of mixed amino acids are provided.
- a polynucleotide encoding a surface receptor C-terminal peptide may be useful in the treatment of various leukocyte activation-associated abnormal conditions. By introducing gene sequences into cells, gene therapy can be used to treat conditions in which leukocytes are activated to result in deleterious consequences.
- a polynucleotide that encodes a PL sequence peptide of the invention is introduced into a cell where it is expressed. The expressed peptide then inhibits the interaction of PDZ proteins and PL proteins in the cell.
- the polypeptides of the invention are expressed in a cell by -introducing a nucleic acid (e.g., a DNA expression vector or mRNA) encoding the desired protein or peptide into the cell.
- a nucleic acid e.g., a DNA expression vector or mRNA
- Expression may be either constitutive or inducible depending on the vector and choice of promoter. Methods for introduction and expression of nucleic acids into a cell are well known in the art and described herein.
- nucleic acids comprising a sequence encoding a peptide disclosed herein, are administered to a human subject.
- the nucleic acid produces its encoded product that mediates a therapeutic effect by inhibiting leukocyte activation.
- Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
- the therapeutic composition comprises a coding sequence that is part of an expression vector.
- a nucleic acid has a promoter operably linked to the coding sequence, said promoter being inducible or constitutive, and, optionaUy, tissue-specific.
- a nucleic acid molecule is used in which the coding sequence and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the nucleic acid (Koller and Smithies, 1989, Proc. Natl. Acad. Sci.
- nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
- the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product.
- a nucleic acid-Ugand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
- the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92 06180 dated April 16, 1992; WO 92/22635 dated December 23, 1992; WO92/20316 dated November 26, 1992; WO93/14188 dated July 22, 1993; WO 93/20221 dated October 14, 1993).
- the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (KoUer and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al, 1989, Nature 342:435-438).
- adenoviruses as viral vectors can be used in gene therapy. Adenoviruses have the advantage of being capable of infecting non- dividing cells (Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503).
- adenoviruses in gene therapy can be found in Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; and MastrangeU et al, 1993, J. CUn. Invest. 91:225-234.
- adenoviral vectors with modified tropism may be used for cell specific targeting (WO98/40508).
- Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al, 1993, Proc. Soc. Exp. Biol. Med.204:289-300).
- retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA.
- the coding sequence to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient.
- More detail about retroviral vectors can be found in Boesen et al, 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
- the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
- introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, lipofection, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
- Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al, 1993, Meth.
- the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
- the cell used for gene therapy is autologous to the patient.
- the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding sequence, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
- Oligonucleotides such as anti-sense RNA and DNA molecules, and ribozymes that function to inhibit the translation of a leukocyte surface receptor mRNA, especiaUy its C- terminus are also within the scope of the invention.
- Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
- antisense DNA oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions of a nucleotide sequence, are prefened.
- the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracU, 5-chloroura l, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylammomemyl-2-tm ⁇ uridine,
- Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
- the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage.
- engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of leukocyte surface receptor RNA sequences.
- ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides conesponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
- RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the RNA molecule.
- DNA sequences may be incorporated into a wide variety of vectors which contain suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
- antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into ceU lines.
- DNA molecules may be introduced as a means of increasing intraceUular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phospho- diesterase linkages within the oligodeoxyribonucleotide backbone.
- compositions comprising the compounds of the invention may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
- Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate processing of the active peptides or peptide analogues into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
- the compounds of the invention may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.
- Systemic formulations include those designed for administration by injection, e.g. subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.
- the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
- physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
- the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. This route of administration may be used to deliver the compounds to the nasal cavity.
- the compounds can be readily formulated by combining the active peptides or peptide analogues with pharmaceutically acceptable carriers weU known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, piUs, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
- suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and or polyvinylpy ⁇ olidone (PVP); granulating agents; and binding agents.
- disintegrating agents may be added, such as the cross-linked polyvinylpynolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
- solid dosage forms may be sugar-coated or enteric-coated using standard techniques.
- suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like may be added.
- the compounds may take the form of tablets, lozenges, etc. formulated in conventional manner.
- the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- the dosage unit may be determined by providing a valve to deUver a metered amount.
- Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
- the compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g, containing conventional suppository bases such as cocoa butter or other glycerides.
- the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
- the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oU) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- suitable polymeric or hydrophobic materials for example as an emulsion in an acceptable oU
- ion exchange resins for example, as a sparingly soluble salt.
