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Title:
NUCLEIC ACID ENCODING PROTEINS INVOLVED IN PROTEIN DEGRADATION, PRODUCTS AND METHODS RELATED THERETO
Document Type and Number:
WIPO Patent Application WO/2000/077207
Kind Code:
A2
Abstract:
In accordance with the present invention, there are provided novel Siah-Mediated-Degradation-Proteins (SMDPs) and/or SCF-Complex Proteins (SCPs). Nucleic acid sequences encoding such proteins and assays employing same are also disclosed. The invention SMDPs and/or SCPs can be employed in a variety of ways, for example, for the production of anti-SMDP and/or SCP antibodies thereto, in therapeutic compositions, and methods employing such proteins and/or antibodies for drug screening, functional genomics and other applications. Also provided are transgenic non-human mammals that express the invention protein.

Inventors:
REED JOHN C (US)
MATSUZAWA SHU-ICHI (US)
Application Number:
PCT/US2000/015873
Publication Date:
December 21, 2000
Filing Date:
June 09, 2000
Export Citation:
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Assignee:
BURNHAM INST (US)
REED JOHN C (US)
MATSUZAWA SHU ICHI (US)
International Classes:
A01K67/027; A61K31/7088; A61K35/76; A61K38/00; A61K39/395; A61K48/00; A61P29/00; A61P35/00; A61P43/00; C07K14/47; C07K16/40; C07K19/00; C12N1/15; C12N1/19; C12N1/21; C12N5/10; C12N9/64; C12N15/09; C12N15/12; C12Q1/02; C12Q1/68; G01N33/15; G01N33/50; G01N33/53; G01N33/566; G01N33/577; G01N33/58; A61K39/00; (IPC1-7): C12N15/12; A01K67/027; A61K38/17; A61K39/395; A61K48/00; C07K14/47; C07K16/18; C07K19/00; C12N5/10; C12N15/00; C12N15/11; C12N15/62; C12Q1/68; G01N33/68
Domestic Patent References:
WO1997022695A21997-06-26
WO1998042741A21998-10-01
WO1999046374A21999-09-16
WO1998041624A11998-09-24
WO1999047540A11999-09-23
WO1999018989A11999-04-22
Other References:
HU GANG ET AL: "Characterization of human homologs of the Drosophila seven in absentia (sina) gene." GENOMICS, vol. 46, no. 1, 15 November 1997 (1997-11-15), pages 103-111, XP002153979 ISSN: 0888-7543 cited in the application
HU GANG ET AL: "Siah-1 N-terminal RING domain is required for proteolysis function, and C-terminal sequences regulate oligomerization and binding to target proteins." MOLECULAR AND CELLULAR BIOLOGY, vol. 19, no. 1, January 1999 (1999-01), pages 724-732, XP002153980 ISSN: 0270-7306
MATSUZAWA SHU-ICHI ET AL: "p53-inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth: Suppression by BAG-1." EMBO (EUROPEAN MOLECULAR BIOLOGY ORGANIZATION) JOURNAL, vol. 17, no. 10, 15 May 1998 (1998-05-15), pages 2736-2747, XP002153981 ISSN: 0261-4189 cited in the application
KINZLER KENNETH W ET AL: "Lessons from hereditary colorectal cancer." CELL, vol. 87, no. 2, 1996, pages 159-170, XP002153982 ISSN: 0092-8674 cited in the application
EMBL DATABASE Accession no AL035305, Sequence ID HS102G201 28 January 1999 RHODES S:"H. sapiens gene from PAC 102G20" XP002160981 -& EMBL DATABASE Accession no O60666, Sequence ID O060666 01 August 1998 TU C ET AL: "Hypothetical 26.2 KDA protein" XP002160982
FILIPEK ANNA ET AL: "Molecular cloning and expression of a mouse brain cDNA encoding a novel protein target of calcylin." JOURNAL OF NEUROCHEMISTRY, vol. 70, no. 5, May 1998 (1998-05), pages 1793-1798, XP000982427 ISSN: 0022-3042
