WO2002066953A2

WO2002066953A2 - High throughput screen for specific inhibition of intramembranous proteolysis

Info

Publication number: WO2002066953A2
Application number: PCT/US2002/004900
Authority: WO
Inventors: Raphael Kopan
Original assignee: Washington University
Priority date: 2001-02-20
Filing date: 2002-02-20
Publication date: 2002-08-29
Also published as: WO2002066953A3; AU2002252006A8; AU2002252006A1

Abstract

The present invention relates to methods of screening treatments for the inhibition of proteolytic enzyme activity for cleavage of a transmembrane domain of a polypeptide of interest. Such methods may be utilized to screen for the inhibition of proteolytic enzyme activity for cleavage of one specific transmembrane domain of a polypeptide or of multiple transmembrane domains of a polypeptide for identification of an enzyme inhibitor that is specific for one substrate, yet does not act upon another substrate.

Description

HIGH THROUGHPUT SCREEN FOR SPECIFIC INHIBITION OF INTRAMEMBRANOUS PROTEOLYSIS

Field of the Invention

This invention relates to methods of screening and identifying specific compounds that inhibit proteolytic activity of enzymes. Specifically this invention relates to the screening and identifying of compounds that inhibit the activity of enzymes responsible for cleavage within the transmembrane domain of proteins such as those responsible for certain diseases such as Alzheimer's disease.

Background of the Invention

Alzheimer's disease (AD) is a late onset neurodegenerative disorder characterized by the extracellular deposition of insoluble aggregates composed of the 40 to 42 amino acid A.beta.peptide (Aβ) in the brain.

Glenner and Wong, Biochem . Biophys . Res . Commun. 120:885-890 (1984); Masters et al . , EMBO J. 4:2757-2763 (1985). A.beta.peptide is derived from an integral membrane protein termed amyloid precursor protein (APP) . Tanzi et al . , Science 235:880 (1987); Kang et al . , Nature 325:733-736

(1987) . The most common known cause of autoso al dominantly inherited early onset AD is mutation in the presenilin genes (collectively PS) . Kopan R. , Goate A. , A common Enzyme connects notch signaling and Alzheimer' s disease, Genes Dev.14(22): 2799-806, (2000 Nov. 15). The PS mutations result in elevated levels of Aβ42, a highly amyloidogenic subspecies of Aβ that is the primary protein deposited in senile plaques, a hallmark of AD pathology. Aβ42 is generated most likely in the Endoplasmic Reticulum from the β-amyloid precursor protein (APP) by two unidentified enzymatic activities, β-secretase, which cleaves in the extracellular domain, and gamma-secretase (γ-secretase) , which cleaves within the transmembrane domain. Kopan R. , Goate A. , A common Enzyme connects notch signaling and Alzheimer' s disease, Genes Dev.14(22): 2799-806, (2000 Nov. 15).

In addition to the role of presenilin 1 (PS1) in APP processing, genetic and biochemical evidence indicates that the PS function in the Notch pathway as well. The

Notch/LIN12 signaling pathway is an evolutionarily conserved mechanism that is used by metazoans to direct cell fate decisions, proliferation and apoptosis at all stages of development and in adult self-renewing cells, for example blood cells. Artavanis-Tsankonas et al . , Notch Signaling: Cell fate control and signal integration in development, Science 284 (5415) : 770-6 (1999 Apr. 30); Milner et al . , Notch as a Mediator of Cell Fate Determination in hematopoeises : Evidence and Speculation, Blood 93 (8) :2431-48 (1999 Apr. 15) ; De Strooper, A presenilin-1 -dependent γ- secretase-like protease mediates release of Notch intracellular domain, Nature, Vol. 398, 8 April, 1999. Signaling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. Mumm et al . , A ligand- induced extracellular cleavage regulates γ-secretase like proteolytic activation of Notchl, Mol . Cell. 5, 197-206 (2000); Huppert et al . , Embryonic Lethali ty in Mice Homozygous for a Processing Deficient Notchl allele, Nature 405, 966-970 (2000); Mumm J. , Kopan R. , Notch Signaling: From the Outside In, Dev. Biol . 228(2): 151-65 (2000 Dec. 15). This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD) , and is reminiscent of γ-secretase mediated cleavage of β-amyloid precursor protein (APP) , a critical event in the pathogenesis of AD. Schroeter et al . , Notch- 1 Signaling requires ligand -induced Proteolytic Release of Intracellular Domain, Nature, Vo. 393 (28 May 1998) ; De Strooper, A presenilin-1-dependent γ-secretase-like protease mediates release of Notch intracellular domain, Nature, Vol. 398, 8 April, 1999. A deficiency in presenilin-1 (PS1) inhibits processing of APP by γ-secretase in mammalian cells. Also, PS1 deficiency reduces the proteolytic release of NICD from^' a truncated Notch construct in mammalian cells. De Strooper, A presenilin-1 -dependent γ~secretase-like protease mediates release of Notch intracellular domain, Nature, Vol. 398, 8 April, 1999. Moreover, several γ-secretase inhibitors block the Notch signaling pathway by preventing proteolytic release of NICD, at the same step affected by PS1 loss, indicating that related proteolytic activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus, the targeting of γ- secretase for the treatment of Alzheimer's disease may risk toxicity caused by reduced Notch signaling. De Strooper, A presenilin-1 -dependent γ-secretase-like protease mediates release of Notch intracellular domain, Nature, Vol. 398, 8 April, 1999.

