GB2376234A

GB2376234A - Trypsin-like serine protease

Info

Publication number: GB2376234A
Application number: GB0106318A
Authority: GB
Inventors: Alan Peter Lewis; Mark Peter Edbrooke
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 2001-03-14
Filing date: 2001-03-14
Publication date: 2002-12-11
Also published as: GB0106318D0

Abstract

A method for the identification of a substance that modulates serine protease activity, which method comprises contacting a cell selected from a neuronal cell, a lung cell and an intestinal cell, which cell expresses a serine protease polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a variant thereof which has trypsin-like activity, with a test substance and monitoring for serine protease activity. The polypeptide of SEQ ID NO:2 or substances that modulate its activity may be used in the treatment of tonsilitis, CNS disease such as Alzheimer's and respiratory disease such as asthma, COPD and fibrotic disease of the lung.

Description

NOVEL PROTEIN Field of the Invention The present invention relates to serine protease polypeptides.

Background of the Invention There are 4 main catalytic types of peptidases ; serine (which includes threonine peptidases) cysteine, aspartic and metallo. The serine, threonine and cysteine peptidases are catalytically very different from the aspartic and metallopeptidases in that the nucleophile of the catalytic site is part of an amino acid, whereas it is an activated water molecule in the other groups.

Serine peptidases are enzymes in which the catalytic mechanism depends upon the hydroxyl group of a serine residue acting as the nucleophile that attacks the peptide bond. Examples of serine peptidases include trypsin, chymotrypsin, thrombin, plasmin, furin, cathepsin G, chymase, tryptase and granzymes A, B, K and M. Threonine-dependent peptidases can be grouped with the serine peptidases and include the proteasome.

The trypsin like serine protease family is, under the MEROPS protease database, classified as Clan PA and Family S IA. The trypsin-like serine proteases include brain serine protease 2, prostarin and trytases I-III.

Summary of the Invention The present invention is based on the novel finding that expression of that a serine protease, referred to herein as HIPHUM 31, is up-regulated in various tissues and in particular in oesophagus and tonsil. HIPHUM 31 is also up-regulated in cerebellum, brain from Alzheimer's and myotonic dystrophy, lung in asthma and COPD, and VEGF endothelial cells. HIPHUM 31 is down-regulated in lung tumour and HSV infection. HIPHUM 31 is a screening target for the identification and development of novel pharmaceutical agents, including modulators of serine protease activity which may be used in the treatment and/or prophylaxis of disorders such as tonsilitis, CNS diseases including Alzheimer's disease and myotonic dystrophy, respiratory disease including asthma and COPD and inflammatory respiratory disease

including fibrotic disease of the lung and IBS. HIPHUM 31 and variants and fragments thereof, HIPHUM 31 polynucleotides and HIPHUM 31 antibodies may also be used in the treatment of these diseases.

Accordingly, the present invention provides: a method for identification of a substance that modulates serine protease activity, which method comprises: (i) contacting (a) a cell selected from a neuronal cell, a lung cell and an intestinal cell, which cell expresses a serine protease polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof which has trypsin-like serine protease activity; and (b) a test substance; and (ii) monitoring for serine protease activity; a method for identification of a substance that modulates serine protease activity, which method comprises: (i) contacting (a) a serine protease polypeptide isolated from a cell selected from a neuronal cell, a lung cell and an intestinal cell, which serine protease polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof which has trypsin-like serine protease activity; and (b) a test substance; and (ii) monitoring for serine protease activity; a modulator of serine protease activity identified by a method according to the invention for use in a method of treatment of the human or animal body by therapy. a modulator of serine protease activity identified by a method according to the invention in the manufacture of a medicament for the treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung. use of a serine protease polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2; or

(ii) a variant thereof which has trypsin-like serine protease activity; or (iii) a fragment of (i) or (ii) which has trypsin-like serine protease activity in the manufacture of a medicament for use in the treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung; use of a polynucleotide encoding a serine protease polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2; or (ii) a variant thereof which has trypsin-like activity; or (iii) a fragment of (i) or (ii) which has trypsin-like activity in the manufacture of a medicament for use in the treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung; use of an antibody specific for a serine protease polypeptide comprising the amino acid sequence of SEQ ID NO: 2 in the manufacture of a medicament for use in the treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung; use of a substance which modulates proteinase activity of a polypeptide with the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof in the manufacture of a medicament for treatment or prophylaxis tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung; a method of treating a subject suffering from tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung, which method comprises administering to said subject an effective amount of a substance which modulates proteinase activity of a polypeptide with the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof; a method for treating tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma.

