USOO6395889B1 (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002 (54) NUCLEIC ACID MOLECULES ENCODING WO WO-98/56804 A1 * 12/1998 ........... CO7H/21/02 HUMAN PROTEASE HOMOLOGS WO WO-99/0785.0 A1 * 2/1999 ... C12N/15/12 WO WO-99/37660 A1 * 7/1999 ........... CO7H/21/04 (75) Inventor: fish E. Robison, Wilmington, MA OTHER PUBLICATIONS Vazquez, F., et al., 1999, “METH-1, a human ortholog of (73) Assignee: Millennium Pharmaceuticals, Inc., ADAMTS-1, and METH-2 are members of a new family of Cambridge, MA (US) proteins with angio-inhibitory activity', The Journal of c: - 0 Biological Chemistry, vol. 274, No. 33, pp. 23349–23357.* (*) Notice: Subject to any disclaimer, the term of this Descriptors of Protease Classes in Prosite and Pfam Data patent is extended or adjusted under 35 bases. U.S.C. 154(b) by 0 days. * cited by examiner (21) Appl. No.: 09/392, 184 Primary Examiner Ponnathapu Achutamurthy (22) Filed: Sep. 9, 1999 ASSistant Examiner William W. Moore (51) Int. Cl." C12N 15/57; C12N 15/12; (74) Attorney, Agent, or Firm-Alston & Bird LLP C12N 9/64; C12N 15/79 (57) ABSTRACT (52) U.S. Cl. .................... 536/23.2; 536/23.5; 435/69.1; 435/252.3; 435/320.1 The invention relates to polynucleotides encoding newly (58) Field of Search ............................... 536,232,235. identified protease homologs. The invention also relates to 435/6, 226, 69.1, 252.3 the proteases. The invention further relates to methods using s s s/ - - -us the protease polypeptides and polynucleotides as a target for (56) References Cited diagnosis and treatment in protease-mediated disorders. The invention further relates to drug-Screening methods using U.S. PATENT DOCUMENTS the protease polypeptides and polynucleotides to identify 5,552,526 A 9/1996 Nakamura et all 530/350 agonists and antagonists for diagnosis and treatment. The 5883.241 A * 3/1999 Docherty et al. r 536/23.2 invention further encompasses agonists and antagonists 5.990.293 A * 11/1999 Docherty et al. .......... 536/23.1 based on the protease polypeptides and polynucleotides. The invention further relates to procedures for producing the FOREIGN PATENT DOCUMENTS protease polypeptides and polynucleotides. EP O874050 A2 * 10/1998 ........... C12N/15/15 WO WO-98/55643 A1 * 12/1998 ........... C12P/21/02 1 Claim, No Drawings US 6,395,889 B1 1 2 NUCLEIC ACID MOLECULES ENCODING The invention also provides an isolated fragment or HUMAN PROTEASE HOMOLOGS portion of any of SEQ ID NOS: 1-33 and the complement of SEQ ID NOS: 1-33. In preferred embodiments, the FIELD OF THE INVENTION fragment is useful as a probe or primer, and/or is at least 15, The invention relates to newly identified polynucleotides more preferably at least 18, even more preferably 20–25, 30, having homology to various protease families. The inven 50, 100, 200 or more nucleotides in length. tion also relates to protease polypeptides. The invention The invention also provides isolated fragments of the further relates to methods using the protease polypeptides polypeptides. and polynucleotides as a target for diagnosis and treatment In another embodiment, the invention provides an isolated in protease-mediated and related disorders. The invention nucleic acid molecule that hybridizes under high Stringency further relates to drug-Screening methods using the protease conditions to a nucleotide Sequence Selected from the group polypeptides and polynucleotides to identify agonists and consisting of SEQ ID NOS: 1-33 and the complements of antagonists for diagnosis and treatment. The invention fur SEO ID NOS: 1-33. ther encompasses agonists and antagonists based on the The invention further provides nucleic acid constructs protease polypeptides and polynucleotides. The invention 15 comprising the nucleic acid molecules described above. In a further relates to procedures for producing the protease preferred embodiment, the nucleic acid molecules of the polypeptides and polynucleotides. invention are operatively linked to a regulatory Sequence. The invention also provides vectors and host cells for BACKGROUND OF THE INVENTION expressing the protease nucleic acid molecules and polypep Proteases are a major target for drug action and develop tides and particularly recombinant vectors and host cells. ment. Accordingly, it is valuable to the field of pharmaceu The invention also provides methods of making the tical development to identify and characterize previously vectors and host cells and methods for using them to produce unknown protease nucleic acids and polypeptides. The the protease nucleic acid molecules and polypeptides. present invention advances the State of the art by providing 25 The invention also provides antibodies or antigen-binding previously unidentified human protease Sequences. fragments thereof that Selectively bind the protease polypep tides and fragments. SUMMARY OF THE INVENTION The invention also provides methods of Screening for It is an object of the invention to identify novel proteases. compounds