- other pharmaceutical delivery systems may be employed.
- Liposomes and emulsions are well known examples of delivery vehicles that may be used to deliver peptides and peptide analogues of the invention.
- Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
- the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent.
- sustained-release materials have been established and are well known by those skilled in the ar Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent additional strategies for protein stabilization may be employed.
- the compounds of the invention may contain charged side chains or termini, they may be included in any of the above-described formulations as the free acids or bases br as pharmaceutically acceptable salts.
- Pharmaceutically acceptable salts are those salts which substantially retain the biologic activity of the free bases and which are prepared by reaction with inorganic acids. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the conesponding free base forms.
- the compounds of the invention will generally be used in an amount effective to achieve the intended purpose.
- the compounds of the invention or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount.
- therapeutically effective amount is meant an amount effective ameliorate or prevent the symptoms, or prolong the survival of, the patient being treated. Determination of a therapeutically effective amount is well vvithin the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
- An "inhibitory amount” or “inhibitory concentration" of a PL-PDZ binding inhibitor is an amount that reduces binding by at least about 40%, preferably at least about 50%, often at least about 70%, and even as much as at least about 90%.
- Binding can as measured in vitro (e.g, in an A assay or G assay) or in situ.
- a therapeutically effective dose can be estimated initially from in vitro assays.
- a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 0 as determined in cell culture i.e., the concentration of test compound that inhibits 50% of leukocyte surface receptor-PDZ domain-containing protein interactions). Such information can be used to more accurately determine useful doses in humans.
- Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.
- Dosage amount and interval may be adjusted individually to provide plasma levels of the compounds that are sufficient to maintain therapeutic effect.
- Usual patient dosages for administration by injection range from about 0.1 to 5 mg kg day, preferably from about 0.5 to 1 mg/kg/day.
- Therapeutically effective serum levels may be achieved by administering multiple doses each day.
- the effective local concentration of the compounds may not be related to plasma concentration.
- One having skiU in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
- the amount of compound administered will, of course, be dependent on the subject being treated, on the subject's weight the severity of the affliction, -the manner of administration and the judgment of the prescribing physician.
- the therapy may be repeated intermittently while symptoms detectable or even when they are not detectable.
- the therapy may be provided alone or in combination with other drugs.
- the drugs that may be used in combination with the compounds of the invention include, but are not limited to, steroid and non-steroid anti-inflammatory agents.
- a therapeutically effective dose of the compounds described herein wiU provide therapeutic benefit without causing substantial toxicity.
- Toxicity of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 5 o (the dose lethal to 50% of the population) or the LD 1 00 (the dose lethal to 100% of the population).
- the dose ratio between toxic and therapeutic effect is the therapeutic index.
- Compounds which exhibit high therapeutic indices are preferred.
- the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
- the dosage of the compounds described herein Ues preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al,
- Tat-CD3 carboxyl terminus fusion peptide (GYGRKKRRQRRRGPPSSSSGL, SEQ ID O:174); Tat- CLASP1 carboxyl terminus fusion peptide, (GYGRKKRRQRRRGSISSSAEV, SEQ ID NO:243); Tat-CLASP2 carboxyl terminus fusion peptide, (GYGRKKRRQRRRGMTSSSSW, SEQ K ) NO:176); and Tat peptide, (GYGRKKRRQRRRG, SEQ ID NO:173); were dissolved at 1 mM in PBS, pH 7, or dH2O.
- RPMI 1640 media supplemented with 10% fetal calf serum (HyClone), 2 mM glutamine, 10 mM Hepes, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 50 ⁇ M beta mercaptoethanol.
- Antigen-specific mouse T cells BR4.2, were activated with the N-terminal 16 amino acid sequences of myelin basic protein (MBPAcl-16) and syngenic mouse splenocytes in 96-well plates. Mitomycin C-treated antigen presenting ceUs, 2 x 10 s B10.BR, were added to each row of serially diluted MBPAcl-16 ranging from 0 to 200 ⁇ M. Next, 10 ⁇ M Tat- peptides or media alone was added to each row. Finally, 2 x 10 4 MBPAcl-16-specific T cell, pre-loaded with 10 ⁇ M Tat-peptides (see above), were added to all wells (Rabinowitz et al,1997,. Proc. Natl.
- CeUs were activated during an overnight incubation at 5% CO2, 37°C. Cell supernatant was collected and stored at -80°C until assayed for cytokine production. The final volume was 200 ⁇ l/well.