COPD and fibrotic disease of the lung, which method comprises: (i) identifying a substance that modulates serine protease activity and/or

expression (ii) administering an effective amount of the substance to a patient suffering from tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung.

Brief Description of the Sequences SEQ ID NO: 1 shows the nucleotide and amino acid sequences of human protein HIPHUM 31.

SEQ ID NO: 2 is the amino acid sequence alone of HIPHUM 31.

Detailed Description of the Invention Throughout the present specification and the accompanying claims the words "comprise"and"include"and variations such as"comprises","comprising", "includes"and"including"are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The present invention relates to the use of a human serine protease, referred to herein as HIPHUM 31. and variants and fragments thereof. Sequence information for HIPHUM 31 is provided in SEQ ID NO: 1 (nucleotide and amino acid) and in SEQ ID NO: 2. A polypeptide of the invention thus consists essentially of the amino acid sequence of SEQ ID NO : 2 or of a variant of that sequence, or of a fragment of either thereof.

The term"variant"refers to a polypeptide which has a same essential character or basic biological functionality as HIPHUM 31. The essential character of HIPHUM 31 can be defined as follows: HIPHUM 31 is a serine protease. Preferably the polypeptide has trypsin-like serine protease activity. Preferably a variant polypeptide is one which cleaves the same substrate as HIPHUM 31. A suitable substrate peptide typically comprises a nitrophenyl amino group, which, when hydrolysed by HIPHUM 31 releases the absorbent nitrophenylamine. Typically, a variant polypeptide comprises a catalytic domain having tryptic substrate specificity, preferably cleaving protein or peptide substrates immediately following an arginine

or lysine residue.

A variant polypeptide typically comprises a signal peptide sequence which is cleaved off during entry of the protien into the endoplasmic reticulum. The resulting protein is preferably an inactive pro-enzyme which can be activated by cleavage to generate the mature proteolytically active form. The signal sequence of HIP HUM 31 comprises residues 1-22 and the pro-peptide constitues residues 23-34 of SEQ ID NO: 2. Thus, preferably, an active variant of HIPHUM 31 is a variant of residues 35- 290 of SEQ ID NO: 2.

Preferably a variant polypeptide comprises catalytic residues of histidine, aspartate and serine residues at positions corresponding to positions 75,124 and 229 of SEQ ID NO: 2.

A polypeptide having the same essential character as HIPHUM 31 may be identified by monitoring for trypsin-like serine protease activity.

In another aspect of the invention, a variant is one which does not show the same activity as HIPHUM 31 but is one which inhibits a basic function of HIPHUM 31. For example, a variant polypeptide is one which inhibits protease activity of HIPHUM 31, for example by binding to the substrate binding site of HIPHUM 31 to prevent cleavage of the natural substrate.

Typically, polypeptides with more than about 65% identity preferably at least 80% or at least 90% and particularly preferably at least 95% at least 97% or at least 99% identity, with the amino acid sequences of SEQ ID NO: 2. are considered as variants of the proteins. Such variants may include allelic variants and the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the peptide maintains the basic biological functionality of HIP HUM 3l.

Amino acid substitutions may be made, for example from 1, 2 or 3 to 10,20 or 30 substitutions. The modified polypeptide generally retains activity as a serine protease. Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

ALIPHATIC Non-polar GAP ILV Polar-uncharged C S T M NQ Polar-charged D E KR AROMATIC H F W Y

Shorter polypeptide sequences are within the scope of the invention. For example, a peptide of at least 20 amino acids or up to 50,60, 70,80, 100,150, 200, or 250 amino acids in length is considered to fall within the scope of the invention as long as it demonstrates a basic biological functionality of HIPHUM 31. In particular, but not exclusively, this aspect of the invention encompasses the situation when the protein is a fragment of the complete protein sequence and may represent a catalytic domain or substrate binding domain. Preferred fragments include fragments comprising the catalytic residues. Such fragments can be used to construct chimeric proteases preferably with another protease, more preferably with another member of the family of serine proteases. Such chimeric proteases may comprise different domains from different serine proteases.

Fragments of HIPHUM 31 or a variant thereof can also be used to raise antiHIPHUM 31 antibodies which may be used in the manufacture of a medicament for use in the treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung. The antibody may be produced in response to a fragment of HIPHUM 31 which comprises an epitope of the HIPHUM 31 polypeptide which may not demonstrate the catalytic, substrate binding or other properties of HIPHUM 31.