CENCIARELLI C ET AL: "Identification of a family of human F-box proteins." CURRENT BIOLOGY, vol. 9, no. 20, 21 October 1999 (1999-10-21), pages 1177-1179, XP000982510 ISSN: 0960-9822 -& EMBL DATABASE Accession no Q9UKT2 Sequence ID Q9UKT2 1 May 2000 XP002160984 -& EMBL DATABASE Accession no O75986 Sequence ID O75986 1 November 1998 XP002160985 -& EMBL DATABASE Accession no Q9UKT4 Sequence ID Q9UKT4 1 May 2000 XP002160991
EMBL DATABASE Accession no Q9Y3I1 Sequence ID Q9Y3I1 1 November 1999 ILYIN GENNADY P ET AL: "cDNA cloning and expression analysis of new members of the mammalian F-box protein family" XP002160986
EMBL DATABASE Accession no Q9Y593 Sequence UD Q9Y593 1 November 1999 SCANLAN M ET AL: "Antigens recognized by autologous antibody in patients with renal carcinoma" XP002160987
EMBL DATABASE Accession no Q9UK97 Sequence ID Q9UK97 1 May 2000 WINSTON J ET AL: " A familiy of mammalian F-box proteins" XP002160988
Attorney, Agent or Firm:
Ramos, Robert T. (CA, US)
Weber-quitzau M. (Uexküll & Stolberg Beselerstr. 4 Hamburg, DE)
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Claims:
That which is claimed is:
1. Isolated nucleic acid encoding a SiahMediated DegradationProtein (SMDP) and/or SFCComplexProtein (SCP), or a functional fragment thereof.
2. Isolated nucleic acid encoding SiahMediated DegradationProtein (SMDP) and/or SFCComplexProtein (SCP), or functional fragments thereof, selected from: (a) DNA encoding the amino acid sequence set forth in SEQ ID Nos: 2,4,6,8,10,12 or 14, or (b) DNA that hybridizes to the DNA of (a) under moderately stringent conditions, wherein said DNA encodes biologically active SMDP and/or SCP, or (c) DNA degenerate with respect to either (a) or (b) above, wherein said DNA encodes biologically active SMDP and/or SCP.
3. A nucleic acid according to claim 2, wherein said nucleic acid hybridizes under high stringency conditions to the SMDP and/or SCP coding portion of any of SEQ ID NOs: l, 3,5,7,9,11 and 13.
4. A nucleic acid according to claim 2, wherein the nucleotide sequence of said nucleic acid is substantially the same as set forth in any of SEQ ID NO: 1,3,5,7,9,11 and 13.
5. A nucleic acid according to claim 2, wherein the nucleotide sequence of said nucleic acid is the same as that set forth in any of SEQ ID NOs: l, 3,5,7,9,11 and 13.
6. A nucleic acid according to claim 2, wherein said nucleic acid is cDNA.
7. A vector containing the nucleic acid of claim 2.
8. Recombinant cells containing the nucleic acid of claim 2.
9. An oligonucleotide comprising at least 15 nucleotides capable of specifically hybridizing with a the nucleotide sequence set forth in any of SEQ ID NOs: l, 3,5,7,9,11 and 13.
10. An oligonucleotide according to claim 9, wherein said oligonucleotide is labeled with a detectable marker.
11. An antisensenucleic acid capable of specifically binding to mRNA encoded by said nucleic acid according to claim 2.
12. A kit for detecting the presence of the SMDP and/or SCP cDNA sequence comprising at least one oligonucleotide according to claim 10.
13. An isolated SiahMediatedDegradationProtein (SMDP) and/or SFCComplexProtein (SCP) characterized by having ability to bind to at least one SMDP and/or SCP.
