Epf receptor assays, compounds and therapeutic compositions

Abstract

The present invention provides assays for the study of the interaction of EPF and its EPF-related peptides with a hDRR receptor. The assays are useful to identify whether a test compound can bind to the hDRR receptor under conditions in which EPF or related peptide can bind to the receptor. The assays are also useful to determine whether the test compound is an agonist or antagonist of hDRRs. The above assays can be performed in a variety of formats including competitive, non-competitive and comparative assays in which the interaction of EPF or EPF-related peptides with hDRRs is assessed as a positive or negative control or compared to the results obtained with the test compound.

1 . An assay that makes use of the interaction of EPF or an EPF-related peptide with a hDRR receptor or hDRR related receptor.  
     
     
         2 . The assay according to  claim 1  wherein EPF or EPF-related peptides are selected from the group consisting of, 
 i) an isolated polypeptide encoding EPF, comprising the amino acid sequence SEQ ID NO:4;  
 ii) an isolated polypeptide derived from EPF and capable of binding to hDRR4; or  
 iii) an isolated polypeptide encoding an EPF-related peptide comprising an amino acid selected from the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21; or  
 iv) an isolated polypeptide comprising the hDRR4 binding fragment encoded by amino acid sequence SEQ ID NO:8.  
 
     
     
         3 . An assay according to  claim 1  wherein the hDRR receptor polypeptide is selected from the group consisting of, 
 i) an isolated polypeptide encoding hDRR4, comprising the amino acid sequence SEQ ID NO:2 or a fragment thereof; or  
 ii) an isolated polypeptide encoding hDRR7, comprising the amino acid sequence SEQ ID NO:12 or a fragment thereof.  
 
     
     
         4 . The isolated and purified EPF-related peptide fragment comprising the hDRR binding fragment encoded by amino acid sequence SEQ ID NO:8.  
     
     
         5 . An isolated and purified nucleic acid molecule encoding an EPF-related peptide fragment comprising the hDRR binding fragment encoded by SEQ ID NO:7.  
     
     
         6 . An isolated and purified EPF-related peptide fragment comprising an amino acid sequence selected from the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO 21.  
     
     
         7 - 34 . (Cancelled).  
     
     
         35 . A method of identifying and obtaining a test compound capable of binding a HDRR receptor comprising: 
 a) incubating a source containing a hDRR or a fragments thereof, with i) EPF or EPF-related peptides ii) said test compound; and    b) measuring the effect of the test compound on the amount of EPF or EPF-related peptides bound to the receptor.    
     
     
         36 . The method according to  claim 35 , wherein the hDRR containing source is selected from the group consisting of; 
 i) an isolated and purified protein having the amino acid sequence of SEQ ID NO:2 or a fragment thereof;    ii) an isolated and purified protein having the amino acid sequence of SEQ ID NO:12 or a fragment thereof;    iii) cells expressing on the surface thereof the hDRR4 polypeptide receptor having the amino acid sequence of SEQ ID NO:2 or a fragments thereof;    iv) cells expressing on the surface thereof the hDRR7 polypeptide receptor having the amino acid sequence of SEQ ID NO:12 or a fragments thereof;    v) membrane preparations of cells expressing on the surface thereof the hDRR4 polypeptide receptor having the amino acid sequence of SEQ ID NO:2 or a fragments thereof; or    vi) membrane preparations of cells expressing on the surface thereof the hDRR7 polypeptide receptor having the amino acid sequence of SEQ ID NO:12 or a fragments thereof.    
     
     
         37 . The method according to  claim 35 , wherein the isolated and purified protein is bound to a solid support.  
     
     
         38 . The method according to  claim 35 , wherein EPF or the EPF-related peptides are labeled and wherein said label is used to measure the effect of the test compound on the amount of EPF or EPF-related peptides bound to the receptor.  
     
     
         39 . The method according to  claim 35 , wherein the method is a method of rational drug design comprising the steps: 
 a) probing the structure of the ligand binding site on hDRR with EPF or EPF-related peptides;    b) identifying contacting atoms in the ligand binding site of the hDRR receptor that interact with the EPF ligand during binding;    c) designing test compounds that interact with the atoms identified in (b) to modulate the activity of the hDRR receptor; and    d) contacting said designed test compound with a source containing hDRR or a functional fragment thereof, to measure the capability of said compound to modulate the hDRR activity.    e) identifying those compounds which modulate the activity of the HDRR receptor.    
     
