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Title:  Caspase 9 activation and uses therefor
United States Patent:  7,118,877
Issued: 
October 10, 2006

Inventors: 
Edris; Wade Allen (New Hope, PA)
Assignee: 
Wyeth (Madison, NJ)
Appl. No.:  10/191,254
Filed: 
July 8, 2002


 

Training Courses -- Pharm/Biotech/etc.


Abstract

The present invention discloses methods for activating Caspase 9 in such a way that it can be used in assays to discover modulators of Caspase 9.

SUMMARY OF THE INVENTION

This application relates to methods for identifying compounds that modulate Caspase 9 activity.

DETAILED DESCRIPTION OF THE INVENTION

Caspase 9 is the proteolytically active member of the multi-component assembly termed the apoptosome (Renatus, et al., (2001) Dimer formation drives the activation of the death protease Caspase 9, PNAS 98, 14250 14255). This complex controls the tissue architecture of the developing nervous system and the deletion of cells injured beyond recovery by environmental stress or ligand-receptor activation. Like other members of the caspase family, Caspase 9 exists in a latent form in of the cell, but unlike the others, simple proteolytic processing is insufficient for activation. Association with an activated form of APAF-1 in a 1-mega dalton complex is required for full catalytic conversion. The difficulties inherent in purifying each member of the complex and assembling them in vitro has greatly hindered efforts in gaining a structural understanding of Caspase 9 activation or using the protease in screens designed for discovering specific modulators. By providing a simple and convenient method for enhancing the activity of Caspase 9 in vitro this invention enables screening, including high throughput screening.

The Hofmeister series (see Original Patent) originates from the ranking of various ions toward their ability to precipitate a mixture of hen egg white proteins.

Hofmeister series salts have been shown to have a much more general utility including showing the graduated effects on the structuring or denaturation of biological macromolecules. More recently the Hofmeister series are usually given in terms of the ability of the ions to stabilize the structure of proteins. A similar effect has been found with the salt-induced activation of lyophilised enzymes (Ru, et al. On the salt-induced activation of lyophilized enzymes in organic solvents: Effect of salt kosmotropicity on enzyme activity, J. Am. Chem. Soc. 122 (2000) 1565 1571). They show opposite correlation for anions and cations with their degree of strong hydration.

The relative positions (mostly corresponding to the degree of strong hydration) in the series should be thought of as indicative only, as there will be variation with protein, pH and temperature, with acetate ions showing pronounced cation-specific effects. Ions destroy the natural hydrogen bonded network of water, having effects similar to increased temperature or pressure; e.g. reduced viscosity. This effect of ions has been successfully approximated by the equivalent osmotic pressure. Ions that have the greatest such effect (exhibiting weaker interactions with water than water itself) are known as structure-breakers or chaotropes, whereas ions having the opposite effect are known as structure-makers or kosmotropes (exhibiting strong interactions with water molecules).

When Caspase 9 samples are prepared in ammonium sulfate rapid conversion of the substrate proteins to small peptides is observed, demonstrating dramatically increased proteolysis in this solution. Without limiting the generality of the mechanism, and without limiting the compositions and methods of the present application, the inventors postulate that the increased polarity of the aqueous solution by the dissolution of ammonium sulfate is responsible for a structural change in Caspase 9 by oligomerization or a conformational change with concomitant activation. A number of viral proteases are known to be activated by the water structuring anions (Kosmotropic agents) of the Hofmeister series (Cacace, M., Landau, E. and Ramsden, J. (1997), The Hofmeister series: salt and solvent effects on interfacial phenomena, Quart. Rev. of Biophysics 30, 241 277). For example, Herpes simplex virus 1 protease, responsible for maturation of the viral capsid, shows a 860-fold increase in activity in 1 M Na citrate (Hall, D. and Darke, P. (1995). Activation of the Herpes Simplex virus type 1 protease. JBC 270, 22697 22700). Treatment with this salt is thought to mimic the native microenvironment of the active protease, possibly in the nucleus with its attendant high concentration of polyanions.

