WO2003008635A2 - η-SECRETASE IN VITRO SCREENING ASSAY - Google Patents
η-SECRETASE IN VITRO SCREENING ASSAY Download PDFInfo
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- WO2003008635A2 WO2003008635A2 PCT/EP2002/007095 EP0207095W WO03008635A2 WO 2003008635 A2 WO2003008635 A2 WO 2003008635A2 EP 0207095 W EP0207095 W EP 0207095W WO 03008635 A2 WO03008635 A2 WO 03008635A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
Definitions
- the invention relates to a novel peptide playing an important role in Alzheimer's disease, to screening assays and test kits based on the detection of said novel peptide and to the use thereof for the identification of modulators of ⁇ -secretase activity.
- the invention further relates to inhibitors identified by the above screening assays and to pharmaceutical compositions comprising the inhibitors.
- AD Alzheimer's disease
- Selkoe a major pathological alteration in the brain of AD patients
- FAD familial AD
- a ⁇ is produced from the ⁇ -amyloid precursor protein ( ⁇ APP) by endoproteolysis, whereby at least two proteolytic activities are required for A ⁇ generation: beta-secretase ( ⁇ -secretase; BACE) mediates the N-terminal cleavage producing a membrane associated C-terminal fragment of ⁇ APP, called C99 or CTF ⁇ (SEQ ID NO:2) (Vassar and Citron, 2000).
- C99 is the immediate precursor for the next proteolytic processing step: the (presumably intramembraneous) cleavage of C99 by ⁇ -secretase to A ⁇ and a C-terminal fragment.
- ⁇ -Secretase cleavage of C99 results in the secretion of A ⁇ into biological fluids.
- the C-terminal product of this cleavage previously described as p7 (Haass and Selkoe, 1993) and also called C-terminal fragment gamma (CTF ⁇ ), has so far not been observed in vivo.
- CTF ⁇ C-terminal fragment gamma
- the C-terminus of A ⁇ is heterogenous in that A ⁇ may end at position 40 or at position 42 of its precursor C99.
- CTF ⁇ /59 C-terminal fragments consisting of 59 amino acids
- CTF ⁇ /57 C-terminal fragments consisting of 57 amino acids
- a peptide supposed to be CTF ⁇ /59 or CTF ⁇ /57 has been observed in vitro (McLendon et al., 2000; Pinnix et al., 2001).
- ⁇ - secretase activity playing a key role in A ⁇ plaque formation, might be a major target for new therapeutical, especially pharmacological, approaches.
- Pinnix et al. (2001 ) describe experiments designed to detect a C-terminal fragment of ⁇ APP derived from guinea pig brain.
- the fragment obtained therein is characterized as a peptide consisting of 57 amino acids.
- the technical problem underlying the present invention is to provide novel methods and means for the development of therapies of Alzheimer's disease and other neurodegenerative disorders in which A ⁇ - plaque formation and/or ⁇ - secretase activity is involved.
- a method for identifying substances capable of modulating the activity of ⁇ -secretase comprising the steps: providing a source of ⁇ -secretase activity, providing a substrate of ⁇ - secretase activity having a cleavage site corresponding to the naturally occuring cleavage site between amino acid 49 and amino acid 50 of the substrate C99, incubating said source and said substrate with a test substance under conditions allowing enzymatic activity of said ⁇ -secretase activity, and determining ⁇ -secretase activity by detecting the C-terminal fragment (and especially the amount thereof) released due to the proteolytic acitivity of said ⁇ - secretase activity.
- the N-terminus of the previously unknown C-terminal fragment corresponds to amino acid 50 of C99.
- the invention is based on the surprising finding that an enzymatic cleavage of C99 between its amino acids 49 and 50 is involved in the processing of C99 to A ⁇ .
- the inventors have discovered and characterized the product of this processing step, now called CTF ⁇ /50 (amino acid sequence: VMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQN; SEQ ID NO:1 ; N-terminus corresponding to amino acid 50 of C99). They have demonstrated that a ⁇ -secretase activity is responsible for this processing step.
- CTF ⁇ /50 is an immediate product of ⁇ - secretase activity acting on C99 and not the product of a further degradation of the previously described CTF ⁇ /57 or CTF ⁇ /59.
- ⁇ -secretase activity acts at a previously unknown cleavage site of C99.
- the identification of this proteolytic activity offers a novel approach for the pharmaceutical modulation of the enzymatic reactions involved in A ⁇ production and, thus, for therapeutical intervention.
- ⁇ -secretase activity has been determined by measuring release of A ⁇ (Wolfe et al., 1999; WO 01/16355).
- formation of the known A ⁇ variants A ⁇ 40 (C-teminus at position 40 of C99) and A ⁇ 42 (C-terminus at position 42 of C99) reflects a cleavage between amino acids 40 and 41 and between amino acids 42 and 43 of C99, respectively.
- these methods do not allow the assessment of the proteolytic activity at the cleavage site between amino acids 49 and 50.
- This difference is of great importance as release of A ⁇ 40 and A ⁇ 42 possibly requires an additional (exo)peptidase activity which might interfere with the ⁇ -secretase activity to be determined.
- screening assays for the detection of modulators of ⁇ -secretase activity that are based on the determination of cleavage products which are not the immediate products of said activity but result from additional cleavage events might give misleading data as it is unclear whether said modulators act on said ⁇ -secretase activity or on other enzymatic activities involved in A ⁇ formation.
