WO2003064681A2

WO2003064681A2 - Assays

Info

Publication number: WO2003064681A2
Application number: PCT/GB2003/000433
Authority: WO
Inventors: Fiona Ralitsa Lucas; Joanne Taylor
Original assignee: Eisai London Research Laboratories Limited
Priority date: 2002-01-31
Filing date: 2003-01-31
Publication date: 2003-08-07
Also published as: AU2003238461A1; GB0202276D0; WO2003064681A3

Abstract

The present invention relates to a protein comprising amyloid precursor protein (APP) and an amino-acid tag, a nucleic acid molecule encoding the protein, a vector comprising the nucleic acid molecule, and a host cell comprising the nucleic acid molecule or the vector. The present invention also relates to assays for measuring Ϝ-secretase activity, and/or simultaneously measuring α-, β-, and Ϝ-secretase activity, which involve utilising the stabilisation of the Ϝ-secretase C-terminal-cleavage product of APP. Such assays are useful in the screening or testing of secretase inhibitors and differentiating such inhibitors from agents that act indirectly, e.g. interface with normal APP biosynthesis and activity.

Description

ASSAYS

The present invention relates to assays for measuring γ-secretase activity, and/or simultaneously measuring -, β-, and γ-secretase activity, which involve utilising the stabilisation of the γ-secretase C-terminal-cleavage product of APP (amyloid precursor protein). The APP containing an amino-acid tag may be expressed in intact cells. Such assays are useful in the screening or testing of secretase inhibitors and differentiating such inhibitors from agents that act indirectly, e.g. interfere with normal APP biosynthesis and activity. This assay is particularly useful for simultaneously measuring β-, α-, and γ- secretase in intact cells i.e. under physiological conditions, and therefore for identifying therapeutic compounds or compositions for the treatment of diseases such as Alzheimer's disease.

Recently there has been a lot of literature interest in the role of γCTF(C-terminal f agment of APP). γCTF is generated by the action of γ-secretase/presenilin 1, an important enzyme in Alzheimer's disease. γCTF is the sister product of beta-amyloid, which is also generated by the action of γ-secretase/presenilin 1 (see Figure 1). Beta-amyloid accumulates in senile plaques in Alzheimer's disease.

Some recent reports suggest γCTF may have a signaling role relevant to the pathology of Alzheimer's disease. However, literature articles report that scientists have struggled with the instability of γCTF. If γCTF could be stabilised in human cells it would provide insight into any potential role of γCTF related to its stability. Literature about γCTF has been scarce due to its instability and therefore difficulty in its study. Recently, literature about γCTF is emerging, although all the reports use sensitive or elaborate measures to detect endogenous γCTF.

β-secretase and γ-secretase action upon APP is considered to be a central event in the pathogenesis of Alzheimer's disease (being involved in the final cleavage that produces the toxic β-amyloid peptide). Therefore, much effort is being directed toward inhibiting these enzymes as a therapeutic approach to prevent Alzheimer's disease progression. Toward this aim it is highly desirable to be able to screen for β-secretase and γ-secretase inhibition under physiological conditions, i.e. in intact cells. Current drug screens for measuring β- and/or γ-secretase inhibition, involve measurement of Aβ production from cultured cells. However, inhibition of Aβ production may be due to a number of different mechanisms, including: inhibition of β-secretase and/or γ-secretase activity, stimulation of α-secretase activity, alterations in the intracellular compartments containing the secretases, alterations in APP transcription, biosynthesis, trafficking, and so on. The ability to simultaneously measure the activity of each secretase relative to the others and total APP in a drug screen would help elucidate the mechanism of action of a potential drug, and importantly, whether it is a specific inhibitor of β-secretase or γ-secretase activity. The intracellular levels of the direct products of α-, β-, and γ-secretase, CTF, βCTF, and γCTF respectively, would provide such an accurate and simultaneous measure of the individual secretase activities in response to a drug. Currently there is no simple method for simultaneously measuring the activity of the individual secretases, partly due to the extreme instability of γCTF, the product of γ-secretase. Therefore the unique stability of γCTF in cells using the method of the present invention, provides a unique and novel assay which can measure the activities of all secretases simultaneously to identify if and/or how an agent is inhibiting the production of Aβ, and thereby differentiate between β-secretase and γ-secretase inhibitors, or otherwise.