Mimicking the activation of Caspase 9 in the apoptosome by modulating the solvent conditions for the protease leads to a simple in vitro screening procedure for discovering inhibitors of this enzyme.

The activation of Caspase 9 by kosmotropic agents is surprising not only in the extent of the activation but also in the fact that Caspase 9 is activated at all. Prior to the present invention there was no reason to believe that kosmotropic salts would activate Caspase 9. This fact is demonstrated by the failure of kosmotropic salts to activate other caspases such as Caspase 3 as described in Example 4.

Caspase 9 inhibitors can be used to treat or reduce the severity of diseases characterized by increased programmed cell death. Using assays as described herein for Caspase 9 activity, various compounds can be screened to discover compounds that inhibit or enhance the expression of Caspase 9 protease activity. Such screening methods are known to those skilled in the art. Such inhibitory molecules can be those contained in synthetic or naturally occurring compound libraries.

Caspase 9 inhibitors include, for example, small molecules and organic compounds that bind and inactivate Caspase 9 protease activity by a competitive or noncompetitive-type mechanism, inhibitors of the conversion of inactive proCaspase 9 into active Caspase 9 protease or other molecules that indirectly inhibit the Caspase 9 pathway. Such Caspase 9 inhibitors can include, for example, suicide inhibitors, anti-Caspase 9 antibodies and proteins, small peptide protease inhibitors, or small non-peptide organic molecule inhibitors. Specific examples of such inhibitors include substrate analogs such as tetrapeptide DEVD-CHO (SEQ ID NO: 1) (Asp-Glu-Val-Asp-aldehyde), fluorescently labeled tetrapeptide: such as DEVD-AMC (SEQ ID NO: 1) (Asp-Glu-Val-Asp-aminomethylcoumarin), YVAD-AMC (SEQ ID NO: 7) (Tyr-Val-Ala-Asp-aminomethylcoumarin), ZEVD-AMC (carbobenzoxy-Glu-Val-Asp-aminomethylcoumarin) and the cowpox virus protein Crm A. Another specific example includes phage display peptide libraries where greater than 10.sup.8 peptide sequences can be screened in a single round of panning (U.S. Pat. No. 6,121,416). Caspase 9 inhibitors can be formulated in a medium that allows introduction into the desired cell type or can be attached to targeting ligands for introduction by cell-mediated endocytosis and other receptor-mediated events.

Caspase 9 substrate antagonists can be used to treat or reduce the severity of diseases mediated by increased programmed cell death. Such substrate antagonists can bind to and inhibit cleavage by Caspase 9. Inhibition of substrate cleavage prevents commitment progression of programmed cell death. Substrate antagonists include, for example, ligands and small molecular compounds.

Caspase 9 inhibitors can also be identified using Caspase 9-encoding nucleic acids and the Caspase 9 polypeptide of the invention in, for example, binding assays such as ELISA or RIA, or enzymatic assays using tetrapeptide substrates, such as courmarin labeled DEVD-AMC (SEQ ID NO: 1) and YVAD-AMC (SEQ ID NO: 7). DEVD-AMC (SEQ ID NO: 1) and YVAD-AMC (SEQ ID NO: 7) represent cleavage sites for the poly(ADP-ribose) polymerase (PARP) and IL-1.beta.: P1 P4 substrate tetrapeptides, respectively (Nicholson et al., Nature 376:37 43 (1995)).

The Caspase 9 polypeptide to be used in such assays can be obtained by, for example, in vitro translation, recombinant expression or biochemical procedures. Such and other methods are known within the art. For example, recombinant Caspase 9 can be expressed by cloning Caspase 9 cDNA into a bacterial expression vector such as pET21b (Novagen Inc., Madison, Wis.). The Caspase 9 can then be expressed and purified using routine molecular biology methods known to those skilled in the art. A purified recombinant Caspase 9 protein can be used to measure hydrolysis rates for various substrates, such as DEVD-AMC (SEQ ID NO: 1) and YVAD-AMC (SEQ ID NO: 7) in a continuous fluorometric assay.

