WO2010029363A2 - Peptide et ses utilisations - Google Patents

Peptide et ses utilisations Download PDF

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Publication number
WO2010029363A2
WO2010029363A2 PCT/GB2009/051169 GB2009051169W WO2010029363A2 WO 2010029363 A2 WO2010029363 A2 WO 2010029363A2 GB 2009051169 W GB2009051169 W GB 2009051169W WO 2010029363 A2 WO2010029363 A2 WO 2010029363A2
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cathepsin
sequence
molecule
propeptide
amino acid
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PCT/GB2009/051169
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English (en)
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WO2010029363A3 (fr
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James Johnston
Richard Buick
Christopher Scott
Roberta Burden
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Fusion Antibodies Limited
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Publication of WO2010029363A3 publication Critical patent/WO2010029363A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22027—Cathepsin S (3.4.22.27)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4873—Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)

Definitions

  • This application relates to peptides and their use in medical treatment.
  • it relates to cathepsin propeptide fragments and their uses.
  • Proteases are a large group of proteins that comprise approximately 2% of all gene products (Rawlings and Barrett, 2004, MEROPS: The peptidase database. Nucleic Acids Res 2004; 32:Dl60-4) . Proteases catalyse the hydrolysis of peptide bonds and are vital for the proper functioning of all cells and organisms. Proteolytic processing events are important in a wide range of cellular processes including bone formation, wound healing, angiogenesis and apoptosis.
  • the lysosomal cysteine proteases were initially thought to be enzymes that were responsible for nonselective degradation of proteins in the lysosomes . Normally associated with localisation in the lysosomes, these proteases were originally thought to be only involved in the non-selective degradation of proteins in endosomal compartments. However, they are now known to be accountable in a number of specific cellular processes, having roles in antigen presentation (Honey and Rudensky, 2003, Nat Rev Immunol 2003 ; 3 : 472-82 ; Bryant & Ploegh, 2004, Curr Opin Immunol 2004 ; 16 : 96-102 ) apoptosis (Zheng et al .
  • Cathepsins are proteolytic enzymes. To date, eleven human cathepsins have been identified, but the specific in vivo roles of each are still to be determined (Katunuma et al , Biol Chem. 2003 Jun; 384 ( 6 ) : 883-90) . Each of these lysosomal proteases has been implicated in the progression of various tumours . It is thought that their abnormally high secretion from tumour cells leads to the degradation of the extracellular matrix (ECM) . This aberrant breakdown of ECM components such as elastin and collagen accelerates the penetration and invasion of these abnormal cells to surrounding normal tissue.
  • ECM extracellular matrix
  • Cathepsin L-like proteases are produced as inactive precursors, containing an N terminal propeptide domain.
  • This propeptide has previously been shown to act as both a chaperone for the folding of the nascent protease and inhibitor of the active species, binding to the active site of the protease in immature lysosomes .
  • Inhibition studies have shown that the Cathepsin S (Cat S) propeptide (CatSPP) has a Ki in the low nanomolar range towards activated Cats and perhaps surprisingly, also has similar properties against both CatK and CatL, although it has also been shown to have no effect on the less homologous CatB, CatH or papain.
  • this property of the CatSPP is unique in that the propeptides of K and L do not have the same uniform inhibition profile to each of its cognate family members .
  • Cat S (Cathepsin S) was originally identified from bovine lymph nodes and spleen and the human form cloned from a human macrophage cDNA library (Shi et al, J Biol Chem. 1992 Apr 15 ; 267 (11) : 7258-62 ) .
  • the gene encoding Cat S is ⁇ located on human chromosome Iq21.
  • the 996 base pair transcript encoded by the Cat S gene is initially translated into an unprocessed precursor protein with a molecular weight of 37.5 kDa.
  • the unprocessed protein is composed of 331 amino acids; a 15 amino acid signal peptide, a 99 amino acid pro-peptide sequence and a 217 amino acid peptide.
  • Cat S is initially expressed with a signal peptide that is removed after it enters the lumen of the endoplasmic reticulum.
  • the propeptide sequence binds to the active site of the protease, rendering it inactive until it has been transported to the acidic endosomal compartments, after which the propeptide sequence is removed and the protease is activated (Baker et al , Protein Expr Purif. 2003 Mar;28(l) :93-10) .
  • Cat S has been identified as a key enzyme in major histocompatibility complex class II (MHC-II) mediated antigen presentation, by cleavage of the invariant chain, prior to antigen loading.
  • MHC-II major histocompatibility complex class II
  • the specificity of Cat S in the processing of the invariant chain Ii allows for Cat S specific therapeutic targets in the treatment of conditions such as asthma and autoimmune disorders (Chapman et al , Annu Rev Physiol 1997;59:63-88) .
  • Cathepsin L was originally isolated from the lysosomes of rat liver before the human form was identified in 1988 (Gal and Gottesman, Biochem J. 1988 JuI 1;253 (1) :303-6; Joseph et al , J Clin Invest. 1988 May;81 (5) :1621-9) .
  • the gene encoding CatL was mapped to human chromosome 9q21-22 (Chauhan et al . , J Biol Chem. 1993 Jan 15 ; 268 (2 ) : 1039-45) and is composed of eight exons and seven introns .
  • the gene product is translated into a preproprotein with a molecular weight of 39 kDa and is processed into two enzymatically active isoforms; a single chain form of 31 kDa and a two-chain form comprised of a 24 kDa heavy chain and a 5kDa light chain.
  • the processing of pro-CatL to the mature active enzyme can occur via various mechanisms including autocatalytic activation (Salminen & Gottesman, Biochem J. 1990 Nov 15;272 (1) :39-44) and by the action of other proteases .
  • CatL has been proposed to have a major role in many biological processes including lysosomal proteolysis and bone resorption, as well as in several diseases such as arthritis and malignancy (Rienheckel et al , Biol Chem. 2001 May; 382 (5) : 735-41) .
  • the role of lysosomal cysteine proteases in antigen presentation has been extensively researched within the past few years .