Polypeptides for use in the invention may be chemically modified, e. g. post-

translationally modified. For example, they may be glycosylated or comprise modified amino acid residues. They may also be modified by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote insertion into the cell membrane. Such modified polypeptides fall within the scope of the term"polypeptide"of the invention.

Nucleotide sequences that encode for HIPHUM 31 or variants thereof as well as nucleotide sequences which are complementary thereto may be used in a method of the invention. The nucleotide sequence may be RNA or DNA including genomic DNA, synthetic DNA or cDNA. Preferably the nucleotide sequence is a DNA sequence and most preferably, a cDNA sequence. Nucleotide sequence information is provided in SEQ ID NO: 1. Such nucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sam brook et aI, 1989.

Typically a polynucleotide for use in the invention comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1.

A polynucleotide for use in the invention can hydridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1 at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA library. The signal level generated by the interaction between a polynucleotide for use in the invention and the coding sequence or complement of the coding sequence of SEQ ID NO: I is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 1. The intensity of interaction may be measured, for example, by radiolabelling the probe, e. g. with 32p.

Selective hybridisation may typically be achieved using conditions of medium to high stringency. However, such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al, 1989. For example, if high stringency is required suitable conditions include from 0.1 to 0.2 x SSC at 60 C up to 65 C. If lower stringency is required suitable conditions include 2 x SSC at 60 C.

The coding sequence of SEQ ID NO : 1 may be modified by nucleotide substitutions, for example from 1, 2 or 3 to 10,25, 50 or 100 substitutions. The

polynucleotide of SEQ ID NO: 1 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends.

A polynucleotide may include one or more introns, for example may comprise genomic DNA. Additional sequences such as signal sequences which may assist in insertion of the polypeptide in a cell membrane may also be included. The modified polynucleotide generally encodes a polypeptide which has a HIPHUM 31 activity.

Alternatively, a polynucleotide encodes a catalytic or substrate-binding portion of a polypeptide or a polypeptide which inhibits HIPHUM 31 activity. Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table above.

A nucleotide sequence which is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO: 1 will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the coding sequence of SEQ ID NO: lover a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably at least 100 contiguous nucleotides or most preferably over the full length of SEQ ID NO : 1.

For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul (1993) J. Mol. Evol.

36 : 290-300; Altschul et al (1990) J. Mol. Biol. 215: 403-10.

Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http ://www. ncbi. nlm. nih. gov/).

This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, 1990). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both

directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value ; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.

The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. NatL Acad Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e. g. , Karlin and Altschul (1993) Proc. Natl. Acad. Sci.

USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N) ), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0. 001.

Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides for use in the invention, with the more stringent combinations (i. e. higher sequence identity over longer lengths) being preferred. Thus, for example a polynucleotide which has at least 90% sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 40 nucleotides.

The nucleotides may be utilised in gene therapy techniques. Nucleotides complementary to those encoding HIPHUM 31, or antisense sequences, may also be used in gene therapy.

The present invention also includes the use of expression vectors that comprise nucleotide sequences encoding the HIPHUM 31 or variants thereof in a method of the invention or in the manufacture of a medicament for use in the treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic

dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung. Expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. 1989.

Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides may also be produced by synthetic means. Such antisense polynucleotides may be used in a method treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung.

Preferably, a polynucleotide for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i. e. the vector is an expression vector. The term "operably linked"refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence, such as a promoter,"operably linked"to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

The vectors may be for example, plasmid or virus vectors provided with a origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. Vectors may be used in vitro, for example to transfect cells which have been removed from a patient. The transfected cells may then be returned to the patient. Vectors may also be adapted to be used in vivo, for example in a method of gene therapy.

Mammalian promoters, such as 3-actin promoters, may be used. Tissuespecific promoters are especially preferred. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR),

the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR). Viral promoters are readily available in the art.

The vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells by homologous recombination. Other examples of suitable viral vectors include herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

The invention also includes cells that have been modified to express the HIPHUM 31 polypeptide or a variant thereof. Such cells may be used in a method of the invention or in a method of treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung or in the manufacture of a medicament for the treatment of any such disease.

According to another aspect, the present invention also relates to the use of antibodies specific for a HIPHUM 31 or a variant thereof in a method of treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung, or in the manufacture of a medicament for the treatment of any such disease.