14. A SMDP and/or SCP according to claim 13, wherein the amino acid sequence of said protein comprises substantially the same sequence as any of SEQ ID Nos: 2, 4,6,8,10,12 or 14.
15. A SMDP and/or SCP according to claim 14 comprising the same amino acid sequence as set forth in any of SEQ ID Nos: 2,4,6,8,10,12 or 14.
16. A SMDP and/or SCP according to claim 13, wherein said protein is encoded by a nucleotide sequence comprising substantially the same nucleotide sequence as set forth in SEQ ID Nos: l, 3,5,7,9,11 or 13.
17. A SMDP and/or SCP according to claim 16, wherein said protein is encoded by a nucleotide sequence comprising the same sequence as set forth in SEQ ID Nos: l, 3,5,7,9,11 or 13.
18. A method for expression of a SMDP and/or SCP protein, said method comprising culturing cells of claim 8 under conditions suitable for expression of said SMDP and/or SCP.
19. An isolated antiSMDP and/or SCP antibody having specific reactivity with a SMDP and/or SCP according to claim 13.
20. Antibody according to claim 19, wherein said antibody is a monoclonal antibody.
21. An antibody according to claim 20, wherein said antibody is a polyclonal antibody.
22. A composition comprising an amount of the antisensenucleic acid according to claim 11 effective to inhibit expression of a human SMDP and/or SCP and an acceptable hydrophobic carrier capable of passing through a cell membrane.
23. A transgenic nonhuman mammal expressing exogenous nucleic acid encoding a SMDP and/or SCP.
24. A transgenic nonhuman mammal according to claim 23, wherein said nucleic acid encoding said SMDP and/or SCP has been mutated, and wherein the SMDP and/or SCP so expressed is not native SMDP and/or SCP.
25. A transgenic nonhuman mammal according to claim 23, wherein the transgenic nonhuman mammal is a mouse.
26. A method for identifying nucleic acids encoding a mammalian SMDP and/or SCP, said method comprising: contacting a sample containing nucleic acids with an oligonucleotide according to claim 9, wherein said contacting is effected under high stringency hybridization conditions, and identifying compounds which hybridize thereto.
27. A method for detecting the presence of a human SMDP and/or SCP in a sample, said method comprising contacting a test sample with an antibody according to claim 19, detecting the presence of an antibodySMDP and/or SCP complex, and therefor detecting the presence of a human SMDP and/or SCP in said test sample.
28. Single strand DNA primers for amplification of SMDP and/or SCP nucleic acid, wherein said primers comprise a nucleic acid sequence derived from the nucleic acid sequences set forth as SEQ ID NOs: l, 3,5,7,9,11 and 13.
29. A method for modulating the activity of an oncogenic protein, comprising contacting said oncogenic proteins with a substantially pure SMDP and/or SCP, or a oncogenic proteinbinding fragment thereof.
30. A bioassay for evaluating whether test compounds are capable of acting as agonists or antagonists for SMDP and/or SCP proteins, or functional fragments thereof, wherein said bioassay comprises: (a) culturing cells containing: DNA which expresses an SMDP and/or SCP or functional fragments thereof, wherein said culturing is carried out in the presence of at least one compound whose ability to modulate an activity of an SMDP and/or SCP is sought to be determined, wherein said activity is selected from a protein: protein binding activity or a protein degradation activity and thereafter (b) monitoring said cells for either an increase or decrease in the level of protein: protein binding or protein degradation.
31. A method for modulating an activity mediated by a SMDP and/or SCP protein, said method comprising: contacting said SMDP and/or SCP protein with an effective, modulating amount of said agonist or antagonist identified by claim 30.
32. The method of claim 31, wherein said modulated activity is the binding of Siah1 to APC.
33. A method for modulating the protein degradation activity mediated by an SMDP and/or SCP protein, said method comprising: contacting said SMDP and/or SCP protein with an effective, modulating amount of said agonist or antagonist identified by claim 30.
