WO2010079340A2 - Assay - Google Patents

Abstract

The invention relates to a method for identifying compounds that act as enhancers or inhibitors of matrix metalloproteinase (MMP) activity. The invention also relates to a method for diagnosing whether or not a subject is at risk of developing, or has, a disease associated with pathogenic APP processing. The invention further relates to the use of type I transmembrane protease inhibitors, in particular inhibitors of MMP 16, MMP 14 or MMP 15 activity, for preventing or treating diseases associated with pathogenic APP processing, such as Alzheimer's disease.

Description

ASSAY

Field of the Invention

The invention relates to a method for identifying compounds that act as enhancers or inhibitors of matrix metalloproteinase (MMP) activity. The invention also relates to a method for diagnosing whether or not a subject is at risk of developing, or has, a disease associated with pathogenic APP processing. The invention further relates to the use of inhibitors of type I transmembrane MMPs, preferably inhibitors of MMP 16, MMP 14 or MMP 15, for preventing or treating diseases associated with pathogenic APP processing, such as Alzheimer's disease.

Background to the Invention

Alzheimer's disease is a common form of dementia found mainly among older people. The disease is reviewed in Selkoe DJ, Physiological Reviews (2001) 81: 741-766, 2001;Cummings JL, New England Journal of Medicine (2004) 351 :56-67 and Kalaria RN., et al., Lancet Neurol. (2008) 9:812-26. A common symptom of the disease is the formation of abnormal amyloid plaques and/or neurofibrillary tangles in brain tissue. Amyloid plaques are formed by aggregation of proteolytic cleavage products of amyloid precursor protein (APP) termed amyloid β (Aβ) peptides. These peptides are toxic to neurons and may also cause aggregation of the microtubule associated protein tau, thereby forming neurofibrillary tangles. The amyloid hypothesis postulates that aberrant production or clearance of Aβ peptide underlies the neurodegeneration and consequent dementia observed in Alzheimer's disease. ^v β-secretase (BACE), γ-secretase and α-secretase proteases are known to be involved in the proteolytic cleavage of APP. BACE and γ-secretases are specifically linked to production of Aβ peptides. Cleavage by γ-secretase generates Aβ peptides of variable lengths, mostly between 37 and 42 amino acids. The 42 amino acid form of Aβ (Aβ 1-42) is known to be highly toxic. N-terminally truncated Aβ peptides are also implicated in the pathology of amyloid plaques, and display enhanced aggregation and toxicity as compared to non-truncated versions. Of these species, Aβ 3-40/42 modified by glutaminyl cyclase to form pyroglutamate Aβ is probably the most significant (Schilling, S., et al., J Neurochem. 2008 Aug; 106(3): 1225-36). While MMP14 and MMP16 have lrecently been identified as inducing shedding of the APP ectodomain when co-expressed with Fe65, it was specifically stated that Aβ production was not significantly affected by these MMPs (Furukawea, M & Sato, H (2006) J Biochem 139, 517-526).

In addition to the well characterised amyloid plaque pathology observed in brain tissue in Alzheimer's disease, other chronic diseases are associated with aberrant processing of APP. For example, cerebral amyloid angiopathy results from deposits of amyloid protein in small blood vessels in the brain which can cause stroke, brain haemorrhage or dementia.

Furthermore, the major genetic risk factor for sporadic, late-onset AD is the inheritance of the epsilon4 (e4) allele of apolipoprotein E (ApoE). The Apoe4 allele is present in 30 to 50% of patients who develop late-onset AD (Science (1993) 261, 921- 923).

ApoE is 299 amino acids long and transports lipoproteins, fat-soluble vitamins, and cholesterol into the lymph system and then into the blood. It is synthesized principally in the liver, but has also been found in other tissues such as the brain, kidneys, and spleen. In the nervous system, non-neuronal cell types, most notably astroglia and microglia, are the primary producers of ApoE, while neurons preferentially express the receptors for ApoE. Three major ApoE iso forms exist in humans that differ at two residues: apoE2 (Cysl 12, Cysl58), apoE3 (Cysl 12, Argl58), and apoE4 (Argl 12, Argl58). The ApoE4 polymorphism is believed to alter the interaction between the N and C terminal domains of ApoE.

Knockout of mouse ApoE in the APPV717F+/- mouse model was found to cause redistribution of Aβ deposits. Cortical and dentate gyrus deposition was decreased whilst CAl and CA3 deposition was increased (Irizarry et al, 2000 Acta Neuropathol 100: 451). In aging APPV717+/- mice, murine ApoE was found to facilitate, but not be required for, Aβ fibril formation in vivo. Human ApoE isoforms markedly delayed Aβ deposition relative to mouse ApoE. ApoE2 (and to a lesser extent) ApoE3 has a prolonged ability to prevent Aβ from converting into fibrillar forms. ApoE4 also prevents this but to a lesser extent than the other isoforms (Fagan et al., 2002 Neurobiology of Disease 9:305). It would appear therefore that the ApoE4 allele represents a loss of function mutation which reduces protection against Aβ pathology. Any therapy which enhanced ApoE levels or activity would be likely to be beneficial in Alzheimer's Disease. ApoE has been shown to form stable complexes with Aβ peptides {Biochemistry.

2000 Dec 26;39(51):16119-24). It would seem that the carboxy-terminal lipid-binding domain of ApoE (residues 200-299) is responsible for this Aβ-binding activity (J Neurochem. 1999 Jan;72(l):230-7). C-terminal fragments of ApoE have been observed in Alzheimer's disease brain {Neuroreport. 1996 Nov 4; 7(15-17):2529-32, Proc Natl Acad Sci USA. 2001 JuI 17;98(15):8838-43). Similar fragments of ApoE are present in amyloid plaques and cerebral amyloid angiopathy (CAA) (J Neuropathol Exp Neurol.

2001 Apr; 60(4): 342-9). More recently, a 13kDa C-terminal fragment of ApoE was found to stabilise the formation of Aβ hexamers and enhance Aβ oligomerisation {Journal of Neurochemistry, 2005, 94, 1351-1360).

Matrix Metalloproteinases have been reported to cleave ApoE (J. Biochemistry (2005) 137(l):95-99). Recently MMP-14 cleavage of ApoE was shown to inactivate the protein {Proteomics. (2008) 8(14): 2926-35). Surprisingly, we have identified that MMP- 16 induces cleavage of over-expressed ApoE3 to produce fragments similar to the toxic 13kDa fragments described previously. Therefore, an inhibitor of matrix metalloproteinases, in particular type-I transmembrane metalloproteinases, preferably MMP 16, would be doubly beneficial for the treatment of diseases which involve abnormal processing of APP, in particular Alzheimer's Disease, by reducing the negative effects due to processing of APP and maintaining the positive effects of ApoE. There is a need to identify new medicines for the treatment of diseases associated with pathogenic APP processing, in particular those which may function by regulating aberrant production of Aβ peptides. Surprisingly, we have identified that inhibitors of type-I transmembrane metalloproteinases, in particular of MMP 16, may provide a new, effective medicine for the treatment of these diseases.

Summary of the Invention

The invention utilises substrate polypeptides comprising amino acid sequence motifs derived from a discrete region of the APP protein to identify enhancers or inhibitors of matrix metalloproteinase (MMP) activity. This is a relatively short polypeptide region comprising APP sequence N-terminal and C-terminal to the BACE cleavage site which corresponds to SEQ ID NO: 7 (EPVDARPAADRGLTTRPGSGLTNIKTEEI SEVKMDAE FRHDSGYEVHHQKLVFFAED). The Inventor has surprisingly shown that MMPs cleave APP within this region and can therefore be used to modulate production of Aβ peptides. The Inventor has discovered that processing carried out by MMPs within this region generates the highly toxic Aβ 3-40 variant. Surprisingly, the Inventor has also discovered that activity of MMPs within this region extends to hitherto uncharacterised processing events for APP. These include processing events which lead to formation of N-terminally extended and C-terminally truncated variants of Aβ. Identification of the involvement of MMPs in new processing events carried out on APP provides a novel method of drug development. In particular, it allows for screening of drugs that can modulate production of Aβ peptides. Screening can be carried out to identify drugs that modulate production of specific Aβ variants. For example, inhibitors of the production of Aβ 3-40 can be used to prevent the aggregation of Aβ that underlies amyloid plaque formation.

The finding that MMPs play a role in novel processing events involving APP also allows MMPs, preferably type-I transmembrane MMPs, even more preferably MMP 16, MMP 14 or MMP 15, most preferably MMP 16, to be used as a biomarker in the identification of diseases associated with pathogenic APP processing. In particular, the invention uses expression level and/or MMP activity to determine whether or not a subject is at risk of, or has, a diseases associated with pathogenic APP processing.

The discovery that MMP 16 can also cleave ApoE and thereby further increases the formation of amyloid plaques and/or neurofibrillary tangles also allows for screening of drugs that modulate the formation of amyloid plaques and/or neurofibrillary tangles by modulating the processing of ApoE.

Given that MMPs cleave APP, the invention also concerns the use of MMP, preferably type-I transmembrane MMP, even more preferably MMP 16, MMP 14 or MMP15, most preferably MMP16, antagonists or inhibitors in the prevention or treatment of diseases associated with pathogenic APP processing, in particular Alzheimer's disease.

Over-expression of MMPs, preferably type-I transmembrane MMPs, even more preferably MMP 16, MMP 14 or MMPl 5, most preferably MMP 16, may also be used to generate non-human animal models of diseases of pathogenic APP processing. Such animal models can not only be used to investigate the pathology of such diseases, but may also be used to screen for compounds which can prevent or treat them. Accordingly, the invention provides a method for identifying a compound that enhances or inhibits matrix metalloproteinase (MMP) activity, preferably type-I transmembrane MMP activity, even more preferably MMP 16, MMP 14 or MMP 15 activity, most preferably MMP 16 activity, the method comprising: a) contacting a type-I transmembrane MMP, preferably an MMP 16, MMP 14 or MMP 15 polypeptide or a variant thereof with the compound; b) contacting said type-I transmembrane MMP polypeptide or variant thereof with a substrate polypeptide comprising a motif of at least ten amino acids from SEQ ID NO: 7 or an equivalent thereof; c) measuring MMP activity by monitoring cleavage immediately following one or more of residues 9, 12, 18, 20, 36 and 48 of SEQ ID NO: 7 or the corresponding residues in an equivalent thereof; d) comparing the MMP activity measured in c) with a control value obtained for said type-I transmembrane MMP polypeptide that has not been contacted with the compound, and thereby determining whether the compound is an enhancer or inhibitor of MMP activity; wherein an increase in MMP activity compared with said control value identifies the compound as being an enhancer of MMP activity; and wherein a decrease in MMP activity compared with said control value identifies the compound as being an inhibitor of MMP activity.

The invention further provides a method for identifying a compound that enhances or inhibits amyloid precursor protein (APP) processing, the method comprising carrying out a method for identifying a compound that enhances or inhibits matrix metalloproteinase (MMP) activity as defined above and thereby identifying a compound that enhances or inhibits APP processing, wherein an increase in MMP activity in the presence of said compound compared with said control value identifies said compound as an enhancer of APP processing; and wherein a decrease in MMP activity in the presence of said compound compared with said control value identifies said compound as an inhibitor of APP processing.

The invention additionally provides a method for identifying a compound that enhances or inhibits Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles, the method comprising carrying out a method for identifying a compound that enhances or inhibits matrix metalloproteinase (MMP) activity as defined above and thereby identifying a compound that enhances or inhibits Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles, wherein an increase in MMP activity in the presence of said compound compared with said control value identifies said compound as an enhancer of Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles; and wherein a decrease in MMP activity in the presence of said compound compared with said control value identifies said compound as an inhibitor of Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles.

Furthermore, the invention provides a method for identifying a compound that enhances or inhibits Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles, the method comprising the steps of a) contacting an MMP 16 polypeptide or a variant thereof with the compound; b) contacting the MMP 16 polypeptide or variant thereof with a substrate polypeptide comprising ApoE or an equivalent thereof; c) measuring MMP activity by monitoring cleavage of the substrate polypeptide; and d) comparing the MMP activity measured in c) with a control value obtained for an MMP polypeptide that has not been contacted with the compound, and thereby determining whether the compound is an enhancer or inhibitor of MMP activity; wherein an increase in MMP activity compared with said control value identifies the compound as being an enhancer of Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles; and wherein a decrease in MMP activity compared with said control value identifies the compound as being an inhibitor of Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles. Preferably, the compound enhances ApoE activity and reduces Aβ oligomerisation. Preferably, the ApoE is selected from ApoE2, ApoE3 and ApoE4 or equivalents thereof.

The invention also provides a method for identifying a compound suitable for the prevention or treatment of a disease associated with pathogenic APP processing, the method comprising carrying out a method for identifying a compound that enhances or inhibits matrix metalloproteinase (MMP) activity as defined above and thereby identifying a compound suitable for the prevention or treatment of a disease associated with pathogenic APP processing, wherein a decrease in MMP activity in the presence of said compound compared with said control value identifies said compound as being suitable for the prevention or treatment of a disease associated with pathogenic APP processing. Preferably, the disease associated with pathogenic APP processing is Alzheimer's Disease. In a related aspect the invention provides a method for identifying whether or not a subject is at risk of developing, or has, a disease associated with pathogenic APP processing, said method comprising: a) measuring the expression level and/or MMP activity of a type-I transmembrane MMP in a sample derived from said subject; b) comparing the type-I transmembrane MMP expression level and/or MMP activity measured in said sample to a normal level of the type-I transmembrane MMP expression and/or activity and thereby identifying whether or not a subject is at risk of developing, or has, a disease associated with pathogenic APP processing; wherein an increased level of type-I transmembrane MMP expression and/or an increased level of type-I transmembrane MMP activity in said sample compared with the normal level identifies the subject as being at risk of developing, or having, a disease associated with pathogenic APP processing. Preferably, the measuring MMP activity in step a) comprises measuring the levels of N- terminally extended Aβ peptides derived from APP, preferably Aβ-25 to +14, Aβ-22 to +14, Aβ-16 to +14 or Aβ-14 to +14 and optionally further measuring the level of Aβ 3-40.

A further aspect of the invention provides an antagonist or inhibitor of type-I transmembrane MMP activity for use in a method of preventing or treating a disease associated with pathogenic APP processing. Preferably, the antagonist or inhibitor inhibits MMP16, MMP14 or MMP15, most preferably, it inhibits MMP16. The disease associated with pathogenic APP processing is preferably Alzheimer's Disease.

The invention further provides a use of an inhibitor or antagonist of an MMP, preferably a type-I transmembrane MMP, even more preferably MMP 16, MMP 14 or MMP15, most preferably MMP16, in the manufacture of a medicament for preventing or treating a disease associated with pathogenic APP processing, preferably Alzheimer's Disease. The invention additionally provides a method of treating or preventing a disease associated with pathogenic APP processing in a subject, comprising administering to the subject an effective amount of an antagonist or inhibitor of MMP, preferably of type-I transmembrane MMPs, even more preferably of MMP 16, MMP 14 or MMP 15 , most preferably of MMP 16, activity.

The invention further provides a non-human animal in which a disease of pathogenic APP processing has been established by over-expression of MMP 16. In a related aspect, the invention also provides a method for establishing a disease of pathogenic APP processing in a non-human animal comprising over-expressing MMP 16 in said animal in an amount sufficient to cause a disease of pathogenic APP processing. The invention additionally provides a method for identifying a compound which prevents or treats a disease of pathogenic APP processing, comprising administering said compound to a non-human animal as defined above and assessing whether or not said compound prevents or treats the disease of pathogenic APP processing. Furthermore, the invention provides a compound identified by the above mentioned method carried out in a non-human animal for use in a method of preventing or treating a disease associated with pathogenic APP processing. In a related aspect, the invention provides a use of a compound identified by the above mentioned method carried out in a non-human animal in the manufacture of a medicament for prevention or treatment of a disease associated with pathogenic APP processing.

Furthermore, the invention provides a kit comprising a type-I transmembrane MMP polypeptide, preferably MMP 16, MMP 14 or MMP 15, most preferably MMP 16, or a variant thereof and a substrate polypeptide comprising a motif of at least ten amino acids from SEQ ID NO: 7 or an equivalent thereof.