Antibodies may be raised against specific epitopes of the polypeptides according to the invention. Such antibodies may be used to block substrate binding to the receptor. An antibody, or other compound,"specifically binds"to a protein when it binds with preferential or high affinity to the protein for which it is specific but does substantially bind not bind or binds with only low affinity to other proteins.

A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of an antibody are well known in the art (see for example Maddox et al, J. Exp. Med. 158,1211-1226, 1993). Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.

Antibodies for use in the invention may be antibodies to human polypeptides or fragments thereof. For the purposes of this invention, the term"antibody", unless specified to the contrary, includes fragments which bind a polypeptide of the invention. Such fragments include Fv, F (ab') and F (ab') 2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.

Antibodies may be used in a method for detecting polypeptides of the invention in a biological sample, which method comprises: I providing an antibody of the invention; II incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and III determining whether antibody-antigen complex comprising said antibody is formed.

A sample may be for example a tissue extract, blood, serum and saliva.

Antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions, etc.

Antibodies may be linked to a revealing label and thus may be suitable for use in methods of in vivo HIPHUM 31 imaging.

Antibodies for use in the invention can be produced by any suitable method.

Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (1988)"Antibodies : A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising antibody in a host animal against the whole polypeptide or a fragment thereof, for example an antigenic epitope thereof, herein after the "immunogen".

A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and

isolating immunogiobuims irom me animal s serum. 1 he ammal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified.

A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256,495-497).

An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat or mouse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier.

The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.

An important aspect of the present invention is the use of polypeptides according to the invention in screening methods to identify modulators of HIPHUM 31 activity. Such modulators may be used in a method of treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD, fibrotic disease of the lung, and lung cancer or in the manufacture of a medicament for the treatment of any such disease. The screening methods may be used to identify substances that bind to serine protease and in particular which bind to HIPHUM 31 such as a substrate for the enzyme. Screening methods may also be used to identify agonists or antagonists which may modulate serine protease activity, inhibitors or activators of HIPHUM 31 activity, and/or agents which up-regulate or down-regulate HIPHUM 31 expression.

Any suitable format may be used for the assay. In general terms such screening methods may involve contacting a polypeptide of the invention with a test

substance and monitoring for binding of the test substance to the polypeptide or measuring protease activity. A polypeptide of the invention may be incubated with a test substance. Modulation of serine protease activity may be determined. In a preferred aspect, the assay is a cell-based assay. The cell is preferably a neuronal cell, a lung cell or an intestinal cell. Preferably the neuronal cell is from the cerebellum or is a cell from a subject suffering from a central nervous system disorder in which expression of HIPHUM 31 is up-regulated or from an animal model of a central nervous system disorder in which expression of HIPHUM 31 is up-regulated. Preferably the assay may be carried out in a single well of a microtitre plate. Assay formats which allow high throughput screening are preferred.

A typical assay for determining whether a test substance acts as an inhibitor or activator of HIPHUM 31 activity comprises monitoring cleavage of a peptidebased substrate using a FRET, HTRF (or TRET) and/or SPA assay. Other assays that may be used to determine whether a test substance acts as an inhibitor or activator of HIPHUM 31 activity include ELISA, zymography, activation or a reporter protease, assays with chimeric proteins and assays with peptides where cleavage is detected by HPLC/LC/MS. Cell based assays may also be used.

A typical peptide-based substrate contains a nitrophenyl amino group. When hydrolyzed by HIPHUM 31 the substrate releases nitrophenylamine which can be

detected by UV/visible absorbance. Typical substrates include Bz-Arg-NHPhNO, Suc-Ala-Ala-Pro-Arg-NHPhN02, Bz-Gly-Pro-Arg-NHMec. Alternatively insulin B chain may be used as a substrate, cleavage being monitored by reverse phase hplc.

Modulator activity can be determined by contacting cells expressing HIPHUM 31 or a variant or fragment thereof with a substance under investigation and by monitoring an effect mediated by the polypeptide. The cells expressing the polypeptide may be in vitro or in vivo. The polypeptide of the invention may be naturally or recombinantly expressed. Preferably, the assay is carried out in vitro using cells expressing recombinant polypeptide. Preferably, control experiments are carried out on cells which do not express the polypeptide of the invention to establish whether the observed responses are the result of activation of the polypeptide.

The binding of a test substance to HIPHUM 31 or a variant of fragment thereof can be determined directly. For example, a radiolabelled test substance can

be incubated with the polypeptide of the invention and binding of the test substance to the polypeptide can be monitored. Typically, the radiolabelled test substance can be incubated with cell membranes containing the polypeptide until equilibrium is reached. The membranes can then be separated from a non-bound test substance and dissolved in scintillation fluid to allow the radioactive content to be determined by scintillation counting. Non-specific binding of the test substance may also be determined by carrying out a competitive binding assay.