The Inventors have developed a means of stabilising and hence causing accumulation of γCTF (see Figure 2) which is otherwise unstable, by the addition of an amino-acid-tag to the C-terminus of APP expressed in cells. This in turn enables an assay in which γCTF is stabilised and is readily detectable. This assay improves on previous assays [e.g. cell-free generation of CTFs; prior artificial elevation of γ-secretase substrates βCTF and αCTF by treating with a γ-secretase inhibitor, (e.g. WO 98/15828); measurement of Aβ level in medium] because it provides a simple one-step assay for simultaneously measuring the specific activities of α-, β- and γ-secretase relative to one another, directly in a physiological system. Thus, it provides a novel assay that differentiates between inhibitors of different secretases and agents that act indirectly, e.g. interfere with normal APP biosynthesis and activity (see Figure 3). Thus in one aspect, the invention provides a protein comprising amyloid precursor (AP) protein and an amino acid tag. The protein is thus a modified AP protein. An AP protein as defined herein may contain all or part of a mammalian AP protein sequence. AP protein (APP) refers to any of the differentially spliced isoforms (inc. 695, 714, 751 and 770 amino acids). Essentially, the modified AP protein carries an amino acid tag positioned between the γ-secretase cleavage site and the C-terminus of APP (including placement of the tag at the C-terminus of APP, i.e. additional to the APP C-terminal sequence). Where the tag is positioned before the C-terminus, it may replace existing sequence such that the C- terminus of the tag forms the C-terminus of the APP. Preferably, the amino acid tag is present at the C-terminus of the APP amino acid sequence.

In a particularly preferred embodiment the amino acid tag is a N5-6His amino-acid-tag (Invitrogen). However, any tag that stabilises the γCTF can be used. Preferably the tag is an amino acid tag. It is usually between 6 and 30 residues in length. It may contain a repeat residue up to 10 times, for example 3, 4, 5, 6, 7, 8, 9 or 10 histidine residues. This repeat residue may be found at the C-terminus of the APP.

All variants and/or fragments of modified APP that are capable of undergoing cleavage by γ-secretase to give at least one detectable cleavage product are considered to fall within the scope of the first aspect of invention. For instance, those skilled in the art would understand that a modified APP can be produced that has been additionally altered such that the wild-type sequence of APP is not present (kept identical) either through additional, missing and/or substituted amino acids. Such additional alterations may be made to improve the usefulness of the modified APP in the assay. Alternatively, such additional alterations may be made simply to provide an equally useful APP for secretase assays which has a different amino acid sequence to that described herein. In a preferred embodiment, a mutation (for example the Swedish mutation of APP - Citron, M. et al. Nature 360, 672-674 (1992) of APP is introduced into or present in the sequence encoding the modified APP, in order to produce a protein which is more sensitive to cleavage by β- secretase. This allows βCTF to be readily produced, therefore making it detectable in simultaneous α-, β- and/or γ-secretase secretase assays. The protein of the first aspect of the invention may be purified. It may consist essentially of AP protein and an amino acid tag, both as described above.

In a second aspect, the invention provides a nucleic acid sequence encoding a protein according to the first aspect of invention. The nucleic acid may be isolated. Essentially, the proteins encoded by such nucleic acid sequences are capable of undergoing cleavage by γ-secretase to give at least one detectable cleavage product. The amino acid tag prevents degradation of at least one cleavage product. The nucleic acid sequence can be present in an expression vector suitable for the expression of the protein. In a preferred embodiment, the protein is encoded by a plasmid suitable for expression in HEK 293 or any other expression system, such as pcDNA3.1/N5-His-TOPO (Invitrogen). In accordance with the present invention, standard vectors for recombinant mammalian cell expression can be used. In one embodiment, the protein of the invention and nucleic acid encoding it are derived from the sequence of APP695, see Figure 4 (accession number Y00264).