  • CatL has been implicated in this process through its ability to perform the final step of Ii proteolysis in cortical thymic epithelial cells.
  • mice were observed to be incapable of the degradation of the invariant chain in cortical epithelial cells of the thymus (Nakagawa et al, Science. 1998 Apr 17 ; 280 (5362 ) : 450-3 ) and exhibited a distinct defect in CD4+ T cell selection (Roth et al, FASEB J. 2000 Oct;14 (13) :2075-86) .
  • Mice lacking cathepsin L also developed periodic hair loss and epidermal hyperplasia due to alterations in hair follicle morphogenesis.
  • CatL in tumour invasion and metastasis has also been studied in great detail due to its ubiquitous expression and its ability to degrade components of the extracellular matrix and basement membrane. Elevated expression levels of CatL have been associated with a wide range of malignancies including breast, colon, prostate, kidney carcinomas and astrocytomas.
  • CatL may function as a transcriptional activator.
  • Alternative isoforms of CatL have previously been reported (Rescheleit et al , FEBS Lett. 1996 Oct 7 ; 394 (3 ) : 345- 8; Seth et al , Gene. 2003 Dec 4; 321 : 83-91) , however an isoform lacking the N-terminal signal peptide has been shown to localise to the nucleus, suggesting a role for CatL in the processing of the CDP/Cux transcription factor.
  • This theory was reinforced by studies on CatL-deficient fibroblasts, which appeared to have a marked reduction in CDP/Cux processing (Goulet et al , MoI Cell. 2004 Apr 23 ; 14 (2 ) : 207-19 ) .
  • CatK Biophys Res Commun. 1995 Jan 5 ; 206 (1) : 89-96
  • the gene encoding CatK is situated on human chromosome Iq21, the same locus as Cats, suggesting that these two proteases may have a common origin.
  • the promoter structure of CatK is similar to that of Cats with the absence of a TATA box but with the presence of two AP-I sites; both common features of genes which show restricted expression patterns . Human CatK expression has been shown to be restricted and is found predominantly in osteoclasts and in the ovary
  • CatK The amino acid sequence of CatK shows high sequence similarity with cathepsins S and L (52% and 46% respectively) and together these three genes form a small subfamily within the mammalian lysosomal cysteine proteases.
  • CatK has been characterised as one of the most potent elastinolytic enzymes, with greater activity that pancreatic elastase at pH5.5 (Bromme et al, J Biol Chem. 1996 Jan 26 ; 271 (4) : 2126- 32; Chapman et al, Annu Rev Physiol 1997 ; 59 : 63-88) . It also has the ability to catalyse the hydrolysis of collagen type I, II and IV (Kafienah et al, Biochem J. 1998 May 1;331 ( Pt 3) :727-32) .
  • the physiological relevance of the collagenolytic activity of CatK is illustrated through its association with the bone disorder, pycnodysostosis (GeIb et al , Biochem MoI Med. 1996 Dec; 59 (2 ) : 200-6) .
  • Pycnodysostosis is an autosomal recessive condition characterised by osteosclerosis and severe skeletal dysplasia. Osteoporosis occurs when the balance between bone resorption and formation has been disrupted, favouring resorption. Resorption is mediated by osteoclasts which generate an acidic environment at their site of attachment where the proteolytic degradation of the matrix occurs .
  • CatK has been implicated in this process due to the identification of nonsense, missense and stop codon mutations in pycnodysostosis patients (GeIb et al, Biochem MoI Med. 1996 Dec; 59 (2) : 200-6) .
  • Cathepsin V was first identified from a human brain cDNA library as a cysteine protease with exceptionally high homology to CatL (78%) (Santamaria et al . , Cancer Res. 1998 Apr 15 ; 58 (8) : 1624-30) .
  • the gene encoding CatV has been mapped to human chromosome 9q21-22, adjacent to CatL.
  • protease expression patterns underlie many human pathological processes.
  • the deregulated expression and activity of cathepsins has been linked to a range of conditions including neurodegenerative disorders, autoimmune diseases and tumourigenesis .
  • Cat S upregulation has been linked to several neurodegenerative disorders. It is believed to have a role in the production of the ⁇ peptide (A ⁇ ) from the amyloid precursor protein (APP) (Munger et al , Biochem J. 1995 Oct 1;311 ( Pt l) :299-305) and its expression has been shown to be upregulated in both Alzheimer's Disease and Down's Syndrome (Lemere et al, Am J Pathol. 1995 Apr ; 146 (4) : 848-60) . Cat S may also have a role in Multiple Sclerosis and Creutzfeldt - Jakob disease through the ability of Cat S to degrade myelin basic protein, a potential autoantigen implicated in the pathogenesis of MS
  • Cat S expression has also been associated with atherosclerosis .
  • Cat S expression is negligible in normal arteries, yet human atheroma display strong immunoreactivity (Sukhova et al, J Clin Invest. 1998 Aug l;102 (3) :576-83) .
  • Further research has linked Cat S expression with inflammatory muscle disease and rheumatoid arthritis .
  • Muscle biopsy specimens from patients with inflammatory myopathy had a 10 fold increase in Cat S expression compared to control muscle sections (Wiendl et al , J Neuroimmunol . 2003 May; 138 (1-2) : 132-43 ), and levels of Cat ⁇ expression were significantly higher in synovial fluid from patients with rheumatoid arthritis compared to those with osteoarthritis (Hashimoto et al, Biochem Biophys Res Commun. 2001 May 4; 283 (2 ) : 334-9 ) .
  • Cat S The role of Cat S has also been investigated in specific malignancies.
  • the expression of Cat S was shown to be significantly greater in lung tumour and prostate carcinomas sections in comparison to normal tissue (Kos et al, Br J Cancer. 2001 Oct 19;85 (8) :1193-200, Fernandez et al , Int J Cancer. 2001 Jan 20; 95 (1) : 51-5) and suggested that Cat S may have a role in tumour invasion and disease progression.