Substances that inhibit the interaction of a polypeptide of the invention with a HIPHUM 31 substrate or with another protease may also be identified through a yeast 2-hybrid assay or other protein interaction assay such as a coimmunoprecipitation or an ELISA based technique.

Assays may be carried out using cells expressing HIPHUM 31, and incubating such cells with the test substance optionally in the presence of a HIPHUM 31 substrate. The results of the assay are compared to the results obtained using the same assay in the absence of the test substance. Cells expressing HIPHUM 31 constitutively may be provided for use in assays for HIPHUM 31 function.

Additional test substances may be introduced in any assay to look for inhibitors or activators of substrate binding or inhibitors or activators of protease activity.

Assays may also be carried out to identify substances which modify HIPHUM 31 expression, for example substances which up-or down-regulate expression. Such assays may be carried out for example by using antibodies for HIPHUM 31 to monitor levels of HIPHUM 31 expression. Other assays which can be used to monitor the effect of a test substance on HIPHUM 31 expression include using a reporter gene construct driven by the HIPHUM 31 regulatory sequences as the promoter sequence and monitoring for expression of the reporter polypeptide.

Additional control experiments may be carried out.

Suitable test substances which can be tested in the above assays include combinatorial libraries, defined chemical entities and compounds, peptide and peptide mimetics, oligonucleotides and natural product libraries, such as display (e. g. phage display libraries) and antibody products.

Typically, organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 daltons. Candidate

products can be biomolecules including, saccharides. fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

Fragments of HIPHUM 31 which do not show HIPHUM 31 peptidase activity may also be screened to monitor their effect on the activity of full length HIPHUM 31 or a functional variant or fragment thereof.

Test substances may be used in an initial screen of, for example, 10 substances per reaction, and the substances of these batches which show inhibition or

activation tested individually. Test substances may be used at a concentration of from InM to 10mM, preferably from, 100nM to lOOOuM or from luM to lOOjM, more preferably from 111M to 101lM. Preferably, the activity of a test substance is compared to the activity shown by a known activator or inhibitor. A test substance which acts as an inhibitor may produce a 50% inhibition of activity of the protease.

Alternatively a test substance which acts as an activator may produce 50% of the maximal activity produced using a known activator.

Another aspect of the present invention is the use of the substances that have been identified by screening techniques referred to above in the treatment of disease states, which are responsive to regulation of serine protease activity. The treatment may be therapeutic or prophylactic. The condition of a patient suffering from such a disease state can thus be improved.

In particular, such substances may be used in the treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung.

Substances that act as inhibitors of HIPHUM 31 activity may be used in the treatment of disease states in which HIPHUM 31 expression is up-regulated such as Alzheimer's disease, myotonic dystrophy (MD), asthma, COPD. Substances that activate HIPHUM 31 activity may be used in the treatment of disease states in which

expression of HIPHUM 31 is down-regulated such as lung tumour and HSV infection.

Substances identified according to the screening methods outlined above and polypeptides, polynucleotides, antibodies and expression vectors described herein may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art. For example, a suitable substance may be dissolved in physiological saline or water for injections. The exact nature of a formulation will depend upon several factors including the particular substance to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania, 17"'Ed. 1985, the disclosure of which is included herein of its entirety by way of reference.

The substances, polypeptides, polynucleotides, antibodies and expression vectors may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, topical or other appropriate administration routes.

A therapeutically effective amount of a modulator, polpypeptide, polynucleotide, antibody or expression vector is administered to a patient. The dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.

Nucleic acid encoding HIPHUM 31 or a variant thereof which inhibits HIPHUM 31 activity may be administered to the mammal. In particular, a nucleic acid encoding a polypeptide with HIPHUM 31 activity may be administered to a subject suffering from a condition in which HIPHUM 31 expression is downregulated, such as lung tumour and HSV infection. A nucleic acid encoding a variant of HIPHUM 31 that inhibits HIPHUM 31 activity may be administered to a patient

suffering from a condition in which HIPHUM 31 expression is up-regulated such as Alzheimer's disease, myotonic dystrophy (MD), asthma and COPD. Nucleic acid, such as RNA or DNA, and preferably, DNA, is provided in the form of a vector, such as the polynucleotides described above, which may be expressed in the cells of the mammal.