Brief Description of the Figures

Figure 1 shows APP cleavage and increase in AβX-40/ Aβ 1 -40/ AβX-42 production induced by over-expression of MMP 14, MMP 15 and MMP 16. A) Generation of novel C-terminal fragments of APP by over-expression of MMP 14 and MMP 16 in HEK293 cells. B) Increase in AβX-40 and Aβ 1 -40 generation by MMP 14, MMP 15 and MMP 16 over-expression in HEK293 cells. C) Increase in AβX-40 and AβX-42 production by MMP 14, MMP 15 and MMP 16 over-expression in ELLIN cells.

Figure 2 shows immunoprecipitation/mass spectroscopy analysis of Aβ species produced by MMP 14 over-expression. A) Immunoprecipitation/mass spectroscopy analysis carried out with the anti-Aβ antibody 4G8. B) Immunoprecipitation/mass spectroscopy analysis carried out with the anti-Aβ antibody 6E10.

Figure 3 shows immunoprecipitation/mass spectroscopy analysis of Aβ species produced by MMP 15 over-expression. Immunoprecipitation/mass spectroscopy analysis was carried out with the anti-Aβ antibody 6E10. Figure 4 shows immunoprecipitation/mass spectroscopy analysis of Aβ species produced by MMP 16 over-expression. A) Immunoprecipitation/mass spectroscopy analysis carried out with the anti-Aβ antibody 4G8. B) Immunoprecipitation/mass spectroscopy analysis carried out with the anti- Aβ antibody 6E10.

Figure 5 shows enhanced clearance/degradation of Aβ 1-40 by MMP 14, MMP 15 and MMP 16. A) Expression of any of MMP 14, MMP 15 and MMP 16 enhances clearance of endogenous Aβl-40 in HEK293 cell cultures in which new Aβ production is inhibited by a γ-secretase inhibitor. This suggests that isolated Aβl-40 is cleaved by MMP 14, MMP 15 and MMP 16. B) AβX-40 is also decreased indicating significant degradation of the Aβl-40 peptide. C) Parallel control experiment demonstrating successful over- expression of MMP 14, MMP 15 and MMP 16 leading to increases in AβX-40 in cells not treated with γ-secretase inhibitor.

Figure 6 shows key MMP cleavage sites in the vicinity of the Aβ peptide. MMP 16 cleavage sites are indicated by downward facing arrows with cleavage positions indicated. BACE, α-secretase and γ-secretase cleavage sites are indicated by upward facing arrows. The Aβ sequence is underlined with rodent amino acid variations shown in italics. Figure 7 shows the cleavage of ApoE by MMPs. A) Cleavage of over-expressed

C-terminally V5 -tagged ApoE3 by MMP co-over-expression. ApoE3 C-terminally tagged with a V5/6His tag was co-over-expressed with MMP 14, MMP 15 or MMP 16. MMP 16 induced significant degradation of ApoE3-V5 with enhanced production of two C-terminal cleavage products in the 17-24 kDa range, identified using an anti-V5 tag antibody. B) Cleavage of untagged over-expressed ApoE3 by MMP co-over-expression. Enhanced degradation of untagged ApoE3 was also seen when co-over-expressed with MMP 16. Production of several cleavage fragments was enhanced. One of these, taking into account the 5kDa size of a V5/6His tag, was similar in size to a C-terminal fragment seen with ApoE-V5 over-expression. C) Increases in AβX-40 production indicating successful MMP over-expression. Successful MMP over-expression was tracked by Aβx-40 ELISA. Strong increases were seen in AβX-40 production confirming expression.

Description of the Sequences

SEQ ID NO: 1 is the nucleic acid sequence of human APP, transcript variant 1. SEQ ID NO: 2 is the amino acid sequence of human APP, transcript variant 1 encoded by SEQ ID NO 1. SEQ ID NO: 3 is the nucleic acid sequence of human APP, transcript variant 2. SEQ ID NO: 4 is the amino acid sequence of human APP, transcript variant 2 encoded by SEQ ID NO: 3.

SEQ ID NO: 5 is the nucleic acid sequence of human APP, transcript variant 3. SEQ ID NO: 6 is the amino acid sequence of human APP, transcript variant 3 encoded by SEQ ID NO: 5.

SEQ ID NO: 7 is the amino acid sequence of human APP within which MMP activity is monitored.

SEQ ID NO: 8 is the amino acid sequence of motif A from human APP. SEQ ID NO: 9 is the amino acid sequence of motif B from human APP.

SEQ ID NO: 10 is the amino acid sequence of motif C from human APP. SEQ ID NO: 11 is the amino acid sequence of motif D from human APP. SEQ ID NO: 12 is the amino acid sequence of motif E from human APP. SEQ ID NO: 13 is the amino acid sequence of motif F from human APP. SEQ ID NO: 14 is the nucleic acid sequence of human MMP14.

SEQ ID NO: 15 is the amino acid sequence of human MMP14 encoded by SEQ ID NO: 14.

SEQ ID NO: 16 is the nucleic acid sequence of human MMP15. SEQ ID NO: 17 is the amino acid sequence of human MMP 15 encoded by SEQ ID NO: 16.

SEQ ID NO: 18 is the nucleic acid sequence of human MMP 16, transcript variant 1.

SEQ ID NO: 19 is the amino acid sequence of human MMP 16, transcript variant 1 encoded by SEQ ID NO: 18. SEQ ID NO: 20 is the nucleic acid sequence of human MMP 16, transcript variant

2.

SEQ ID NO: 21 is the amino acid sequence of human MMP 16, transcript variant 2 encoded by SEQ ID NO: 20.

SEQ ID NOs: 22-70 are the amino acid sequences of peptides derived from human APP of SEQ ID NO: 2.

SEQ ID NO: 71 is the nucleic acid sequence of human ApoE3. SEQ ID NO: 72 is the amino acid sequence of human APOE3 encoded by SEQ ID NO: 71.

Detailed Description of the Invention It is to be understood that different applications of the disclosed methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a compound" includes "compounds", reference to "a polypeptide" includes two or more such polypeptides, and the like. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Method for identifying compounds that enhance or inhibit MMP activity

The invention provides a method for identifying a compound that acts as an enhancer or inhibitor of MMP activity. MMP activity as used herein refers typically to metalloproteinase, or protease activity. MMP activity is thus typically measured as cleavage of a substrate polypeptide. Such methods allow the screening of one or more compounds for their ability to act as enhancers or inhibitors of MMP activity. The methods are preferably carried out in vitro or ex vivo. The method can also be used to confirm that a known enhancer or inhibitor of MMP activity enhances or inhibits cleavage of a substrate polypeptide as defined herein by an MMP. Thus, the method can be used to confirm the effects of an enhancer or inhibitor of MMP activity identified by any other means.

Techniques for determining the effect of compound(s) on an enzyme-substrate reaction are well known in the art. Any of those techniques may be used in accordance with the invention.

An enhancer increases the protease activity and/or expression of an MMP. An inhibitor decreases the protease activity and/or expression of an MMP. The enhancer or inhibitor preferably increases or decreases the activity of MMP on the substrate polypeptide defined herein. An enhancer or inhibitor of MMP activity may enhance or inhibit MMP activity by any mechanism. For instance, an enhancer or inhibitor may act directly by binding to an MMP polypeptide. It may bind directly at the enzyme active site or may bind at another site and exert allosteric effects on enzyme function. The enhancer or inhibitor may act in a non-competitive or a competitive manner with respect to the substrate polypeptide.

An enhancer or inhibitor may also act indirectly on MMP activity. It may have effects on activation of MMP activity, for example by acting via secondary messenger systems. An enhancer or inhibitor of MMP activity may also act at the level of MMP expression so as to increase or decrease MMP mRNA or protein levels. It may also act to regulate the stability of the expressed mRNA or protein.

An enhancer or inhibitor of MMP activity may also act by altering substrate specificity. For example, inhibitors of MMP activity may shift the substrate specificity from APP-type substrates (as discussed below) towards other substrate polypeptides.

The terms "inhibitor" and "antagonist" are intended to have the same meaning in the context of the invention, and are used interchangeably throughout.

The method can be carried out using any MMP polypeptide in any form. Suitable MMP polypeptides are discussed in more detail below. The MMP polypeptide can be in solution. The solution may comprise a purified or substantially purified recombinant MMP polypeptide in a suitable buffer. Such buffers are known in the art. Alternatively, the solution may be a culture medium or a cell lysate from a cell culture expressing a MMP polypeptide. The MMP may be anchored to a lipid-containing membrane. The membrane may be natural or artificial. Suitable membranes are known in the art. The MMP polypeptide is preferably expressed in a cell or cell culture. Suitable cell types are discussed in more detail below. The cell or cell culture may additionally express one or more non-MMP proteases that cleave APP and/or the substrate polypeptide. Alternatively, the MMP polypeptide can be immobilised on a platform or surface. Suitable platforms or surfaces are known in the art. An example is a standard 96 or 384 well plate.

The substrate polypeptide can also be used in any form. The substrate polypeptide can be in solution. The solution may comprise a purified or substantially purified recombinant substrate polypeptide in a suitable buffer. The solution may also comprise a synthetic substrate polypeptide in a suitable buffer. The solution may be a culture medium or a cell lysate from a cell culture expressing a substrate polypeptide. The substrate polypeptide may be anchored to a lipid-containing membrane. The membrane may be the same one to which the MMP polypeptide is anchored. The substrate polypeptide is preferably expressed in a cell or cell culture. The cell or cell culture may be the same cell or cell culture expressing MMP polypeptide or may be a different one. The cell or cell culture may additionally express the MMP polypeptide and/or one or more additional proteases that cleave APP. Alternatively, the substrate polypeptide can be provided immobilised to a platform or surface. The substrate polypeptide may be immobilised such that cleavage products derived from it are retained or released from the surface.

The substrate polypeptide can also be provided in the form of conditioned medium isolated from cell lines expressing the substrate polypeptide. In particular, conditioned medium isolated from HEK293 or ELLIN cells (patent application WO2008084254) may be a good source of substrate polypeptide. The term "conditioned" is intended to refer to a difference in the chemical composition of the medium used in culture of the cells, as a result of substances secreted by the cells. In such embodiments, the cells providing the conditioned media may express one or more proteases which cleave APP, such as α- secretases, β-secretase (BACE), and γ-secretases. The conditioned medium may also be used to contact purified recombinant MMP polypeptide.

Where the MMP polypeptide and/or substrate polypeptide are derived from a cell or cell culture, a processing step will typically be required prior to measurement of MMP activity. For example, cell medium may be processed by centrifugation or by passage through a membrane that filters out unwanted molecules or cells. A solution derived from a cell may be stored prior to measurement of MMP activity, preferably below -70°C. Similarly, a cell culture expressing MMP polypeptide or substrate polypeptide may be stored, prior to harvesting cell lysate, preferably below -70°C. Where MMP polypeptide or substrate polypeptide are expressed in a cell or cell culture, expression of one or both polypeptides can be transient or stable. Expression of one or both polypeptides may result from an endogenous gene or from an exogenous polynucleotide. Expression may be inducible or constitutive. Methods of providing a cell or cell culture expressing a MMP polypeptide or a substrate polypeptide are described below.

As will be appreciated, a cell-based method of the invention can be carried out in many ways. Both the MMP polypeptide and substrate polypeptide may be expressed in the same cell or cell culture and the compound may be contacted therewith. Alternatively, the MMP polypeptide alone may be expressed in the cell or cell culture and the compound and substrate may then be contacted therewith. Similarly, the substrate polypeptide alone may be expressed in a cell or cell culture and the compound and MMP polypeptide may then be contacted therewith. Furthermore, the MMP polypeptide, the substrate polypeptide and the compound may be expressed in a cell or cell culture.

Typically, only one MMP polypeptide is used. However, in some embodiments, two or more, such as 3, 4 or 5 or more, different MMP polypeptides are used. Typically, only one substrate polypeptide is used. However, two or more, such as 3, 5, 10, 15, 30 or more, different substrate polypeptides can be used. For example, two or more distinct amyloid precursor protein (APP) or amyloid β (Aβ) species may be used, or two or more ApoE species may be used. Substrate polypeptides are discussed in more detail below.

If the contacting takes place in solution or on a surface or platform, the method is carried out under conditions that allow the enzyme to function. Suitable conditions include, but are not limited to a temperature in the range of room temperature (such as

25°C) to 37⁰C, a buffer comprising Tris-HCl, Hepes, or phosphate at pH in the range of pH 7-8 and containing moderate amounts of CaCl₂ (10 millimolar). If the MMP polypeptide and/or the substrate polypeptide are expressed in a cell or cell culture, the method is carried out under conditions that maintain viability of the cell or the cell culture. Suitable conditions include, but are not limited to, a humidified atmosphere of 5% CO₂ at 37⁰C in appropriate culture media. Suitable conditions for culture of HEK293 and ELLIN cells are described in the materials and methods section.

The MMP polypeptide, compound and substrate polypeptide can be contacted in any order. The MMP polypeptide may be contacted first with compound and then with the substrate polypeptide. This type of pre-incubation may be necessary to allow sufficient time for a compound to have an effect on MMP activity. The MMP polypeptide may be contacted first with the substrate polypeptide and then with the compound. This order is useful for determining how quickly the compound can exert its effect on MMP activity. The MMP polypeptide may be contacted with the substrate polypeptide and the compound at the same time. Contacting is carried out for a sufficient period to allow for MMP activity on the substrate polypeptide to be measured by the methods described below. The MMP polypeptide is preferably contacted with the substrate polypeptide in the presence of the compound.

The MMP polypeptide and the substrate polypeptide are contacted in a manner that allows a physical interaction between the two polypeptides. This is necessary for the MMP polypeptide to cleave the substrate polypeptide. However, it should be understood that, in some embodiments of the method, the MMP polypeptide will not physically interact with the substrate polypeptide. For instance, the compound may abolish cleavage of the substrate polypeptide by irreversibly binding to the active site of the MMP polypeptide or by effects on MMP expression or stability. Under such circumstances, there may be no interaction between the MMP polypeptide and the substrate.

The MMP polypeptide and the compound are contacted in any manner that allows the compound to have an effect on MMP activity. This may not necessarily involve a physical interaction between the compound and the MMP polypeptide. For instance, the compound may affect expression of the MMP polypeptide via RNA interference. A person skilled in the art will be able to determine appropriate techniques for contacting the MMP polypeptide with the substrate polypeptide and the compound.

The method of the invention can further comprise contacting the substrate polypeptide with one or more, such as 2, 3 or 4, non-MMP proteases which are able to cleave the substrate polypeptide. Such proteases include, but are not limited to, α- secretases, β-secretase (BACE), and γ-secretases. Inclusion of a γ-secretase is particularly preferred. Additional proteases may result in the formation of more than two, such as 3, 4, 5 or more, cleavage products. Where the substrate polypeptide is APP or a fragment thereof, this will allow for measurement of the effects of the compound on the pattern of Aβ species which can be generated by the simultaneous, concurrent or sequential action of the one or more proteases. The additional protease(s) may be used in any of the forms discussed above for the MMP polypeptide and substrate polypeptide. In a preferred embodiment, the additional protease(s) are expressed in the same cell or cell culture as the MMP polypeptide and/or substrate polypeptide.

The method of the invention can be carried out in a single reaction (i.e. one which contains a compound, a MMP polypeptide and a substrate polypeptide). For instance, the method of the invention can be used to identify whether or not a single or an individual compound is an enhancer or inhibitor of MMP activity. Alternatively, the method of the invention can be used to identify whether or not two or more compounds in combination are capable of enhancing or inhibiting MMP activity.

As will be appreciated, the method of the invention is preferably carried out in multiple simultaneous or concurrent reactions, such as 5, 10, 15, 20, 30, 40, 50, 100, 150, 200 or more simultaneous or concurrent reactions. Each reaction contains at least one compound, at least one MMP polypeptide and at least one substrate polypeptide. This allows a variety of aspects of MMP activity to be investigated.

