Novel genes related to glutaminyl cyclase

Abstract

Novel glutaminyl-peptide cyclotransferase-like proteins (QPCTLs), which are isoenzymes of glutaminyl cyclase (QC, EC 2.3.2.5), and to isolated nucleic acids coding for these isoenzymes, all of which are useful for the discovery of new therapeutic agents, for measuring cyclase activity, and for determining the inhibitory activity of compounds against these glutaminyl cyclase isoenzymes.

1 . An isolated nucleic acid comprising
 (a) a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 22, or an amino acid sequence having at least about 90% sequence identity thereto and having glutaminyl cyclase activity;   (b) a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, and SEQ ID NO: 20, or an alternative splice variant thereof;   (c) a probe comprising at least 14 contiguous nucleotides of said nucleic acid sequence of (a) or (b); or   (d) a nucleic acid sequence complementary to any one of (a), (b), or (c).   
     
     
         2 . The isolated nucleic acid of  claim 1  wherein the nucleic acid is DNA or RNA. 
     
     
         3 . The isolated nucleic acid of  claim 1 , wherein the nucleic acid sequence encodes a polypeptide having (i) at least about 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO. 18, SEQ ID NO: 21, and SEQ ID NO: 22 and (ii) glutaminyl cyclase activity. 
     
     
         4 . The isolated nucleic acid of  claim 1 , wherein the probe of (c) comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61. 
     
     
         5 . The isolated nucleic acid of  claim 1  further comprising an expression-control element operably linked with said nucleic acid, wherein the expression-control element drives the expression of a polypeptide encoded by said nucleic acid. 
     
     
         6 . An antisense oligonucleotide directed against the isolated nucleic acid of  claim 1 . 
     
     
         7 . An expression vector comprising:
 a nucleic acid comprising
 (a) a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 22, oran amino acid sequence having at least about 90% sequence identity thereto and having glutaminyl cyclase activity; or 
 (b) a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, and SEQ ID NO: 20, or an alternative splice variant thereof; and 
   a promoter;   wherein the nucleic acid is operably linked to the promoter.   
     
     
         8 . A transgenic host cell transformed with a nucleic acid comprising
 (a) a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 22, or an amino acid sequence having at least about 90% sequence identity thereto and having glutaminyl cyclase activity; or   (b) a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, and SEQ ID NO: 20, or an alternative splice variant thereof;   wherein the host cell is a mammalian, insect, or bacterial host cell.   
     
     
         9 . A process for producing a polypeptide comprising
 culturing the host cell of  claim 8  under conditions sufficient for the production of said polypeptide; and   recovering the polypeptide or a fragment thereof from the culture;   wherein said polypeptide is expressed at the surface of said cell.   
     
     
         10 . An isolated polypeptide comprising:
 (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 22, or an amino acid sequence having at least about 90% sequence identity thereto and having glutaminyl cyclase activity;   (b) an amino acid sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, and SEQ ID NO: 20, or an alternative splice variant thereof; or   (c) a fragment of (a) or (b) wherein said fragment is immunologically reactive and has glutaminyl cyclase activity;   wherein the isolated polypeptide is optionally glycosylated.   
     
     
         11 . The polypeptide of  claim 10 , wherein the polypeptide comprises an amino acid sequence having at least about  95 % sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, or SEQ ID NO: 22 and has glutaminyl cyclase activity. 
     
     
         12 . An antibody directed to the polypeptide of  claim 10 . 
     
     
         13 . A method of screening for a compound capable of inhibiting the enzymatic activity of at least one polypeptide of  claim 10 , comprising:
 incubating at least one polypeptide of  claim 10  and a suitable substrate for the at least one polypeptide of  claim 10  in the presence of at least one test compound or salt thereof;   measuring an enzymatic activity of the at least one polypeptide;   comparing said activity with enzymatic activity determined in the absence of the at least one test compound or salt thereof; and   selecting a test compound that reduces the enzymatic activity of the at least one polypeptide of  claim 10 .   
     
     
         14 . The method of  claim 13  wherein the enzymatic activity is glutaminyl cyclase activity. 
     
     
         15 . The method of  claim 14  further comprising:
 incubating a polypeptide comprising SEQ ID NO: 10 (wild type human glutaminyl cyclase) and a suitable substrate for wild type human glutaminyl cyclase in the presence of the at least one test compound or salt thereof;   measuring the glutaminyl cyclase activity of the polypeptide comprising SEQ ID NO: 10; and   selecting a test compound that reduces the glutaminyl cyclase activity of the at least one polypeptide of  claim 10  but does not reduce the glutaminyl cyclase activity of the polypeptide comprising SEQ ID NO: 10.   
     
     
         16 . A QPCTL antagonist, which inhibits the glutaminyl cyclase activity of at least one polypeptide of  claim 10 . 
     
     
         17 . The QPCTL antagonist of  claim 16 , wherein the antagonist is (i) a QPCTL competitive inhibitor or (ii) a QPCTL inhibitor that binds to an active-site bound metal ion of the QPCTL. 
     
     
         18 . The QPCTL antagonist of  claim 16 , which is a small molecule inhibitor identified by the screening method of  claim 14 . 
     
     
         19 . The QPCTL antagonist of  claim 16 , which is a small molecule inhibitor identified by the screening method of  claim 15 . 
     
     
         20 . A pharmaceutical composition for parenteral, enteral or oral administration, comprising at least one QPCTL inhibitor of  claim 16 , or a pharmaceutical acceptable salt thereof, optionally in combination with customary carriers and/or excipients. 
     
     
         21 . A method of prevention or treatment of a disease selected from Alzheimer's disease, Familial British Dementia, Familial Danish Dementia, Down Syndrome, Huntington's disease, Kennedy's disease, ulcer disease, duodenal cancer with or w/o  Helicobacter pylori  infections, colorectal cancer, Zolliger-Ellison syndrome, gastric cancer with or without  Helicobacter pylori  infections, pathogenic psychotic conditions, schizophrenia, infertility, neoplasia, inflammatory host responses, cancer, malign metastasis, melanoma, psoriasis, rheumatoid arthritis, atherosclerosis, impaired humoral and cell-mediated immune responses, leukocyte adhesion and migration processes in the endothelium, impaired food intake, impaired sleep-wakefulness, impaired homeostatic regulation of energy metabolism, impaired autonomic function, impaired hormonal balance or impaired regulation of body fluids, multiple sclerosis, the Guillain-Barré syndrome and chronic inflammatory demyelinizing polyradiculoneuropathy, comprising administering to a subject in need thereof a QPCTL inhibitor of  claim 16 , or a pharmaceutical acceptable salt thereof. 
     
     
         22 . A method of diagnosing any one of the diseases and/or conditions as defined in  claim 21  in a subject, comprising:
 collecting a sample from a subject who is suspected to be afflicted with said disease and/or condition,   contacting said sample with an inhibitor of a glutaminyl peptide cyclotransferase; and   determining whether said subject is afflicted by said disease and/or condition.   
     
     
         23 . The method of  claim 22 , wherein said sample is selected from the group consisting of a blood sample, a serum sample, a sample of cerebrospinal liquor, and a urine sample. 
     
     
         24 . A diagnostic kit for carrying out the method of  claim 22  comprising a detection means and a determination means.