As a consequence of the central role Notch signaling plays in the adult self renewing tissues, such as glia differentiation, hematopoiesis, skin, gut and pancreas, and neurite outgrowth there is a need to identify γ-secretase inhibitors that will target only APP proteolysis and spare Notch signaling in the adult. Similarly, there is a need for a method of screening treatments capable of identifying substrate-specific inhibitors of proteolytic enzyme activity targeting transmembrane domains of polypeptides in a cell. For example, a known transmembrane domain proteolytic activity, Site2 protease (S2P) , cleaves the Sterol Response- element binding protein (SREBP) as well as ATF6. Ye et al . , ER Stress Induces Cleavage of Membrane -Bound ATF6 by the Same Proteases that Process SREBPs, Moll. Cell, Vol. 6, 1355-1364 (2000) . However, because SREBP is involved in cholesterol metabolism, inhibition of SREBP may not be wise. See U.S. Patent #5,527,690. Other substrates of S2P may be clinical targets. Thus, there exists a need to screen for specific inhibitors that block the cleavage of those other substrates but not of SREBP or ATF6. Summary of the Invention

Accordingly, it is an object of the present invention to provide a method for screening for pharmaceutical agents, chemical compositions, genetic products, growth conditions, nutritional supplements, physical treatments and conditions, or any other additives that will target specific enzymes for proteolysis of specific substrates.

The present invention provides a method for screening a candidate treatment for the ability to inhibit proteolytic cleavage of a transmembrane domain of a polypeptide comprising providing host cells wherein each cell comprises a dual address polypeptide containing a transmembrane domain including a cleavable sequence, cleavage of which induces a cell death; preventing the expression of said dual address polypeptide in the cells; exposing the cells to the candidate treatment for enzyme inhibition under conditions conducive to the proteolytic cleavage of said transmembrane domain; activating the expression of the polypeptide in conjunction with the continued exposure to the candidate treatment; and determining the presence of detectable change in the cells.

Furthermore, the present invention provides a method of screening treatments for inhibition of proteolytic enzyme activity for cleavage of a first transmembrane domain of a polypeptide and for tolerance of proteolytic cleavage of a second transmembrane domain of a polypeptide comprising screening two sets of cells by the method described above wherein the first set of cells comprises the first transmembrane domain in the absence of the second transmembrane domain and the second set of cells comprises the second transmembrane domain in the absence of the first transmembrane domain.

Brief Description of the Drawings

Figure 1 (A) is a graphic representation of the effects of γ-secretase inhibitor, the Wolfe inhibitor compound #11, on T cell differentiation in fetal thymic organ culture. The graph shows that the application of γ-secretase inhibitors to fetal thymus organ cultures interferes with T- cell development in a manner consistent with loss or reduction of Notchl function. Progression from an immature CD4-/CD8- state to an intermediate CD4+/CD8+ double positive (DP) stae was blocked. These results establish that γ- secretase inhibition indeed leads to Notch insufficiency as predicted.

Figure 1 (B) is a graphic representation of the effects of γ-secretase inhibitor, the Wolfe inhibitor compound #11, on T cell differentiation in fetal thymic organ culture with a CD3 HI Gate: Differentiated T cells. Here we show that the inhibitor specifically blocks the development of mature CD8+ cells but not of CD4+ cells, demonstrating that the effects are not due to general toxicity.

Figure 2 is a schematic representation of one use of this invention. Here the TKO screen is utilized to identify cellular activities whose loss will block proteolysis. The γ-secretase releases Bax, and library clones block the release. The schematic representation shows a leader peptide and the Notch transmembrane domain in frame to the protein Bax, and added a HA tag amino-terminal to the transmembrane domain (HA-N-Bax) . Upon cleavage of the transmembrane domain tether by the presenilin dependent proteolytic apparatus, Bax will be targeted to the mitochondria where it will initiate the death cascade.

Figure 3 is a graphic representation of the results of a test that show the ability of Cho cells to survive transfections with high levels of Bax or HA-N-Bax. The graph shows that the ability of Bax to kill Cho cells was unaffected by the presence of absence of an active PS protein. In contrast, as shown in the graph by the blue triangle line, HA-N-Bax only killed transfected Cho cells that contained active PS1 protein. Cho cells expressing a dominant negative PS1^D38SA protein became refractory to HA-N- Bax as shown in the line with the blue square, but were still sensitive to Bax as shown in the line with the pink square. The Y axis reflects the average numbers of transfected, LacZ positive cells/field; the X axis reflects Bax DNA concentration. This figure confirms that our method will work to protect cells from death by specifically blocking cleavage of the Notch transmembrane domain.