  • Cat S has been shown to be active in the degradation of ECM macromolecules such as laminin, collagens, elastin and chondroitin sulphate proteoglycans (Liuzzo et al , MoI Med. 1999 May; 5 (5) : 320-33 ) and invasion assays using the U251MG grade IV glioblastoma cell line showed up to 61% reduction in invasion in the presence of a Cat S inhibitor LHVS29 (Flannery et al , Am J Pathol. 2003 JuI; 163 (1) : 175- 82) . This would suggest that Cat S may have an important role in the process of tumour invasion in astrocytomas and therefore may be a target for anti- invasive therapy.
  • ECM macromolecules such as laminin, collagens, elastin and chondroitin sulphate proteoglycans
  • CatL has also been found to have important roles in a range of different pathological conditions including tumourigenesis .
  • the generation of CatL knockout mice revealed a critical role in epidermal homeostasis, regulation of the hair cycle, and MHC class II- mediated antigen presentation in cortical epithelial cells of the thymus (Reinheckel et al , Biol Chem. 2001 May;382 (5) :735-41) .
  • Cat K expression has previously been correlated with a range of different pathologies including osteoporosis and specific malignancies .
  • the rare skeletal condition, pycnodysostosis is caused by a deficiency in CatK.
  • CatK normally functions to degrade type-1 collagen and other bone proteins (Motyckova and Fisher, Curr MoI Med. 2002 Aug;2 (5) :407-21)
  • the osteoclasts from patients with Pycnodysostosis are dysfunctional due to mutations within the cathepsin K gene (GeIb et al , Biochem MoI Med. 1996 Dec;59 (2) :200-6) .
  • CatK expression is associated with lung adenocarcinomas yet absent from the non-invasive bronchioalveolar carcinomas, acting as a potential marker of the invasive growth of lung carcinomas (Rapa et al, Am J Clin Pathol. 2006 Jun,- 125 (6) : 847- 54) .
  • CatK has also been identified as the principal protease in giant cell tumour of the bone (Lindeman et al , Am J Pathol. 2004 Aug; 165 (2 ): 593-600) and an association with breast carcinomas (Littlewood-Evans et al , Cancer Res. 1997 Dec 1;57 (23) :5386-90) has been shown. Therefore, the development of CatK inhibitors has great potential, particularly in pathological conditions where excess osteoclast activation and bone resorption occurs such as osteoporosis, bone metastasis and multiple myeloma.
  • Cat V was originally identified in colorectal and breast carcinomas, as well as certain ovarian and renal cell carcinomas as a cysteine protease with exceptionally high homology to CatL (78%) (Santamaria et al., Cancer Res. 1998 Apr 15 ; 58 (8 ) : 1624-30) .
  • the increase in expression and activity of the cathepsin L-like proteases has been observed in a range of diseases and implicated in their pathogenesis. Therefore, the generation of inhibitors specifically targeting these proteases have the potential as therapeutic agents .
  • CatSPP has the most interesting inhibitory kinetic profile, as it is an equally effective inhibitor of both CatL and CatK in addition to Cats. Maubach and co-workers showed in competitive enzyme binding assays that CatSPP is an equipotent inhibitor of Cats (Ki of 0.27 nM) and CatL (Ki of 0.36nM) (Maubach et al . , Eur J Biochem.
  • CatSPP is actually a more potent inhibitor of CatL (K 1 of 0.46 nM) than it is of Cats (Ki of 7.6 nM) and has almost identical efficacy against CatK (K 1 of 7.0 nM) (Guay et al . , Eur J Biochem. 2000 Oct; 267 (20) : 6311-8 ) .
  • the present inventors have investigated the effects of a number of specific propeptide fragments and have surprisingly shown that such fragments, despite missing particular motifs previously thought to be essential for inhibitory activity nevertheless retain sufficient activity to inhibit cathepsin activity and to be of use therapeutically.
  • a method of inhibiting activity of a cathepsin L-like protease in cells or tissue comprising administration of a cathepsin propeptide molecule or a nucleic acid encoding said cathepsin propeptide fragment to said cells or tissue, wherein the cathepsin propeptide molecule is a fragment consisting of the amino acid sequence as shown as any one of Sequence ID Nos : 1 to 7, or a derivative of said fragment.
  • the method is in vitro. In another the method is in vivo.
  • Activity may be inhibited completely or partially.
  • the method may be used to reduce aberrant activity to normal activity i.e. within the normal range in non-pathological states.
  • a method of inhibiting overexpression of a cathepsin L-like protease in cells or tissue comprising administration of a cathepsin propeptide molecule or a nucleic acid encoding said cathepsin propeptide molecule to said cells or tissue, wherein the cathepsin propeptide molecule is a fragment consisting of the amino acid sequence as shown as any one of Sequence ID Nos: 1 to 7, or a derivative of said fragment.
  • a method of treating a condition associated with overexpression and/or aberrant activity of a cathepsin L-like protease in a patient in need of treatment thereof comprising administration of a cathepsin propeptide molecule or a nucleic acid encoding said cathepsin propeptide molecule, wherein the cathepsin propeptide molecule is a fragment consisting of the amino acid sequence as shown as any one of Sequence ID Nos: 1 to 7 , or a derivative of said fragment.
  • cathepsin propeptide molecule or a nucleic acid encoding said cathepsin propeptide molecule, wherein the cathepsin propeptide molecule is a fragment consisting of the amino acid sequence as shown as any one of Sequence ID Nos: 1 to 7 , , or a derivative of said fragment, for use in medicine.
  • the invention further provides a cathepsin propeptide molecule or a nucleic acid encoding a cathepsin propeptide molecule for use in treatment of a condition associated with overexpression and/or aberrant activity of a cathepsin L-like protease, wherein the cathepsin propeptide molecule is a fragment consisting of the amino acid sequence as shown as any one of Sequence ID Nos: 1 to 7 , or a derivative of said fragment.