- CTF ⁇ /50 being a processing product not only in the BACE / ⁇ -secretase pathway but also in the ⁇ -secretase / ⁇ -secretase pathway, can be easily assessed in in vitro assays, e.g. by immunoblotting or ELISA. Thus, determination of the amount of CTF ⁇ /50 released can be used as a simple and cost-effective read-out in methods for the identification of substances that modulate ⁇ -secretase activity.
- a ⁇ 40 production vis-a-vis A ⁇ 42 production is an unclarified issue.
- candidate inhibitors seem to inhibit A ⁇ 40 release but stimulate A ⁇ 42 secretion. It may be hypothesized that such substances might specifically inhibit an (exo)peptidase acting downstream of ⁇ - secretase activity, i.e. further degrading A ⁇ 42 to A ⁇ 40.
- the method according to the invention would not be affected by this phenomenon as only substances specifically inhibiting ⁇ -secretase activity would be detected.
- the method according to the invention allows the specific identification of substances which interfere with the previously unknown cleavage between amino acids 49 and 50 of C99 but not with other endo- or exoproteolytic activities that might be involved in the ⁇ APP processing pathway.
- Fig. 1 Identification of in vivo produced CTF ⁇ in human cells and mouse brain according to Example 1 :
- A Membrane fractions of HEK 293 cells stably transfected with Swedish mutant ⁇ APP ⁇ gs (swAPP) were analyzed by combined immunoprecipitation/immunoblotting with antibody 6687 to the C-terminus of ⁇ APP.
- the same ⁇ APP CTFs including the approximately 6 kDa CTF ⁇ were also observed by immunoblotting with antibody 6687 in mouse brain.
- B The ⁇ -secretase inhibitor DAPT inhibits CTF ⁇ production.
- HEK 293 cells stably transfected with swAPP were treated with the indicated concentrations of DAPT for 4h.
- Upper and middle panel Membrane fractions were prepared and analyzed for ⁇ APP CTFs by combined immunoprecipitation/immunoblotting with antibody 6687. Increasing concentrations of DAPT lead to a build up of CTF ⁇ and CTF ⁇ (upper panel) with a concomitant significant block of CTF ⁇ generation (middle panel).
- Lower panel Conditioned media were analyzed for secreted A ⁇ by combined immunoprecipitation/immunoblotting with antibodies 3926/6E10. The same dose dependent inhibition of CTF ⁇ production by DAPT was observed for A ⁇ generation.
- FIG. 2 In vitro generation of CTF ⁇ according to Example 1 : (A) Time dependent in vitro production of CTF ⁇ . Membrane preparations were incubated at 37°C for the indicated time points. The reaction mixtures were then separated in a soluble fraction (S100; lower panel) and a pellet fraction (P100; upper panel) by ultracentrifugation.
- Compound 1 inhibit the in vitro production of CTF ⁇ .
- Membrane preparations were incubated with (+) or without (-) 250 nM DAPT (left panel) or 50 ⁇ M CM256 (right panel).
- the S100 fractions of the reaction mixtures were immunoblotted with antibody 6687. Note that both inhibitors significantly reduce CTF ⁇ generation.
- C In vitro generation of CTF ⁇ depends on biologically active presenilins. Left panel: time dependent in vitro production of CTF ⁇ by membrane preparations derived from cells expressing PS1 wt. Right panel: Inhibition of CTF ⁇ production in membrane preparations derived from cells expressing the biologically inactive PS1 D385N mutation.
- ⁇ APP CTFs were identified by immunoblotting with antibody 6687.
- D The ⁇ -secretase inhibitor DAPT reduces the remaining in vitro CTF ⁇ production observed in C (right panel).
- Left panel time dependent in vitro production of CTF ⁇ by membrane preparations derived from cells expressing PS1 wt in the presence (+) or absence (-) of 250 nM DAPT.
- Right panel Inhibition of CTF ⁇ production in membrane preparations derived from cells expressing the biologically inactive PS1 D385N mutation in the presence (+) or absence (-) of 250 nM DAPT.
- ⁇ APP CTFs were identified by immunoblotting with antibody 6687.
- Fig. 3 Radiosequencing and mass spectrometry analysis of CTF ⁇ as described in Example 1.
- A CTF ⁇ generated in vitro from membrane preparations of 35 S- methionine labeled HEK 293 cells stably expressing swAPP was subjected to radiosequencing. A major methionine peak was observed at cycle 2 and a second peak at cycle 32 of the Edman degradation. The corresponding amino acid sequence of CTF ⁇ starting at valine 50 is shown below.
- B In vivo detection of a truncated CTF ⁇ in living HEK 293 cells stably overexpressing swAPP.
- Fig. 4 Illustration of the similarity of endoproteolytic processing of ⁇ APP and Notch as mentioned in Example 1. Human ⁇ APP is presumably cleaved after position 40, 42 and 49 of the ⁇ -amyloid domain. Mouse Notchl is cleaved PS dependent after amino acid 1743 (Schroeter et al., 1998).
- the amino acid abbreviations are according to the standard one or three letter code.
- the numbering of the amino acids in A ⁇ , in other fragments of C99 and in C99 itself is based on C99, i.e. the N-terminal amino acid of C99 is amino acid no. 1 and so on.