Figure 4 is a direct assay for Notch proteolysis, using a transmembrane domain mutation in Notch which prevents translation within the transmembrane domain (M1726V) . This figure demonstrates that a single, conservative amino acid substitution in Notch dramatically affects proteolysis. The amino acid substitution is underlined in the sequence in the figure. The accompanying gel shows that while the Notch peptide containing the transmembrane domain is a substrate for γ-secretase (two bands on the gel, a precursor on top and a product below) , the mutated Notch transmembrane domain at V1744L is resistant to proteolytic cleavage as shown by a single precursor band indicating lack of proteolytic activity. The transmembrane domain of Notch in which M1727 was mutated to V is LHIVYVAAAFVLLFFVGCGVLL and is SEQ. ID 1. The protease resistant mutant transmembrane domain of Notch

LHIVYVAAAFVLLFFVGCGLLLL is SEQ. ID. 2.

Definitions

Generally, the nomenclature used hereafter, and the laboratory procedures are those well known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. To facilitate understanding of the invention, a number of terms as used herein are defined below: The term "treatment" or "treatments" as used herein refers to the use of chemical compounds, chemical libraries, pharmaceutical agents, genetic alterations and products, growth conditions, nutritional supplements or additives, biological agents including proteins, protein fragments, RNA fragments and DNA fragments, minerals, or any other organic or inorganic matter, physical conditions, such as radiation, sound waves, light waves, electromagnetic waves, heat or any other physical property manipulated to produce a therapeutic result .

The term "HA" refers to hemagglutinin. The term "TM" refers to transmembrane domain portion of a polypeptide.

The term "LP" refers to the leader peptide portion of a polypeptide.

The term "Tag" refers to a marker sequences of a polypeptide.

The term "Effector" refers to the subdomain of a dual address protein that translocates to a second site of action. An example of an effector is the Bax protein.

The use of dashes between the terms and abbreviations, such as "LP-TM-Effector" refers to a continuous polypeptide containing at least those portions of the polypeptide indicated by the terms and abbreviations in the order of their appearance in the dashed phrase. For example, "LP-TM- Effector" refers to a continuous polypeptide that contains at least a leader peptide connected to a transmembrane domain connected to an effector protein.

Detailed Description of Invention

The present invention is directed to methods for the screening of potential inhibitors of specific proteolytic activity in a cell. Specifically, this invention is particularly useful for the screening of inhibitors of enzymes responsible for the proteolysis of transmembrane proteins. One particular transmembrane protein is APP, which is responsible for the production of A.beta.peptide, which is characteristically present in Alzheimer's disease. Also, one proteolytic enzyme that is of particular interest is γ-secretase, which is known to cleave APP causing A.beta.peptide build up in the brain.

The screening method of this invention utilizes cell cultures obtained from mammalian tissues by methods commonly known in the art. Human tissue cultures are preferred for use as the source of the cell cultures for this invention. A number of suitable host cell lines have been developed in the art, and include the human derived HeLa cells, myeloma cell lines, Jurkat cells, and so forth, as well as non-human cells such as CHO cell lines and various COS cell lines. Other cells known in the art may be used.

In preferred embodiments, the medium for which the cell culture is grown for this invention includes a selective growth medium, which is a medium in which the cells grow normally until an agent is added that triggers the expression of the substrate whose cleavage initiates a detectable change in the cell, preferably the death cascade in expressing cells. An example of this type of cell growth is a tetracyclin (tet) "on/off" system. Rossi et al . , Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibi tion by pi 6 , Nat. Genet. 20(4): 389-93 (Dec. 1998). This type of system is described in U.S. Patents #6,015,709, #5,589,362, #5,654,168, and #5,789,156. Growth in a tet containing control medium suppresses expression of the HA-TM-Bax. Suitable cell lines will grow in the media containing tetracyclin, but the same cells will be killed by the death cascade initiated in the mitochondria by the dual address protein originating from Bax when tetracyclin is removed from the media. The tetracyline system in this embodiment can be replaced by any method that allows tight control over the expression of the particular polypeptide of interest containing the transmembrane domain tethered to the dual address protein. Other currently available systems include Ecdyson induced expression, and rapamycin regulatable gene switch. Pollock et al . , Delivery of a stringent dimerizer- regulated gene expression system in a single retroviral vector, Proc. Natl . Acad. Sci . USA, Vol. 97, Issue 24, 13221-13226 (Nov. 21, 2000) . Other media known in the art may be used that would accomplish this same purpose.