  • cathepsin propeptide molecule or a nucleic acid encoding a cathepsin propeptide molecule in the preparation of a medicament for the treatment of a condition associated with overexpression and/or aberrant activity of a cathepsin L-like protease, wherein the cathepsin propeptide molecule is a fragment consisting of the amino acid sequence as shown as any one of Sequence ID Nos: 1 to 7 , or a derivative of said fragment.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a cathepsin propeptide molecule or a nucleic acid encoding a cathepsin propeptide molecule, wherein the cathepsin propeptide molecule is a fragment consisting of the amino acid sequence as shown as any one of Sequence ID Nos: 1 to 7 , or a derivative of said fragment.
  • a cathepsin propeptide molecule is devoid of the alpha helix 2 motif of the wild type propeptide.
  • the cathepsin propeptide molecule can be devoid of both the alpha helix 2 motif and the alpha helix 1 motif of the wild type propeptide.
  • the cathepsin propeptide molecules for use in the invention may optionally be truncated at the C terminal compared to the full length propeptide molecule, for example by 30 or less, for example, by 20 or less, or by 10 or less amino acids.
  • Cathepsin L -like proteases comprise cathepsin L protease, cathepsin S protease, cathepsin K protease and cathepsin V proteases.
  • the cathepsin propeptide molecules for use in the present invention may be a propeptide fragment, or derivative thereof, of any Cathepsin L- like protease.
  • the cathepsin propeptide molecule is a fragment of a Cathepsin S propeptide.
  • the full length cathepsin propeptide domain has the amino acid sequence as shown as Sequence ID No : 8:
  • the cathepsin S propeptide fragment is a fragment consisting of the amino acid sequence as shown as Sequence ID No: 1:
  • the cathepsin S propeptide fragment is a fragment consisting of the amino acid sequence as shown as Sequence ID No: 2:
  • the cathepsin S propeptide fragment is a fragment consisting of the amino acid sequence as shown as Sequence ID No : 3:
  • the cathepsin S propeptide fragment is a fragment consisting of the amino acid sequence as shown as Sequence ID No: 4:
  • the cathepsin S propeptide fragment is a fragment consisting of the amino acid sequence as shown as Sequence ID No: 5: SYDLGMNHLGDMTSEEVMSLXSSLRVPSQWQRNITY (Sequence ID No:
  • the cathepsin S propeptide fragment is a fragment consisting of the amino acid sequence as shown as Sequence ID No : 6:
  • NHLGDMTSEEVMSLXSS (Sequence ID No : 6 ) ; where X is M or T.
  • the cathepsin S propeptide fragment is a fragment consisting of the amino acid sequence as shown as Sequence ID No : 7:
  • a "derivative" of one such cathepsin propeptide fragment of and for use in the invention typically means a polypeptide which, compared with one of the cathepsin propeptide fragments having the amino acid sequence consisting of any one of Sequence ID No : 1 to Sequence ID No : 7, is modified by varying the amino acid sequence, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself.
  • said derivative does not comprise an amino acid sequence corresponding to the alpha helix 2 motif and/or an amino acid sequence corresponding to the alpha helix 1 motif of a cathepsin S propeptide.
  • Such derivatives may involve insertion, addition, deletion and/or substitution of one or more amino acids.
  • derivatives may involve the insertion, addition, deletion and/or substitution of 25 or fewer amino acids, for example 15 or fewer, typically 10 or fewer, such as 5 or fewer for example of 1 or 2 amino acids only.
  • Derivatives of the cathepsin propeptide peptide may contain amino acids other than the natural amino acids or substituted amino acids .
  • Derivatives of the propeptide fragments of and for use in the invention maintain cathepsin propeptide fragment biological activity i.e. the ability to inhibit cathepsin activity. Such activity may be determined by any means as taught herein or as available in the art. For example, in one embodiment, biological activity may be determined by a fluorometric assay, In another embodiment, biological activity may be determined by a tumour invasion assay.
  • cathepsin propeptide fragments contemplated for use in the present invention include, but are not limited to, the following:
  • ID No : 1 may include, but are not limited to, a derivative consisting of the amino acid sequence selected from the group as shown as Sequence ID No:
  • Derivatives of the cathepsin S propeptide fragment having the amino acid sequence as shown as Sequence ID No : 2 may include, but are not limited to, a derivative consisting of the amino acid sequence selected from the group as shown as Sequence ID No: 14-22, where X is M or T:
  • derivatives of or for use in the invention are derivatives of the cathepsin S propeptide fragment having the amino acid sequence as shown as Sequence ID No : 3 and may include, but are not limited to, a derivative consisting of the amino acid sequence selected from the group as shown as Sequence ID No: 23-33:
  • derivatives of or for use in the invention are derivatives of the cathepsin S propeptide fragment having the amino acid sequence as shown as Sequence ID No : 4 and may include, but are not limited to, a derivative consisting of the amino acid sequence selected from the group as shown as Sequence ID No : 34-36:
  • derivatives of or for use in the invention are derivatives of the cathepsin S propeptide fragment having the amino acid sequence as shown as Sequence ID No : 5 and may include, but are not limited to, a derivative consisting of the amino acid sequence selected from the group as_ shown as
  • Sequence ID No: 37-56 where X is M or T: MLHNLEHSMGMHSYDLGMNHLGDMTSEEVMSLXSSLRVPSQWQRNITY (Sequence ID No : 37)
  • derivatives of or for use in the invention are derivatives of the cathepsin S propeptide fragment having the amino acid sequence as shown as Sequence ID No : 6 and may include, but are not limited to, a derivative consisting of the amino acid sequence selected from the group as shown as Sequence ID No: 57-71, where X is M or T:
  • NHLGDMTSEEVMSLXSS (Sequence ID No : 66)
  • derivatives of or for use in the invention are derivatives of the cathepsin S propeptide fragment having the amino acid sequence as shown as Sequence ID No : 7 and may include, but are not limited to, a derivative consisting of the amino acid sequence selected from the group as shown as Sequence ID No: 72-77 , where X is M or T:
  • a derivative of or for use in the invention is a derivative consisting of the amino acid sequence selected from the group as shown as Sequence ID No: 78-95, where X is M or T:
  • KQYKEKNEEAVRRLIWEKNLKFVMLHNLEHSMGMHSYDLGMNHLGDMTSEEVM SLXSSLRVPSQWQRNITYK (Sequence ID No: 89) KQYKEKNEEAVRRLIWEKNHLGDMTSEEVMSLXSSLRVPSQWQRNITYKSNPN R (Sequence ID No : 90);
  • a Cathepsin propeptide fragment of or for use in the invention may be a cathepsin propeptide fragment of any species.