- the sequence of C99 is:
- ⁇ APP is meant to encompass naturally occuring human full length ⁇ APP, any known splice variant thereof (including e.g. splice variants APP 6 95, APP751 and APP770; cf. e.g. NCBI database entry P05067), any naturally occuring variation or mutation thereof (such as the so-called “Swedish mutation” ⁇ APP, swAPP (Citron et al., 1992), and corresponding molecules of other species, as long as they may serve as a substrate for ⁇ -secretase activity in an assay as described hereafter, i.e.
- ⁇ APP is also meant to encompass the genuine substrates of ⁇ -secretase activity, i.e. C99 and C83 and respective variants and derivatives.
- C99 and C83 can be provided to the assay either directly in the form of C99 or C83 or, alternatively, in the form of a full-length ⁇ APP being processed "in situ" to C99 or C83.
- ⁇ APP may also stand for a fragment and for a modified (e.g. genetically engineered or chemically modified) derivative of full-length ⁇ APP, C99 or C83, as long as such a fragment or derivative may serve as substrate for ⁇ -secretase activity in an assay as described hereafter.
- Genetically engineered ⁇ APP may be a ⁇ APP in the above defined sense and modified by amino acid substitutions, deletions or insertions, under the proviso that the screening method according to the invention can still be performed therewith.
- a modified ⁇ APP not comprising a cleavage site corresponding to the cleavage site identified by the inventors or a modified ⁇ APP targeted to cellular compartments where no ⁇ -secretase activity is present would not be suited to the method of the invention.
- genetically engineered ⁇ APP might be a ⁇ APP fused with a reporter protein such as green fluorescent protein, luciferase, ⁇ -galactosidase and so on.
- swAPP 695 is especially preferred in an assay as described hereafter, because it is a better substrate for ⁇ -secretase than wild type APP, thus providing high levels of C99, the immediate substrate of ⁇ -secretase activity.
- ⁇ -secretase activity means in the context of this invention any proteolytic activity that is capable of cleaving the substrate C99 between amino acids 49 and 50 and whose physiological substrate is C99 in its physiological environment (e.g. in cell membranes).
- ⁇ -secretase activity may be the proteolytic activity presently ascribed to the not yet fully characterized ⁇ - ' secretase per se, a proteolytic activity of ⁇ -secretase in combination with one or more presenilins or, if applicable, presenilin activity per se.
- the term "source of ⁇ -secretase activity” comprises any biological material such as cells, homogenized cells, enriched membrane fractions, purified membranes, protein fractions, proteins reconstituted in membranes etc. which possess ⁇ - secretase activity.
- the source are human embryonic kidney (HEK) 293 cells, more preferably membrane preparations thereof that can be obtained e.g. as described in Example 1.
- HEK human embryonic kidney
- the methods for the purification of membranes are known in the art (cf. e.g. Methods in Enzymology, Vol. 219: “Reconstitution of intracellular Transport” and T.G. Cooper: “Biochemische vonmethoden", De Gruyter Verlag, 1981).
- substrate of ⁇ -secretase activity is meant to encompass “ ⁇ APP” according to the definition above as well as artificially designed proteins that encompass the cleavage site detected by the inventors.
- the cleavage site may be defined by the sequence ITLVML (SEQ ID NO: 3) or VIVITLVMLKKK (SEQ ID NO: 4).
- swAPP 695 over)expressed in e.g. HEK 293 cells (and endogenously cleaved to C99) is the substrate of ⁇ -secretase activity.
- substance capable of modulating ⁇ -secretase activity means naturally occurring and synthetic compounds capable of activating or inhibiting enzymatic ⁇ -secretase activity as defined above, whereby substances unspecifically interfering with enzymatic reactions (such as e.g. agents that cause denaturation of proteins) should, of course, not be encompassed. Persons skilled in the art will be able to differentiate between specific and unspecific inhibition or activation of enzymatic activity.
- fragment beginning with a sequence corresponding to the N-terminal sequence of CTF ⁇ /50 encompasses, of course, CTF ⁇ /50 itself.
- a ⁇ APP modified C-terminal of the cleavage site identified by the inventors is used as a substrate for ⁇ -secretase activity, a modified C- terminal fragment will result that will have to be detected according to the method of the invention.
- said fragment originates from proteolytic cleavage of a ⁇ APP as defined above at the cleavage site identified by the inventors, i.e. the site corresponding to amino acids 49 and 50 of C99.
- condition allowing enzymatic activity of ⁇ -secretase activity means that reaction conditions are chosen under which proteolytic cleavage by ⁇ - secretase activity is enabled. Examples for such conditions are given below.
- the substrate of ⁇ -secretase activity is an endogenous ⁇ APP that is constitutively expressed in the cell.
- cells expressing an exogenous ⁇ APP are used. Especially preferred is exogenous swAPP 695 . Expression of exogenous ⁇ APP may be achieved by transfecting cells with a gene coding for ⁇ APP in a suitable expression vector. Endogenous and exogenous substrates are described in detail in WO 01/16355.
- ⁇ APP substrate is a fusion protein of a reporter protein with e.g. wild-type ⁇ APP, swAPP or C99.
- reporter proteins are green fluorescent protein, luciferase, ⁇ - galactosidase, etc.
- the cell line used in the example below is HEK 293.
- other cells and cell lines such as H4, U373, NT2, PC12, COS, CHO, fibroblasts, myeloma cells, neuroblastoma cells, hybridoma cells, oocytes, empryonic stem cells and so on can be used as well. Cells and cell lines of neuronal or glial origin or fibroblasts are especially preferred.
- cells and tissues of the brain as well as homogenates and membrane preparations thereof may be used.