The present invention also requires the use of a dual address protein. A dual address protein is a protein which exists at two discrete subcellular locations. The full- length dual address protein is first held at a docking site where, in response to stimulus/ligand, it undergoes intramembranous proteolysis to release a subdo ain that then translocates to a second site of action, typically the nucleus. Mumm et al . , Notch Signaling: From the Outside In, Developmental Biology, Vo. 228, No. 2, pp. 151-165 (De . 2000) . The dual address protein may be any dual address protein that initiates a detectable change in the cell. The preferred dual address protein for use in this invention initiates the death cascade in the mitochondria of the cell when released from its tether. Particularly useful dual address proteins for this invention are death inducers, including the Bax protein as an effector. Gopeing et al . , Regulated Targeting of BAX to mi tochondria, J. Cell Biol . 143 (1) :207-15 (1998 Oct. 1); Korsmeyer et al . , BCL-2 Gene family and regulation of programmed Cell death, Cancer Res. 59(7 Suppl) : 1693s-1700s (1999 Apr. 1). The Bax protein is fused to a leader peptide and a transmembrane domain of interest acting as a membrane tether, which constitutes the first "address" . (LP-TM-Effector) The use of a tag attached between the leader peptide and the transmembrane domain may be helpful for the purpose of confirmation that the transmembrane domain of interest is being produced in the cell and targeted properly, but is not necessary to this invention. (LP-Tag-TM-Effector) . Any tag known in the art may be used for the purpose of confirming that the cell is manufacturing the polypeptide of interest, such as LP-TM- Bax. Some examples of tags that may be used are the HA tag, the Myc tag and the FLAG tag. Kopan et al . , Signal transduction by activated mNotch : importance of proteolytic processing and its regulation by the extracellular domain, Proc. Natl. Acad. Sci . USA 93(4):1683-8 (Feb. 20, 1996). Other tags may be fluorescent proteins (i.e. GFP, RFP, CFP, and YFP. See Feng, et al . , Imaging Neuronal Subsets in Transgenic mice Expressing Mul tiple Spectral Variants of GFP, Neuron 28, 41-51 (2000) . Alternatively, if no tag is used, then biochemical or immunohistochemical methods known in the art may be used to detect the effector polypeptide to obtain an accurate accounting of the cells that are producing the polypeptide of interest. An effector, protein is released when cleaved by the proteolytic apparatus, thus initiating a selectable effect. If Bax is used, it will initiate the death cascade in the mitochondria of the cell. Other dual address proteins that may be used are DNA recombinase enzymes such as CRE and FLP, bacterial or yeast transcription factors, or any dual address protein with a biological activity of interest. Nagy A., Cre recombinase : the universal reagent for genome tailoring, Genesis 26(2) : 99-109 (2000 Feb) ; Fiering et al . , Analysis of Mammalian eis -regulatory DNA elements by homologous recombination, Methods Enzymol 306: 42-66 (1999). Other proteolytic enzymes, such as S2P, may be screened for inhibition by the methods of this invention. Brown et al . , Regulated Intramembrane Proteolysis : A Control Mechanism Conserved from Bacteria to Humans, Cell Vol. 100, 391-398 (Feb. 18, 2000) . Regulated intramembranous proteolysis (RIP) is a recently appreciated signal transduction mechanism for which this invention will permit the identification of compounds capable of inhibiting any RIP regulated pathway. Brown et al . , Regulated Intramembrane Proteolysis : A Control Mechanism Conserved from Bacteria to Humans, Cell Vol. 100, 391-398 (Feb. 18, 2000) .

Transmembrane domains of interest to be used in this invention may be either in native form or modified, e.g., so as to prevent any mechanism that would allow the release of the tethered dual address protein by a means other than the proteolytic activity sought to be inhibited by the screen. In order for a transmembrane domain to be useful for this invention, amino acid substitutions may be necessary in order to prevent any mechanism that would allow the release of the tether without the proteolytic activity which is sought to be inhibited. An example of a mechanism that would prevent the use of the polypeptide containing the Notch transmembrane domain tethered to the Bax protein is the utilization of AUG codons for alternative translation initiation. Lauring et al . , Evidence that an IRES wi thin the Notch2 coding region can direct expression of a nuclear form of the protein, Mol . Cell 6(4): 939-45 (Oct. 2000); Kopan et al . , Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain, Proc . Natl. Acad. Sci. USA 93(4): 1683-8 (Feb. 20, 1996) . The presence of an internal ribosome entry site on the RNA responsible for the production of the polypeptide containing Notch2 was recently reported. Lauring et al . , Evidence that an IRES wi thin the Notch2 coding region can direct expression of a nuclear form of the protein, Mol. Cell 6(4): 939-45 (Oct. 2000). Ribosome scanning and the internal ribosome entry site would initiate translation of the RNA at a downstream codon rather than the correct codon, which could produce the effector without a leader peptide or the transmembrane of interest . Therefore, AUG codons may be changed to produce an alternative amino acid to prevent this problem. A particular transmembrane domain of interest for this invention is the APP transmembrane domain. Another particular transmembrane domain of interest for this invention is the Notch transmembrane domain. One modified Notch tramsmembrane domain that is useful for this invention is shown in figure 4 (note: the underlined V represents the altered AUG codon in the Notch transmembrane domain) . Other modifications of the Notch transmembrane domain that prevent any mechanism that would allow the release of the tethered dual address protein by a means other than the proteolytic activity sought to be inhibited by the screen may also be used. Specific transmembrane domains that may be used for this invention include, but are not limited to the transmembrane domains of APP, mutated Notch, sterol response element binding protein, ATF6, and IRE1. Brown et al . , Regulated Intramembrane Proteolysis : A Control Mechanism Conserved from Bacteria to Humans, Cell Vol. 100, 391-398 (Feb. 18, 2000) . Some transmembrane domains of interest include the APP protein of GAIIGIMVGGVIATVIVITLVML, which is SEQ. ID 3. Other transmembrane domains of interest include modified APP proteins of GAIIGIWGGVIATVIVITLVLL , which is SEQ. ID. 4, and GAIIGIMVGGVIATVIVITLLML, which is SEQ. ID.