  • the species is a mammalian species, for example, mouse, rat, human etc.
  • the cathepsin propeptide fragment is a human cathepsin propeptide fragment, for example of the human cathepsin propeptide having amino acid sequence corresponding to amino acid residues 17 to 113 of the cathepsin S protease as disclosed in accession no M90696, (reproduced as amino acid residues 13 to 109 of the amino acid sequence shown in Figure 1) .
  • the cathepsin propeptide molecule may incorporate a tag, for example a polyHis tag.
  • the cathepsin propeptide molecule comprises an antibody Fc portion or any other fusion protein moiety such as GST.
  • the Fc portion is an IgG Type b Fc portion, for example a murine IgG Type b Fc portion. Accordingly, in one embodiment of the invention there is provided a fusion protein comprising a cathepsin propeptide molecule according to the invention.
  • Such a fusion protein is considered, in one embodiment, to be a derivative of the cathepsin propeptide fragment.
  • the cathepsin propeptide molecule may be fused to any suitable peptide molecule, such as a polyHistag, an Fc portion or GST.
  • the fusion protein does not comprise a cathepsin peptide derived or cathepsin propeptide derived peptide sequence; for example, in one embodiment such a peptide sequence is considered not to comprise a cathepsin peptide derived or cathepsin propeptide derived peptide sequence if it comprises no more than ten, for example no more than five contiguous amino acids corresponding to a contiguous cathepsin propeptide sequence, e.g. a cathepsin S propeptide sequence.
  • Cathepsin propeptide molecules for use in the invention may be used in the treatment of any condition with which aberrant expression of a cathepsin L -like protease is associated.
  • conditions in which the invention may be used include, but are not limited to, neoplastic disease, inflammatory disorders, neurodegenerative disorders, autoimmune disorders, asthma, or atherosclerosis .
  • the condition is a condition associated with overexpression and/or aberrant activity of cathepsin S.
  • cathepsin propeptide fragments for example, but not limited, to fragments which can lack the alpha helix 2 motif and/or which are C terminal truncated as compared to full length propeptides, nevertheless retain inhibitory effects . It is believed that this suggests that, in common with the full length propeptides, such propeptide fragments may be used therapeutically to inhibit the activity of cathepsins L-type proteases, and may be used to attenuate the progression of invasive or metastatic cancer cells .
  • Cathepsin propeptide fragment derivatives A "derivative" of cathepsin propeptide fragment for use in the invention typically means a polypeptide which, compared with a fragment having the amino acid sequence shown as Sequence ID No : 1, Sequence ID No : 2 , Sequence ID No : 3 , Sequence ID No : 4 , Sequence ID No : 5 , Sequence ID No : 6 , or Sequence ID No : 7 , is modified by varying the amino acid sequence, but retains inhibitory activity as described above. Such derivatives may involve insertion, addition, deletion and/or substitution of one or more amino acids.
  • derivatives may involve the insertion, addition, deletion and/or substitution of 25 or fewer amino acids, for example 15 or fewer, typically 10 or fewer, such as 5 or fewer for example of 1 or 2 amino acids only.
  • Derivatives of the cathepsin propeptide fragments peptide may contain other amino acids than the natural amino acids or substituted amino acids.
  • such derivatives are such that they lack an alpha helix 2 motif and/or an alpha helix 1 motif.
  • derivatives can be obtained from peptidomimetics .
  • the cathepsin propeptide derivative comprises an Fc portion.
  • Derivatives of cathepsin propeptide fragments of and for use in the invention which may be used in the invention preferably retain cathepsin propeptide fragment biological activity i.e. the ability to inhibit cathepsin activity.
  • activity may be determined by any means as taught herein or as available in the art.
  • biological activity may be determined by a fluorometric assay, for example as employed in the Examples, for example using Cats and the fluorigenic substrate Cbz-Val-Val-Arg-AMC in the presence of predetermined concentrations of the derivative .
  • a derivative is considered to maintain cathepsin propeptide fragment biological activity if it has cathepsin inhibitory activity of at least 1%, for example at least 10%, such as at least 25%, for example at least 50%, at least 75% or at least 90% of the cathepsin propeptide fragment of which it is a derivative.
  • a derivative is considered to maintain cathepsin propeptide fragment biological activity if it has a Ki value, for example as determined above for a true Ki value, for the inhibition of Cats, of ⁇ lOmM, such as ⁇ lmM, such as ⁇ 500 ⁇ M, for example ⁇ 500 ⁇ M, ⁇ 250 ⁇ M, ⁇ 100 ⁇ M, ⁇ 50 ⁇ M or ⁇ 20 ⁇ M.
  • Ki value for example as determined above for a true Ki value, for the inhibition of Cats, of ⁇ lOmM, such as ⁇ lmM, such as ⁇ 500 ⁇ M, for example ⁇ 500 ⁇ M, ⁇ 250 ⁇ M, ⁇ 100 ⁇ M, ⁇ 50 ⁇ M or ⁇ 20 ⁇ M.
  • biological activity may be determined by ability to inhibit tumour invasion, for example, in a tumour model, for example using a modified Boyden chamber invasion assay.
  • the cathepsin propeptide fragment derivatives retain at least 50%, for example at least 75%, at least 85%, or at least 90% of the tumour invasion inhibition activity of the cathepsin propeptide fragment having the amino acid sequence shown as Sequence ID No : 1, Sequence ID No : 2 , Sequence ID No : 3 , Sequence ID No : 4 , Sequence ID No : 5 , Sequence ID No : 6 , or Sequence ID No : 7.