All preferred features of the first aspect of the invention, also apply to the second aspect.

A third aspect of the invention thus provides a host cell comprising the nucleic acid of the second aspect of the invention. The host cell may be transfected or transformed with the nucleic acid. The host cell is preferably in vitro. The host cell may be mammalian (e.g. human, rat, mouse, hamster or other) or non-mammalian.

All preferred features of the first and second aspects also apply to the third aspect.

A fourth aspect of the invention provides a method of making a protein according to the first aspect of the invention or a nucleic acid encoding such a protein according to the second aspect of the invention. Such methods are standard in the art. A method of making the protein comprises expressing the protein in an expression system which comprises nucleic acid encoding the protein. The expression system is preferably in a number of host cells which comprise nucleic acid encoding the protein (as described according to the second and third aspects of the invention). The protein can be produced by introducing APP cDNA into a plasmid to incorporate a tag or by cloning APP cDNA and adding a tag sequence before introducing into a plasmid.

A method of making the nucleic acid encoding the protein includes coupling together nucleic acid residues to produce a coding sequence.

The fourth aspect of the invention also provides a method of making a host cell according to the third aspect of the invention. Again, such a method itself is standard in the art. Generally, it includes transforming or transfecting a suitable host cell with nucleic acid according to the second aspect of the invention.

In a fifth aspect, the invention provides a method for determining the activity of γ-secretase, which comprises the steps of

1) providing a protein according to the first aspect of the invention;

2) bringing said protein into contact with a γ-secretase; and

3) determining the presence and/or amount of a γ-secretase product derived from said protein.

In step 1, the protein is preferably produced by introducing APP cDNA into a plasmid to incorporate a tag or cloning APP cDNA and adding a tag sequence before introducing into a plasmid.

The protein is preferably produced by transfecting the plasmid containing modified APP encoding nucleic acid into a suitable cell line, using standard transfection procedures.

Step 3 may, of course, be carried out in a number of different ways. Method 1 (preferred because it is the simplest) involves taking whole cell lysates of cells expressing the protein (APP+tag), electrophoresing on a low MW (molecular weight) resolving gel, Western blotting, and detecting all CTFs including γCTF using antibody directed against the tag or APP C-terminus. Finally, quantitation of γCTF relative to αCTF and βCTF as a measure of γ-secretase activity (see Fig 3); Method 2 involves immunoprecipitation of all CTFs including γCTF, from whole cell lysates of cells expressing the protein, using antibody directed against the tag or APP C-terminus, followed by standard PAGE and detection methods. Finally, quantitation of γCTF relative to αCTF and βCTF as a measure of γ- secretase activity (see Fig 3).

In a sixth aspect, the invention provides a method of simultaneously measuring the activities of α-, β- and γ-secretases comprising the steps of

1) providing a protein according to the first aspect of the invention;

2) bringing said protein into contact with α-, β- and γ-secretase enzymes; and

3) determining the presence and/or relative amounts of α-, β- and γ-secretase products derived from said protein.

In step 1, the protein is preferably produced by introducing APP cDNA into a plasmid to incorporate a tag; or cloning APP cDNA and adding a tag sequence before introducing into a plasmid and by transfecting the plasmid containing APP+tag sequence into a suitable cell line, using standard transfection procedures;

Step 3 may, of course, be carried out in a number of different ways. Method 1 (preferred because it is the simplest) involves taking whole cell lysates of cells expressing the protein (APP+tag), electrophoresing on a low MW resolving gel, Western blotting, and detecting αCTF, βCTF and γCTF using antibody directed against the tag or APP C-terminus. Finally, quantitation of relative amounts of, αCTF, βCTF and γCTF as a measure of their respective secretase activities (see Fig 3); Method 2 involves immunoprecipitation of all CTFs from whole cell lysates of cells expressing the protein, using antibody directed against the tag or APP C-terminus, followed by standard PAGE and detection methods. Finally, quantitation of relative amounts of, αCTF, βCTF and γCTF as a measure of their respective secretase activities (see Fig 3). In a seventh aspect, the invention provides a method of screening a test compound for its ability to modulate α-, β- and/or γ-secretase activity comprising the steps of