5. Further, transmembrane domains of interest include SREBP-1, which is LALCTLVFLCLSCNPLASLLGA, which is SEQ. ID.

6, and SREBP-2, which is ILLCVLTFLCLSFNPLTSLL, which is SEQ. ID. 7. Additionally, the ATF6 transmembrane domain may be used for this invention and includes WCVMIVLAFIILNYGPMSML, which is SEQ. ID. 8. Also, the transmembrane domain of Irel may be used for this invention and includes LLAATFTAILLGAWVLYLM, which is SEQ. ID. 9. This invention provides a systematic screen to identify genes, chemical agents, physical conditions, growth conditions, nutritional supplements, waves (light, electromagnetic, etc.), or any other additives having the ability to prevent or inhibit the proteolysis of a membrane tether. This screen can be used to identify specific pharmacological inhibitors of proteolytic activity directed to specific transmembrane domain targets. Candidate treatments to be screened may be generated by a variety of methods. One technique, termed Technical Knock Out (TKO) , is based on random inactivation of genes mediated by expression of an anti-sense cDNA library, followed by selection for a specific phenotypic alteration. Diess et al., Science 252, 117-120 (1991); A. Kimchi, Biochem Biophys Acta 1377, F13-33 (1998); A. Kimchi, Ann Rheum Dis 58 Suppl 1, 14-19 (1999); Kissil et al . , Mol Med Today 4, 268-274 (1998); Levy-Strumpf et al . , Oncogene 17, 3331-40 (1998) . The TKO method is commonly known in the art and other methods known in the art may be used to accomplish this purpose. Other genetic screens based on gain of function can also be applied.

The screening of the treatment for inhibition of proteolysis of a transmembrane domain is performed under conditions that are conducive to the proteolytic cleavage of the transmembrane domain. In order to have a condition that is conducive to the proteolytic cleavage of the transmembrane domain, there must be a proteolytic enzyme present in the cell that would cleave the transmembrane domain at the time the treatment is introduced to the cell. Thus, the enzyme may be present in the cell because it is an endogenous enzyme to the cell, or alternatively the enzyme may be introduced into the cell by alternative means. For example, the proteolytic enzyme itself may be introduced into the cells. Alternatively, electroporation or other DNA transfection techniques known in the art may be used to insert the gene coding for the enzyme into the cell. The proteolytic enzyme may be introduced into the cell by any other method known in the art. However, it is preferred that the proteolytic enzyme is endogenous to the cell and is produced by the cells selected for this method.

By performing parallel screens with different transmembrane domains that are specifically chosen, this invention can be used to identify specific inhibitors that block cleavage of one transmembrane domain, such as APP for example, while permitting the cleavage of another transmembrane domain, such as Notch. An alternative use for this method is based on inserting a randomized library coding for the transmembrane domain position of Tag-TM-Bax. This variation of the method can be used to characterize protease specificity to any transmembrane domain sequence. For example, the transmembrane domain coding sequence may be (NNK)₂₃ wherein N is any nucleotide (A,T,C,or G) and K is either the nucleotide G or T. The TKO method is but one method commonly known in the art that may be utilized to screen for gene products that inhibit proteolytic activity of certain transmembrane domains . Other methods known in the art may also be used.

Additionally, other candidate treatments may be screened, including chemical compounds. For example, chemical libraries may be utilized to test for their effectiveness for the inhibition of proteolytic activity on a specific transmembrane domain of interest in order to locate a pharmaceutical agent for treatment of certain disease states. This invention may also be used to screen for growth conditions, such as nutritional supplements or additives, that may inhibit proteolytic activity of a transmembrane domain of interest. Also, physical conditions such as the use of radiation, such as gamma, X, Beta, positron emission, or any other form of radiation, sound waves, light waves, heat or any other physical property may be used in this invention to screen for such physical conditions that inhibit proteolytic activity of any transmembrane domain.