     
         40 . A method for identifying and obtaining a test compound capable of modulating the activity of the hDRR receptor comprising: 
 a) incubating a source containing hDRR or functional fragments thereof, with said test compound;    b) measuring the effect of the test compound on the activity of the HDRR receptor; and    c) comparing this effect with the activity of the hDRR receptor upon binding of the EPF ligand or EPF-related peptides and identifying those compounds which have an effect comparable to the EPF ligand or EPF related peptides.    
     
     
         41 . The method according to  claim 40 , wherein the hDRR containing source is a cell expressing on the surface thereof the polypeptide receptor having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:12.  
     
     
         42 . The method according to  claim 40 , wherein the effect of the modulator on the HDRR receptor is modulation of an intracellular second messenger formation that is mediated by hDRR receptors.  
     
     
         43 . The method according to  claim 42 , wherein the intracellular second messenger is cAMP, calcium or a reporter gene product.  
     
     
         44 . A compound identified and obtained by the method of  claim 35 ,  39 , or  40  wherein said compound is capable of binding and/or modulating hDRR receptor activity.  
     
     
         45 . A compound identified and obtained by the method of  claim 35 ,  39  or  40 , wherein said compound is an agonist or antagonist of hDRR.  
     
     
         46 . A compound identified by the method of  claim 35 ,  39  or  40  for use as a medicine.  
     
     
         47 . A pharmaceutical composition comprising a compound identified by the methods of claims  35 ,  39  or  40  and a pharmaceutically acceptable excipient or carrier.  
     
     
         48 . A pharmaceutical composition comprising a compound comprising the hDRR binding fragment encoded by SEQ ID NO: 8 or an EPF-related peptide derived therefrom having at least 70% sequence identity therewith.  
     
     
         49 . A pharmaceutical composition comprising an EPF-related peptide encoded by an amino acid sequence selected from the group consisting of the nucleotides of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.  
     
     
         50 . A pharmaceutical composition for use as anticonceptives or for the treatment of certain diseases, including but not limited to; cancers like transitional cell carcinoma, liposarcoma, adenocarcinoma, diffuse large B-cell lymphoma, lymphocytic leukemia, lymphoblastic leukemia, myeloblastic leukemia, myelomonocytic leukemia, osteosarcoma; refractory anemia when said compound; for the treatment of autoimmune diseases like rheumatoid arthritis, multiple sclerosis or other conditions where immunosuppressive actions are desired like Inflammatory Bowel Disease (IBD) or to prevent transplant rejection, wherein the pharmaceutical composition comprises the hDRR binding fragment encoded by SEQ ID NO:8 or an EPF-related peptide derived therefrom having at least 70% sequence identity therewith.  
     
     
         51 . A pharmaceutical composition for the treatment of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis or other conditions where immunosuppresive actions are desired like in IBD, to prevent transplant rejection, for use as contraceptive, in the prevention of foetal loss or for the treatment of cancers, comprising an hDRR7 agonist which is a compound identified by the method of claims  35 ,  39  or  40 .  
     
     
         52 . A pharmaceutical composition for the treatment of pain wherein said compound is an hDRR4 agonist identified by the method of claims  35 ,  39  or  40 .  
     
     
         53 . A method for isolating hDRR from a cellular fraction containing the same comprising contacting the cellular fraction with EPF or a hDRR binding fragment thereof immobilized to a solute substrate and eluting hDRR therefrom.  
     
     
         54 . A monoclonal antibody directed to the hDRR binding fragment encoded by SEQ ID NO:8 or and EPF-related peptide derived therefrom having at least 70% sequence identity therewith.  
     
     
         55 . A diagnostic kit comprising; 
 i) a polypeptide selected from the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, and the hDRR4 binding fragment encoded by SEQ ID NO:8 sequences derived therefrom having at least 70% sequence identity therewith; or    ii) an antibody directed to a polypeptide selected from the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, and the hDRR4 binding fragment encoded by SEQ ID NO:8 sequences derived therefrom having at least 70% sequence identity therewith.