- Antibody against mouse CD3 (Pharmigen #14S-2C11) was coated ovemight at 4°C using 96-well flat bottom Elisa plates at a final concentration of 0.5 ⁇ g/ml, diluted in PBS. Just prior to use, plates were washed three times with 200 ⁇ l/well PBS to remove excess anti- CD3. To ensure that cells were given sufficient time to transduce Tat-peptides before activation, T cells (5x10 5 cells/ml) were pre-treated with or without 10 ⁇ M Tat-peptides for two hours at 5% CO2, 37°C and then diluted in media with or without 10 ⁇ M Tat-peptides to a final concentration of 2x10 4 cells per well in a final volume of 200 ⁇ l. Cells were then treated as described above.
- EFN ⁇ was measured from cell supematants, described above, at ambient temperature using the Endogen, Inc. ELISA protocol 3. Briefly, 96-well, flat bottom, high binding ELISA plates were preincubated overnight with coating antibody (MM700). Plates were washed with50 mM TRIS, 0.2% tween-20, pH 8 and they blocked for one hour with PBS plus 2% BSA. Washed plates were then incubated one hour with 25 ⁇ l of ceU supernatant and 25 ⁇ l blocking buffer, or with 50 ⁇ l EFN ⁇ standard. The presence of EFN ⁇ was detected with a biotin-labeled anti-mouse EFN ⁇ monoclonal antibody (MM700B, Endogen, Inc.,).
- MM700B biotin-labeled anti-mouse EFN ⁇ monoclonal antibody
- Quantitative amounts of detection antibody are revealed with horseradishperoxidase-conjugated streptavidin.
- the absorbance at 450 nm was measured using a microtiter plate reader (Thermo Max, Molecular Devices) and the concentration of unknown IFN ⁇ from cell supematants was calculated from a standard curve generated by Softmax Pro. software (Molecular Devices).
- FIGURE 4 A shows that the Tat-CD3 fusion peptide inhibits T Cell activation mediated by ⁇ eptide:MHC as compared to controls of Tat-peptide alone or no peptide.
- FIGURE 4B shows that Tat-CLASP2 carboxyl terminus fusion peptide inhibited T ceU activation mediated by monoclonal anti-CD3 as compared to Tat-peptide alone. Tat-CLASPl fusion peptide did not inhibit T ceU activation in this experiment.
- peptides containing potential inhibitory sequences can be transported into T cells through transporter peptide such as Tat to disrupt surface receptor organization mediated by PDZ proteins. Disruption of PDZ-mediated surface receptor organization leads to blockage of T ceU activation in response to antigen.
- 21 uM labeled CLASP-2 peptide AA2L filled 50% of the binding sites for CLASP-2 on DLGl.
- 21 uM unlabeled CLASP-2 peptide should be able to block the binding of a given Ugand to DLGl by approximately 50%, assuming that the given ligand (1) binds to the same site(s) on DLGl as Qasp ⁇ 2 and (2) is not added at sufficient concentration to reduce significantly the binding of the CLASP-2 peptide (i.e. cannot out-compete the CLASP-2 peptide).
- an effective inhibitor of DLGl -ligand binding i.e. the eight amino acid CLASP-2 peptide MTSSSSW, SEQ ED NO: 191 with a known potency range (order of magnitude 21 uM) was designed based on knowledge of the affinity, 21 uM, with which a particular labeled ligand, the CLASP-2 peptide AA2L, bound to DLGl .
- This example describes the cloning of PDZ domain containing genes or portions of PDZ domain containing genes were into eukaryotic expression vectors in fusion with red fluorescent protein (RFP).
- RFP red fluorescent protein
- DNA fragments conesponding to PDZ domain containing genes were generated by RT-PCR from jurkat cell line (transformed T-cells) derived RNA. Primers were designed to create restriction nuclease recognition sites at the PCR fragment's ends, to allow cloning of those fragments into the appropriate vectors. Subsequent to RT-PCR, DNA samples submitted to agarose gel electrophoresis. Bands conesponding in size to the expected size were excised,
- DNA extracted and treated with appropriate restriction endonuclease DNA samples were purified once more by gel electrophoresis, and gel extracted DNA fragments were coprecipitated and ligated with the appropriate linearized cloning vector. After transformation into E.coli, bacterial colonies were screened by PCR for the presence and conect orientation of insert. Positive clones were picked for large scale DNA preparation and the insert including the flanking vectors sites were sequenced to ensure correct sequence of fragments and junctions with the vectors and fusionproteins.