This invention may be used to screen a particular treatment for inhibition of proteolytic activity against a particular transmembrane domain while simultaneously performing the same screen for the same particular treatment against a different transmembrane domain. Such parallel screens will enable the identification of specific treatments for inhibition that block cleavage of one transmembrane domain while permitting the cleavage of another transmembrane domain. Multiple transmembrane domains may be screened against each other simultaneously. These parallel screens will be useful, for example, for identifying inhibitors of APP proteolysis that do not inhibit the proteolysis of Notch.

Other features, objects and advantages of the present invention will be apparent to those skilled in the art. The explanations and illustrations presented herein are intended to acquaint others skilled in the art with the invention, its principles, and its practical application. Those skilled in the art may adapt and apply the invention in its numerous forms, as may be best suited to the requirements of a particular use. Accordingly, the specific embodiments of the present invention as set forth are not intended as being exhaustive or limiting of the present invention. All references cited herein are explicitly incorporated by reference as if fully stated herein.

The following examples illustrate the invention.

EXAMPLE 1

The Genetic screen: The γ-secretase complex (secretasome) contains additional proteins besides Presenilin and Nicastrin. Kopan et al . , Genes & Development 14, 2799-2806 (2000); C. Goutte et al . , Development 127, 2481-2492 (2000); Yu et al . , Nature 407, 48-54 (2000) . Most in the field are pursing a biochemical strategy of cloning interacting proteins, a strategy unlikely to identify regulators. Genetic screens in invertebrates take time, are only clinically useful if the vertebrate secretasome and its regulation have been conserved throughout evolution, and cannot be directed at APP since no phenotype is associated with its loss. To identify genes involved in APP proteolysis and not in Notch signaling in human cells we have taken a rapid, unbiased, tissue culture based genetic approach. This genetic approach allows for the identification of genes based solely on their function. This will allow us to uncover genes and potentially regulatory mechanisms not previously imagined to be involved in APP and Notch cleavage or secretasome function. Identifying these genes is useful for the identification of novel pharmacological targets. One particular genetic screen, termed Technical Knock

Out (TKO) is based on random inactivation of genes mediated by expression of an anti-sense cDNA library, followed by selection for a specific phenotypic alteration as discussed on page 12 in the specification. The TKO is based on a library expressed from an episomal shuttle vector that is optimized for efficient introduction, expression, selection (hygromycin resistance) and retrieval from human cells of the specific anti-sense construct responsible for the phenotypic change. Diess et al . , Science 252, 117-120 (1991); Angell et al . , J^". Biol . Chem. (2000); Hofmann et al., Mol . Cell Biol . 18, 6493-6504 (1998).

Experimental Data for Screen:

Anti-sense constructs that inhibit APP proteolysis are contemplated as being useful for the screen of this invention. For this invention to succeed, a selection must be designed that can identify rare cells that have lost APP cleavage activity while in parallel, retain the ability to cleave the Notch transmembrane domain. This selection is now described using Notch proteolysis as an example. We reasoned that coupling the minimal domain required for intramembranous proteolysis to a downstream component of the apoptotic machinery would provide us with a strong selection tool to identify components and regulators of the secretasome. As proof of principal, we fused the Notch transmembrane domain in frame to the protein Bax, and added a leader peptide and an HA tag amino-terminal to the transmembrane domain (HA-N-Bax). Oltvai et al . , Cell 74, 609-19 (1993) . Thus, we converted Bax to a dual address protein which, upon cleavage by the presenilin dependent protelytic apparatus, will be targeted to the mitochondria where it will initiate the death cascade. Goping et al . , J. Cell Biol . 143, 207-215 (1998); Gross et al . , Embo J. 17, 3878-85 (1998); S. J. Korsmeyer, Tredns Genet 11, 101-5 (1995) .

Dual Address Bax Kills Cells in a Presenilin Dependent Manner :

Double blind experiments were conducted to test the ability of Cho cells to survive transfections with high levels of Bax or HA-N-Bax. We show here that the ability of Bax to kill Cho cells was unaffected by the presence or absence of an active PS protein. Results are shown in Figure 3. In contrast, HA-N-Bax only killed transfected Cho cells that contained active PS1 protein. Cho cells co- expressing a dominant negative psi^D385A protein became refractory to HA-N-Bax but were still sensitive to Bax. This result demonstrates that these constructs can be used to screen for loss of γ-secretase activity establishing the feasibility of a loss of function screen with a tethered Bax.