Preferably, the method of the invention involves simultaneously or concurrently identifying multiple compounds that enhance or inhibit MMP activity. In other words, the method of the invention may involve high-throughput screening of more than one compound. High-throughput screening is typically carried out using 5, 10, 15, 20, 30, 40, 50, 100, 150, 200 or more different compounds. Typically, each compound is screened in a different reaction. However, two or more compounds may be assayed in the same reaction. The method of the invention can be used to identify the concentration at which a compound optimally enhances or inhibits MMP activity. In such an embodiment, multiple reactions are simultaneously or concurrently carried out using different concentrations of the compound in each reaction. The method of the invention can be used to identify whether a compound affects MMP activity in a substrate specific manner. In such an embodiment, multiple reactions are simultaneously or concurrently carried out using different substrate polypeptides in each reaction. The method of the invention can be used to determine the extent to which the compound's effects may be saturated out by substrate concentration. In such an embodiment, multiple reactions are simultaneously or concurrently carried out using different concentrations of substrate polypeptides in each reaction.

Multiple reactions can be carried out in the wells of a flat plate. The wells typically have a capacity of from about 25 microlitres to about 250 microlitres, from about 30 microlitres to about 200 microlitres, from about 40 microlitres to about 150 microlitres or from about 50 microlitres to 100 microlitres. 96 or 384 reactions may be simultaneously or concurrently carried out in the wells of a standard 96 or 384 well plate. Such plates are commercially available for example from Greiner Labortechnik Ltd and Corning BV. Binding proteins or antibodies may be immobilised on a surface of one or more, preferably all, of the wells where required. Such binding proteins or antibodies can be used to immobilise the MMP polypeptide and/or the substrate polypeptide to the surface of the wells.

Where multiple reactions are performed, each reaction will typically be carried out under a set of similar conditions to allow for comparison of results obtained. Suitable conditions are set out above. As appropriate, each reaction is also typically carried out using the same molar concentration of the reaction constituents, namely the compound, the substrate polypeptide and/or the MMP polypeptide, to allow for comparison of results obtained. As described above, the concentration of one or more of the constituents may vary between reactions depending on the purpose of the assay.

Suitable enzyme and substrate concentrations may be approximately 0.1 - 50 nanomolar of enzyme and a 1-20 micromolar concentration of substrate, for example as described in FEBS Lett. 1992 296(3):263-6. The amount of the MMP polypeptide in each reaction may also be measured in enzyme units of MMP activity, preferably metalloproteinase activity or protease activity. Enzyme units may be determined using the E.coli expressed and purified catalytic domains of human MMP14 (Catalogue #475935 ), MMP15 (Catalogue #475938) and MMP16 (Catalogue #475939) which are commercially available from Calbiochem. One activity unit is defined by Calbiochem for all three proteases as the amount of enzyme that will hydrolyze 1.0 micromol MCA-Pro-Leu-Gly- Leu-Dpa-Ala-Arg-NH2 (#03-32-5032) per min at 37°C, pH 7.5. A suitable reference substrate for determining MMP activity is the fluorogenic peptide MCA-Pro-Leu-Gly-Leu- Dpa-Ala-Arg-NH₂, which is commercially available (R&D Systems #ES001). Related substrate sequences can also be used (Biochemical and Biophysical Research Communications 266, 308-313 (1999)). The concentration of the compound contacted with the MMP polypeptide will vary depending on the nature of the compound. A person skilled in the art can determine an appropriate concentration. Typically, from about 0.01 nanomolar to 10 micromolar concentrations of compound may be used, for example from 0.1 nanomolar to 1 micromolar, from 0.5 to 100 nanomolar or from 1 to 10 nanomolar. Where cells or cell cultures are used, each reaction typically involves the same number of cells. For instance, cells are typically seeded with approximately the same number of cells in each well of a plate, and each reaction is performed after the same time period. Typically 3-5xlO⁴ cells are seeded per well of a 96-well plate.

For each of the embodiments discussed above, the precise conditions used in the assay may vary. Experimental conditions may be optimised as a matter of routine by the person skilled in the art on the basis of their general knowledge to improve sensitivity and reliability of the method of the invention.

In order to allow for a determination of whether or not the compound is an enhancer or inhibitor of MMP activity, a comparison is made with a control value. The MMP activity value obtained following contacting of MMP polypeptide with the compound and the substrate polypeptide is compared with the control value. The control value is the MMP activity observed under conditions where the MMP polypeptide has been contacted with the substrate polypeptide, but has not been contacted with the compound. Preferably, the conditions are otherwise identical to those used to obtain the protease activity value following contacting with the compound. Following the comparison with the control value, the effect of the compound may be identified in terms of an increase in MMP activity or a decrease in MMP activity with respect to the control value. An increase is indicative of an enhancer. A decrease is indicative of an inhibitor.

Preferably, the control value is obtained while carrying out the method of the invention. For example, a control reaction is performed at the same time as reaction(s) where the MMP polypeptide is contacted with the substrate polypeptide and the compound. This ensures that the control value is obtained under the same conditions as the MMP activity measured following contacting of MMP polypeptide with the substrate polypeptide and the compound. The control value can also be obtained separately from the method of the invention. For instance, the control value may be obtained beforehand and recorded, for instance on a computer. The control value may be used for multiple repetitions of the method. The control value can be derived from more than one control reaction. For instance, the control value may be the arithmetic mean of the measurement obtained from several, such as 2, 5, 10, 15 or more, control reactions. In order to allow for an effective comparison, the control value has the same units as the measurement in the test sample with which it is being compared. A person skilled in the art is capable of obtaining such a value. The type of control value referred to above is commonly known in the art as a "negative control". The method of the invention can also be carried out in conjunction with one or more positive controls for MMP activity. This involves carrying out reactions using one or more compounds which are known enhancers or inhibitors of MMP activity. A positive control allows for validation or measurement of the protease activity of MMP polypeptide that is used in the method of the invention. For instance, this may be useful to allow comparison of results that have been obtained using different sources of MMP polypeptide. A positive control also allows the extent to which the compound enhances or inhibits MMP activity to be determined. Suitable known inhibitors of MMPs include, but are not limited to the broad spectrum MMP inhibitors Batimastat and BB25-16. For example, these are known to inhibit MMP14 with nM potency (Chem. Soc. Rev., 2004, 33, 401 - 409). Where MMP14 is used in the method of the invention, certain sulfate-containing natural products have been shown to act as inhibitors and thus can act as suitable reference compounds for inhibitory activity (J Nat Prod. 2003 66(4):569-71, Tetrahedron, 2001, 57, 3885).. Where MMP 15 or MMP 16 are used in the method of the invention, ADAM protease inhibitors are useful as reference compounds for inhibitory activity (Bioorganic & Medicinal Chemistry, (2008) 16:19(1): 8781-8794). Examples of MMP inhibitors which effect a decrease in MMP expression include MMP antisense oligo- or polynucleotides, siRNAs, transcriptional inhibitors that bind to the MMP 5' promoter/regulatory region and hammerhead ribozymes.

The incubation period of the reaction constituents prior to measurement of MMP activity will be selected on the basis of the time required to generate a signal of appropriate strength. Measurement of MMP activity can be performed at one or more timepoints following contacting of a MMP polypeptide with the test compound. This may allow for a determination of the duration and stability of the effect of the compound. Similarly, MMP activity can be measured at one more timepoints subsequent to addition of substrate polypeptide to allow for determination of the effects of the compound on the kinetics of MMP activity. As discussed above, the substrate polypeptide can be contacted with the MMP polypeptide prior to contacting with the compound. This may allow for a determination of how quickly the compound exerts its effect on pre-existing MMP activity. Techniques for measuring MMP, metalloproteinase and protease activity are well known in the art. Any technique may be used. The method preferably involves detecting one or more specific cleavage products derived from the substrate polypeptide. Preferred methods of measuring MMP activity involve fluorescence, an immunoassay or mass spectrometry. Measuring substrate cleavage using fluorescence is well known in the art. For example, a substrate polypeptide may be labelled with a fluorescent moiety and cleavage can be monitored by a change in the fluorescence spectrum or a decay in the fluorescent signal. The fluorogenic peptide MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ described above is suitable for use as a reference substrate for fluorescence-based methods. A preferred fluorescence-based method that may be used to measure MMP activity is fluorescence resonance energy transfer (FRET). This uses two fluorophores, a donor and an acceptor. Excitation of the donor by an energy source (e.g. flash lamp or fluorometer laser) triggers an energy transfer to the acceptor if they are within a given proximity to each other. The acceptor in turn emits light at its given wavelength. The use of long-lived fluorophores combined with time-resolved detection (a delay between excitation and emission detection) is preferred to minimize interference. A particularly preferred fluorescence-based method is homologous time resolved fluorescence (HTRP). This uses lanthanides which have large Stake's shifts and extremely long emission half- lives (from microseconds to milliseconds) when compared to more traditional fluorophores (Mathis G J Biomol Screen. 1999; 4(6): 309-314).

Measuring substrate cleavage using an immunoassay is also well known in the art. The immunoassay can involve specific detection of one or more cleavage products derived from the substrate polypeptide. Conversely, an immunoassay can be used to detect clearance or degradation of the substrate polypeptide by measuring the amount of uncleaved substrate polypeptide remaining after the action of MMP on the substrate polypeptide. Any suitable immunoassay which allows for detection of cleavage products or uncleaved substrate polypeptide by an antibody may be used. Any suitable commercially available antibody for a given target may be used.

Preferred antibodies for use in detection of MMP activity according to the invention are antibodies which are capable of selective binding to N-terminally extended Aβ, N-terminally truncated Aβ, and C-terminally truncated Aβ. Preferably, antibodies binding to N-terminally extended Aβ bind to an epitope N-terminal to residue 1 of Aβ. For example, such epitopes may be comprised in regions Aβ-25 to -1 (DRGLTTRPGSGLTNIKTEEISEVKM, SEQ ID NO: 63), Aβ-22 to -1 (LTTRPGSGLTNIKTEEISEVKM, SEQ ID NO: 64), Aβ-16 to -1 (SGLTNIKTEEISEVKM, SEQ ID NO: 65) or Aβ-14 to -1 (LTNIKTEEISEVKM, SEQ ID NO: 66). Such antibodies may be routinely generated on the basis of the general knowledge of the skilled person.

A suitable antibody for detection of N-terminally truncated Aβ is one which binds an epitope following residue 1 of Aβ. A preferred example is the anti-Aβ antibody 4G8, commercially available from Signet (#9220-02). 4G8 binds an epitope within amino acids 17-24 of Aβ and can therefore bind to N-terminally truncated Aβ, such as Aβ 3-40. Use of this antibody is described below in the Examples. A suitable antibody for detection of C- terminally truncated Aβ is one which binds an epitope preceding residue 14 of Aβ. A preferred example is the anti-Aβ antibody 6E10, also commercially available from Signet (#9320-02). The 6E10 antibody binds an epitope within amino acids 1-16 of Aβ, and can be used to detect C-terminally truncated Aβ, for example C-terminally truncated variants terminating at residue 14 of Aβ.

A preferred immunoassay is Enzyme-Linked Immunosorbent Assay (ELISA). For example, production of specific cleavage products of APP such as Aβl-40, Aβ3-40, or Aβ6-40 can be measured using antibodies specific to those peptides. Suitable ELISA assay kits for detection of Aβ species are commercially available, for example from WAKO. In some embodiments, the ELISA assay may be performed in flat plates where wells are coated with binding proteins or antibodies which can bind and allow for detection of the cleavage product or uncleaved substrate polypeptide. Other types of immunoassay include immunoprecipitation and Western blotting.

Whilst immunoassays are preferred, any other high-affinity ligand-receptor interaction, such as streptavidin-biotin, could be used to measure MMP activity.

Measuring substrate cleavage using mass spectrometry is also well known in the art. Cleavage products derived by the action of MMP on the substrate polypeptide can be separated on the basis of their mass and charge to allow for a determination of the relative proportions of each specific cleavage product in the reaction mixture. In such embodiments, the reaction mixture may be concentrated prior to analysis by use of a suitable antibody which precipitates all cleavage products. Preferred cell based assays include reporter assays for cleavage of a protein substrate. For example, APP can be N-terminally tagged with secreted alkaline phosphatase (SEAP) or a similar enzymatically active protein tag such as luciferase or beta-galactosidase. Shedding of APP ectodomain can be measured by accumulation of enzyme activity in conditioned media. Alternatively, the C-terminus of APP can be tagged with a GaW reporter element. On cleavage of APP by MMPs the APP C-terminal fragment/Gal4 chimera migrates to the nucleus where it activates an artificial reporter gene by binding to a UAS promoter element upstream of a reporter gene expressing luciferase/SEAP/β-galactosidase.

MMP polypeptide

Matrix metalloproteinases (MMPs) are Zn²⁺ binding endopeptidases that degrade various components of the extracellular matrix (ECM). The MMPs are enzymes implicated in normal and pathologic tissue remodeling processes, wound healing, angiogenesis, and tumor invasion. Membrane-type MMPs are a subclass in the MMP family since the other members lack a C-terminal transmembrane domain and are secreted as soluble forms. Membrane-type MMPs include MMP 14, MMP 15 and MMP 16.

The method of the invention uses an MMP14, an MMP15, or an MMP16 polypeptide. An MMP 14, MMP 15, or MMP 16 polypeptide is a polypeptide which can cleave a substrate polypeptide comprising a motif of at least ten amino acids from SEQ ID NO: 7 or an equivalent thereof. Cleavage must occur immediately following one or more of, such as two, three, four, five or all of, residues 9, 12, 18, 20, 36 and 48 of SEQ ID NO: 7 or the corresponding residues in an equivalent thereof. In other words, the MMP 14, MMP 15, or MMP 16 polypeptide cleaves the above-mentioned substrate polypeptide between one or more of, such as two, three, four, five or all of, residues 9 and 10; residues 12 and 13; residues 18 and 19; residues 20 and 21; residues 36 and 37; or residues 48 and 49 of SEQ ID NO: 7. It is particularly preferred that the MMP14, MMP15, or MMP16 polypeptide cleaves the substrate polypeptide between one or more of, such as two, three, four, five or all of, residues 5 and 6 of the sequences ARPAADRGLT (motif A); AADRGLTTRP (motif B); TTRPGSGLTN (motif C); RPGSGLTN I K (motif D); VKMDAEFRHD (motif E); and YEVHHQKLVF (motif F). Where an MMP 16 polypeptide is used, in one embodiment the method of the invention monitors cleavage immediately following one or more of, such as two, three, four, five or all of, residues 9, 12, 18, 20 and 36 of SEQ ID NO: 7 or the corresponding residues in an equivalent thereof. In an alternative embodiment where an MMP 16 polypeptide is used, the method of the invention monitors cleavage immediately following more than one of, such as two, three, four, five or all of, residues 9, 12, 18, 20, 36 and 48 of SEQ ID NO: 7 or the corresponding residues in an equivalent thereof. In other words, in this embodiment, a single cleavage event by an MMP 16 polypeptide at position 48 of SEQ ID NO: 7 or the corresponding residue in an equivalent thereof is not monitored. It is particularly preferred that the MMP 16 polypeptide cleaves the substrate polypeptide between one or more of, such as two, three, four, or all of, residues 5 and 6 of the sequences ARPAADRGLT (motif A); AADRGLTTRP (motif B); TTRPGSGLTN (motif C); RPGSGLTNIK (motif D); VKMDAEFRHD (motif E). In an alternative embodiment, it is particularly preferred that the MMP 16 polypeptide cleaves the substrate polypeptide between more than one of, such as two, three, four, five or all of motifs A to E and YEVHHQKLVF (motif F).

The ability of an MMP 14, MMPl 5, or MMP 16 polypeptide to cleave a substrate polypeptide as defined above may be routinely determined by a person skilled in the art. The ability of a polypeptide to cleave a substrate polypeptide in the manner defined above may be determined by any of the methods disclosed above for measuring MMP activity. The ability of an MMP 14, MMP 15, or MMP 16 polypeptide to cleave a substrate polypeptide in the manner defined above is preferably determined as described in the Examples.

The cDNA sequence for MMP 14 is shown in SEQ ID NO: 14 and encodes the protein shown in SEQ ID NO: 15. Various animal and insect genes/cDNAs and their encoded proteins are known and can be found in various public databases. Database accession numbers for various animal and insect homologue proteins are as follows: Mmpl4 Mus musculus (NP 032634); Mmpl Drosophila melanogaster (NP 726473, NP 523852); mmpl 4a Danio rerio (NP_919397); MMP 14 Pan troglodytes (XP OO 1157686, XP_001157566); MMP 14 Bos taurus (NP_776815); MMP14 Canis familiaris (XP 856947, XP_848757); Mmpl4 Rattus norvegicus (NP_112318); AgaP_AGAP006904 Anopheles gambiae str. PEST (XP_001688108, XP_001688107). It should be understood that the skilled person would also be able to identify an animal or insect homologue of SEQ ID NO: 15 in a database using conventional sequence analysis methods.