  • Cathepsin propeptide fragments and derivatives for use in the invention may be produced using any method known in the art.
  • the cathepsin propeptide molecule is produced by a method involving a purification step involving metal ion affinity chromatography (IMAC) .
  • IMAC metal ion affinity chromatography
  • IMAC interleukin-binding-maleic anhydride-semiconductor
  • adsorption is predicated on the formation of a metal coordination complex between a metal ion, immobilized by chelation on the adsorbent matrix, and accessible electron donor amino acids on the surface of the protein to be bound.
  • poly-histidine tags to recombinant proteins is well known in the art (for example, see U.S. Pat. No. 4,569,794.
  • nucleic acid encoding propeptide fragments or derivatives thereof may be used.
  • Nucleic acid of and for use in the present invention may comprise DNA or RNA. It may be produced recombinantIy, synthetically, or by any means available to those in the art, including cloning using standard techniques.
  • the nucleic acid may be inserted into any appropriate vector.
  • the vector is an expression vector and the nucleic acid is operably linked to a control sequence which is capable of providing expression of the nucleic acid in a host cell.
  • suitable vectors may include viruses (e. g. vaccinia virus, adenovirus, baculovirus etc); yeast vectors, phage, chromosomes, artificial chromosomes, plasmids, or cosmid DNA.
  • the vectors may be used to introduce the nucleic acids into a host cell.
  • a wide variety of host cells may be used for expression of the nucleic acid for use in the invention.
  • Suitable host cells for use in the invention may be prokaryotic or eukaryotic. They include bacteria, e.g. E. coli, yeast, insect cells and mammalian cells.
  • Mammalian cell lines which may be used include Chinese hamster ovary cells, baby hamster kidney cells, NSO mouse melanoma cells, monkey and human cell lines and derivatives thereof and many others .
  • a host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used. Such processing may involve glycosylation, ubiquination, disulfide bond formation and general post-translational modification.
  • the cathepsin propeptide molecules, nucleic acids and methods of and for use in the invention may be used in the treatment of a number of medical conditions. These include inflammatory disorders neurodegenerative disorders, autoimmune diseases, cancer, asthma and atherosclerosis. In particular, they may be used in the treatment of conditions associated with overexpression (i.e. greater than in similar comparable normal healthy cells) and/or aberrant activity (eg greater than in similar comparable normal healthy cells) of cathepsin proteases .
  • Treatment includes any regime that can benefit a human or non-human animal .
  • the treatment may be in respect of an existing condition or may be prophylactic (preventative treatment) .
  • Treatment may include curative, alleviation or prophylactic effects .
  • the propeptide fragments, derivatives, nucleic acids and methods of and for use in the invention may be used in the treatment of cancers.
  • Treatment of cancer includes treatment of conditions caused by cancerous growth and includes the treatment of neoplastic growths or tumours.
  • the invention may be particularly useful in the treatment of existing cancer and in the prevention of the recurrence of cancer after initial treatment or surgery.
  • tumours examples include, for instance, sarcomas, including osteogenic and soft tissue sarcomas, carcinomas, e.g., breast-, lung-, bladder-, thyroid-, prostate-, colon-, rectum-, pancreas-, stomach-, liver-, uterine-, prostate , cervical and ovarian carcinoma, lymphomas , including Hodgkin and non-Hodgkin lymphomas, neuroblastoma, melanoma, myeloma, Wilms tumor, and leukemias, including acute lymphoblastic leukaemia and acute myeloblastic leukaemia, astrocytomas, gliomas and retinoblastomas.
  • sarcomas including osteogenic and soft tissue sarcomas, carcinomas, e.g., breast-, lung-, bladder-, thyroid-, prostate-, colon-, rectum-, pancreas-, stomach-, liver-, uterine-, prostate , cervical and
  • the cancer is selected from breast cancer, colon cancer, prostate cancer and astrocytomas .
  • Inflammatory and/or autoimmune disorders which may be treated using the invention include multiple sclerosis, Grave's Disease, inflammatory muscle disease and rheumatoid arthritis.
  • Neurodegenerative disorders which may be treated using the binding members, nucleic acids and methods of the invention include, but are not limited to, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis and Creutzfeldt-Jakob disease.
  • Atherosclerosis and tuberculosis Other conditions which may be treated using the methods of the invention include atherosclerosis and tuberculosis.
  • Evidence has been shown linking atherosclerosis and obesity with aberrant Cats.
  • Cathepsin L has been shown to process TB antigens in infections, thus perhaps preventing their proper processing.
  • compositions according to the present invention, and for use in accordance with the present invention may comprise, in addition to active ingredients, a pharmaceutically acceptable excipient, a carrier, buffer stabiliser or other materials well known to those skilled in the art (see, for example, Remington: The Science and Practice of Pharmacy, 21st edition, Gennaro AR, et al, eds , . Lippincott Williams
  • Such materials may include buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants; preservatives; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates; chelating agents; tonicifiers; or surfactants.
  • buffers such as acetate, Tris, phosphate, citrate, and other organic acids
  • antioxidants such as antioxidants
  • preservatives proteins, such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such aspolyvinylpyrrolidone
  • amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine
  • carbohydrates such as glycine, glutamine, asparagine, histidine, arginine,
  • the composition may also contain one or more further active compounds selected as necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect the activity of the propeptide, nucleic acid or composition of the invention.
  • the formulation in addition to an a cathepsin propeptide, may comprise an antibody which binds one or more cathepsin L-type proteases, or an antibody to some other target such as a growth factor that e.g. affects the growth of the particular cancer, and/or a chemotherapeutic agent.
  • the active ingredients may be administered via microspheres, microcapsules liposomes, other microparticulate delivery systems.
  • active ingredients may be entrapped within microcapsules which may be prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions , nano-particles and nanocapsules) or in macroemulsions .