Nucleic acid encoding the polypeptide may be administered by any available technique. For example, the nucleic acid may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated gene delivery. The nucleic acid may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration.

Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents.

Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the nucleic acid to be administered can be altered. Typically the nucleic acid is administered in the range of Ipg to lmg, preferably to Ipg to 10u. g nucleic acid for particle mediated gene delivery and 10flg to Img for other routes.

The following Examples illustrate the invention.

Example 1: Characterisation of the sequence A serine protease, designated as HIPHUM 31 has been identified. The nucleotide and amino acid sequences of the receptor have been determined. These are set out below in SEQ ID NOs: 1 and 2. Suitable primers and probes were designed and used to analyse tissue expression. HIPHUM 31 was found to be primarily expressed in oesophagus and tonsil and also in cerebellum. HIPHUM 31 was found to be upregulated in brain from Alzheimer's disease and myotonic dystrophy (MD), lung from asthma and COPD and in VEGF treated endothelial cells. HIPHUM 31 was found to be downregulated in lung tumour and in HSV infection.

The chromosomal localization was also mapped. Human HIPHUM 31 has

been mapped to 16p 13. 3. 1 his locus is linked to diseases such as polycystic kidney disease, tuberous sclerosis and pseudoxanthoma elasticum.

Example 2: Screening for substances which exhibit protein modulating activity Preparations of a purified HIPHUM 31 polypeptide or a variant are generated for screening purposes. 96 and 384 well plate, high throughput screens (HTS) are employed using fluorescence or colourimetric indicator molecules. Secondary screening involves the same technology. Tertiary screens involve the study of modulators in rat, mouse and guinea-pig models of disease relevant to the target.

A brief screening assay protocol is as follows :Plate Preparationfor Determination ofinhibitory Potency at a Single Concentration of Test Compound. Test compounds are placed in columns 1-10 of a 96-well plate.

The average enzymatic activity, from the wells in column 11, is used as the control value for calculating percent inhibition. In most cases assay plates contain 1/-lL/well of test compound. The control well (column 11) contains 1/-lL/well of solvent (100 % DMSO). For assays conducted in 384-well plates, 1 uL of test compound is added to columns 1-20 and the same volume of 100% DMSO is added to column 21. The average enzymatic activity, from the wells in column 21, is used as the control value for calculating percent inhibition.

Assay Due to the inherent stability of the enzyme at room temperature and the low concentration of enzyme used, the enzyme, substrate and buffer are combined prior to addition to the assay plate containing test compounds. Both the stock solution of enzyme, 0.2 mg/mL, and the stock solution of substrate, 500 mM, are diluted with the solution of assay buffer to yield 3.3 ng/mL enzyme and 100 uM substrate in 25 mM Tris, pH 7.5, 10 mM KCl, 140 mM NaCl. A volume of 100 aL is added to the 96-well assay plate or a volume of 50 L is added to the 384-well assay plate. The absorbance is monitored every 3.7 hours for 18.5 hours at 405 nm using a Wallac Victor filter-based plate reader coupled with a Zymark Twister. A maximum of 50 assay plates are monitored at one time using this automated system in order to have the 3.7 hour cycle per plate. An empty plate is added at the top and bottom of each of the 3 stacks of plates to reduce evaporation in the plates that would be exposed to air during the run. Data for a single plate at a single time point is stored in each file.

SEQUENCE LISTING < 110 > GLAXO GROUP LIMITED < 120 > NOVEL PROTEIN < 130 > QG1044 (P80915) < 160 > 2 < 170 > PatentIn version 3.0 < 210 > 1 < 211 > 873 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1). (873) < 400 > 1