- Cloning vectors were pDsREDl-Nl (purchased from CLONTECH, # 6921-1) and pDsREDl-Nl(+ATG), a derivative of pDsREDl-Nl generated by recombinant DNA technology. DNA fragments to clone that contained the ATG-start codon were cloned into pDsREDl-Nl. Fragments void of a proper translation initiation codon were cloned into pDsREDl-N-(+ATG), since this vector includes an translation initiation start codon. Vector pDsREDl-Nl(+ATG) differs from pDsREDl only with regard to the multiple cloning sites.
- Linker sequences between the cloned inserts and RFP vary depending on the vectors and on the restriction endonuclease used for cloning. Deduced linker amino acid sequences are listed in the table below; For some constructs, the first N-terminal and / or last
- Construct (N-J) [includes the third in frame-methionin (putative start) codon in (GI: 3882222) and extends c-terminal of the PDZ domain to the region on sequence divergency between KIAA 0751 (GI: 3882222) and hypothetical 41.8 Kd protein (AF007156 / GI: 3882222)]; primers: 318 KEF and 320 KIR; vector: pDsREDl-Nl
- N-P Construct (N-P) [Covers the methionin start codon and extends over the C-terminal boundary of the PDZ domain]; primers: 322 PAF and 324 PAR; vector: pDsREDl-Nl
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US6942981B1 (en) | 1999-05-14 | 2005-09-13 | Arbor Vita Corporation | Method of determining interactions with PDZ-domain polypeptides |
US20060100132A1 (en) * | 2003-06-06 | 2006-05-11 | Brit Corneliussen | Method for diagnosing inflammatory bowel disease |
US7410950B2 (en) | 2004-04-01 | 2008-08-12 | Board Of Regents, The University Of Texas System | Peptides of CaV2.2 that inhibit pain |
KR101236450B1 (en) | 2004-12-21 | 2013-02-25 | 무스크 파운데이션 포 리서치 디벨롭먼트 | Compositions and methods for promoting wound healing and tissue regeneration |
AU2012202117B2 (en) * | 2004-12-21 | 2014-06-26 | Musc Foundation For Research Development | Compositions and methods for promoting wound healing and tissue regeneration |
US9408381B2 (en) | 2004-12-21 | 2016-08-09 | Musc Foundation For Research Development | Alpha Connexin c-Terminal (ACT) peptides for use in transplant |
US8633160B2 (en) | 2005-12-30 | 2014-01-21 | Nono Inc. | Small molecule inhibitors of PDZ interactions |
WO2012048298A2 (en) | 2010-10-08 | 2012-04-12 | Caridianbct, Inc. | Methods and systems of growing and harvesting cells in a hollow fiber bioreactor system with control conditions |
MX351881B (en) | 2012-03-01 | 2017-11-01 | Firststring Res Inc | Topical gels containing alpha connexin c-terminal (act) peptides. |
CN105793411B (en) | 2013-11-16 | 2018-04-17 | 泰尔茂比司特公司 | Cell amplification in bioreactor |
WO2015148704A1 (en) | 2014-03-25 | 2015-10-01 | Terumo Bct, Inc. | Passive replacement of media |
EP3183346A4 (en) | 2014-08-22 | 2018-10-24 | Auckland Uniservices Limited | Channel modulators |
WO2016049421A1 (en) | 2014-09-26 | 2016-03-31 | Terumo Bct, Inc. | Scheduled feed |
WO2017004592A1 (en) | 2015-07-02 | 2017-01-05 | Terumo Bct, Inc. | Cell growth with mechanical stimuli |
EP3464565A4 (en) | 2016-05-25 | 2020-01-01 | Terumo BCT, Inc. | Cell expansion |
US11104874B2 (en) | 2016-06-07 | 2021-08-31 | Terumo Bct, Inc. | Coating a bioreactor |
US11685883B2 (en) | 2016-06-07 | 2023-06-27 | Terumo Bct, Inc. | Methods and systems for coating a cell growth surface |
EP3656841A1 (en) | 2017-03-31 | 2020-05-27 | Terumo BCT, Inc. | Cell expansion |
US11624046B2 (en) | 2017-03-31 | 2023-04-11 | Terumo Bct, Inc. | Cell expansion |
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