- CTF ⁇ /50 or of a fragment beginning with a sequence corresponding to the N-terminal sequence of CTF ⁇ /50 can be performed by immunoblotting/western blotting, by ELISA and other suitable peptide or protein detection methods known in the art.
- Antibodies useful for the detection of CTF ⁇ /50 are, e.g., polyclonal antibody 6687 binding to the last 20 C-terminal amino acids of ⁇ APP (Steiner et al., 2000) and SAD3128 available from LABGEN®.
- a ⁇ APP fused to a reporter protein as mentioned above it is possible to estimate the amount of cleaved substrate by detection of the reporter protein. In this case, a separation of cleaved and uncleaved substrate followed by the respective detection method depending on the reporter protein might be performed.
- a source of ⁇ -secretase activity and a substrate thereof are incubated with a test substance under conditions allowing enzymatic activity of the source of ⁇ -secretase activity.
- the amount of substrate cleaved in presence of the test substance is determined by measuring the amount of CTF ⁇ /50 (or a derivative thereof, respectively, if a derivative of C99 or ⁇ APP has been used as the substrate) produced.
- the amount of CTF ⁇ /50 (or derivative) released during the incubation step reflects the activity of ⁇ -secretase activity acting at the cleavage site of ⁇ APP as defined above.
- control experiments will be included in the assay, wherein the experiment described before will be performed under essentially the same conditions as above but without addition of said test substance or with addition of a substance known to have no effect on ⁇ -secretase activity.
- the results of both experiments will be compared and a reduced amount of released CTF ⁇ /50 (or the derivative) will be indicative of an inhibitory effect of the test substance on ⁇ -secretase activity (inhibitor), whereas an increased amount of CTF ⁇ /50 indicates that the test substance activates said activity (activator).
- said test substance is classified as "modulator".
- a broad range of buffers can be used.
- a reaction buffer having a pH in the range of 5 to 10, more preferably in the range of 6 to 8 and most preferably in the range of 6.3 to 6.9 may be used.
- a suitable buffer is 150 mM sodium citrate, pH 6.4.
- the reaction temperature may be selected, for example, in the range of 20 °C to 37 °C. A higher temperature might result in denaturation of proteins, a lower temperature will result in a decrease of the speed of the reaction.
- the reaction mixture may additionally comprise membrane stabilizing agents such as sucrose and sorbitol, preferably in an amount of 200 to 1000 mM, more preferably in an amount of 200 to 500 mM and most preferably in an amount of 200 to 300 mM.
- the reaction buffer may contain proteinase inhibitors, e.g. the "PI Complete" mix, available from Roche®, and EDTA. EDTA serves to inhibit metalloproteinases responsible for the degradation of CTF ⁇ .
- Said proteinase inhibitor mix does not include pepstatin which is an inhibitor of ⁇ -secretase activity.
- an assay based on the method according to the invention will be used in a two step analysis of test substances:
- several assays are known in the art in which ⁇ -secretase activity is measured by assessing release of A ⁇ (Wolfe et al., 1999; "A ⁇ release based assays").
- a ⁇ release might be the result of an at least two-step cleavage process: one cleavage occuring at the site identified by the inventors (amino acid 49 / 50 of C99), the other at site 40 / 41 (resulting in A ⁇ 40) or site 42 / 43 (resulting in A ⁇ 42) of C99.
- a ⁇ release based assays do not discriminate between inhibitors (or, more generally, modulators) of these two cleavage activities.
- the known A ⁇ release based assay By coupling the known A ⁇ release based assay to the assay according to the invention, it is possible to make said discrimination. For example, it is possible to first identify inhibitors by performing the A ⁇ release based assay and, afterwards, including test substances having shown to be effective in said first assay in the second assay based on the method according to the invention. Substances not being effective in said second assay can be classified as substances modulating the proteolytic activity acting at amino acids 40 / 41 or 42 / 43, whereas substances found to be effective in both assays act on cleavage site 49 / 50.
- the two-step assay outlined above is of great importance because of the following reason: as discovered by the inventors, the cleavage site 49 / 50 in C99 shows similarity to the known S3 cleavage site of Notch protein (Schroeter et al., 1998). If both cleavage events are mediated by the same or closely related proteinases, it is expected that substances inhibiting cleavage of C99 at site 49 / 50 might also inhibit Notch protein cleavage, possibly resulting in severe and undesirable side-effects of medicaments ultimately derived from a respective substance.
- the novel screening method has the additional advantage that it provides a means for the discrimination between substances that are suspected to affect Notch protein proteolysis and substances not expected to do so.
- HTS high throughput screening
- a method according to the invention is a high throughput screening (HTS) method.
- HTS relates to an experimental setup wherein a large number of compounds is tested simultaneously.
- said HTS setup may be carried out in microplates, may be partially or fully automated and may be linked to electronic devices such as computers for data storage, analysis, and interpretation using bioinformatics.
- said automation may involve robots capable of handling large numbers of microplates and capable of carrying out several thousand tests per day.
- a test compound which is known to show the desired modulating or inhibitory function will also be included in the assay as a positive control.
- the term HTS also comprises ultra high throughput screening formats (UHTS).
- said UHTS formats may be carried out using 384- or 1536-well microplates, sub-microliter or sub-nanoliter pipettors, improved plate readers and procedures to deal with evaporation.
- HTS methods are described e.g. in US 5,876,946 and US 5,902,732.
- the expert in the field can adapt the method described below to a HTS or UHTS format without the need of carrying out an inventive step.