Experimental Design: We will place HA-APP™-Bax under control of tetracycline operator sequences (Clontech) and a minimal promoter from human cytomegalovirus (tetHA-APP-Bax) . The APP™ supports Aβ production. Tetracycline-regulated transactivator (tTA) - expressing Hela cell lines will be transfected to produce clones that grow in media containing tetracycline but are killed by Bax when tetracycline is removed from the media. Similar construct, containing the Notch transmembrane domain is also being prepared. The TKO antisense library will be used to induce loss of function: HeLa tTA cells containing tetHA-APP-Bax will be transfected with TKO library. Deiss et al . , Science 252, 117-20 (1991). The forward selection (ex. death induced by expression of APP-Bax) will result in the recovery of cells that have either lost secretasome activity or acquired resistance to Bax induced apoptosis. Loss of "cleavage pathway" genes will be identified by expanding the surviving clones and directly measuring Aβ levels in the overnight culture supernant from 96 or 48 well plates by ELISA (Biosource International, Camarillo, CA) . Alternatively, a precursor of Ab (APP C99) transfection will be used to measure Aβ levels. This analysis will produce a good approximation of the rate of false positives (secretasome loss/ death surviving colonies) in the screen output. Episomes from surviving, Aβ40 and Aβ42 deficient cells will be rescued. We will follow the original TKO protocol as described above. Deiss et al . , Science 252, 117-20 (1991) . Sequencing the anti-sense cDNA inserts of the positive episomes will be done next, followed by bio- informatic and biochemical analyses. Direct measurement of Aβ will miss clones that do not block Notch proteolysis while blocking APP cleavage. If the number of clones is small, Notch proteolysis will be examined directly in each clone by transfecting the TM/Myc construct (Fig. 4) . If many surviving colonies were generated in the first screen, unique episomes will be transfected into cells containing inducible HA-N-Bax. Stable transfectants will be plated in 96 well plates such that episomes occupy the same grid position in HA-N-Bax proteolysis to produce viable colonies, but fail to protect HA-N-Bax cells from death will be preferentially rescued as they target APP proteolysis but do fail to block Notch proteolysis.

Determination of Substrate Specificity:

It was postulated that the secretasome lacks specificity for substrate sequence. Struhl et al . , Molecular Cell 6, 625-63 (2000) . However, our observations suggest that this is an oversimplification. Figure 4 demonstrates that a single, conservative amino acid substitution in Notch dramatically affects proteolysis. We want to establish substrate preferences for the secretasome by a modified site selection approach. Blackwell et al . , Science 250, 1104-10 (1990); Huang et al . , Mol Cell Biol 16, 3893-900 (1996) . In place of the transmembrane domain in Figure 2 we will insert a random transmembrane domain, composed from an oligonucleotide library of the reiterated codon (NNK) ₂₂-Bax/Puro vector, will code for all possible amino acid combinations in the putative transmembrane domain in that open reading frame and will be transfected into HeLa cells. DNA from puro resistant, HA tag positive cells will be prepared and PCR amplification of the transmembrane domains will be performed. A small number of surviving clones is expected if the secretasome lacks sequence specificity. In this case, sequencing of individual PCR products will be done to identify amino acid compositions resistant to secretasome mediated proteolysis. Thousands of clones are expected if the only few specific substrates are recognized by γ-secretase. If the secretasome is a selective protease, pool sequencing will reveal bias against amino acids at specific positions, defining preferences.

Experimental Design and Preliminary Results

The information required for P"¹ dependent proteolysis is contained within the Notch transmembrane domain. Using a series of deletion mutants we determined that the Notch transmembrane domain contains all the information required for V1744 dependent proteolysis. Proteins containing the sequence: IEAVKSEPVEPPLPSQLHIVYVAAAFVLLFFVGCGVLLSRKRR-6 Myc Tag (in bold is the transmembrane domain, underlined is a mutation eliminating translation initiation, in italics is V1744L) were cleaved, but the proteins containing the peptide : IEAVKSEPVEPPLPSQGAIIGIMVGGVIATVIVITLVMLSRKRR, containing the APP transmembrane domain in bold and the V mutant in italics: IEAVKSEPVEPPLPSQGAIIGIMVGGVIATVIVITLI.MLSRKRR are effectively cleaved. Furthermore, the transmembrane domain of Notch4 is also cleaved at V1463 at an equivalent position to NlVall744. These results clearly demonstrate that in human HEK293 cells presenilin dependent proteolysis can distinguish between the two Notch substrates while continuing to cleave APP. These observations suggest that all Notch proteins create a distinct class of PS substrates.

In preliminary tests, we constructed two variants of HA-TM-Bax molecule: one harboring the intact transmembrane domain of Notch and the other in which critical amino acids (GCGV) containing S3 (underlined) was substituted to (LLFF) .

We demonstrated previously that this substitution reduced S3 proteolysis. In titration experiments we found that Bax killed Hela cells, as did our HA-N-Bax constructs. The HA- N^LLFF-Bax was less efficient at killing. We then tested in double blind experiments the ability of Cho cells to survive transfection with high levels of Bax or HA-N-Bax. Results are shown in Figure 3.