1) providing a -, β- and/or γ-secretase;

2) bringing said α-, β- and or γ₇secretase into contact with a protein according to the first aspect of the invention;

3) bringing said α-, β- and/or γ-secretase and said protein into contact with a test substance; and

4) determining the presence and/or relative amounts of α-, β- and γ-secretase products derived from said protein.

In step 1, the α-, β- and/or γ-secretase is preferably expressed in a cell line (endogenously or otherwise). The protein is preferably produced by transfecting encoding nucleic acid in the cell line. The cell line may express both said protein and one or all of α-, β- and γ- secretase.

If necessary or desired, the test substance can be introduced to said protein before the secretase(s) in step 2. Alternatively, said protein, secretase(s) and test substance can be brought into contact simultaneously.

In an eighth aspect, the invention provides the use of an amino acid tag for the stabilisation of γ-secretase cleavage products of APP.

In a ninth aspect, the invention provides a method of assaying the ability of an amino acid tag to prevent or reduce degradation of a γ-secretase cleavage product of APP in a cell, comprising the steps of 1) expressing a nucleic acid sequence encoding a protein according to the first aspect of the invention (for example by introducing APP cDNA into a plasmid to incorporate a tag; or cloning APP cDNA and adding a tag sequence before introducing into a plasmid);

2) bringing said protein into contact with γ-secretase; (for example by transfecting the plasmid containing the protein (APP+tag) sequence into a suitable cell line, using standard transfection procedures) and

3) determining the presence and/or amount of said γ-secretase cleavage product. [For example by Method 1 (preferred because it's the simplest) - taking whole cell lysates of cells expressing said protein, electrophoresing on a low MW resolving gel, Western blotting, and detecting all CTFs including γCTF using antibody directed against the tag or APP C-terminus. Finally, detection and/or quantitation of γCTF relative to αCTF and βCTF as a measure of γCTF stability (see Fig 3); Method 2 — immunoprecipitation of all CTFs including γCTF, from whole cell lysates of cells expressing said protein, using antibody directed against the tag or APP C-terminus, followed by standard PAGE and detection methods. Finally, detection and/or quantitation of γCTF relative to αCTF and βCTF as a measure of γCTF stability (see Fig 3).

Generally the methods of the invention will be carried out in a host cell, for example in HEK293 cells expressing the protein having a C-terminal tag. In some situations it may be preferable to carry out assays wherein some or all of the assay components have been at least partly purified or isolated from cellular material, for example a cell-free assay, using methods well known in the art.

hi addition, the Inventors demonstrate dose-response inhibition of the γCTF fragment in the presence of known γ-secretase inhibitors. This provides strong evidence that the fragment is indeed γCTF and it demonstrates usefulness in an assay to determine the potency of compounds.

Preferred features of each aspect of the invention are as defined for each other aspect, mutatis mutandis. The proteins, nucleic acids and assays of the present invention facilitate the detection of the specific intracellular cleavage products of -, β- and unusually γ-secretase (see Figure 1). Without wishing to be bound by theory, the Inventors hypothesise that the amino acid tag interferes with the normal degradation of the γCTF. This may be due to interference with trafficking signals which normally direct the fragment to the degradation machinery. The amino acid tag may prevent/enable interaction with cellular proteins which influence the stabilization of the γCTF. Until now, the γCTF has been very elusive due to its instability, therefore it has been difficult to study and not much is known about its degradation.

As described herein, the Inventors have shown that the detection of γCTF is useful in secretase assays (see Figure 3). In addition, the Inventors demonstrate dose-response inhibition of the γCTF fragment in the presence of known γ-secretase inhibitors. This provides strong evidence that the fragment is indeed γCTF and it demonstrates usefulness in an assay to determine the potency of compounds.