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions , nano-particles and nanocapsules
  • macroemulsions for further details, see Remington: The Science and Practice of Pharmacy, 21st edition, Gennaro AR, et al, eds, . Lippincott Williams & Wilkins, 2005.
  • Sustained-release preparations may be used for delivery of active agents.
  • suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e. g. films, ⁇ suppositories or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly (2- hydroxyethyl-methacrylate) , or poly (vinylalcohol) ) , polylactides (U. S. Pat. No.
  • copolymers of L-glutamic acid and ethyl-L glutamate copolymers of L-glutamic acid and ethyl-L glutamate, non- degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D- (-)-3- hydroxybutyric acid.
  • propeptide fragments, derivatives and nucleic acids described herein are intended, at least in some embodiments, to be administered to a human or other mammal for medical treatment.
  • Peptides are typically administered parenterally, and may be readily metabolized by plasma proteases. Oral administration, which is perhaps the most attractive route of administration, may be even more problematic.
  • acid degrades and enzymes break down peptides .
  • Those peptides that survive to enter the intestinal intact are subjected to additional proteolysis as they are continuously barraged by a variety of enzymes, including gastric and pancreatic enzymes, exo- and endopeptidases , and brush border peptidases.
  • passage of peptides from the lumen of the intestine into the bloodstream can be severely limited.
  • various prodrugs have been developed that enable parenteral and oral administration of therapeutic peptides.
  • Peptides can be conjugated to various moieties, such as polymeric moieties, to modify the physiochemical properties of the peptide drugs, for example, to increase resistance to acidic and enzymatic degradation and to enhance penetration of such drugs across mucosal membranes.
  • moieties such as polymeric moieties
  • Abuchowski and Davis have described various methods for derivatizating enzymes to provide water-soluble, non- immunogenic, in vivo stabilized products ("Soluble polymers-Enzyme adducts," Enzymes as Drugs, Eds. Holcenberg and Roberts, J. Wiley and Sons, New York, N. Y. (1981)) .
  • Abuchowski and Davis discuss various ways of conjugating enzymes with polymeric materials, such as dextrans , polyvinyl pyrrolidones , glycopeptides, polyethylene glycol and polyamino acids.
  • the resulting conjugated polypeptides retain their biological activities and solubility in water for parenteral applications.
  • US 4,179,337 teaches coupling peptides to polyethylene glycol or polypropropylene glycol having a molecular weight of 500 to 20,000 Daltons to provide a physiologically active non-immunogenic water soluble polypeptide composition.
  • the polyethylene glycol or polypropylene glycol protects the polypeptide from loss of activity and the composition can be injected into the mammalian circulatory system with substantially no immunogenic response.
  • a prodrug can be prepared using this chemistry, by first preparing a maleic anhydride reagent from polydispersed MPEG5000 and then conjugating this reagent to the peptides disclosed herein. The reaction of amino acids with maleic anhydrides is well known. The hydrolysis of the maleyl-amide bond to reform the amine-containing drug is aided by the presence of the neighbouring free carboxyl group and the geometry of attack set up by the double bond. The peptides can be released (by hydrolysis of the prodrugs) under physiological conditions .
  • Such strategies may be employed to deliver the propeptides for use in the present invention.
  • the peptides can also be coupled to polymers, such as polydispersed PEG, via a degradable linkage, for example, the degradable linkage shown (with respect to pegylated interferon ⁇ -2b) in Roberts, M.J., et al., Adv. Drug Delivery Rev. , 2002, 54, 459-476.
  • the peptides can also be linked to polymers such as PEG using 1,6 or 1,4 benzyl elimination (BE) strategies (see, for example, Lee, S., et al . , Bioconjugate Chem. , (2001), 12, 163-169; Greenwald, R. B., et al., US 6,180,095, 2001; Greenwald, R.
  • Some approaches involve using enzyme inhibitors to slow the rate of degradation of proteins and peptides in the gastrointestinal tract and may be used for the propeptides described herein; manipulating pH to inactivate local digestive enzymes; using permeation enhancers to improve the absorption of peptides by increasing their paracellular and transcellular transports; using nanoparticles as particulate carriers to facilitate intact absorption by the intestinal epithelium, especially, Peyer's patches, and to increase resistance to enzyme degradation; liquid emulsions to protect the drug from chemical and enzymatic breakdown in the intestinal lumen; and micelle formulations for poorly water-solubulized drugs .
  • the peptides can be provided in a suitable capsule or tablet with an enteric coating, so that the peptide is not released in the stomach.
  • the peptide can be provided as a prodrug.
  • the peptides are present in these drug delivery devices as prodrugs .
  • Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy or carboxylic acid groups of various polymers, for example, polyalkylene glycols such as polyethylene glycol.
  • Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters are covalently bonded to the above peptides through the C-terminal carboxylic acids.
  • Prodrugs comprising the peptides (propeptide fragments or derivatives) of the invention or prodrugs from which peptides of the invention (including analogues and fragments) are released or are releasable are also considered, in one embodiment, to be derivatives of the invention.
  • the present invention further encompasses the use of mimetic propeptides which can be used as therapeutic peptides .
  • Mimetic pro peptides are short peptides which mimic the biological activity of the cathepsin propeptide fragments described herein.
  • Such mimetic peptides can be obtained from methods known in the art such as, but not limited to, phage display or combinatorial chemistry. For example, the method disclosed by Wrighton, et al . , Science 273:458-463 (1996) can be used to generate mimetic cathepsin propeptide fragments .
  • nucleic acids encoding cathepsin propeptide fragments may also be used in methods of treatment. Such nucleic acids may be delivered to cells of interest using any suitable technique known in the art.
  • Nucleic acid (optionally contained in a vector) may be delivered to a patient's cells using in vivo or ex vivo techniques .
  • in vivo techniques transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno- associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example) may be used (see for example, Anderson et al .
  • nucleic acid is introduced into isolated cells of the patient with the modified cells being administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, e. g. U. S. Patent Nos . 4, 892, 538 and 5, 283, 187) .