The human protein of SEQ ID NO: 15 contains a PGBD-like domain, a cysteine switch domain, a metalloproteinase "zincin" catalytic domain, a hemopexin-like domain and a transmembrane region. The domain positions in the sequence of the human protein of SEQ ID NO: 15 are approximately as follows: PGBD-like: aa34-98; cysteine switch domain: aa91-98; metalloprotease ("zincin") catalytic domain: aal 14-284; hemopexin-like domain: aa311-508; transmembrane region: aa542-562. An MMP14 polypeptide may comprise the amino acid sequence of SEQ ID NO: 15 or a variant of any thereof. The MMP 14 polypeptide more preferably consists of the amino acid sequence of SEQ ID NO: 15 or a variant thereof.

The human cDNA sequence for MMP 15 is shown in SEQ ID NO: 16 and encodes the protein shown in SEQ ID NO: 17. Various animal and insect genes or cDNAs and their encoded proteins are known and can be found in public databases. Database accession numbers for various animal and insect homologue proteins are as follows: Mmpl5 Mus musculus (XP_001002215, XP OO 1002209); MMP15 Pan troglodytes (XP OOl 150678, XP_523380); MMP15 Bos taurus (XP_597651); MMP15 Canis familiaris (XP_544383); Mmpl5 Rattus norvegicus (NP OO 1099638, XPJ)01057363). It should be understood that the skilled person would also be able to identify an animal or insect homologue of SEQ ID NO: 17 in a database using conventional sequence analysis methods. The human protein of SEQ ID NO: 17 contains a PGBD-like domain, a ZN Metallopeptidase catalytic domain, a hemopexin domain and a transmembrane region. The domain positions in the sequence of the human protein of SEQ ID NO: 17 are approximately as follows: PGBD-like: aa52-l 16; ZN Metallopeptidase catalytic domain: aal35-305; hemopexin domain: aa362-559; transmembrane region: aa 624-645.

An MMP 15 polypeptide may comprise the amino acid sequence of SEQ ID NO: 17 or a variant of any thereof. The MMP 15 polypeptide more preferably consists of the amino acid sequence of SEQ ID NO: 17 or a variant thereof. The human cDNA sequence for MMP 16, transcript variant 1 is shown in SEQ ID

NO: 18 and encodes the protein shown in SEQ ID NO: 19. The human cDNA sequence for MMP 16, transcript variant 2 is shown in SEQ ID NO: 20 and encodes the protein shown in SEQ ID NO: 21. Various animal and insect homologue proteins are as follows : Mmplό Mus museums (NP_062698); mmpl 6a Danio rerio (XP 692849); MMP16 Pan troglodytes (XP OOl 136415, XPJ)Ol 136497) ; MMP16 Bos taurus (XP 604345); MMP 16 Gallus gallus (NP_990528); MMP 16 Canis familiaris (XP_544165); MMP 16 Rattus norvegicus (NP 542954). It should be understood that the skilled person would also be able to identify an animal or insect homologue of SEQ ID NO: 19 or 21 in a database using conventional sequence analysis methods. Any such transcript variants are suitable for use according the invention.

The human protein of SEQ ID NO: 19 contains a PGBD-like domain, ZN protease domain, a hemopexin-like domain and a transmembrane region. The domain positions in the sequence of the human protein of SEQ ID NO: 19 are approximately as follows: PGBD-like: 24-106; ZN protease domain: aal26-291 ; Hemopexin-like domain: aa335-532; transmembrane region: aa564-584.

The human protein of SEQ ID NO: 21 is a soluble isoform of MMP 16 and contains a PGBD-like domain, a Cysteine switch domain, a ZN protease domain, and a hemopexin- like domain. The domain positions in the sequence of the human protein of SEQ ID NO: 21 are approximately as follows: PGBD-like: aa24-106; Cysteine switch domain: aa99- 106; ZN protease domain: aal23-292; Hemopexin-like domain: aa335-407. An MMP16 polypeptide may comprise the amino acid sequence of SEQ ID NOs: 19 or 21 or a variant of either thereof or the amino acid sequence of an animal or insect homologue of MMP 16, such as the animal and insect homologues whose accession numbers are listed above or a variant of any thereof. It is preferred that an MMP 16 polypeptide comprises the amino acid sequence of human MMP 16 of SEQ ID NO: 19 or 21 or a variant of either thereof. The MMP 16 polypeptide more preferably consists of the amino acid sequence of SEQ ID NO: 19 or 21 or a variant of either thereof.

A variant of any of these MMP 14, MMP 15 or MMP 16 polypeptides (hereinafter also described as the native protein(s)) may comprise truncations, mutants or homologues thereof. Variants also include any transcript variants thereof. A variant must cleave a substrate polypeptide as described above. Any homologues mentioned herein are typically at least 40% homologous to the relevant region of the native protein. Homology can be measured using known methods. 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, 387-395). 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 S. F. (1993) J MoI Evol 36:290-300; Altschul, S, F et al (1990) J MoI Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

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. A variant MMP polypeptide comprises (or consists of) a sequence which has at least 40% identity to the native protein. In preferred embodiments, a variant sequence may be at least 55%, 65%, 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 97% or 99% homologous to a particular region of the native protein over at least 20, preferably at least 30, for instance at least 40, 60, 100, 200, 300, 400 or more contiguous amino acids, or even over the entire sequence of the variant. Alternatively, the variant sequence may be at least 55%, 65%, 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 97% or 99% homologous to full-length native protein. Typically the variant sequence differs from the relevant region of the native protein by at least, or less than, 2, 5, 10, 20, 40, 50 or 60 mutations (each of which can be substitutions, insertions or deletions). A variant sequence of the invention may have a percentage identity with a particular region of the full-length native protein which is the same as any of the specific percentage homology values (i.e. it may have at least 40%, 55%, 80% or 90% and more preferably at least 95%, 97% or 99% identity) across any of the lengths of sequence mentioned above. Variants of the native protein also include truncations. Any truncation may be used so long as the variant is still able to cleave a substrate polypeptide as described above. Truncations will typically be made to remove sequences that are non-essential for protease activity and/or do not affect conformation of the folded protein, in particular folding of the active site. Truncations may also be selected to improve solubility of the MMP polypeptide. Appropriate truncations can routinely be identified by systematic truncation of sequences of varying length from the N- or C-terminus. Preferred truncations are N- terminal and may remove all other sequences except for the protease domain (also referred to as Zn protease, Zn metalloprotease or Zn metallopeptidase domain). Such truncations are particularly preferred where the assay is carried in vitro. Other preferred truncations are those which remove the transmembrane domain to produce soluble MMP polypeptides. Such truncations are preferred where full-length APP is used as a substrate in a cell-based assay. In such assays, the truncated variant may additionally comprise the transmembrane domain. Domain positions for the protease and transmembrane domains in SEQ ID NOs: 15, 17, 19 or 21 are described above.

Variants of the native protein further include mutants which have one or more, for example, 2, 3, 4, 5 to 10, 10 to 20, 20 to 40 or more, amino acid insertions, substitutions or deletions with respect to a particular region of the native protein. Deletions and insertions are made preferably outside of the protease domain as described below. Insertions are typically made at the N- or C-terminal ends of a sequence derived from the native protein, for example for the purposes of recombinant expression as detailed below. Another common N-terminal insertion is a signal peptide to assist secretion in a cell system where the variant sequence derived from the native protein does not contain a signal peptide.

Substitutions are also typically made in regions that are non-essential for protease activity and/or do not affect conformation of the folded protein. Such substitutions may be made to improve solubility or other characteristics of the enzyme. Although not generally preferred, substitutions may also be made in the active site or in the second sphere, i.e. residues which affect or contact the position or orientation of one or more of the amino acids in the active site. These substitutions may be made to improve turnover of substrate polypeptide or to map the binding site of a test compound. This is discussed in more detail below.

Substitutions preferably introduce one or more conservative changes, which replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative change may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table A. Where amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains in Table B.

Table A - Chemical properties of amino acids

Table B. Hydropathy scale

Side Chain Hydropathy

He 4.5

VaI 4.2

Leu 3.8

Phe 2.8

Cys 2.5

Met 1.9

Ala 1.8

GIy -0.4

Thr -0.7

Ser -0.8

Trp -0.9

Tyr -1.3

Pro -1.6

His -3.2

GIu -3.5

GIn -3.5

Asp -3.5

Asn -3.5

Lys -3.9

Arg -4.5

It is preferred that a variant of SEQ ID NO: 15 comprises residues 114-284 of SEQ ID NO: 15. It is preferred that a variant of SEQ ID NO: 17 comprises residues 135-305 of SEQ ID NO: 17. It is preferred that a variant of SEQ ID NO: 19 comprises residues aal24-291 of SEQ ID NO: 19. It is preferred that a variant of SEQ ID NO: 21 comprises residues aal 23-292 of SEQ ID NO: 21. These regions correspond to the protease domains of SEQ ID NOs: 15, 17, 19, and 21 respectively. However, a variant can comprise amino acid insertions, substitutions or deletions in the protease domains as long as the variant is still able to cleave a substrate polypeptide as described above.

For mstance, a variant may contain conservative amino acid substitutions in residues 114-284 of SEQ ID NO: 15, residues 135-305 of SEQ ID NO: 17 or residues 124- 291 of SEQ ID NO: 19, or residues 123-292 of SEQ ID NO: 21, as long as the variant is still able to cleave a substrate polypeptide as described above. Suitable conservative substitutions are discussed above. If the variant does comprise amino acid insertions, substitutions or deletions in the protease domain, it is preferred that the variant is able to cleave a substrate polypeptide as described above with an efficiency that is comparable to, or the same as, the native protein.

It is particularly preferred that the variant is able to cleave the substrate polypeptide as described above with an efficiency that is comparable to, or the same as the native protein. Where a variant does comprise amino acid insertions, substitutions or deletions in the protease domain, it is preferred that the variant comprises a sequence that is at least 90%, at least 95%, at least 97% or at least 99% homologous to residues 114-284 of SEQ ID NO: 15, residues 135-305 of SEQ ID NO: 17 or residues 124-291 of SEQ ID NO: 19 or residues 123-292 of SEQ ID NO: 21.

Substrate polypeptide

The inventor has surprisingly identified a discrete region of human APP where MMP-mediated proteolytic cleavage occurs. Human APP, transcript variant 1 is shown in SEQ ID NO: 2. The Inventor has shown that MMP14, MMPl 5 and MMP16 cleave human APP between residues 646 and 647 (alanine and aspartic acid), residues 649 and 650 (glycine and leucine), residues 655 and 656 (glycine and serine), residues 657 and 658 (glycine and leucine), residues 673 and 674 (alanine and glutamic acid), and residues 685 and 686 (histidine and glutamine) of human APP of SEQ ID NO: 2. With respect to the Aβ 1 -40 peptide generated by BACE cleavage, these cleavage events occur between residues -26 and -25, residues -23 and -22, residues -17 and -16, residues +2 and +3 and residues +14 and +15.

The Inventor has shown that typical cleavage products derived from human APP by the action of MMP14, MMP15 and MMP16 are A/? -25 to +14, Aβ -22 to +14, Aβ -16 to +14 and Aβ -14 to +14. These are shown below respectively as SEQ ID NO:s 67 to 70. The inventors have also surprisingly shown that isolated A/H-40 peptide acts as a substrate for MMP 14, MMP 15 and MMP 16.

The substrate polypeptide comprises, consists or consists essentially of a motif of at least ten amino acids from SEQ ID NO: 7 or an equivalent thereof. SEQ ID NO: 7 is the amino acid sequence EPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAED.

The motif has at least ten contiguous amino acids from SEQ ID NO: 7 or an equivalent thereof. The motif from SEQ ID NO: 7 allows for MMP activity to be measured by monitoring cleavage of the substrate polypeptide immediately following one or more of residues 9, 12, 18, 20, 36 and 48 of SEQ ID NO: 7. In other words, the motif from SEQ ID NO: 7 comprises at least one of residues 9, 12, 18, 20, 36 and 48 from SEQ ID NO: 7. In some embodiments, the motif may comprise more than one, preferably two, three, four, five or all of, residues 9, 12, 18, 20, 36 and 48 from SEQ ID NO: 7. As outlined below, the substrate polypeptide may comprise more than one motif from SEQ ID NO: 7, allowing for monitoring of cleavage immediately following more than one of residues 9, 12, 18, 20, 36 and 48 from SEQ ID NO: 7.

Any motif from SEQ ID NO: 7 comprising at least one of residues 9, 12, 18, 20, 36 and 48 of SEQ ID NO: 7 may be used. Particularly preferred motifs from SEQ ID NO: 7 include ARPAADRGLT (motif A, SEQ ID NO:8); AADRGLTTRP (motif B, SEQ ID NO: 9); TTRPGSGLTN (motif C, SEQ ID NO: 10); RPGSGLTNIK (motif D, SEQ ID NO: 11 ); VKMDAEFRHD (motif E, SEQ ID NO: 12); and YEVHHQKLVF (motif F, SEQ ID NO: 13). A substrate polypeptide for use in the method of the invention must comprise a motif of least ten contiguous amino acids from SEQ ID NO: 7 or an equivalent thereof. However, longer amino acid motifs are particularly preferred. In some embodiments, the substrate polypeptide comprise, consists or consists essentially of at least eleven, at least twelve, at least thirteen, or at least fourteen contiguous amino acids from SEQ ID NO: 7 or an equivalent thereof. Examples include ARPAADRGLTT (SEQ ID NO: 22), ARPAADRGLTTR (SEQ ID NO: 23), ARPAADRGLTTRP (SEQ ID NO: 24), ARPAADRGLTTRPG (SEQ ID NO: 25); AADRGLTTRPGS (SEQ ID NO: 26), AADRGLTTRPGSG (SEQ ID NO: 27), AADRGLTTRPGSGL (SEQ ID NO: 28); TTRPGSGLTNI (SEQ ID NO: 29), TTRPGSGLTNIK (SEQ ID NO: 30), TTRPGSGLTNIKT (SEQ ID NO: 31), TTRPGSGLTNIKTE (SEQ ID NO: 32); RPGSGLTNIKT (SEQ ID NO: 33), RPGSGLTNIKTE (SEQ ID NO: 34),

RPGSGLTNIKTEE (SEQ ID NO: 35), RPGSGLTNIKTEEI (SEQ ID NO: 36); VKMDAEFRHDS (SEQ ID NO: 37), VKMDAEFRHDSG (SEQ ID NO: 38), VKMDAEFRHDSGY (SEQ ID NO: 39), VKMDAEFRHDSGYE (SEQ ID NO: 40); YEVHHQKLVFF (SEQ ID NO: 41), YEVHHQKLVFFA (SEQ ID NO: 42), YEVHHQKLVFFAE (SEQ ID NO: 43), YEVHHQKLVFFAED (SEQ ID NO: 44).

The substrate polypeptide of the invention will now be further described by ^"■reference to ten amino acid motifs from SEQ ID NO: 7 or equivalents thereof. It should be understood that the general principles established in description of motifs are equally applicable to longer motifs, for example eleven, twelve, thirteen, or fourteen amino acid motifs.

The preferred motifs A-F comprise four amino acids N-terminal and C-terminal to the residues immediately preceding and following the cleavage site in SEQ ID NO: 7 (A/D for motif A; G/L for motif B; G/S for motif C; G/L for motif D, A/E for motif E; H/Q for motif F). Therefore, cleavage is monitored between residues 5 and 6 of each motif. However, it should be understood that a motif from SEQ ID NO: 7 may include a varying number of residues N-terminal and C-terminal to a particular cleavage site, providing the residues immediately preceding and following the cleavage site are included.

In some embodiments, the motif solely comprises N-terminal sequence preceding the cleavage site i.e E PVDARPAAD (SEQ ID NO: 45), DARPAADRGL (SEQ ID NO: 46), DRGLTTRPGS (SEQ ID NO: 47), ELTTRPGSGL (SEQ ID NO: 48), EI SEVKMDAE (SEQ ID NO: 49), HDSGYEVHHQ (SEQ ID NO: 50). In other embodiments, the motif solely comprises C-terminal sequence following the cleavage site i.e ADRGLTTRPG (SEQ ID NO: 51), GLTTRPGSGL (SEQ ID NO: 52), GSGLTNIKTE (SEQ ID NO: 53), GLTNIKTEEI (SEQ ID NO: 54), AEFRHDSGYE (SEQ ID NO: 55), HQKLVFFAED (SEQ ID NO: 56).