- the source of ⁇ -secretase activity e.g. membrane preparations
- the substrate of ⁇ -secretase activity e.g. C99 or a modified derivative as outlined above
- a reaction buffer and a means for specifically detecting the reaction product CTF ⁇ /50 may be assembled to a test kit.
- the detection means may be a monoclonal or a polyclonal antibody or derivative specifically reacting with CTF ⁇ /50.
- the test kit may additionally comprise microtiter plates, labeled second antibodies used for immunoblotting / western blotting techniques (known in the art), reaction vessels, blotting membranes, buffers etc.
- inhibitors such at DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S- phenylglycin t-butyl ester) and "Compound 1" (N-[(1 ,1-dimethylethoxy)carbonyl]- L-valyl-(4S,5S)-4-amino-2,2-difluoro-5-methyl-3-oxoheptanoyl-L-valyl-L- Isoleucine methyl ester; also called CM256) will be excluded from the scope of protection.
- compositions comprising substances identified with the method according to the invention and pharmaceutical acceptable carriers or excipients.
- a pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of a substance capable of inhibiting ⁇ -secretase activity.
- physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients (as disclosed e.g. in Remington's Pharmaceutical Sciences (1990), 18 th ed., Mack Publ., Easton).
- the person skilled in the art will know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition.
- a substance identified with the method according to the invention is used for the preparation of a medicament for the treatment of neurodegenerative diseases, especially Alzheimer's disease.
- the invention provides the previously unknown CTF ⁇ /50 (SEQ ID No. 1 ). It is clear that allelic variations and functionally equivalent derivatives thereof will also be encompassed by the invention. Examples
- Human embryonic kidney 293 (HEK 293) cells expressing swAPP were generated and cultured as described (Steiner et al., 2000).
- HEK 293 cells were transiently transfected with cDNA encoding recombinant CTF ⁇ 57 using DOTAP (liposome formulation of the monocationic lipid N-[1-(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propane methylsulfate in water; Roche) according to the supplier ' s instructions.
- DOTAP liposome formulation of the monocationic lipid N-[1-(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propane methylsulfate in water; Roche
- a cDNA encoding recombinant CTF ⁇ 57 was amplified by PCR and cloned into pcDNA3 vector (Invitrogen) as an Hindlll and Xbal fragment.
- Recombinant CTF ⁇ 57 consists of an N-terminal methionine followed by the C-terminus of ⁇ APP starting at position 43 of the ⁇ -amyloid domain.
- Antibodies The polyclonal antibodies 6687 to the last 20 C-terminal amino acids of ⁇ APP (Steiner et al., 2000) and 3926 to A ⁇ 1-42 (Wild-Bode et al., 1997) have been described.
- DAPT N-[N-(3,5-difluorophenacetyl)-L- alanyl]-S-phenylglycin t-butyl ester
- CM256 N-[(1,1- dimethylethoxy)carbonyl]-L-valyl-(4S,5S)-4-amino-2,2-difluoro-5-methyl-3- oxoheptanoyl-L-valyl-L-lsoleucine methyl ester; gift from Dr. M. Wolfe), previously designated "Compound 1" (Esler et al., 2000), that were diluted from stock solutions in DMSO to the concentrations described.
- a ⁇ was immunoprecipitated from conditioned media collected for 4 h with antibody 3926, separated on Tris-Tricine gels and detected by immunoblotting with antibody 6E10 using a chemiluminescent detection system (Tropix, USA).
- CTF ⁇ was analyzed by combined immunoprecipitation/westernblotting with antibody 6687 of membrane extracts from stably transfected HEK 293 cells or from mouse brain. Briefly, homogenates of cells or brain tissue were prepared in hypotonic buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 7.0) containing 1x protease inhibitors (PI) (Complete, Roche) as described (Steiner et al., 1998). Following homogenization membranes were isolated from the postnuclear supernatant (PNS) by centrifugation at 16.000 g for 45 min at 4°C.
- PPS postnuclear supernatant
- the membranes were resuspended in RIPA buffer (150 mM NaCI, 10 mM Tris, 1% NP-40, 0.5% cholic acid, 0.1% SDS, 5 mM EDTA, pH 8.0, 1x PI) following a clarifying spin at 16.000 g for 10 min at 4°C, and subjected to immunoprecipitation with antibody 6687.
- RIPA buffer 150 mM NaCI, 10 mM Tris, 1% NP-40, 0.5% cholic acid, 0.1% SDS, 5 mM EDTA, pH 8.0, 1x PI
- lysates were prepared with RIPA buffer 48 h after transfection and subjected to immunoprecipitation with antibody 6687.
- CTF ⁇ was generated in vitro from membrane preparations of HEK 293 cells stably transfected with swAPP following previously described procedures (McLendon et al., 2000; Pinnix et al., 2001 ). In brief, cells were resuspended (0.5 ml/10-cm dish) in homogenization buffer (10 mM MOPS, pH 7.0, 10 mM KCI, 1x PI). Cell homogenates and a PNS were prepared as described (Steiner et al., 1998).
- Membranes were pelleted from the PNS by centrifugation for 20 min at 16.000 g at 4°C, washed with homogenization buffer and resuspended (50 ⁇ l/10-cm dish) in assay buffer (150 mM sodium citrate, pH 6.4, 1x PI). To allow generation of CTF ⁇ , samples were incubated at 37°C for the indicated time points in a volume of 25 ⁇ l/assay. Control samples were kept on ice. After termination of the assay reactions on ice, samples were separated into pellet (P100) and supernatant (S100) fractions by ultracentrifugation for 1 h at 100.000 g at 4°C. Following SDS-PAGE on 10-20% Tris-Tricine gels (Invitrogen) samples were analyzed for CTF ⁇ by immunoblotting with antibody 6687.