Claims

What is claimed is :

1. A method for screening a candidate treatment for the ability to inhibit proteolytic cleavage of a transmembrane domain of a polypeptide comprising:

(a) providing host cells wherein each cell comprises a dual address polypeptide containing a transmembrane domain including a cleavable sequence, cleavage of which induces cell death;

(b) preventing the expression of said dual address polypeptide in the cells of step (a) ; (c) exposing the cells of step (b) to the candidate treatment for proteolytic cleavage inhibition under conditions conducive to the proteolytic cleavage of said transmembrane domain;

(d) activating the expression of the polypeptide of step (a) in conjunction with the continued exposure of step (c) ;

(e) determining the presence of a detectable change in the cells.

2. A method of claim 1 wherein the detectable change is cell death.

3. A method of claim 1 wherein the transmembrane domain of the polypeptide contains a modification which prevents or substantially inhibits the release of the dual address protein from the transmembrane domain tether without proteolytic activity.

4. A method of claim 2 wherein cell death is induced by cleavage of the transmembrane polypeptide to release a dual address protein that subsequently initiates death in the host cell.

5. A method of claim 1 wherein the cells are grown in a medium that silences expression of said polypeptide of step

(a) .

6. A method of claim 5 wherein the cell medium is modified to activate expression of said polypeptide of step (a) .

7. A method of claim 1 wherein the cells of step (c) contain an endogenous proteolytic enzyme for the cleavage of the transmembrane domain.

8. A method of claim 1 wherein the polypeptide of step (a) further comprises an extracellular tag.

9. A method of claim 8 wherein the extracellular tag is an HA tag.

10. A method of claim 8 wherein the extracellular tag is a Myc tag.

11. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises the transmembrane domain of Amyloid precursor protein.

12. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises the transmembrane domain of a modified Amyloid precursor protein.

13. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises the transmembrane domain of a modified Notch protein.

14. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises SEQ. ID. 1.

15. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises SEQ. ID. 2.

16. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises SEQ. ID. 3.

17. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises SEQ. ID. 4.

18. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises SEQ. ID. 5.

19. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises SEQ. ID. 6.

20. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises SEQ. ID. 7.

21. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises SEQ. ID. 8.

22. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises SEQ. ID. 9.

23. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises the transmembrane domain of sterol response element binding protein.

24. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises the transmembrane domain of ATF6.

25. A method of claim 1 wherein the transmembrane domain of the polypeptide of step (a) comprises Irel.

26. A method of claim 1 wherein the host cells of step (a) are mammalian cells.

27. A method of claim 1 wherein the host cells of step (a) are human cells.

28. A method of claim 1 wherein the host cells of step (a) are tetracycline-regulated transactivator expressing human cervical carcinoma derived cells.

29. A method of claim 1 wherein the enzyme comprises gamma- secretase.

30. The method of claim 1 wherein the enzyme comprises a presenilin protein.

31. The method of claim 1 wherein the enzyme comprises S2P.

32. The method of claim 1 wherein the dual address protein comprises the Bax protein.

33. The method of claim 1 wherein the dual address protein comprises a DNA recombinase enzyme.

34. The method of claim 1 wherein the dual address protein comprises CRE.

35. The method of claim 1 wherein the dual address protein comprises FLP.

36. The method of claim 1 wherein the dual address protein comprises a transcription factor.

37. The method of claim 1 wherein the silencing of step (b) occurs on the presence of tetracyclin.

38. The method of claim 1 wherein the activating of step (d) occurs in the absence of tetracyclin.

39. The method of claim 1 wherein the treatment is administration of a chemical composition.

40. The method of claim 1 wherein the treatment is inducement of a genetic alteration.

41. The method of claim 1 wherein the treatment is administration of a nutritional supplement.

42. The method of claim 1 wherein the treatment is administration of pharmaceutical agent.

43. The method of claim 1 wherein the treatment is administration of radiation..

44. The method of claim 1 wherein the treatment is administration of a biological agent.

45. A method for screening a candidate treatment for the ability to inhibit proteolytic cleavage of a first transmembrane domain of a polypeptide and for tolerance of proteolytic cleavage of a second transmembrane domain of a polypeptide comprising screening two sets of cells by the method of claim 1 wherein the first set of cells comprises the first transmembrane domain in the absence of the second transmembrane domain and the second set of cells comprises the second transmembrane domain in the absence of the first transmembrane domain.

46. The method of claim 45 wherein the first and second set of cells are mammalian cells.

47. The method of claim 45 wherein the first and second set of cells are human cells.

48. The method of claim 45 wherein the first and second set of cells are tetracycline-regulated transactivator expressing human cervical carcinoma derived cells.