In further embodiments, at least one residue N-terminal to the cleavage site and at least one residue C-terminal to the cleavage site are present. Non-limiting examples are PVDARPAADR (SEQ ID NO: 57) and HHQKLVFFAE (SEQ ID NO: 58).

Motifs may be based on any possible combination of sequences N-terminal and C- terminal to the cleavage site, for example two N-terminal residues and six C-terminal residues; three C-terminal residues and five N-terminal residues. Non-limiting examples of these particular combinations are respectively VHHQKLVFFA (SEQ ID NO: 59) and EVKMDAEFRH (SEQ ID NO: 60). It is preferred that two or more, preferably three or more residues are included from the sequences N-terminal to the cleavage site and C- terminal to the cleavage site in the motif. The most preferred motifs include four residues from the sequences N-terminal to the cleavage site and C-terminal to the cleavage site, and are shown as motifs A-F above.

An equivalent of a motif from SEQ ID NO: 7 is any sequence of at least ten amino _jacids that is at least 60% identical to a motif from SEQ ID NO: 7 as described above. For instance, an equivalent is a sequence often amino acids that is at least 60% identical to ten contiguous amino acids of SEQ ID NO: 7. An equivalent is cleaved by any of the MMP polypeptides described herein. Such cleavage may be determined using any method disclosed herein. An equivalent thereby allows the effect of a compound on MMP activity to be determined. An equivalent is preferably cleaved by a MMP polypeptide described herein with similar or comparable efficiency to a substrate polypeptide comprising a motif from SEQ ID NO: 7. A reduced cleavage efficiency for the equivalent is acceptable, so long as a detectable signal is generated.

The equivalent preferably comprises 1, 2 or 3 conservative substitutions with respect to a motif from SEQ ID NO: 7. Hence, if the motif from SEQ ID NO: 7 is ten amino acids in length, the equivalent will have 66.6% identity, preferably 77.7% or 88.8% identity to the ten amino acid motif from SEQ ID NO: 7. The 1, 2 or 3 conservative substitutions are preferably made at the residues surrounding or adjacent to the cleavage site. However, it is particularly preferred that the residues corresponding to residues 9, 12, 18, 20, 36 and 48 of SEQ ID NO: 7 are unchanged i.e for example that conservative substitutions are made with respect to residues 7, 8, 10, 11, 13, 16, 17, 19, 21, 34, 35, 37, 46, 47, 49. For example, for VKMDAEFRHD (motif E), an equivalent sequence having three conservative substitutions could be VKCEADFRHD (SEQ ID NO: 61), and cleavage would be monitored immediately following residue 5 of the equivalent. To take another example, where a ten amino acid motif from SEQ ID NO: 7 is VHHQKLVFFA, an equivalent sequence having two conservative substitutions could be I HHNKLVFFA (SEQ ID NO: 62), and cleavage would be monitored immediately following residue 3 of the equivalent, corresponding to residue 48 of SEQ ID NO: 7. When using equivalents of motifs from SEQ ID NO: 7, the method involves monitoring cleavage at those amino acid(s) in the motif that correspond to residues 9, 12, 18, 20, 36 and 48 of SEQ ID NO: 7.

These types of equivalents may be used to improve catalytic activity of MMP by enhancing binding contacts at the active site. Conservative substitutions may be made at other positions than those described above so long as the equivalent has 66.6% identity, preferably 77.7% or 88.8% identity to a ten amino acid motif from SEQ ID NO: 7. Conservative substitutions may be selected according to the options presented above, including those in Tables A and B. It is straightforward to identify which residues in a motif from SEQ ID NO: 7 can be conservatively substituted without loss of cleavage by an MMP polypeptide described herein. For instance, the residues in SEQ ID NO: 7 which are involved in binding with an MMP polypeptide could be identified. Structural studies or chemical cross-linking experiments can be carried out to identify enzyme-substrate contacts. Substitution of contact residues would typically result in a loss of cleavage by an MMP polypeptide. Alternatively, each residue of SEQ ID NO: 7 may be substituted in a systematic manner and cleavage by an MMP polypeptide may be determined for each mutant.

Substrate polypeptides for use in the method of the invention may comprise, consist or consist essentially of a motif from SEQ ID NO: 7 or an equivalent thereof.

Alternatively, substrate polypeptides comprising, consisting or consisting essentially of more than one, such as two, three, four, five or more, motifs from SEQ ID NO: 7 or an equivalent thereof may be used. Where multiple motifs from SEQ ID NO: 7 or equivalents are present, it is preferred that the substrate polypeptide comprises more than one, preferably two, three, four, five or all of, residues 9, 12, 18, 20, 36 and 48 of SEQ ID NO: 7 or the corresponding residues in an equivalent thereof. This allows for multiple cleavage events carried out by MMP14, MMP15, or MMP16 to be monitored. The multiple motifs may be in any orientation and/or spacing in the substrate polypeptide, but it is preferred that they are present in the same orientation as SEQ ID NO: 7. As outlined above, where MMP 16 is used, in one embodiment of the invention, the single cleavage event at position 48 of SEQ ID NO: 7 or the corresponding residue in an equivalent thereof is not monitored in the method of the invention. If MMP 16 is used in this embodiment to monitor the cleavage event at position 48 of SEQ ID NO: 7 or the corresponding residue in an equivalent thereof, one or more cleavage events at positions 9, 12, 18, 20 and 36 will also be monitored. In this situation, the substrate polypeptide will necessarily comprise at least two, preferably three, four or five or ten motifs from SEQ ID NO: 7 or equivalents thereof. One of these motifs will comprise residue 48 of SEQ ID NO: 7 or the corresponding residue in an equivalent thereof, and the other motif(s) will comprise one of residues 9, 12, 18, 20 or 36 of SEQ ID NO: 7 or the corresponding residues in an equivalent thereof. Particularly preferred substrate polypeptides comprise, consist or consist essentially of SEQ ID NO: 7. Other preferred substrate polypeptides comprise SEQ ID NO: 7 and additional C-terminal sequence from human APP of SEQ ID NO: 2.

In addition to comprising a motif of at least 10 amino acids from SEQ ID NO: 7 or an equivalent thereof, the substrate polypeptide may further comprise any other amino acid sequence, so long as cleavage by an MMP polypeptide is still observed. Where the substrate polypeptide comprises SEQ ID NO: 7, the additional amino acid sequence is preferably derived from residues 695 onwards of human APP of SEQ ID NO: 2. Particularly preferred substrate polypeptides are those which additionally comprise sequence of Aβ. For example, preferred substrate polypeptides additionally comprise residues 696-701, 696-706, 696-712, 696-714, 696-722 of SEQ ID NO: 2. This corresponds to residues 24-29, 24-34, 24-40, 24-42 or 24-50 of Aβ. Even more preferred substrate polypeptides are those which comprise cleavage sites for α-secretases, β- secretases (BACE) and/or γ-secretases. Cleavage sites for these proteases are well known in the art.

Most preferred substrate polypeptides are those which comprise, consist, or consist essentially of SEQ ID NO: 2. Use of such substrates is particularly preferred where the method comprises contacting the substrate polypeptide with a MMP polypeptide and one or more additional APP proteases. Substrate polypeptides which comprise, consist, or consist essentially of SEQ ID NO: 4 or SEQ ID NO: 6 may also be used in the methods of the invention.

As described above, the inventor has also identified that ApoE is cleaved by MMP 16. ApoE and equivalents thereof can therefore also be used as substrate polypeptides for MMP 16. The human ApoE3 nucleic acid sequence is shown in SEQ ID NO: 71, the corresponding protein sequence is shown in SEQ ID NO: 72. Human ApoE2 (Cysl 12, Cysl58) and ApoE4 (Argl 12, Argl58) are variants that only differ in the residues at positions 112 or 158 as indicated from ApoE3 (Cysl 12, Argl58). Other equivalents can be easily identified by the skilled person, and include, for example, the following database entries: NP 033826 (Mus musculus), NP 620183 (Rattus norvegicus), NP_001009007 (Pan troglodytes), NP_001018401 (Dario rerio), and NP_776416 (Bos taurus). The skilled person will be able to identify positions where substitutions can be made without affecting cleavage by MMP 16, in particular conservative substitutions as described above.

Preparation of reagents The MMP polypeptide can be produced by recombinant expression in a suitable host system. It is preferred that the recombinant polypeptide be produced by prokaryotic expression. For example, a bacterial expression vector may be prepared containing a polynucleotide sequence encoding a MMP polypeptide as defined above. The polynucleotide sequence may further comprise a protein tag at the C- or N-terminus which is suitable for purification (such as a His tag, HA tag, V5 tag, VSVG tag, GST or similar). The MMP polypeptide may be fused at the N- or C- terminus to another protein to increase stability. Multiple suitable bacterial expression vectors may be used. The pET vector series is an example of such a vector, where recombinant protein expression is induced by the addition of IPTG (isopropyl β-D thiogalactoside). The construct may typically be transformed into a suitable bacterial host such as

BL21 (DE3) bacterial cells (or equivalent) and recombinant protein expression induced as described. Soluble, tagged MMP will be column-purified as per the purification protocol appropriate to the protein tag. For example, with a His tag, the protein would be purified on a nickel resin column. Alternatively, purified MMP polypeptide may be produced recombinantly in a eukaryotic system, for example in insect cells or mammalian cells. For example, a human stable cell line expressing the MMP polypeptide may be used. MDA-MB-231 and ZR-75- 1 cells are preferred host cell lines which express MMP 14 and MMP 15 at high levels (Anticancer Res. 2004 Nov-Dec;24(6):4025-30). Rat smooth muscle cells are a preferred host cell line for expression of MMP16 (J Biol Chem. 1997 Apr 1 1 ; 272(15): 9749-54). Alternatively, a cell line transfected with a MMP expression construct and transiently expressing a MMP polypeptide may be used. The MMP polypeptide may be purified by immunoprecipitation from the conditioned media of transfected or stable MMP-expressing cells using an anti-MMP antibody. Such antibodies are commercially available. Purity of the MMP polypeptide may be assessed by SDS/PAGE and protein staining by colloidal blue or a similar method. The amount of purified protein and total _^enzyme activity may be determined by routine procedures known in the art. This will then allow for similar or identical molar amounts or enzyme units of recombinantly expressed MMP polypeptide to be provided in each reaction.

The same protein production methodology outlined above may be applied to preparation of the substrate polypeptide. A bacterial expression vector may be prepared containing a polynucleotide sequence encoding a substrate polypeptide. The substrate polypeptide will then typically be purified, for example as described above for the MMP polypeptide. The amount of purified substrate polypeptide may then be determined by routine procedures known in the art. This will then allow for similar or identical molar amounts of recombinantly expressed substrate polypeptide to be provided in each reaction. The method of the invention may use cells or cell cultures expressing MMP polypeptide and/or substrate polypeptide. In such embodiments, the cells will generally harbour a polynucleotide sequence encoding a MMP polypeptide and/or a substrate polypeptide. Additional polynucleotide sequences encoding other APP proteases may also be provided. The discussion below applies to provision of any of these polynucleotide sequences. The polynucleotide sequences may be provided transiently in the cell, for example in the form of a cDNA housed in a vector that has been transfected into the cell by methods known in the art. Examples of such transfection methods include the use of cationic lipids or liposomes and calcium phosphate. Alternatively, the polynucleotide sequence may be stably expressed in the cell. Techniques for generating stable cell lines are also well known in the art. For example, cells may be transiently transfected with a linearised vector comprising the polynucleotide sequence. The linearised vector can integrate into the genome of the cell, providing for stable expression. Alternatively, the cells may be infected with a virus comprising the polynucleotide sequence, where the virus provides for integration of the sequence into the genome. Efficient transfection and generation of stable cell lines will typically require selection of the cell population for uptake of vector or virus comprising the polynucleotide sequence. In these situations, the vector or virus will further comprise a selectable marker that expresses a protein conferring resistance to a compound which is toxic to mammalian cells. Cells that have taken up the vector or virus will be resistant to the compound, whilst other cells will be eliminated from cell culture by the toxic effects of the compound. Suitable selectable markers are known in the art. The polynucleotide sequence will be operably linked to a promoter allowing for expression in mammalian cells. A variety of suitable promoters are known in the art, and may be selected according to the specific cell system used to express the MMP polypeptide, and according to the level of expression that is required in the cell. Examples of suitable promoters include CMV, SV40, and RSV. The promoter may be inducible in response to presence of an inducer compound in the cell culture, allowing for timed regulation of expression of the MMP polypeptide.

In further embodiments, the MMP polypeptide may be expressed from an endogenous MMP-encoding gene in a suitable cell line, such as MDA-MB-231 and ZR- 75-1 cells (for MMP 14 and MMP 15) or rat smooth muscle cells (for MMP 16). Suitable cell lines expressing substrate polypeptide from an endogenous gene include, but are not limited to, HEK293 and ELLIN (patent application WO2008084254) neuroblastoma cell lines. Examples of suitable cells expressing other APP proteases include, but are not limited to HeLa, CHO. Suitable cell lines for use in exogenous expression of substrate and/or MMP polypeptides include HeLa, HEK293 and CHO.

Compound(s)

Any compound(s) can be used in the method of the invention. The compound(s) are preferably ones that are suspected of enhancing or inhibiting MMP activity. The compound may be suspected of enhancing or inhibiting activity of MMP 14, MMP 15 and MMP 16. Alternatively, the compound may be suspected of enhancing or inhibiting activity of MMP 14 alone, MMP 15 alone or MMP 16 alone i.e as being a specific or selective inhibitor of MMP 14, MMP 15 or MMP 16. For example, a specific or selective enhancer or inhibitor of MMP 16 activity may be characterised as having no effect on activity of other MMPs.

The compound(s) can be in any suitable form. It is typically in solution. The solution typically comprises a suitable buffer. The solution may be cell medium or cell lysate from a cell culture expressing the compound(s). A polynucleotide encoding the compound may be provided in the cell in the same way as described above for MMP polypeptide and substrate polypeptide. The compound may be expressed in a cell together with the MMP polypeptide and/or the substrate polypeptide. The compound may be expressed in an inducible manner. The compound(s) may be any chemical compound(s) used in drug screening programmes. They may be natural or synthetic. Extracts of plants which contain several characterised or uncharacterised components may also be used. Typically, organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 Daltons. Compounds can be biomolecules including peptide and peptide mimetics, oligonucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate compounds may be obtained from a wide variety of sources including libraries of synthetic or natural substances. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. The compound(s) may be the product(s) of a combinatorial library such as are now well known in the art (see e.g. Newton (1997) Expert Opinion Therapeutic Patents, 7(10): 1183-1 194). Natural product libraries, such as display (e.g. phage display libraries), may also be used. Antibodies directed to the site of interaction between the MMP polypeptide and the substrate polypeptide are another class of suitable compounds. For example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and humanized antibodies may be used. The antibody may be an intact immunoglobulin molecule or a fragment thereof such as a Fab, F(ab')₂ or Fv fragment. Candidate inhibitor antibodies may be characterised and their binding regions determined to provide single chain antibodies and fragments thereof which are responsible for disrupting the interaction between the MMP polypeptide and the substrate polypeptide. A suitable antibody may bind to either the MMP polypeptide or the substrate polypeptide, and thereby prevent or block the interaction between these molecules. Antibodies may be raised against specific epitopes of the MMP polypeptide or the substrate polypeptide. For example, antibodies may be raised specifically against those regions which are involved in the interaction between the MMP polypeptide and the substrate polypeptide.