- assay buffer 150 mM sodium citrate, pH 6.4, 1x PI
- CTF ⁇ was then generated in vitro from membrane preparations as described above except that after termination of CTF ⁇ generation the assay reactions were separated into pellet (P16 ⁇ and supernatant (S16) fractions by centrifugation at 16.000 g for 30 min at 4°C. After isolation of CTF ⁇ from S16 by immunoprecipitation with antibody 6687 immunocomplexes were separated by SDS-PAGE on 10-20% Tris-Tricine gels (Invitrogen) and blotted onto a PVDF membrane. After autoradiography, the CTF ⁇ band was excised and subjected to radiosequencing by automated Edman degradation as described (Haass et al., 1992).
- PS mediated ⁇ -secretase cleavage does not only result in the generation of soluble A ⁇ but also in the generation of a ⁇ APP C-terminal fragment (CTF ⁇ ) representing the counterpart of A ⁇ .
- CTF ⁇ C-terminal fragment
- two species of CTF ⁇ have been postulated, CTF ⁇ /57 and CTF ⁇ /59, respectively.
- C-terminal fragments of ⁇ APP were immunoprecipitated from membrane fractions of human embryonic kidney 293 (HEK 293) cells stably transfected with ⁇ APP ⁇ gs carrying the Swedish mutation (swAPP) (Citron et al., 1992).
- PS1 D385N acts like a dominant negative mutation that inhibits the biological function of PSs required for the ⁇ -secretase cleavage of ⁇ APP.
- a strong increase of ⁇ APP CTF ⁇ and CTF ⁇ was observed, indicating a significant inhibition of ⁇ - secretase cleavage (Fig. 1C).
- generation of CTF ⁇ was strongly reduced (Fig. 1C).
- Membranes from HEK 293 cells stably expressing swAPP were incubated under the conditions described under item "Generation and analysis of CTF ⁇ in vitro" above in the presence of a protease inhibitor cocktail (McLendon et al., 2000; Pinnix et al., 2001). After termination of the in vitro assay, membranes were separated by ultracentrifugation. The pellet (P100) and the supernatant (S100) fraction were analyzed for the presence of ⁇ APP CTFs. CTF ⁇ and CTF ⁇ were predominantly observed within the P100 fraction (Fig. 2A; upper panel), whereas CTF ⁇ was significantly enriched in the S100 fraction (Fig. 2A; lower panel).
- the inventors then used the in vitro assay to isolate sufficient amounts of CTF ⁇ to allow the further structural characterization of this peptide.
- attempts to perform mass spectroscopy with the isolated peptide were unsuccessful for unknown reasons.
- the inventors tried to determine the sequence of the peptide's N-terminus by radiosequencing.
- HEK 293 cells stably expressing swAPP were metabolically labeled with 35 S- methionine. Radiolabeled CTF ⁇ was generated in vitro as described above. After ultracentrifugation, CTF ⁇ was immunoprecipitated from the S100 fraction with antibody 6687 to the last 20 amino acids of ⁇ APP.
- Radiolabeled CTF ⁇ was then subjected to automated Edman degradation (Haass et al., 1992). Surprisingly, this revealed a major peak of radioactivity in fraction 2 and not in fractions 9 and 11 as one would have expected for a CTF ⁇ beginning at position , 41 or 43 (Fig. 3A). This indicates that CTF ⁇ is generated by a proteolytic
- the N-terminus of CTF ⁇ is located at a position, which is homologous to the PS dependent S3 cleavage of Notch (Fig. 4).
- the S3 cleavage of Notch occurs right at the cytoplasmic boarder of the membrane (Schroeter et al., 1998).
- the novel cleavage site of ⁇ APP may also occur close to the cytoplasmic boarder of the membrane. Based on the results by Tischler et al. (Tischler and Cordell, 1996), the cut between amino acid 49 and 50 would indeed occur within the cytoplasmatic domain.
- cytoplasmatic cleavage would be more likely than an intramembraneous proteolytic cut, which is likely to be inhibited by the higly hydrophobic environment within a phospholipid bilayer.
- a cytoplasmic cleavage may facilitate a shift of the remaining stub into the cytoplasm, where it could easily be attacked by the final ⁇ -secretase cut. Indeed, Murphy et al., 1999, provided evidence for such a model.
- cytoplasmic tail of ⁇ APP requires "shedding" before/during it undergoes the final ⁇ -secretase cut, a phenomenon which would be very similar to the required ectodomain shedding of ⁇ -secretase substrates (Struhl and Adachi, 2000).
- the identification of CTF ⁇ in vivo may also raise the interesting possibility that this fragment similar the Notch intracellular cytoplasmic domain (NICD) may have a biological function in signal transduction. Based on the striking similarity of the biological mechanisms involved in the generation of NICD and CTF ⁇ as well as potentially similar functions in signal transduction, the term AICD for the Amyloid precursor protein intracellular domain is proposed.
- ⁇ -secretase may cut first at position 40/42 of the ⁇ -amyloid domain followed by a second cleavage after position 49 releasing CTF ⁇ from the membrane.
- the data may indicate simultaneous cleavage at all three sites.