Numerous methods are known in the art for measuring caspase activity including using fluorogenic substrates of the caspase, enzyme activity assays, immunoblotting, and affinity labeling as described in Current Protocols in Cell Biology, Chapter 18 which is hereby incorporated by reference in its entirety. Prior to the present invention these methods were not useful for Caspase 9 because of its low activity level in vitro.

Once Caspase 9 is activated using the techniques disclosed herein its activity can be quantified using a fluorescent assay. In one embodiment 7-amino-trifluoromethyl coumarin (AFC) is used. AFC fluoresces when cleaved from a peptide, such as Ac-LEHD-AFC (SEQ ID NO: 5) because it is no longer quenched by the acetyl (Ac) blocking group. AFC excitation occurs at 400 nm and its fluorescence is 505 nm. When the peptide is intact this fluorescence is quenched by the presence of the blocking Ac (acetyl) group on the N-terminal of the peptide because the both groups are so close in distance. Caspase 9 cleaves after the D (asp) and releases the AFC group which is then not quenched by the blocking group.

High-throughput screening of caspases other than Caspase 9 is known in the art. For example, Caspase 8 was screened by Smith et al. (Expression, preparation, and high-throughput screening of Caspase-8: discovery of redox-based and steroid diacid inhibition, Arch Biochem Biophys 2002 Mar. 15; 399(2):195 205, incorporated herein by reference in its entirety) as follows: an Escherichia coli expression construct for Caspase-8 was constructed in which a His-tag sequence is inserted 5' of codon 217 of Caspase-8. The strain produced a significant amount of soluble His-tagged 31-kDa inactive single-chain enzyme precursor. This 31-kDa protein was purified to 98% purity. Hydroxyapatite column chromatography resolved the enzyme into two species, one with the appropriate 31,090 relative mass and the other with 178 additional mass units (i.e., 31, 268). The latter proved to result from E. coli-based modification of the His-tag with one equivalent of glucono-1,5-lactone. The purified proteins were activated by autoproteolysis to the appropriate 19-plus 11-kDa enzyme by the addition of dithiothreitol in appropriate buffer conditions. This yielded an enzyme with specific activity of 4 5 units/mg (U/mg) against 200 microM Ac-IETD-pNA (SEQ ID NO: 2) at 25 degrees Celcius (C). The fully active protein was used in a high-throughput screen for inhibitors of Caspase-8. A preliminary robustness screen demonstrated that Caspase-8 is susceptible to reactive oxygen-based inactivation in the presence of dithiothreitol (DTT) reducing agent but not in the presence of cysteine. Investigation into the mechanism of this inactivation showed that quinone-like compounds were reduced by DTT establishing a reactive oxygen generating redox cycle the products of which (likely H(2)O(2)) inactivated the enzyme. Caspase-8 inhibitors and steroid-derived diacids with affinity in the low micromolar range were uncovered in the screen. Structure-activity investigation of the inhibitors showed that both the steroid template and the acid moieties were required for activity. One skilled in the art will recognize how the present invention can be used to modify such existing screening methods to accommodate Caspase 9 which previously could not be screened in a meaningful way.

 

Claim 1 of 14 Claims

1. A method for identifying a compound that modulates Caspase 9 activity, comprising: a) contacting a sample containing Caspase 9 with a test compound; b) adding a purified kosmotropic agent to the sample containing Caspase 9 such that the sample comprises one or more kosmotropic agents and the final concentration of the kosmotropic agent or agents exceeds 0.3M, and c) detecting the activity of Caspase 9, wherein a change in activity indicates a compound which modulates Caspase 9 activity.

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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