34. A therapeutic composition comprising a compound selected from an SMDP and/or SCP, or functional fragment thereof, a SMDP and/or SCP modulating compound identified according to claim 30, or an antiSMDP and/or SCP antibody; and a pharmaceutically acceptable carrier.
35. A method of treating a pathology characterized by abnormal cell proliferation or abnormal inflammation, said method comprising administering an effective amount of the composition according to claim 34.
36. A method of inducing the degradation of the function of a target protein, said method comprising: expressing, in a cell, a chimeric protein comprising a targetprotein binding domain operatively linked to a proteindegradation binding domain of a protein member of the ubiquitinmediated proteindegradation family.
37. A method of determining the function of a target protein, said method comprising: expressing, in a first cell, a chimeric protein comprising a targetprotein binding domain operatively linked to a proteindegradation binding domain of a protein member of the ubiquitinmediated protein degradation family; and comparing the phenotype of said first cell to the phenotype of a control second cell.
38. A method of identifying a nucleic acid molecule encoding a protein that modulates a cellular phenotype, said method comprising: (a) expressing, in a cell, a chimeric nucleic acid comprising a member of a nucleic acid library fused to nucleic acid encoding a protein degradation binding domain of a protein member of the ubiquitin mediated protein degradation family; and (b) screening said cells for a modulation of said phenotype.
39. The method of claim 38, wherein the phenotype is selected from the group consisting of: cell proliferation, cell survival, cell death, cell secretion, and cell migration.
40. A chimeric nucleic acid identified according to claim 38.
41. A nucleic acid library comprising a plurality of chimeric nucleic acids, wherein each chimeric nucleic acid comprises an SMDP and/or SCP or functional fragment thereof.
42. The method of claim 38 wherein said nucleic acid encoding a protein degradation binding domain is selected from the group consisting of Siala, SIPL, SIP S, SAF1, SAF2, and SAD, or functional fragments thereof.
43. A method for treating a disease by degrading the function of a target protein comprising: introducing, into a cell, a chimeric protein comprising a targetprotein binding domain operatively linked to a proteindegradation binding domain of a protein member of the ubiquitinmediated protein degradation family.
44. A chimeric protein comprising the SMDP and/or SCP of claim 13.
Description:
INTERNATIONAL SEARCH REPORT Inter, nal Application No PCT/US 00/15873 C. (Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X HU GANG ET AL :"Siah-1 N-terminal RING 1-4, domain is required for proteolysis 6-14,16, function, and C-terminal sequences 18-28, regulate oligomerization and binding to 30,31,33 target proteins." MOLECULAR AND CELLULAR BIOLOGY, vol. 19, no. 1, January 1999 (1999-01), pages 724-732, XP002153980 ISSN: 0270-7306 the whole document X MATSUZAWA SHU-ICHI ET AL :"p53-inducible 1-4, human homologue of Drosophila seven in 6-14,16, absentia (Siah) inhibits cell growth: 18-28, Suppression by BAG-1."30, 31,33 EMBO (EUROPEAN MOLECULAR BIOLOGY ORGANIZATION) JOURNAL, vol. 17, no. 10,15 May 1998 (1998-05-15), pages 2736-2747, XP002153981 ISSN: 0261-4189 cited in the application the whole document A KINZLER KENNETH W ET AL:"Lessons from 32 hereditary colorectal cancer." CELL, vol. 87, no. 2,1996, pages 159-170, XP002153982 ISSN: 0092-8674 cited in the application the whole document X WO 98 42741 A (GENETICS INST) 1-35 1 October 1998 (1998-10-01) the whole document especially SEQ ID NO 1 and 2 X EMBL DATABASE Accession no