Further features and details of the invention will be apparent from the following description of assays for γ-secretase activity and inhibitors thereof which is given by way of example with reference to the accompanying drawings, in which:-

Figure 1 shows the products of APP processing in cells.

Amyloid precursor protein is cleaved by the action of α-, β-, and γ-secretases (arrows).

Intracellular levels of αCTF, βCTF and γCTF are indicative of the respective secretase activity. However, until now, γCTF has proved very elusive. Figure 2 shows the unique abundance of γCTF in human cells.

The inventors have generated HEK 293 cells overexpressing APP695sw-N5-6His. The APP is tagged at the C-terminus with the extra amino acids indicated. The tagging was achieved by cloning APP695sw into pcDΝA3.1/N5-His plasmid from Invifrogen. A

Western Blot of whole cell lysates reveals 3 dominant low molecular weight bands corresponding to βCTF, αCTF, and γCTF respectively. (The blot was probed with anti-N5 antibody (Invitrogen))

Figure 3 shows a convenient assay based on measuring CTF levels, for determining secretase activities:

β-secretase inhibition - expected results: Decrease in βCTF fragment Increase in αCTF fragment (seen at 0.5μM. Higher concentrations of AEBSF were toxic)

No change in γCTF fragment

γ-secretase inhibition - expected results: Increase in βCTF fragment Increase in αCTF fragment

Decrease in γCTF fragment

Figure 4 shows the human peptide sequence for amyloid A4 precursor of Alzheimer's disease ACCESSION Y00264. Bold-underlined sequence - KM mutated to NL in the Swedish mutant.

Figure 5 shows the mammalian expression vector, pcDNA3.1/N5-His-TOPO (Invitrogen), used for expression in HEK 293 cells

Figure 6 shows the tagged APP695 expressed in HEK 293 cells fransfected with pcDΝA3.1N5-His-TOPO (APP695 minus stop codon). Underlined sequences: KM mutated to ΝL in the Swedish mutation GKP3PNPLLGLDST - V5 epitope tag HHHHHH - Polyhistidine tag

Figure 7 shows the N-terminal amino acid sequence of the 'yCTF band derived from HEK 293 cells overexpressing tagged APP695. The N-terminal sequence was determined by Edman degradation to be NMLKK....

The invention will now be described with reference to the following non-limiting Examples.

EXAMPLES

Example 1 - Preparation of Assay Cell Line

WT APP695 was obtained from ρcDNA9-110 (APP695 - accession Y00264), see Figure 4.

The Inventors engineered the APP659 sequence minus the stop codon, into pcDNA3.1N5- His-TOPO (Invitrogen, see Figure 5). The resulting protein expressed is therefore tagged (see Figure 6)

The pcDΝA3.1N5-His-TOPO (APP695) was mutated to the Swedish mutant using Stratagene Quickchange Site-directed mutagenesis kit (see Figure 4). Ν.B. the Swedish mutation of APP is much more sensitive to cleavage by β-secretase, therefore the βCTF is readily produced and is therefore detectable.

Transfection procedure Approximately 15 million HEK 293 cells were electroporated with 30/ g DΝA

(pcDΝA3.1-N5-His-TOPO-swAPP695, at 500μF capacitance at 350N. Stable transfectants were selected by addition of 800μg/ml G418 into the culture medium.