- Example 2 Screening assay - medium throughput
- HEK 293 cells stably transfected with ⁇ APP 69 5 carrying the Swedish mutation (swAPP) were grown as described in Citron et al., 1992, i.e. in DMEM with Glutamax (Gibco BRL) containing 10 % FCS, 1 % Penicillin/Streptavidin, 200 ⁇ g/ml G418.
- the cells were resuspended (0.5 ml/10-cm dish) in homogenization buffer (10 mM MOPS, pH 7.0, 10 mM KCI, 1x PI).
- PPS post nuclear supernatant
- Membranes were pelleted from the PNS by centrifugation for 20 min at 16.000 g at 4°C, washed with homogenization buffer and resuspended (50 ⁇ l/10-cm dish) in assay buffer (150 mM sodium citrate, pH 6.4, 1x PI). To allow generation of CTF ⁇ , control samples without added test substance and samples comprising different concentrations of substances to be screened were incubated at 37°C for e.g. 1 h. The reactions were stopped by cooling them to 4 °C.
- the samples are separated into pellet (P100) and supernatant (S100) fractions by ultracentrifugation for 1 h at 100.000 g al 4°C.
- CTF ⁇ /50 is detected and quantified by ELISA using an antibody specific for the N-terminal sequence of CTF ⁇ /50.
- a reduced signal compared to the control sample indicates that the respective test compound inhibits ⁇ -secretase activity acting at amino acid 49 / 50 of C99.
- Example 3 Screening assay - medium throughput
- the screening assay is carried out as described in Example 2 with the exception that instead of the ultracentrifugation step membranes are solubilized by adding STEN - lysis buffer (50 mM Tris, pH 7.6; 150 mM NaCI; 2 mM EDTA; 1 % NP-40 (final)). CTF ⁇ /50 is detected and quantified in this solution by ELISA as described in Example 2.
- STEN - lysis buffer 50 mM Tris, pH 7.6; 150 mM NaCI; 2 mM EDTA; 1 % NP-40 (final)
- the screening assay is carried out as described in Example 2 with the exception that the release of CTF ⁇ /50 is determined by immunoprecipitation and immunoblotting of CTF ⁇ /50 and densitometric analysis of the resulting bands. A reduced signal compared to the control sample indicates that the respective test compound inhibits ⁇ -secretase activity acting at amino acid 49 / 50 of C99.
- Amyloid ⁇ -peptide is produced by cultured cells during normal metabolism. Nature, 359, 322-325.
- Notch-1 signalling requires iigand-induced proteolytic release of intracellular domain. Nature, 393, 382-386.
- Selkoe, D.J. (1999) Translating cell biology into therapeutic advances in Alzheimer's disease. Nature, 399, A23-31.
- Glycine 384 is required for presenilin-1 function and is conserved in polytopic bacterial aspartyl proteases. Nature Cell Biol, 2, 848-851.
- Beta-amyloid precursor protein Location of transmembrane domain and specificity of gamma-secretase cleavage. J Biol Chem. 1996 Sep 6;271(36):21914-9.
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CA002452832A CA2452832A1 (en) | 2001-07-04 | 2002-06-27 | .gamma.-secretase in vitro screening assay |
JP2003514950A JP2004535206A (en) | 2001-07-04 | 2002-06-27 | In vitro screening assay for γ-secretase |
EP02745414A EP1407043A2 (en) | 2001-07-04 | 2002-06-27 | Gamma-secretase in vitro screening assay |
MXPA03011898A MXPA03011898A (en) | 2001-07-04 | 2002-06-27 | gamma-SECRETASE IN VITRO. |
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Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2396415A (en) * | 2002-12-19 | 2004-06-23 | Merck Sharp & Dohme | Assay for gamma secretase modulators |
WO2009012237A1 (en) * | 2007-07-13 | 2009-01-22 | Elan Pharmaceuticals, Inc. | Compositions and methods for identifying substrate specificity of inhibitors of gamma secretase |
WO2009052334A1 (en) | 2007-10-19 | 2009-04-23 | Janssen Pharmaceutica, N.V. | Carbon linked modulators of y-secretase |
US7557244B2 (en) | 2006-04-21 | 2009-07-07 | Ortho-Mcneil Pharmaceutical, Inc. | Substituted biphenyl carboxylic acids and derivatives thereof |
US7897643B2 (en) | 2007-10-17 | 2011-03-01 | Janssen Pharmaceutica N.V. | Biphenyl carboxylic acids and derivatives thereof |
US7951843B2 (en) | 2007-10-19 | 2011-05-31 | Janssen Pharmaceutica N.V. | Amide linked modulators of γ-secretase |
US7968725B2 (en) | 2008-07-22 | 2011-06-28 | Janssen Pharmaceutica N.V. | Pyridinyl modulators of γ-secretase |
US8119669B2 (en) | 2007-03-07 | 2012-02-21 | Janssen Pharmaceutica N.V. | Substituted phenoxy N-alkylated thiazolidinediones as estrogen related receptor-α modulators |
US8247610B2 (en) | 2007-10-19 | 2012-08-21 | Janssen Pharmaceutica N.V. | Amine linked modulators of γ-secretase |
US8252935B2 (en) | 2007-10-19 | 2012-08-28 | Janssen Pharmaceutica N.V. | Piperidinyl and piperazinyl modulators of γ-secretase |