Further classes of compounds include oligonucleotides which act at the level of transcription of the MMP polypeptide. In one aspect, decreased functional expression of the MMP polypeptide may be achieved by inhibiting the expression from the MMP gene. For example, down-regulation of expression of MMP may be achieved using anti-sense technology or RNA interference. When using anti-sense genes or partial gene sequences to downregulate gene expression, a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene. See, for example, Smith et al, (1988) Nature 334, 724-726. Such methods would use a nucleotide sequence which is complementary to the coding sequence. Further options for down regulation of gene expression include the use of transcriptional inhibitors that bind to the MMP 5' promoter/regulatory region and ribozymes, e.g. hammerhead ribozymes, which can catalyse the site-specific cleavage of RNA, such as mRNA (see e.g. Jaeger (1997) Curr Opin Struct Biol 7:324-335, or Gibson & Shillitoe (1997) MoI Biotechnol 7: 242-251). RNA interference is based on the use of small double stranded RNA (dsRNA) duplexes known as small interfering or silencing RNAs (siRNAs). Such molecules are capable of inhibiting the expression of a target gene that they share sequence identity or homology to. Typically, the dsRNA may be introduced into cells by techniques such as microinjection or transfection. Methods of RNA interference are described in, for example, Hannon (2002) Nature 418: 244-251 and Elbashir et al (2001) Nature 411: 494- 498; Aigner A., J Biotechnol. 2006 Jun 124(1): 12-25.

The compounds may be specific or selective enhancers or inhibitors of MMP 14, MMP 15 or MMP 16 as described above. Alternatively, the compounds may be specific or selective enhancers or inhibitors of both MMP 14 and MMP 15 ; both MMP 14 and MMP 16; or both MMP 15 and MMP 16. In other embodiments, the compounds may enhance or inhibit MMP activity for all of MMP 14, MMP 15 and MMP 16. In further embodiments, the compounds may be broad-spectrum enhancers or inhibitors of MMP activity i.e enhance or inhibit MMP activity for all MMPs or for all membrane-bound MMPs, or for all type I transmembrane MMPs..

Methods for identifying potential therapeutic agents

The inventor has surprisingly shown that MMP 14, MMP 15 and MMP 16 cleave APP in a discrete region. In particular, MMP activity is shown by the Inventor to be specifically involved in generation of N-terminally extended and/or C-terminally truncated forms of Aβ, and N-terminally truncated Aβ species, in particular Aβ 3-40. Given that MMP is capable of cleaving APP to form variant Aβ species, such as Aβ 3-40, the method of the invention described above can be used for identifying compounds that enhance or inhibit APP processing, that enhance or inhibit Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles or that are suitable for the prevention or treatment of a disease associated with pathogenic APP processing.

In one embodiment, the invention provides a method for identifying a compound that enhances or inhibits Amyloid Precursor Protein (APP) processing. "Processing" is intended to refer to the proteolytic cleavage of APP into peptide fragments. In this embodiment, any method described above for identifying an enhancer of inhibitor of MMP activity is carried out. An increase in MMP activity in the presence of the compound compared with said control value identifies said compound as an enhancer of APP processing. A decrease in MMP activity in the presence of the compound compared with said control value identifies said compound as an inhibitor of APP processing.

Compounds that enhance or inhibit APP processing will typically enhance or inhibit the formation of Aβ species. They may enhance or inhibit formation of Aβ 1-40 or Aβ 1-42, or enhance or inhibit formation of variant Aβ species. Variant Aβ species include N-terminally truncated, N-terminally extended and/or C-terminally truncated Aβ species. N-terminally extended Aβ species include any peptides derived from APP which comprise additional N-terminal residues with respect to Aβ 1-40. Typically, such species comprise 14 to 25 additional N-terminal residues. C-terminally truncated Aβ species include any peptides derived from APP which lack one or more residues comprised in Aβ 3-40. Typically, such species lack 23 to 26 residues comprised in Aβ 3-40. Preferred N- terminally extended and C-terminally truncated Aβ species include: Aβ-25 to +14 (DRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHH, SEQ ID NO: 67), Aβ-22 to +14 (LTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHH, SEQ ID NO:

68), Aβ-16 to +14 (SGLTNIKTEEISEVKMDAEFRHDSGYEVHH, SEQ ID NO: 69) or Aβ- 14 to +14 (LTNIKTEEISEVKMDAEFRHDSGYEVHH, SEQ ID NO: 70).

N-terminally truncated Aβ species include any peptides derived from APP which lack one or more residues comprised in Aβ 1-14. Typically, such species lack from 3 to 14 residues comprised in Aβ 1-14. A particularly preferred N-terminally truncated Aβ species is Aβ 3-40. In another embodiment, the invention provides a method for identifying a compound that enhances or inhibits Aβ aggregation or the formation of amyloid plaques and/or neurofibrillary tangles. The compound may enhance or inhibit Aβ aggregation or the formation of amyloid plaques and/or neurofibrillary tangles via effects on formation of Aβ 1-40 or Aβ 1-42. Alternatively, the compound may enhance or inhibit Aβ aggregation or the formation of amyloid plaques and/or neurofibrillary tangles via effects on formation of variant Aβ species as defined above. Where the compound inhibits Aβ aggregation or the formation of amyloid plaques and/or neurofibrillary tangles, it is preferred that the compound reduces formation of Aβ 3-40. In this embodiment, any method described above for identifying an enhancer of inhibitor of MMP activity is carried out. An increase in MMP activity in the presence of the compound compared with said control value identifies said compound as an enhancer of Aβ aggregation or the formation of amyloid plaques and/or neurofibrillary tangles. A decrease in MMP activity in the presence of the compound compared with said control value identifies said compound as an inhibitor of Aβ aggregation or the formation of amyloid plaques and/or neurofibrillary tangles. The ability of the compound to enhance or inhibit Aβ aggregation or the formation of amyloid plaques and/or neurofibrillary tangles may be subsequently tested using known assays for Aβ aggregation and the formation of amyloid plaques and/or neurofibrillary tangles. Any assays of this type known in the art may be used.

Examples of in vitro Aβ aggregation assays include LeVine H., Arch Biochem Biophys. 1997 342(2):306-16, Okuno H et al, Chem Biol Drug Des. 2006 Nov; 68(5): 273-5 , and Curr Alzheimer Res. 2007 Dec;4(5):544-6. In vivo animal models are reviewed in Gδtz J., et al., (2008) Nature Reviews Neuroscience 9, 532-544. Specific examples of in vivo models include Taconic model 001349 (which overexpresses APP containing the Swedish familial Alzheimer's disease mutation, where the APP BACE cleavage site is mutated from KMDA to NLDA), and Taconic model 001638 (which overexpresses the P301L tau mutant). These two types of mutations may be used alone or in combination. Mutations of presenilin genes 1 and 2 may also be included. In a further embodiment, the invention provides a method for identifying a compound suitable for the prevention or treatment of a disease associated with pathogenic APP processing. In this embodiment, any method described above for identifying an enhancer of inhibitor of MMP protease activity is carried out. A decrease in MMP activity in the presence of the compound compared with the control value identifies the compound as being suitable for treating a disease associated with pathogenic APP processing. It is preferred that a decrease in MMP activity involves a reduced formation of Aβ 3-40. Compounds which inhibit MMP 14, MMP 15 or MMP 16 activity are likely to be inhibitors of MMP-associated APP cleavage in vivo. They therefore have the ability to inhibit pathogenic APP processing and Aβ aggregation in vivo and thus prevent or treat a disease associated with pathogenic APP processing. The prevention or treatment of diseases associated with pathogenic APP processing is discussed in more detail below. Animal models in which a disease associated with pathogenic APP processing has been established can also be used to identify such compounds. Suitable animal models are described below.

Diseases of pathogenic APP processing involve altered APP processing. APP processing can be increased or decreased. Such diseases typically result from the toxic effects of peptide species derived from APP, commonly known as Aβ species. These peptides have a propensity to aggregate and form pathogenic deposits in tissue and/or blood vessels. Aβ peptides may be toxic to cells per se, and/or have additional toxicity or pathogenicity resulting from formation of deposits or aggregates. Preferred diseases associated with pathogenic APP processing include, but are not limited to, Alzheimer's disease cerebral amyloid angiopathy, and Parkinson's disease. The disease is preferably Alzheimer's disease.

Diagnostic method

As discussed above, the inventor has surprisingly demonstrated the involvement of MMP 14, MMP 15 and MMP 16 in the cleavage of APP to form variant Aβ peptides. Thus, altered MMP 14, MMP 15 or MMP 16 function may be used as a biomarker for diseases associated with pathogenic APP processing, particularly those involving the formation of the toxic N-terminally truncated Aβ peptide Aβ 3-40.

The invention therefore provides a method for identifying whether or not a subject is at risk of developing, or has, a disease associated with pathogenic APP processing. The method involves measuring the expression level and/or activity of MMP 14, MMP 15 or MMP 16 in a subject. The MMP 14, MMP 15 or MMP 16 polypeptide may be any MMP polypeptide described herein. It is preferred that the MMP polypeptide is selected from SEQ ID NOs: 15, 17, 19 or 21 or variants thereof. The activity is MMP activity, preferably metalloprotease or protease activity. The method may involve measuring the expression level of MMP 14, MMP 15 or MMP 16, the MMP activity of MMP 14, MMP 15 or MMP 16 or both the expression level and the MMP activity for MMP 14, MMP 15 or MMP 16. In some embodiments, expression level and/or MMP activity are measured for all three of MMP 14, MMP 15 and MMP 16. In other embodiments, expression level and/or MMP activity are measured for both MMP 14 and MMPl 5; both MMP 14 and MMP 16; or both MMP 15 and MMP 16. The expression level and/or activity of MMP 14, MMP 15 or MMP 16 is then compared with a normal value for MMP 14, MMP 15 or MMP 16 expression or activity. An increased level of MMP 14, MMP 15 or MMP 16 expression and/or an increased level of MMP 14, MMP 15 or MMP 16 activity in the sample compared with the normal level identifies the subject as being at risk of developing, or having, a disease associated with pathogenic APP processing. The disease associated with pathogenic APP processing can be any of those discussed above. It is preferably Alzheimer's disease.

In one embodiment, the invention relates to identifying whether or not the subject is at risk of developing the disease. The invention therefore relates to the diagnosis of susceptibility of a subject to the disease. This may allow for an early prophylactic or palliative treatment to prevent development of the disease. The invention may be used to confirm susceptibility in subjects already suspected as being at risk or selected as being predisposed to developing the disease. Risk factors that increase susceptibility to developing diseases associated with pathogenic APP processing include, but are not limited to, aging, lifestyle risk factors, genetic risk factors and environmental risk factors. The major genetic risk factors for early onset Alzheimer's disease are APP and presenilin mutations, and APP gene dosage (such as in Downs' syndrome). The main genetic risk factor for late onset Alzheimer's disease is the presence of the ApoE4 allele. Genetic risk factors are reviewed in Nat Genet. 2007 Jan;39(l): 17-23. Lifestyle risk factors are reviewed in Am J Epidemiol. 2002 Sep l;156(5):445-53. Biomarkers that may be used to identify susceptibility include: phospho tau in CSF (cerebrospinal fluid) (Hansson O., et ai, Lancet Neurol. 2006 Mar;5(3):228-34), Aβ40:Aβ42 CSF ratio (Shoji M, Kanai M. J Alzheimers Dis. (2001) (3):313-321), brain shrinkage as measured by MRI reference scanning (Sluimer JD., et ah, Neurology. 2008 May 6;70(19 Pt 2):1836-41), and PIB compound in vivo amyloid imaging (Forsberg A., Neurobiol Aging. 2008, Oct. 29(10): 1456-65).

In another embodiment, the invention relates to identifying whether or not the subject has the disease. The invention therefore relates to the diagnosis of the disease. Typically, the subject has the disease or displays symptoms of the disease. The method may therefore be carried out on subjects who display preliminary symptoms of the disease.

The method of the invention is carried out in vitro or ex vivo on a sample derived from the subject. The sample is preferably a fluid sample. The sample typically comprises a body fluid. The sample may be urine, lymph, saliva, cerebrospinal fluid, peritoneal fluid, pericardial fluid, vitreous or other ocular sample, pleural fluid, vaginal fluid, mucus, pus or amniotic fluid but is preferably blood, plasma or serum. The sample can be a cell or tissue sample, such as lung, brain, liver, skin or nails. The sample is preferably a brain tissue or cell sample. The sample is typically processed prior to its use in measurement of MMP expression level or activity.

Typically, the subject is human. However, it may be non-human. For instance, the subject can be a commercially farmed animal, such as a horse, cow, sheep or pig, or may be a pet such as a cat or a dog. Preferred non-human animals include, but are not limited to, primates, such as a marmoset or monkey. The subject can be a human or non-human animal undergoing treatment for a disease associated with pathogenic APP processing.

The activity of MMP in the sample may be measured by any method known in the art or described herein. Measuring MMP activity for MMP 14, MMP 15 or MMP 16 may comprise measuring the levels of N-terminally extended Aβ peptides derived from APP, preferably Aβ-25 to +14, Aβ-22 to +14, Aβ-16 to +14 or Aβ-14 to +14 (SEQ ID NOs: 67- 70) and optionally further measuring the level of Aβ 3-40.

Measurement of MMP expression level is typically performed at the mRN A or protein level, but MMP gene copy number may also be measured. Standard mRNA detection methodology is based on a quantitative or semi -quantitative measurement of the presence of a specific RNA molecule in the sample by a PCR technique, using one or more primers comprising a sequence derived from the molecule of interest i.e. MMP. Standard protein detection methodology may comprise use of an antibody specific to MMP in an immunological assay where binding of the antibody to MMP polypeptide generates a quantitative or semi-quantitative signal, for example ELISA.

A person skilled in the art will be able to determine a normal level of expression and/or MMP activity for a MMP polypeptide. It will typically be the average level of MMP expression and/or activity observed in a representative sample of a healthy population. The control population may be of similar age as the subject population.

Specifically, the control population does not have a disease associated with pathogenic

APP processing or any other disease or condition that is likely to result in altered MMP expression or MMP activity. This will allow for a statistically significant diagnosis to be performed on the basis of comparison with the normal level i.e. one which takes into account natural variation in MMP expression level or activity that is observed in the sample population.

The expression level and/or activity of MMP in a sample from a subject can also be used to monitor the progression of a disease associated with APP processing in a subject or the suitability of a treatment. The expression level and/or protease activity may be measured at suitable time intervals after diagnosis as described above. An increase in

MMP expression level and/or activity with time is indicative of a worsening of the disease.

A decrease in MMP expression level and/or activity with time is indicative of successful treatment of the disease.

Method of treatment and medical use

The invention also provides a method of preventing or treating a disease associated with pathogenic APP processing by administering an effective amount of an antagonist of MMP 14, MMP 15 or MMP 16 activity. The invention also provides an inhibitor or antagonist of MMP 14, MMP 15 or MMP 16 activity for use in a method of preventing or treating of a disease associated with pathogenic APP processing. The invention further provides use of an inhibitor or antagonist of MMP 14, MMP 15 or MMP 16 activity in the manufacture of a medicament for preventing or treating a disease associated with pathogenic APP processing. In all the above embodiments, the inhibitor or antagonist of MMP 14, MMP 15 or

MMP 16 activity may be administered in order to prevent the onset of one or more symptoms of the disease. In this embodiment, the subject can be asymptomatic. The subject may have a predisposition to the disease as described above. A prophylactically effective amount of the inhibitor or antagonist is administered to such a subject. A prophylactically effective amount is an amount which prevents the onset of one or more symptoms of the disease. Alternatively, the inhibitor or antagonist of MMP 14, MMP 15 or MMP 16 activity may be administered once the symptoms of the disease have appeared in a subject i.e. to cure existing symptoms of the disease. A therapeutically effective amount of the inhibitor or antagonist is administered to such a subject. A therapeutically effective amount is an amount which is effective to ameliorate one or more symptoms of the disease. Typically such an amount reduces the production of toxic Aβ variant peptides, such as Aβ 3-40, in the subject. This can be confirmed as described above.

The subject may be any of those discussed above in relation to the diagnostic method. The subject is preferably identified as being at risk of, or having, the disease using the method of the invention described above. The disease associated with pathogenic APP processing can be any of those discussed above. It is preferably Alzheimer's disease. As outlined above, Alzheimer's disease is a common form of dementia found mainly among older people. A common symptom of the disease is the formation of abnormal amyloid plaques and neurofibrillary tangles in brain tissue. This underlies the neurodegenerative phenotype. Antagonists or inhibitors of MMP 14, MMP 15 or MMP 16 activity may be used to prevent or delay the neurodegeneration observed in Alzheimer's disease or to ameliorate symptoms of dementia. Inhibitors/antagonists of MMP 14, MMP 15 or MMP 16 activity may also be used to prevent or slow growth of existing amyloid plaques and neurofibrillary tangles, or prevent or slow growth of new instances of these lesions, thereby stabilising an existing condition. In these embodiments, inhibitors/antagonists of MMP 14, MMP 15 or MMP 16 activity may primarily exert their effects through enhancing clearance of Aβ peptides.