  • Techniques available for introducing nucleic acids into viable cells may include the use of retroviral vectors, liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • the propeptide fragments, derivatives, nucleic acid, agent, product or composition may be administered in a localised manner to a tumour site or other desired site or may be delivered in a manner in which it targets tumour or other cells .
  • Targeting therapies may be used to deliver the active agents more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons, for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
  • propeptides, fragments, derivatives, nucleic acids or compositions of the invention are preferably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual .
  • the actual dosage regimen will depend on a number of factors including the condition being treated, its severity, the patient being treated, the agent being used, and will be at the discretion of the physician.
  • the optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration.
  • Figure 1 illustrates the DNA and protein sequence for the complete reading frame of the recombinant cathepsin S propeptide as inserted in pQE30;
  • FIG. 1 shows schematically cathepsin S propeptide and seven propeptide fragments of the invention
  • Figure 3 illustrates a graph summarising the effects of PPl (Sequence ID No : 1) at various concentrations on Cathepsin S activity (top panel) , and a graph of non-linear regression analysis (Morrison and Walsh, 1988) allowing for the determination of the inhibition constant (Ki)
  • Figure 4. illustrates a graph summarising the effects of PP2 (Sequence ID No : 2) at various concentrations on Cathepsin S activity (top panel), and a graph of non-linear regression analysis (Morrison and Walsh, 1988) allowing for the determination of the inhibition constant (Ki);
  • Figure 5 illustrates a graph summarising the effects of PP3 (Sequence ID No : 3) at various concentrations on Cathepsin S activity (top panel), and a graph of non-linear regression analysis (Morrison and Walsh, 1988) allowing for the determination of the inhibition constant (Ki);
  • Figure 6 illustrates a graph summarising the effects of PP4 (Sequence ID No : 4) at various concentrations on Cathepsin S activity (top panel), and a graph of non-linear regression analysis (Morrison and Walsh, 1988) allowing for the determination of the inhibition constant (Ki);
  • Figure 7 illustrates a graph summarising the effects of PP5 (Sequence ID No : 5) at various concentrations on Cathepsin S activity (top panel) , and a graph of non-linear regression analysis (Morrison and Walsh, 1988) allowing for the determination of the inhibition constant (Ki);
  • Figure 8. illustrates a graph summarising the effects of PP ⁇ (Sequence ID No: 6) at various concentrations on Cathepsin S activity (top panel) , and a graph of non-linear regression analysis (Morrison and Walsh, 1988) allowing for the determination of the inhibition constant (Ki);
  • Figure 9. illustrates a graph summarising the effects of PP7 (Sequence ID No : 7) at various concentrations on Cathepsin S activity (top panel) , and a graph of non-linear regression analysis (Morrison and Walsh, 1988) allowing for the determination of the inhibition constant (Ki) ;
  • Figure 10 illustrates a graph summarising the effects of PP8 (Sequence ID No : 8) at various concentrations on Cathepsin S activity (top panel), and a graph of non-linear regression analysis (Morrison and Walsh, 1988) allowing for the determination of the inhibition constant (Ki) ; and
  • Figure 11. comprises a table summarising the Ki of each of fragments PPl to PP8.
  • Cats activity was monitored using the fluorigenic substrate carbobenzloxy-L-valinyl-L- valinyl-L-arginylamido-4-methyl coumarin ( Z-VaI-VaI- Arg-AMC, 25 ⁇ M) , assays for cathepsins L, K and V were performed using carbobenzloxy-L-phenylalanyl-L- arginylamido-4-methyl coumarin (Z-Phe-Arg-AMC, 25 ⁇ M) and assays for CatB were performed using carbobenzloxy-L- arginylamido -L-arginylamido-4- methyl coumarin (Z-Arg-Arg-AMC, 25 ⁇ M) as substrates.
  • rCatSPP Purified rCatSPP was added to assays as required at various concentrations (0-1000 nM) . All experiments were performed using a Cytofluor® 4000 spectrofluorimeter with excitation at 395 nm and emission at 460 nm.
  • Each propeptide fragment (PPl to PP8) was tested for its biological activity.
  • the biological activity of each fragment was ascertained by fluorometric assay using Cats and the fluorigenic substrate Cbz-Val-Val- Arg-AMC in the presence of predetermined concentrations of the propeptide fragment .
  • Progress curves for the hydrolysis of Cbz-VaI-VaI-Arg-AMC in the presence of rCatSPP were plotted and the dose- dependent inhibition of Cats activity was observed (figures 3 to 10) .
  • PP8 was used as a control. Assays were all performed in triplicate.
  • the progress curves are indicative of the action of a slow-binding reversible inhibitor.
  • Ki values were calculated for inhibition of Cats with the propeptide fragment.
  • the Ki values are summarised in Figure 11.

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Abstract

Cette invention concerne de nouveaux fragments de propeptides de la cathepsine et leur utilisation dans des procédés destinés à traiter des maladies telles que le cancer. Des dérivés de ces propeptides et des compositions pharmaceutiques contenant ces propeptides et leurs dérivés sont également décrits.
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WO2007099348A2 (fr) * 2006-03-02 2007-09-07 Fusion Antibodies Limited Peptide et ses utilisations

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WO2007099348A2 (fr) * 2006-03-02 2007-09-07 Fusion Antibodies Limited Peptide et ses utilisations

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Title
BURDEN ROBERTA E ET AL: "Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L-like proteases in tumor microenvironment" MOLECULAR CANCER THERAPEUTICS, vol. 7, no. 3, March 2008 (2008-03), pages 538-547, XP002597506 ISSN: 1535-7163 *
KAULMANN GUIDO ET AL: "The crystal structure of a Cys25 -> Ala mutant of human procathepsin S elucidates enzyme-prosequence interactions" PROTEIN SCIENCE, vol. 15, no. 11, November 2006 (2006-11), pages 2619-2629, XP002597507 ISSN: 0961-8368 *

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