AL035305,1-28 Sequence ID HS102G201 28 January 1999 RHODES S:"H. sapiens gene from PAC 102G20" XP002160981 the whole document -& EMBL DATABASE Accession no 060666, Sequence ID 0060666 01 August 1998 TU C ET AL:"Hypothetical 26.2 KDA protein" XP002160982 4 4 INTERNATIONAL SEARCH REPORT PCT/US 00/15873 C. (Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT Category ° Citation of document, with indication, where appropnate, of the relevant passages Relevant to claim No. X FILIPEK ANNA ET AL:"Molecular cloning 1-28 and expression of a mouse brain cDNA encoding a novel protein target of calcylin." JOURNAL OF NEUROCHEMISTRY, vol. 70, no. 5, May 1998 (1998-05), pages 1793-1798,XP000982427 ISSN: 0022-3042 the whole document P, X WO 99 46374 A (SCHMITT ARMIN; SPECHT 1-35 THOMAS (DE); DAHL EDGAR (DE); HINZMANN BERND) 16 September 1999 (1999-09-16) the whole document especially SEQ ID No 142 and 143 X WO 98 41624 A (WALLACH DAVID ; YEDA RES & 1-4, DEV (IL) ; KOVALENKO ANDREI (IL)) 6-14,16, 24 September 1998 (1998-09-24) 18-35 the whole document P, X CENCIARELLI C ET AL:"Identification of a 1-28 family of human F-box proteins." CURRENT BIOLOGY, vol. 9, no. 20, 21 October 1999 (1999-10-21), pages 1177-1179,XP000982510 ISSN: 0960-9822 the whole document P, X-& EMBL DATABASE Accession no Q9UKT2 1-4, Sequence ID Q9UKT2 6-14,16, 1 May 2000 18-28 XP002160984 X-& EMBL DATABASE Accession no 075986 1-28 Sequence ID 075986 1 November 1998 XP002160985 P, X-& EMBL DATABASE Accession no Q9UKT4 1-28 Sequence ID Q9UKT4 1 May 2000 XP002160991 P, X EMBL DATABASE Accession no Q9Y3I1 Sequence 1-28 IDQ9Y3I1 1 November 1999 ILYIN GENNADY P ET AL :"cDNA cloning and expression analysis of new members of the mammalian F-box protein family" XP002160986 the whole document P, X WO 99 47540 A (ENDRESS GREGORY A; HUMAN 1-4, GENOME SCIENCES INC (US); FENG PING (US);) 6-14,16, 23 September 1999 (1999-09-23) 18-28 SEQ ID NO 197 4 _/__ INTERNATIONAL SEARCH REPORT Inte ional Application No PCT/US 00/15873 C. (Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. P, X EMBL DATABASE Accession no Q9Y593 Sequence 1-4, UD Q9Y593 6-14,16, 1 November 1999 18-28 SCANLAN M ET AL:"Antigens recognized by autologous antibody in patients with renal carcinoma" XP002160987 the whole document P, X EMBL DATABASE Accession no Q9UK97 Sequence 1-4, ID Q9UK97 6-14,16, 1 May 2000 18-28 WINSTON J ET AL:"A familiy of mammalian F-box proteins" XP002160988 the whole document X WO 99 18989 A (BAYLOR COLLEGE MEDICINE) 1-4, 22 April 1999 (1999-04-22) 6-14,16, 18-28 F16 (SEQ ID NO 41 and 43) 4 international application No. INTERNATIONAL SEARCH REPORT PCT/US 00/15873 Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) This International Search Report has not been established in respect of certain claims under Article 17 (2) (a) for the following reasons: 1.3 Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely: Although claims 29 31-33 35 36 and 43 are (partially) directed to a method of treatment of the human/animal body, the search has been carried out and based on the alleged effects of the compound/composition. 2. E Claims Nos.: 40 because they relate to parts of the International Application that do not comply with the prescribed requirements to such an extent that no meaningful International Search can be carried out, specifically: see FURTHER INFORMATION sheet PCT/ISA/210 3. Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4 (a). Box 11 Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows: see additional sheet 1. 1. As all required additional search fees were timely paid by the applicant, this International Search Report covers all searchable claims. 