As outlined above, cerebral amyloid angiopathy results from deposits of amyloid protein in small blood vessels in the brain which can cause stroke, brain haemorrhage or dementia. According to the invention, inhibitors/antagonists of MMP 14, MMP 15 or MMP 16 activity may be used to prevent or slow formation of such deposits, thereby treating or preventing cerebral amyloid angiopathy prior to an instance of a stroke or brain haemorrhage. Inhibitors/antagonists of MMP 14, MMP 15 or MMP 16 activity include all compounds which are inhibitors of MMP 14, MMP 15 or MMP 16 expression and/or MMP activity, preferably metalloprotease or protease activity. The inhibitors/antagonists may be specific or selective inhibitors of MMP 14, MMP 15 or MMP 16 activity as described above. Alternatively, the compounds may be specific or selective inhibitors of both MMP 14 and MMP 15 activity; both MMP 14 and MMP 16 activity; or both MMP 15 and MMP 16 activity. In other embodiments, the compounds inhibit MMP activity for all of MMP 14, MMP 15 and MMP 16. In further embodiments, the compounds may be broad-spectrum enhancers or inhibitors of MMP activity i.e enhance or inhibit MMP activity for all MMPs or for all membrane-bound MMPs or for all type I transmembrane MMPs.

An inhibitor of MMP 14, MMP 15 or MMP 16 activity is preferably identified in accordance with the invention. Inhibitors of MMP 14, MMP 15 or MMP 16 activity also specifically include any compound previously known in the art to act as an inhibitor of MMP protease activity or MMP expression. An inhibitor may inhibit MMP activity for any MMP polypeptide described hrein. It is preferred that the inhibitor inhibits MMP 14 of SEQ ID NO: 15, MMP 15 of SEQ ID NO: 17, or MMP 16 of SEQ ID NOs: 19 or 21 or a variant of any thereof as described above. The effect of a compound on expression and/or MMP activity may be measured as described above.

Preferred inhibitors/antagonists of MMP 14, MMP 15 or MMP 16 activity for use in accordance with the invention include, but are not limited to small organic molecules which have a molecular weight of from 50 to 2500 Daltons, antibodies directed to the site of interaction between the MMP polypeptide and the substrate polypeptide, and oligonucleotides which act to reduce transcription of the MMP polypeptide e.g. siRNAs. Inhibitors/antagonists of MMP 14, MMP 15 or MMP 16 activity may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other materials from their source or origin. Where used herein, the term "isolated" encompasses all of these possibilities. They may optionally be labelled or conjugated to other compounds.

Inhibitors/antagonists of MMP 14, MMP 15 or MMP 16 activity can be formulated into pharmaceutical compositions. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non- toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes. Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

For delayed release, the inhibitor/antagonist of MMP 14, MMP 15 or MMP 16 activity may be included in a pharmaceutical composition which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art. For continuous release of peptides, the peptide may be covalently conjugated to a water soluble polymer, such as a polylactide or biodegradable hydrogel derived from an amphipathic block copolymer, as described in U.S. Pat. No. 5,320,840. Collagen-based matrix implants, such as described in U.S. Pat. No. 5,024,841, are also useful for sustained delivery of peptide therapeutics. Also useful, particularly for subdermal slow-release delivery to perineural regions, is a composition that includes a biodegradable polymer that is self-curing and that forms an implant in situ, after delivery in liquid form. Such a composition is described, for example in U.S. Pat. No. 5,278,202.

The dose of an inhibitor/antagonist of MMP 14, MMP 15 or MMP 16 activity 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. Again, 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 inhibitor/antagonist, the age, weight and conditions of the subject to be treated and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g. That dose may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered daily.

Non-human animal and model of diseases of pathogenic APP processing

The inventors have surprisingly shown that MMP 14, MMP 15 and MMP 16 play a role in the formation of Aβ variant peptides, which are strongly implicated in the pathology of diseases of APP processing. MMP 14, MMP 15 and MMP 16 over-expression may therefore be used to generate an animal that displays symptoms similar to those displayed by a human subject that has been diagnosed with a disease of pathogenic APP processing. Such an animal is suitable for use as a model for studying diseases of pathogenic APP processing. The animal will also be suitable for identifying compounds that prevent or treat diseases of pathogenic APP processing.

The invention provides a non-human animal in which a disease of pathogenic APP processing has been established by over-expression of MMP 14, MMP 15 and MMP 16 and a method of generating such an animal. One or more of MMP 14, MMP 15 and MMP 16 may be over-expressed. Both MMP 14 and MMP 15 ; both MMP 15 and MMP 16 or both MMP 14 and MMP 16 may be expressed. Alternatively, all of MMP 14, MMP 15 and MMP 16 maybe over-expressed. Any MMP polypeptide described herein may be overexpressed. The MMP 14 may be that shown in SEQ ID NO: 15 or a variant thereof as described above. The MMPl 5 may be that shown in SEQ ID NO: 17 or a variant thereof as described above. The MMP 16 may be that shown in SEQ ID NO: 19 or 21 or a variant of either thereof as described above. The disease of pathogenic APP processing may be any of those discussed above.

The non-human animal can comprise further mutations or modifications to establish the disease. The disease may also be established by the coordinate expression of other genes or mutant forms thereof which impact on diseases of APP processing.

Preferred mutants include APP, presenilin and tau mutants either alone or in combination. Examples of suitable animal models in which MMP overexpression may be established include Taconic model 001349 (which overexpresses APP containing the Swedish familial Alzheimer's disease mutation, where the APP BACE cleavage site is mutated from KMDA to NLDA), and Taconic model 001638 (which overexpresses the P301L tau mutant). These two types of mutations may be present alone or in combination. Mutations of presenilin genes 1 and 2 may also be included. Other suitable animal models are described in Gotz J, et al cited above.

In one embodiment, an APP-type substrate of MMP which is not normally present in the animal is also expressed. A preferred example of such a substrate is human APP. Providing human APP in the context of MMP over-expression may allow for generation of a mouse disease model that more faithfully replicates the pathology of human diseases of APP processing.

MMP 14, MMP 15 or MMP 16 is expressed in a sufficient amount to cause or generate symptoms of pathogenic APP processing in the animals. The sufficient amount typically varies between animals and will depend on a number of factors, for example the age of the animal, and whether or not additional genes contributing to the disease of pathogenic APP processing are also present. The animal is non-human. The non-human animal is typically of a species commonly used in biomedical research, for example a mammal, and is preferably a laboratory strain. Suitable animals include non-human primates, dogs, cats, sheep and rodents. It is preferred that the animal is a rodent, particularly a mouse, rat, guinea pig, ferret, gerbil or hamster. Most preferably the animal is a mouse.

The animal over-expresses MMP 14, MMP 15 or MMP 16, optionally in combinations as described above. Techniques for generating transgenic non-human animals are well known in the art. Any such technique may be used. Any MMP polynucleotide described herein may be used. For instance, a polynucleotide construct comprising a promoter operably linked to a coding sequence for MMP 14, for example SEQ ID NO: 14 or a variant thereof, is produced. A coding sequence for MMPl 5, for example SEQ ID NO: 16 or a variant thereof may be used. Alternatively, a coding sequence for MMP 16, for example SEQ ID NO: 18 or 20 or a variant of either thereof may be used. The polynucleotide construct may be randomly integrated in the genome of the animal or may be targeted to a particular site. Targeting may be achieved by flanking the promoter and coding sequence with genomic sequences, which correspond to genomic sequences at the locus where insertion is required. Thus, if the polynucleotide construct is contacted with the locus of interest, homologous recombination events may lead to replacement of the chromosomal sequence with the promoter operably linked to the coding sequence for MMP. Targeting may take place to swap an endogenous MMP gene with a polynucleotide construct that allows for over-expression of the exogenous MMP gene. Alternatively, both an endogenous and an exogenous MMP gene may be present in the animal.

The polynucleotide construct is typically transferred into a fertilized egg of a mammalian animal by pronuclear microinjection. Alternative approaches may also be used. For example, embryonic stem cells or retroviral mediated gene transfer into germ lines may be used. Whichever approach is taken, transgenic animals are then generated. For example, microinjected eggs may be implanted into a host female and the progeny may be screened for the expression of the marker gene. The success of targeting may be monitored by use of an appropriate selection marker. The founder animals that are obtained may be bred. Preferred animals are mice in which the endogenous MMP gene has been replaced with a polynucleotide driving high level expression of an exogenous MMP gene. For example, the endogenous MMP 14 gene may be replaced with a polynucleotide driving high level expression of SEQ ID NO: 15 or a variant thereof as defined above. The endogenous MMP 15 gene may be replaced with a polynucleotide driving high level expression of SEQ ID NO: 17 or a variant thereof as described above. The endogenous MMP 16 gene may be replaced with a polynucleotide driving high level expression of SEQ ID NO: 19 or 21 or a variant of either thereof as described above. The invention provides a method of establishing a disease of pathogenic APP processing in a non-human animal comprising over-expressing MMP 14, MMPl 5, or MMP 16 in said animal in an amount sufficient to cause a disease of pathogenic APP processing. The method may involve the use of a transgenic technology as described above. The method may comprise over-expressing all of MMP 14, MMP 15 and MMP 16; or both MMP 14 and MMP 15; both MMP 15 and MMP 16; or both MMP 14 and MMP 16. The method may further comprise expression or over-expression in the non-human animal of one or more of genes that also contribute to onset and progression of the disease. Such genes are discussed above. The disease may be established in varying levels of severity by regulation of the expression levels of MMP 14, MMP 15, MMP 16 and the other genes mentioned above.

Known therapeutic compounds which are used to treat diseases of pathogenic APP processing, in particular Alzheimer's disease may be administered to the animal to investigate their impact on the disease model. In a related aspect, the invention provides a method for identifying a compound which prevents or treats a disease of pathogenic APP processing. It is preferred that the method identifies a compound which prevents or treats Alzheimer's disease. The method comprises administering a compound to a non-human animal of the invention and assessing whether or not the compound prevents or treats the disease of pathogenic APP processing. Compounds which prevent a disease of pathogenic APP processing reduce, prevent or delay the appearance of any symptoms of the disease. For example, where the disease is Alzheimer's disease, symptoms of dementia or appearance of amyloid plaques may be prevented or delayed. Substances which treat diseases of pathogenic APP processing may alleviate or abolish the symptoms of the disease in the animal.

The method of identifying compounds is typically carried out before or after the symptoms of the disease have developed in the animal. The method of identifying substances that prevent the disease is typically carried out before its symptoms have developed in the animal. The method of identifying substances that treat the disease is typically carried out after the symptoms have developed in the animal. Suitable compounds that can be tested in the above method include any of those described above.

Where a compound has been identified by the above method as being able to prevent or treat a disease of pathogenic APP processing in a non-human animal of the invention, it may then be used in medical applications. Thus, in one aspect, the invention provides a compound identified by the above method for use in a method of preventing or treating a disease associated with pathogenic APP processing. In a related aspect, the invention also provides for use of a compound identified by the above method in the manufacture of a medicament for prevention or treatment of a disease associated with pathogenic APP processing. In both of these aspects, it is preferred that the disease is Alzheimer's disease. A more detailed discussion of treating diseases associated with pathogenic APP processing can be found above. The invention also provides a kit that may be used to carry out the screening method of the invention. The kit comprises a type I transmembrane MMP, preferably MMP 14, MMP 15 or MMP 16, most preferably MMP 16, polypeptide and a substrate polypeptide. Any of the MMP polypeptides and substrate polypeptides described herein may be used. The kit may comprise MMP 14, MMP 15 and MMP 16 polypeptides; MMP 14 and MMP 15 polypeptides; MMP 15 and MMP 16 polypeptides; or MMP 14 and MMP 16 polypeptides.

The kit may additionally comprise one or more other reagents or instruments which enable any of the embodiments of the method mentioned above to be carried out. Such reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions), antibodies conjugated to detection moieties, substrates for enzymatically active tags, means to obtain a sample from a subject (such as a vessel or an instrument comprising a needle), means to measure MMP activity and/or expression or cell culture apparatus. Reagents may be present in the kit in a dry state such that a fluid sample resuspends the reagents. The kit may also, optionally, comprise instructions to enable the kit to be used in the method of the invention.

The following Example illustrates the invention, but are not intended to limit the invention in any way. The skilled person will be well aware of alternative ways of carrying out these and similar experiments.

Example 1: Cleavage of APP by MMP14, MMP15 and MMP16

L Materials and methods

1.1 Sources of cDNAs All human cDNAs were purchased from Origene in the pCMV6-XL4 mammalian expression vector:

Human MMP 14 (Accession #NM_004995, Origene #SC116990) Human MMP 15 (Accession #NM_002428, Origene #SC118648) Human MMP 16 (Accession #NM_005941, Origene #SC316625)

pCMV6-XL4 lacking any gene transcript was used as the empty- vector control.

1.2 HEK293 cell growth conditions

HEK293 cells were cultured in a humidified atmosphere of 5% CO2 at 37°C in Dulbecco's Modified Eagle's Medium with 4500 mg/L glucose (Sigma #D6546) supplemented with 5% (v/v) bovine foetal calf serum (PAA laboratories #A 15-003), 100 units/mL penicillin and 100 micrograms/mL streptomycin (Invitrogen #15140-114) and 2 millimolar L-glutamine (Invitrogen #25030-032).

1.3 ELLIN cell growth conditions

ELLIN cells (described in patent application WO2008084254) were cultured in a humidified atmosphere of 5% CO2 at 37⁰C in a 1:1 mix of Minimum Essential Medium (Sigma- Aldrich #M2279) and HAM'S Fl 2 medium (Invitrogen #21765-029) supplemented with 15% (v/v) bovine foetal calf serum (PAA laboratories #A15-003), a 1 :100 dilution of IOOX Non-Essential Amino Acids (Sigma- Aldrich #M7145), 100 units/mL penicillin and 100 micrograms/mL streptomycin (Invitrogen #15140-114). 1.4 cDNA transfection protocol

HEK293 cells or ELLIN cells were transfected using Lipofectamine 2000 (Invitrogen #1 1668-027) using the standard manufacturer's protocol. 48 hours after transfection cell media were harvested for analysis of Aβ levels. Aβl-40 levels were determined using an HTRF kit purchased from CisBio (#62B40PEB). Aβx-40 and Aβx-42 ELISAs were carried out using ELISA kits purchased from WAKO (#294-62501 and #290-62601 respectively). Cell viability was determined using Alamar Blue (Biosource #D ALl 025). Cell lysates were also harvested and both full-length APP and APP C-terminal fragment levels analysed by Western blotting using an anti-APP antibody (1 :2000 Invitrogen #51-2700).

1.5 Immunoprecipitation Mass-Spec Protocol HEK293 cells were transfected with cDNAs as described. Media samples were harvested after 48hrs and Aβ species then immunoprecipitated as follows. A slurry of G- Plus/Protein A agarose beads (Calbiochem #IP05) was prepared and 5mL of beads per sample were combined with 5 mL of 1 mg/mL 4G8 (Signet labs #9220-02) or 6E10 (Signet #9320-02) anti-human Aβ monoclonal antibody. 10 mL of conditioned media taken from the transfected cells were supplemented with protease inhibitors (Roche

#11697498001) and then spiked with 10 picomoles of Aβ 12-28 (Bachem #H-7865.1000) as an internal control for success of the immunoprecipitation. The cell media were then mixed with the beads/antibody mixture and CHAPS detergent added to a final concentration of 1% v/v. Media samples were rotated overnight at 4°C. The following day the beads were collected by centrifugation and washed twice with 500 mL ice cold 10 millimolar Tris-HCl pH 8, 140 millimolar NaCl, 0.01% w/v NOG (Anatrace, Inc. #0331). The beads were then washed once with 500 mL ice cold 10 millimolar Tris-HCl pH 8 and once with distilled water. Aβ peptides were eluted with 50% acetonitrile, 2.5% TFA, 0.2% NOG and spotted onto a MALDI target. MALDI-TOF analysis was then carried out. 1.6 A31-40 Clearance Assay

HEK293 cells were transfected with cDNAs as described. 24hrs later transfected cells were treated with conditioned media from ELLIN neuroblastoma cells containing Aβ species accumulated over 48hrs. The HEK293 cells were also treated with an excess of γ- secretase inhibitor (1OnM LY411,575) to prevent any further Aβ production. Aβl-40 levels were tracked over 72hrs by HTRF assay (Cis-Bio # 62B40PEB). Cell viability was determined at 72hrs using Alamar Blue (Biosource #DAL1025).