atg agg egg ccg gcg gcg gtg ccg etc ctg ctg ctg ctg tgt ttt ggg 48 Met Arg Arg Pro Ala Ala Val Pro Leu Leu Leu Leu Leu Cys Phe Gly 1 5 10 15 tct cag agg gcc aag gca gca aca gcc tgt ggt cgc ccc agg atg ctg 96 Ser Gln Arg Ala Lys Ala Ala Thr Ala Cys Gly Arg Pro Arg Met Leu 20 25 30 aac cga atg gtg ggc ggg cag gac acg cag gag ggc gag tgg ccc tgg 144 Asn Arg Met Val Gly Gly Gln Asp Thr Gln Glu Gly Glu Trp Pro Trp 35 40 45 caa gtc age ate cag cgc aac gga age cac ttc tgc ggg ggc age etc 192 Gln Val Ser Ile Gln Arg Asn Gly Ser His Phe Cys Gly Gly Ser Leu 50 55 60 ate gcg gag cag tgg gtc ctg acg get gcg cac tgc ttc cgc aac ace 240 Ile Ala Glu Gln Trp Val Leu Thr Ala Ala His Cys Phe Arg Asn Thr i 65 70 75 80 tct gag acg tec ctg tac cag gtc ctg ctg ggg gca agg cag cta gtg 288 Ser Glu Thr Ser Leu Tyr Gln Val Leu Leu Gly Ala Arg Gln Leu Val 85 90 95 cag ccg gga cca cac get atg tat gcc egg gtg agg cag gtg gag age 336 Gln Pro Gly Pro His Ala Met Tyr Ala Arg Val Arg Gln Val Glu Ser 100 105 110 aac ccc ctg tac cag ggc acg gcc tec age get gac gtg gcc ctg gtg 384 Asn Pro Leu Tyr Gln Gly Thr Ala Ser Ser Ala Asp Val Ala Leu Val 115 120 125 gag ctg gag gca cca gtg ccc ttc ace aat tac ate etc ccc gtg tgc 432 Glu Leu Glu Ala Pro Val Pro Phe Thr Asn Tyr Ile Leu Pro Val Cys

130 135 140 ctg cct gac ccc teg gtg ate ttt gag acg ggc atg aac tgc tgg gtc 480 Leu Pro Asp Pro Ser Val Ile Phe Glu Thr Gly Met Asn Cys Trp Val 145 150 155 160 act ggc tgg ggc age ccc agt gag gaa gac etc ctg ccc gaa ccg egg 528 Thr Gly Trp Gly Ser Pro Ser Glu Glu Asp Leu Leu Pro Glu Pro Arg 165 170 175 ate ctg cag aaa etc get gtg ccc ate ate gac aca ccc aag tgc aac 576 Ile Leu Gln Lys Leu Ala Val Pro Ile Ile Asp Thr Pro Lys Cys Asn 180 185 190 ctg etc tac age aaa gac ace gag ttt ggc tac caa ccc aaa ace ate 624 Leu Leu Tyr Ser Lys Asp Thr Glu Phe Gly Tyr Gln Pro Lys Thr Ile 195 200 205 aag aat gac atg ctg tgc gcc ggc ttc gag gag ggc aag aag gat gcc 672 Lys Asn Asp Met Leu Cys Ala Gly Phe Glu Glu Gly Lys Lys Asp Ala 210 215 220 tgc aag ggc gac teg ggc ggc ccc ctg gtg tgc etc gtg ggt cag teg 720 Cys Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Leu Val Gly Gln Ser 225 230 235 240 tgg ctg cag gcg ggg gtg ate age tgg ggt gag ggc tgt gcc cgc cag 768 Trp Leu Gln Ala Gly Val Ile Ser Trp Gly Glu Gly Cys Ala Arg Gln 245 250 255 aac cgc cca ggt gtc tac ate cgt gtc ace gcc cac cac aac tgg ate 816 Asn Arg Pro Gly Val Tyr Ile Arg Val Thr Ala His His Asn Trp Ile 260 265 270 cat egg ate ate ccc aaa ctg cag ttc cag cca gcg agg ttg ggc ggc 864 His Arg Ile Ile Pro Lys Leu Gln Phe Gln Pro Ala Arg Leu Gly Gly 275 280 285 cag aag tga 873 Gln Lys 290 < 210 > 2 < 211 > 290 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Arg Arg Pro Ala Ala Val Pro Leu Leu Leu Leu Leu Cys Phe Gly 1 5 10 15 Ser Gln Arg Ala Lys Ala Ala Thr Ala Cys Gly Arg Pro Arg Met Leu 20 25 30 Asn Arg Met Val Gly Gly Gln Asp Thr Gln Glu Gly Glu Trp Pro Trp

35 40 45

Gln Val Ser Ile Gln Arg Asn Gly Ser His Phe Cys Gly Gly Ser Leu 50 55 60 Ile Ala Glu Gln Trp Val Leu Thr Ala Ala His Cys Phe Arg Asn Thr 65 70 75 80 Ser Glu Thr Ser Leu Tyr Gln Val Leu Leu Gly Ala Arg Gln Leu Val 85 90 95 Gln Pro Gly Pro His Ala Met Tyr Ala Arg Val Arg Gln Val Glu Ser 100 105 110 Asn Pro Leu Tyr Gln Gly Thr Ala Ser Ser Ala Asp Val Ala Leu Val 115 120 125