that modulate expression or activity of the It is a further object of the invention to provide novel protease polypeptides or nucleic acid (RNA or DNA). protease polypeptides that are useful as reagents or targets in The invention also provides a process for modulating protease assays applicable to treatment and diagnosis of protease polypeptide or nucleic acid expression or activity, protease-mediated disorders. especially using the Screened compounds. Modulation may It is a further object of the invention to provide poly 35 be used to treat conditions related to aberrant activity or nucleotides corresponding to the novel protease polypep expression of the protease polypeptides or nucleic acids. tides that are useful as targets and reagents in protease assays The invention also provides assays for determining the applicable to treatment and diagnosis of protease-mediated presence or absence of and level of the protease polypep disorders and useful for producing novel protease polypep tides or nucleic acid molecules in a biological Sample, tides by recombinant methods. 40 including for disease diagnosis. Aspecific object of the invention is to identify compounds The invention also provides assays for determining the that act as agonists and antagonists and modulate the expres presence of a mutation in the protease polypeptides or Sion of the novel proteases. nucleic acid molecules, including for disease diagnosis. A further specific object of the invention is to provide In still a further embodiment, the invention provides a compounds that modulate expression of the proteases for 45 computer readable means containing the nucleotide and/or treatment and diagnosis of protease- related disorders. amino acid Sequences of the nucleic acids and polypeptides The present invention is based on the identification of of the invention, respectively. novel nucleic acid molecules that are homologous to pro DETAILED DESCRIPTION OF THE tease Sequences. INVENTION 50 Thus, in one aspect, the invention provides an isolated The following text describes classes of proteins with nucleic acid molecule that comprises a nucleotide Sequence which the Sequences described herein have been compared selected from the group consisting of SEQ ID NOS: 1-33 with respect to homology. and the complements of SEQ ID NOS: 1-33. Eukaryotic and Viral Aspartyl Active Sites In another aspect, the invention provides isolated proteins 55 Aspartyl proteases, also known as acid proteases, (EC and polypeptides encoded by nucleic acid molecules of the 3.4.23.-), are a widely distributed family of proteolytic invention. enzymes (Foltman (1981) Essays Biochem 17:52–84; Davis In another embodiment, the invention provides an isolated (1990) Annu. Rev. Biophys. Chem. 19:189–215; Rao et al. nucleic acid molecule that comprises a nucleotide Sequence (1991) Biochemistry 30: 4663-4671) that exist in that is at least about 60% identical, preferably at least about 60 vertebrates, fungi, plants, retroviruses and Some plant 80% identical, preferably at least about 85% identical, more Viruses. ASpartate proteases of eukaryotes are monomeric preferably at least about 90% identical, and even more enzymes which consist of two domains. Each domain con preferably at least about 95% identical, and most preferably tains an active site centered on a catalytic aspartyl residue. about 98% or more identical to a nucleotide sequence The two domains most probably evolved from the duplica selected from the group consisting of SEQ ID NOS: 1-33 65 tion of an ancestral gene encoding a primordial domain. and the complements of SEQ ID NOS: 1-33. Currently known eukaryotic aspartyl proteases include, but The invention also provides isolated variant polypeptides. are not limited to: US 6,395,889 B1 3 4 Vertebrate gastric pepsins A and C (also known as Eukaryotic Thiol (Cysteine) Proteases Active Sites Eukary gastricsin). otic thiol proteases (EC 3.4.22.-) (Dufour (1988) Biochimie Vertebrate chymosin (rennin), involved in digestion and 70: 1335–1342) are a family of proteolytic enzymes which used for making cheese. contain an active Site cysteine. Catalysis proceeds through a Vertebrate lysosomal cathepsins D (EC 3.4.23.5) and E thioester intermediate and is facilitated by a nearby histidine (EC 3.4.23.34). Side chain; an asparagine completes the essential catalytic Mammalian renin (EC 3.4.23.15) whose function is to triad. Proteases that belong to this family include, but are not generate angiotensin I from angiotensinogen in the limited to: plasma. Vertebrate lysosomal cathepsins B (EC 3.4.22. 1), H (EC Fungal proteases Such as aspergillopepsin A (EC 3.4.22.16), L (EC 3.4.22.15), and S (EC 3.4.22.27) 3.4.23.18), candidapepsin (EC 3.4.23.24), mucoropep (Kirschke et al. (1995) Protein Prof. 2:1587–1643). sin (EC 3.4.23.23) (mucor rennin), endothiapepsin (EC Vertebrate lysosomal dipeptidyl peptidase I (EC 3.4.14.1) 3.4.23.22), polyporopepsin (EC 3.4.23.29), and rhizo (also known as cathepsin C) (Kirschke et al.