2. As aii searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. As only some of the required additional search fees were timely paid by the applicant, this International Search Report covers only those claims for which fees were paid, specifically claims Nos.: 4. No required additional search fees were timely paid by the applicant. Consequently, this International Search Report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: Remark on Protest The additional search fees were accompanied by the applicant's protest. Fx-l No protest accompanied the payment of additional search fees. u FURTHER INFORMATION CONTINUED FROM PCT/ISAJ 210 This International Searching Authority found multiple (groups of) inventions in this international application, as follows: 1. Claims: 1-39 41-44 partially Siah-1-alpha (SEQ ID NO 2), its encoding nucleotides, antibodies against the protein and their use 2. Claims: 1-39 41-44 partially SIP proteins (SEQ ID NO 4 and 6), their encoding nucleotides, antibodies against the proteins and their use 3. Claims: 1-39 41-44 partially SAF-1 proteins (SEQ ID NO 8 and 10), their encoding nucleotides, antibodies against the proteins and their use 4. Claims: 1-39 41-44 partially SAF-2 protein (SEQ ID NO 12), its encoding nucleotides, antibodies against the protein and their use 5. Claims: 1-39 41-44 partially SAD protein, its encoding nucleotides, antibodies against the protein and their use FURTHER INFORMATION CONTINUED FROM PCT/ISA/210 Continuation of Box 1. 2 Claims Nos.: 40 Present claim 40 relate to a chimeric nucleic acid defined by reference to a desirable characteristic or property, namely encoding a chimera of a protein degradation binding domain and a polypeptide being able to modulate a cellular phenotype. The claim covers all products having this characteristic or property, whereas the application provides no support within the meaning of Article 6 PCT and/or disclosure within the meaning of Article 5 PCT for any such product. In the present case, the claims so lack support, and the application so lacks disclosure, that a meaningful search over the whole of the claimed scope is impossible. Independent of the above reasoning, the claims also lack clarity (Article 6 PCT). An attempt is made to define the product by reference to a result to be achieved. Again, this lack of clarity in the present case is such as to render a meaningful search over the whole of the claimed scope impossible. Consequently, no search has been carried out. The applicant's attention is drawn to the fact that claims, or parts of claims, relating to inventions in respect of which no international search report has been established need not be the subject of an international preliminary examination (Rule 66.1 (e) PCT). The applicant is advised that the EPO policy when acting as an International Preliminary Examining Authority is normally not to carry out a preliminary examination on matter which has not been searched. This is the case irrespective of whether or not the claims are amended following receipt of the search report or during any Chapter II procedure. INTERNATIONAL SEARCH REPORT Intei Wal Application No Information on patent family members PCT/US 00/15873 Patent document Publication Patent family Publication cited in search report date member (s) date WO 9722695 A 26-06-1997 FR 2742766 A 27-06-1997 FR 2747691 A 24-10-1997 CA 2240449 A 26-06-1997 EP 0868512 A 07-10-1998 JP 2000502893 T 14-03-2000 WO 9842741 A 01-10-1998 AU 6777298 A 20-10-1998 EP 0998490 A 10-05-2000 WO 9946374 A 16-09-1999 DE 19811193 A 16-09-1999 EP 1068317 A 17-01-2001 WO 9841624 A 24-09-1998 AU 6634798 A 12-10-1998 BR 9808915 A 01-08-2000 CN 1250479 T 12-04-2000 EP 0972033 A 19-01-2000 NO 994524 A 11-11-1999 WO 9947540 A 23-09-1999 AU 3451799 A 11-10-1999 EP 1064297 A 03-01-2001 AU 3072799 A 11-10-1999 WO 9947538 A 23-09-1999 WO 9918989 A 22-04-1999 AU 1088399 A 03-05-1999