Glu Leu Glu Ala Pro Val Pro Phe Thr Asn Tyr 11 e Leu Pro Val Cys 130 135 140 Leu Pro Asp Pro Ser Val Ile Phe Glu Thr Gly Met Asn Cys Trp Val 145 150 155 160 Thr Gly Trp Gly Ser Pro Ser Glu Glu Asp Leu Leu Pro Glu Pro Arg 165 170 175

Ile Leu Gln Lys Leu Ala Val Pro Ile Ile Asp Thr Pro Lys Cys Asn 180 185 190 Leu Leu Tyr Ser Lys Asp Thr Glu Phe Gly Tyr Gln Pro Lys Thr Ile 195 200 205

Lys Asn Asp Met Leu Cys Ala Gly Phe Glu Glu Gly Lys Lys Asp Ala 210 215 220 Cys Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Leu Val Gly Gln Ser 225 230 235 240 Trp Leu Gln Ala Gly Val Ile Ser Trp Gly Glu Gly Cys Ala Arg Gln 245 250 255 Asn Arg Pro Gly Val Tyr Ile Arg Val Thr Ala His His Asn Trp Ile 260 265 270 His Arg Ile Ile Pro Lys Leu Gln Phe Gln Pro Ala Arg Leu Gly Gly 275 280 285 Gln Lys 290

Claims

1. A method for identification of a substance that modulates serine protease activity, which method comprises: (i) contacting (a) a cell selected from a neuronal cell, a lung cell and an intestinal cell, which cell expresses a serine protease polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof which has trypsin-like serine protease activity with (b) a test substance; and (i) monitoring for serine protease activity.

2. A method for identification of a substance that modulates serine protease activity, which method comprises: (i) contacting (a) a serine protease polypeptide isolated from a cell selected from a neuronal cell, a lung cell and an intestinal cell, which serine protease comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof which has trypsin-like serine protease activity; with (b) a test agent; and (ii) monitoring for serine protease activity.

3. A method according to any one of the preceding claims wherein the variant has at least 80% sequence identity to SEQ ID NO: 2.

4. A modulator of serine protease activity identified by a method according to any one of the preceding claims for use in a method of treatment of the human or animal body by therapy.

5. Use of a modulator of serine protease activity identified by a method according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung.

6. Use of a serine protease polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2; or

(11) a variant thereot which has trypsin-like senne protease activity ; or (iii) a fragment of (i) or (ii) which has trypsin-like serine protease activity in the manufacture of a medicament for use in the treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung.

ZDI

7. Use of a polynucleotide encoding a serine protease polypeptide comprising (i) the amino acid sequence of SEQ ID NO : 2 ; or (ii) a variant thereof which has trypsin-like serine protease activity ; or (iii) a fragment of (i) or (ii) which has trypsin-like serine protease activity in the manufacture of a medicament for use in the treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung.

8. Use according to claim 7 wherein the polynucleotide comprises : (a) the nucleic acid sequence of SEQ ID NO : 1 and/or a sequence complementary thereto ; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a) ; (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b) ; or (d) a sequence having at least 80% identity to a sequence as defined in (a), (b) or (c).

9. Use of an antibody specific for a serine protease polypeptide comprising the amino acid sequence of SEQ ID NO : 2 in the manufacture of a medicament for use in the treatment of tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung.

10. Use of a substance which modulates proteinase activity of a polypeptide with the amino acid sequence shown in SEQ ID NO : 2 or a variant

thereot in the manutacture ot a medicament tor treatment or prophylaxis ot tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung.

11. A method of treating a subject suffering from tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung which method comprises administering to said subject an effective amount of a substance which modulates proteinase activity of a polypeptide with the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof.

12. A method for treating tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung, which method comprises: (i) identifying a substance that modulates serine protease activity and/or expression (ii) administering an effective amount of the substance to a patient suffering from tonsilitis, CNS disease such as Alzheimer's disease and myotonic dystrophy, respiratory and inflammatory respiratory disease such as asthma, COPD and fibrotic disease of the lung.

13. A method according to claim 12 wherein step (i) comprises: (a) contacting a test substance and a serine protease polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2 or (ii) a variant thereof which has trypsin-like serine protease activity or (iii) a fragment of (i) or (ii) which has trypsin-like serine protease activity, and (b) determining the effect of the test substance on the activity of the polypeptide, thereby determining whether the test substance modulates serine protease activity.