2 Results

2.1 Effects of MMP 14, MMP 15 or MMP 16 over-expression on APP cleavage

MMP 14, MMP 15 or MMP 16 was over-expressed in HEK293 cells by transient transfection. Cell lysate was taken from control cells transfected with empty vector and cells over-expressing MMP 14, MMP 15 or MMP 16. The cell lysate was processed by SDS-PAGE and Western blotting was performed with an anti-APP antibody (Invitrogen # 1-7300). The results are shown in Fig. IA. The Western blot indicates that MMP14 and MMP 16 cleave APP upstream of the BACE cleavage site generating novel C-terminal fragments.

An ELISA assay (WAKO # 294-62501) was then used to investigate further the effects of MMP 14, MMP 15 or MMP 16 over-expression on APP cleavage in HEK293 cells. The results are shown in Fig. IB. MMP 14, MMP 15 or MMP 16 over-expression in parental HEK293 cells significantly increased secretion of AβX-40.

The effects of MMP 14, MMP 15 or MMP 16 over-expression were also investigated in ELLIN neuroblastoma cells. These cells secrete relatively high levels of endogenous Aβ. The results shown in Fig. 1 C indicate that MMP 14, MMP 15 or MMP 16 upregulate production of AβX-42 similarly to AβX-40 (as measured by WAKO ELISAs #290-62601 and #294-62501). The overall increases in AβX-40/42 production were comparatively lower in this cell line compared to HEK293 due to reduced efficiency of cDNA transfection. 2.2 Identification of cleavage sites for MMP 14, MMP 15 or MMP 16 in a discrete region of human APP

The nature of the Aβ species produced by MMP 14, MMP 15 or MMP 16 transfected HEK293 cells was further investigated by mass spectrometry. Lysates from empty vector control cells and cells over-expressing MMP 14, MMP 15 or MMP 16 were immunoprecipitated using the anti-Aβ antibodies 4G8 (Signet #9220-02) and 6E10 (Signet #9320-02). The anti-Aβ antibody 4G8 is also described in Kim KS, et al. Neurosci Res. (1988) Comm 2: 121-130. The 4G8 antibody binds an epitope within amino acids 17-24 of the Aβ peptide sequence. N-terminally truncated Aβ can hence be immunoprecipitated with this antibody. The 6E10 antibody binds an epitope within amino acids 1-16 of Aβ. C-terminally truncated Aβ can hence be immunoprecipitated with this antibody.

The immunoprecipitated species were then analysed by mass spectroscopy. MMP 14, 15 and 16 were shown to cleave APP at a variety of sites including after 3 and 14 amino acids downstream of the BACE cleavage site and before 14, 22 and 25 amino acids upstream of the BACE cleavage site. These cleavages are summarised in Figures 2, 3 and 4. The cleavage events result in the production of several N-terminally extended Aβ fragments with the major species being Aβ -25 to + 14, Aβ -22 to +14 and Aβ -14 to + 14.

In addition the production of Aβ 3-40 was increased. This species is known to form highly toxic pyroglutamate Aβ (Biological Chemistry (2008), 389(8): 993-1006) and is potentially of great significance in Alzheimer's disease. Inhibition of the formation of pyroglutamate Aβ was recently shown to ameliorate both cognition and pathology in an animal model of Alzheimer's disease (Nat Med. (2008) 14(10):l 106-11). Inhibition of MMP-mediated Aβ 3-40/42 formation may hence provide an alternative therapeutic strategy for Alzheimer's disease.

2.3 Effects of MMP 14, MMP 15 or MMP 16 over-expression on clearance of Aβ

It was tested whether MMP 14, MMP 15 or MMP 16 is capable of cleaving isolated Aβ peptide. The results are shown in Fig. 5. Expression of MMP 14, MMP 15 or MMP 16 increased clearance/degradation of endogenous Aβl-40 from conditioned cell media

Herived from HEK293 cells indicating that it is possible for these MMPs to cleave isolated Aβ. MMP 16 for example is highly expressed in the hippocampus (Nature. 2007 Jan 11 ;445(7124): 168-76) and has been found to be particularly expressed in microglia (Acta Neuropathol. 1998 Oct;96(4):347-50) which have been shown to be responsible for Aβ clearance (FEBS Lett. 2007 Feb 6;581(3):475-8).

Example 2: Degradation of ApoE by MMP16

Methods

Experimental Details

Sources of cDNAs All human MMP cDNAs were purchased from Origene in the pCMV6-XL4 mammalian expression vector:

Human MMP 14 (Accession #NM_004995, Origene #SC1 16990)

Human MMP 15 (Accession #NM_002428, Origene #SC1 18648)

Human MMP 16 (Accession #NM_005941, Origene #SC316625) Human ApoE4 (Accession # NM_000041 , Origene # SC319433) pCMV6-XL4 lacking any gene transcript was used as the empty-vector control.

The ApoE4 cDNA was mutated to ApoE3 by changing Argl 12 to Cy s (CGC→TGC) using the QuikChange Multi Site-Directed mutagenesis kit (Stratagene Europe #200513). A V5/6His tag was added to the C-terminus by PCR using the pcDNA3.1/V5-His TOPO TA cloning kit (Invitrogen #K4800-01).

HEK293 cell growth conditions

HEK293 cells were cultured in a humidified atmosphere of 5% CO2 at 37°C in Dulbecco's Modified Eagle's Medium with 4500 mg/L glucose (Sigma #D6546) supplemented with 5% (v/v) bovine foetal calf serum (PAA laboratories #A 15-003), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen #15140-1 14) and 2 mM L- glutamine (Invitrogen #25030-032).

cDNA transfection protocol

HEK293 cells or ELLIN cells were transfected using Lipofectamine 2000 Clnvitrogen #1 1668-027) using the standard manufacturer's protocol. 48 hours after transfection cell media were harvested for analysis of Aβ levels. Aβx-40 ELISA measurements were carried out using an ELISA kit purchased from WAKO (#294-62501). Cell viability was determined using Alamar Blue (Biosource #DAL1025).

Cell lysates also harvested and ApoE degradation analysed by Western blotting using either a polyclonal anti-ApoE antibody (1 : 1000 Sant Cruz Biotechnology # sc-6383) or a monoclonal anti-V5 tag antibody (Invitrogen #R960-25).

Results

MMP 14, MMP 15 or MMP 16 were co-over-expressed with either wild- type or

C-terminally V5-tagged ApoE3 in HEK293 cells by transient transfection. Cell lysates were taken 48hrs after transfection. Cell lysates were processed by SDS-PAGE and Western blotting was performed with either either a polyclonal anti-ApoE antibody (1 :1000 Santa Cruz Biotechnology # sc-6383) or a monoclonal anti-V5 tag antibody (Invitrogen #R960-25).

The results are shown in Figure 7. ApoE3 C-terminally tagged with a V5/6His tag was co-over-expressed with MMP14, MMP15 or MMP16. MMP16 induced significant degradation of ApoE3-V5 with enhanced production of two C-terminal cleavage products in the 17-24 kDa range, identified using an anti-V5 tag antibody (Figure 7A). Enhanced degradation of untagged ApoE3 was also seen when co-over-expressed with MMP 16. Production of several cleavage fragments was enhanced. One of these, taking into account the 5kDa size of a V5/6His tag, was similar in size to a C-terminal fragment seen with ApoE-V5 over-expression (Figure 7B).

An ELISA assay (WAKO # 294-62501) was used to confirm successful over- expression of MMP 14, MMP15and MMP 16. The results are shown in Fig. 7C. All MMPs significantly increased secretion of AβX-40 indicating successful over-expression.

Claims

1. A method for identifying a compound that enhances or inhibits matrix metalloproteinase (MMP) activity, the method comprising: a) contacting a type-I transmembrane MMP polypeptide or a variant thereof with the compound; b) contacting the type-I transmembrane MMP polypeptide or variant thereof with a substrate polypeptide comprising a motif of at least ten amino acids from SEQ ID NO:7 or an equivalent thereof; c) measuring MMP activity by monitoring cleavage of the substrate polypeptide immediately following one or more of residues 9, 12, 18, 20, 36 and 48 of SEQ ID NO: 7 or the corresponding residues in an equivalent thereof; and d) comparing the MMP activity measured in c) with a control value obtained for an MMP polypeptide that has not been contacted with the compound, and thereby determining whether the compound is an enhancer or inhibitor of MMP activity; wherein an increase in MMP activity compared with said control value identifies the compound as being an enhancer of MMP activity; and wherein a decrease in MMP activity compared with said control value identifies the compound as being an inhibitor of MMP activity.

2. A method according to claim 1 , wherein said motif is selected from one or more of: a) ARPAADRGLT (motif A); b) AADRGLTTRP (motif B); c) TTRPGSGLTN (motif C); d) RPGSGLTNIK (motif D); e) VKMDAEFRHD (motif E); f) YEVHHQKLVF (motif F); or g) an equivalent of any of motifs A to F; and wherein MMP activity is measured by monitoring cleavage immediately following residue 5 of said motifs A to F or the corresponding residue in an equivalent thereof.

3. A method according to claim 1 or 2, wherein the substrate polypeptide comprises SEQ ID NO: 7 or an equivalent thereof.

4. A method according to any one of claims 1 to 3, wherein said substrate polypeptide comprises the amino acid sequence of human APP of SEQ ID NO: 2, 4 or 6, or a fragment thereof.

5. A method according to claim 1, wherein said type-I transmembrane MMP polypeptide is an MMP 16 polypeptide and comprises the amino acid sequence of SEQ ID NO: 19 or 21 or a variant of either thereof.

6. A method according to claim 1, wherein said type-I transmembrane MMP polypeptide is an MMP 14 polypeptide and comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof, or said type-I transmembrane MMP polypeptide is an MMP 15 polypeptide and comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof. -_^

7. A method according to any one of the preceding claims, wherein measuring MMP activity involves fluorescence, an immunoassay or mass spectrometry.

8. A method according to any one of the preceding claims, wherein measuring MMP activity involves detecting one or more specific cleavage products derived from said substrate polypeptide.

9. A method according to claim 8, wherein the specific cleavage products are selected from: a) N-terminally extended Aβ peptides or variants thereof, where the substrate polypeptide comprises one or more of motifs A to E or equivalents thereof; b) Aβ 3-40 or a variant thereof, where the substrate polypeptide comprises motif E or an equivalent thereof.

10. A method for identifying a compound that enhances or inhibits amyloid precursor protein (APP) processing, the method comprising carrying out a method according to any one of the preceding claims and thereby identifying a compound that enhances or inhibits APP processing, wherein an increase in MMP activity in the presence of said compound compared with said control value identifies said compound as an enhancer of APP processing; and wherein a decrease in MMP activity in the presence of said compound compared with said control value identifies said compound as an inhibitor of APP processing.

11. A method for identifying a compound that enhances or inhibits Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles, the method comprising carrying out a method according to any one of the preceding claims and thereby identifying a compound that enhances or inhibits Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles, wherein an increase in MMP activity in the presence of said compound compared with said control value identifies said compound as an enhancer of Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles; and wherein a decrease in MMP activity in the presence of said compound compared with said control value identifies said compound as an inhibitor of Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles.

12. A method for identifying a compound that enhances or inhibits Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles, the method comprising the following steps: a) contacting an MMP 16 polypeptide or a variant thereof with the compound; b) contacting the MMP 16 polypeptide or variant thereof with a substrate polypeptide comprising ApoE or an equivalent thereof; c) measuring MMP activity by monitoring cleavage of the substrate polypeptide; and d) comparing the MMP activity measured in c) with a control value obtained for an MMP polypeptide that has not been contacted with the compound, and thereby determining whether the compound is an enhancer or inhibitor of MMP activity; wherein an increase in MMP activity compared with said control value identifies the compound as being an enhancer of Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles; and wherein a decrease in MMP activity compared with said control value identifies the compound as being an inhibitor of Aβ aggregation or formation of amyloid plaques and/or neurofibrillary tangles.

13. The method of claim 12, wherein the compound enhances ApoE activity and reduces Aβ oligomerisation.

14. The method of claim 12 or 13, wherein the ApoE is selected from ApoE2, ApoE3 and ApoE4 or equivalents thereof.

15. A method for identifying a compound suitable for the prevention or treatment of a disease associated with pathogenic APP processing, the method comprising carrying out a method according to any one of the preceding claims and thereby identifying a compound suitable for the prevention or treatment of a disease associated with pathogenic APP processing, wherein a decrease in MMP activity in the presence of said compound compared with said control value identifies said compound as being suitable for the prevention or treatment of a disease associated with pathogenic APP processing.

16. A method for identifying whether or not a subject is at risk of developing, or has, a disease associated with pathogenic APP processing, said method comprising: a) measuring the expression level and/or MMP activity of a type-I transmembrane MMP in a sample derived from said subject; b) comparing the type-I transmembrane MMP expression level and/or MMP activity measured in said sample to a normal level of the type-I transmembrane MMP expression and/or activity and thereby identifying whether or not a subject is at risk of developing, or has, a disease associated with pathogenic APP processing; wherein an increased level of type-I transmembrane MMP expression and/or an increased level of type-I transmembrane MMP activity in said sample compared with the normal level identifies the subject as being at risk of developing, or having, a disease associated with pathogenic APP processing.

17. A method according to claim 16, wherein measuring MMP activity in step a) comprises measuring the levels of N-terminally extended Aβ peptides derived from APP, preferably Aβ-25 to +14, Aβ-22 to +14, Aβ-16 to +14 or Aβ-14 to +14 and optionally further measuring the level of Aβ 3-40.

18. An inhibitor of type-I transmembrane MMP activity for use in a method of preventing or treating a disease associated with pathogenic APP processing.

19. An inhibitor of type-I transmembrane MMP activity according to claim 18 wherein said disease is Alzheimer's disease.

20. Use of an inhibitor of type-I transmembrane MMP activity in the manufacture of a medicament for preventing or treating a disease associated with pathogenic APP processing.

21. Use according to claim 20 wherein said disease is Alzheimer's disease.

22. A method of treating or preventing a disease associated with pathogenic APP processing in a subject, comprising administering to the subject an effective amount of an inhibitor of type-I transmembrane MMP activity.

23. A method according to claim 22, wherein said subject has been identified as being at risk of developing, or having, a disease associated with pathogenic APP processing using a method according to claim 16 or 17.

24. A method according to any one of claims 15, 16, 17, 22, and 23, wherein said disease is Alzheimer's disease.

25. A non-human animal in which a disease of pathogenic APP processing has been established by over-expression of a type-I transmembrane MMP.

26. A non-human animal according to claim 25, wherein said disease is Alzheimer's disease.

27. A method for establishing a disease of pathogenic APP processing in a non-human animal comprising over-expressing a type-I transmembrane MMP in said animal in an amount sufficient to cause a disease of pathogenic APP processing.

28. A method for identifying a compound which prevents or treats a disease of pathogenic APP processing, comprising administering said compound to a non-human animal as defined in claim 25 or 26 and assessing whether or not said compound prevents or treats the disease of pathogenic APP processing.

29. A method according to claim 27 or 28, wherein said disease is Alzheimer's disease.

30. A compound identified by the method of claim 28 for use in a method of preventing or treating a disease associated with pathogenic APP processing.

31. A compound according to claim 30, wherein said disease is Alzheimer's disease.

32. Use of a compound identified by the method of claim 28 in the manufacture of a medicament for prevention or treatment of a disease associated with pathogenic APP processing.

33. Use according to claim 32, wherein said disease is Alzheimer's disease.

34. A kit comprising a type-I transmembrane MMP polypeptide or a variant thereof and a substrate polypeptide comprising a motif of at least ten amino acids from SEQ ID NO: 7 or an equivalent thereof.

35. The method, use, compound, non-human animal or kit of any of claims 16 to 34, wherein the type-I transmembrane MMP is selected from MMP 16, MMP 14 and MMP 15 or variants thereof.