Conducting the Assay APP695sw-N5 stably expressing HEK 293 cells cell were seeded into a 6 well plate at a cell density of 3 x 10⁴ cells/cm². After overnight culture, medium was replaced with fresh medium +10%FCS, ± inhibitor at the indicated concentrations (μM). After 2 hours cell lysates were harvested in 50μl Triton lysis buffer (150mM ΝaCl, 1% Triton X-100, 0.1% SDS, 50mM Tris pH8, 5mM EDTA) containing a cocktail of protease inhibitors (Calbiochem in #539134) and EDTA. Samples were assayed for protein (Pierce BCA) for equal loading onto 12% Bis-Tris gel (Invitrogen/ Νovex). A minimum of 50μg of protein was loaded per well. After electrophoresis in MES buffer (Invitrogen), proteins were transferred from gel to nitrocellulose membranes. Nitrocellulose membranes were blocked in 5% milk in PBST, before incubating with anti-V5 antibody (Invitrogen, 1:5000 in 5% milk) for 2hrs at RT. After washing in PBST, secondary antibody was added (anti-mouse IgG-HRP, 1:2000 in 5% milk). The membrane was developed using ECL reagents. Quantitative densitometry of the gel bands is used to determine relative amounts of each CTF per treatment. Example 2 - Identification of β-secretase or γ-secretase inhibitors

AEBSF (Sinha (1999) Proc. Natl. Acad. Sciences. 96:11049) and Z-LF-CHO (Citron M et al. Neuron 17, 171-179 (1996)) were used as β-secretase and γ-secretase inhibitors respectively (see Figure 3). Before the identity of β-secretase was known, it was previously found that AEBSF inhibits the production of β-cleavage products of APP with an IC₅₀ of ImM. The Inventors now know that it is highly unlikely that β-secretase is directly inhibited with AEBSF as AEBSF is a broad-spectrum serine protease inhibitor whereas, β-secretase is an aspartyl protease. However, in the absence of a better compound, the Inventors used AEBSF as a control for detecting a reduction in βCTF in this assay as shown in Figure 3.

Claims

1. A protein comprising amyloid precursor protein (APP) and an amino acid tag.

2. The protein as claimed in claim 1, wherein the amino acid tag is present at the C- terminus of the APP amino acid sequence.

3. The protein as claimed in claim 1 or claim 2, wherein the amino acid tag is a N5- 6His amino-acid-tag.

4. The protein as claimed in any one of claims 1 to 3 wherein the APP is the K670M671→ Ν670L671 Swedish mutation.

5. A nucleic acid molecule encoding the protein as claimed in any one of claims 1 to 4.

6. A vector comprising the nucleic acid molecule as claimed in claim 5.

7. A host cell comprising with the nucleic acid of claim 5 or the vector of claim 6.

8. A method of making a protein according to any one of claims 1 to 4, nucleic acid according to claim 5 or 6, or a host cell according to claim 7.

9. A method for determining the activity of γ-secretase, which comprises the steps of: 1) providing a protein as defined in any one of claims 1 to 4;

2) bringing said protein into contact with a γ-secretase; and

10. A method of simultaneously measuring the activities of α-, β- and γ-secretases comprising the steps of

1) providing a protein as defined in any one of claims 1 to 4;

2) bringing said protein into contact with α-, β- and γ-secretase enzymes; and 3) determining the presence and/or relative amounts of α-, β- and γ-secretase products derived from said protein.

11. A method of screening a test compound for its ability to modulate -, β- and/or γ- secretase activity comprising the steps of:

1) providing a α-, β- and/or γ-secretase;

2) bringing said a-, β- and/or γ-secretase into contact with a protein as defined in any one of claims 1 to 4 and with a test substance; and

12. A method, as claimed in claim 11, wherein the α-, β- and/or γ-secretase are brought into contact with the protein before the test substance is introduced.

13. Use of an amino acid tag as defined in any one of claims 1 to 4 for the stabilisation of γ-secretase cleavage products of APP.

14. A method of assaying the ability of an amino acid tag to prevent or reduce degradation of a γ-secretase cleavage product of APP in a cell, comprising the steps of 1) expressing a nucleic acid sequence encoding a protein as defined in claim 5;

2) bringing said protein into contact with γ-secretase; and

3) determining the presence and/or amount of said γ-secretase cleavage product.

15. A protein comprising amyloid precursor protein and an amino acid tag, substantially as herein described with reference to one or more of the examples.

16. A method of screening a test compound for its ability to modulate -, β- and/or γ-secretase activity, substantially as herein described with reference to one or more of the examples.