US8580832B2 (en) | 2007-03-07 | 2013-11-12 | Janssen Pharmaceutica N.V. | Substituted phenoxy aminothiazolones as estrogen related receptor-α modulators |
WO2014062771A1 (en) * | 2012-10-16 | 2014-04-24 | The Johns Hopkins University | An inducible reconstitution and real-time quantitative kinetic system for the study of intramembrane enzymes |
US9023767B2 (en) | 2009-05-07 | 2015-05-05 | Memorial Sloan-Kettering Cancer Center | γ-Secretase substrates and methods of use |
US9632088B2 (en) | 2010-09-07 | 2017-04-25 | Memorial Sloan-Kettering Cancer Center | Methods and compositions for gamma-secretase assay |
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WO1998015828A1 (en) * | 1996-10-07 | 1998-04-16 | Scios Inc. | Method to identify direct inhibitors of the beta-amyloid forming enzyme gamma-secretase |
DE19941039A1 (en) * | 1999-08-28 | 2001-03-01 | Boehringer Ingelheim Pharma | gamma-secretase in vitro test system |
-
2001
- 2001-07-04 DE DE10131899A patent/DE10131899A1/en not_active Withdrawn
-
2002
- 2002-06-27 MX MXPA03011898A patent/MXPA03011898A/en unknown
- 2002-06-27 JP JP2003514950A patent/JP2004535206A/en active Pending
- 2002-06-27 WO PCT/EP2002/007095 patent/WO2003008635A2/en not_active Application Discontinuation
- 2002-06-27 CA CA002452832A patent/CA2452832A1/en not_active Abandoned
- 2002-06-27 EP EP02745414A patent/EP1407043A2/en not_active Withdrawn
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WO1998015828A1 (en) * | 1996-10-07 | 1998-04-16 | Scios Inc. | Method to identify direct inhibitors of the beta-amyloid forming enzyme gamma-secretase |
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GB2396415B (en) * | 2002-12-19 | 2006-05-31 | Merck Sharp & Dohme | Novel assay for modulation of GAMMA secretase |
US7285393B2 (en) | 2002-12-19 | 2007-10-23 | Merck Sharp & Dohme Limited | Assay for modulation of γ secretase |
GB2396415A (en) * | 2002-12-19 | 2004-06-23 | Merck Sharp & Dohme | Assay for gamma secretase modulators |
US7557244B2 (en) | 2006-04-21 | 2009-07-07 | Ortho-Mcneil Pharmaceutical, Inc. | Substituted biphenyl carboxylic acids and derivatives thereof |
US8119669B2 (en) | 2007-03-07 | 2012-02-21 | Janssen Pharmaceutica N.V. | Substituted phenoxy N-alkylated thiazolidinediones as estrogen related receptor-α modulators |
US9034852B2 (en) | 2007-03-07 | 2015-05-19 | Janssen Pharmaceutica N.V. | Substituted phenoxy N-alkylated thiazolidinediones as estrogen related receptor-α modulators |
US8580832B2 (en) | 2007-03-07 | 2013-11-12 | Janssen Pharmaceutica N.V. | Substituted phenoxy aminothiazolones as estrogen related receptor-α modulators |
WO2009012237A1 (en) * | 2007-07-13 | 2009-01-22 | Elan Pharmaceuticals, Inc. | Compositions and methods for identifying substrate specificity of inhibitors of gamma secretase |
US7897643B2 (en) | 2007-10-17 | 2011-03-01 | Janssen Pharmaceutica N.V. | Biphenyl carboxylic acids and derivatives thereof |
WO2009052334A1 (en) | 2007-10-19 | 2009-04-23 | Janssen Pharmaceutica, N.V. | Carbon linked modulators of y-secretase |
US8247610B2 (en) | 2007-10-19 | 2012-08-21 | Janssen Pharmaceutica N.V. | Amine linked modulators of γ-secretase |
US8252935B2 (en) | 2007-10-19 | 2012-08-28 | Janssen Pharmaceutica N.V. | Piperidinyl and piperazinyl modulators of γ-secretase |
US8692017B2 (en) | 2007-10-19 | 2014-04-08 | Janssen Pharmaceutica, N.V. | Carbon linked modulators of gamma-secretase |
US7951843B2 (en) | 2007-10-19 | 2011-05-31 | Janssen Pharmaceutica N.V. | Amide linked modulators of γ-secretase |
US7968725B2 (en) | 2008-07-22 | 2011-06-28 | Janssen Pharmaceutica N.V. | Pyridinyl modulators of γ-secretase |
US9023767B2 (en) | 2009-05-07 | 2015-05-05 | Memorial Sloan-Kettering Cancer Center | γ-Secretase substrates and methods of use |
US9632088B2 (en) | 2010-09-07 | 2017-04-25 | Memorial Sloan-Kettering Cancer Center | Methods and compositions for gamma-secretase assay |
WO2014062771A1 (en) * | 2012-10-16 | 2014-04-24 | The Johns Hopkins University | An inducible reconstitution and real-time quantitative kinetic system for the study of intramembrane enzymes |
US10030260B2 (en) | 2012-10-16 | 2018-07-24 | The Johns Hopkins University | Inducible reconstitution and real-time quantitative kinetic system for the study of intramembrane enzymes |
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CA2452832A1 (en) | 2003-01-30 |
MXPA03011898A (en) | 2004-03-26 |
WO2003008635A8 (en) | 2003-10-30 |
DE10131899A1 (en) | 2003-02-27 |
JP2004535206A (en) | 2004-11-25 |
WO2003008635A3 (en) | 2004-01-29 |
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