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WO1997031018A1 - Structure cristalline d'apopaine et son utilisation - Google Patents

Structure cristalline d'apopaine et son utilisation Download PDF

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Publication number
WO1997031018A1
WO1997031018A1 PCT/US1997/002578 US9702578W WO9731018A1 WO 1997031018 A1 WO1997031018 A1 WO 1997031018A1 US 9702578 W US9702578 W US 9702578W WO 9731018 A1 WO9731018 A1 WO 9731018A1
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WIPO (PCT)
Prior art keywords
apopain
ice
crystal
protein
dna
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PCT/US1997/002578
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English (en)
Inventor
Donald W. Nicholson
Kimberly A. Fazil
Michel Gallant
Yves Gareau
Marc Labelle
Dita M. Rasper
Rejean Ruel
John P. Vaillancourt
Joseph W. Becker
Jennifer Rotonda
Nancy A. Thornberry
Erin P. Peterson
Original Assignee
Merck & Co., Inc.
Merck Frosst Canada Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from GBGB9607206.1A external-priority patent/GB9607206D0/en
Application filed by Merck & Co., Inc., Merck Frosst Canada Inc. filed Critical Merck & Co., Inc.
Priority to EP97907690A priority Critical patent/EP0885243A4/fr
Priority to JP9530285A priority patent/JP2000505457A/ja
Publication of WO1997031018A1 publication Critical patent/WO1997031018A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C12N9/6475—Interleukin 1-beta convertase-like enzymes (3.4.22.10; 3.4.22.36; 3.4.22.63)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to crystalline apopain, the structure of apopain as determined by X-ray crystallography, the use of that structure to solve the structure of apopain homologues and other crystal forms of apopain, mutants and co-complexes of apopain, and the use of the apopain structure and that of its homologues, mutants, and co- complexes to design modulators of apopain.
  • One aspect of the invention resides in the obtaining of crystals of apopain of sufficient quality to determine the three dimensional (tertiary) structure of the protein by X-ray diffraction methods. Obtaining such crystals is in fact very much an unexpected result. It is well known in the protein crystal lographic art that obtaining crystals of quality sufficient for determining the structure of the apopain has not been achievable until the present application. Accordingly, one object of the present invention is to provide crystals of sufficient quality to obtain a determination of the three-dimensional structure of apopain to high resolution. The value of the crystals of apopain extends beyond merely being able to obtain a structure for apopain alone.
  • the knowledge obtained concerning apopain may be used to model the tertiary structure of related proteins.
  • Another aspect of the present invention is to provide a starting material for use in the determination of the structure of other members of the apopain family of proteins.
  • the knowledge of the structure of the apopain family of proteins provides a means of investigating the mechanism of action of these proteins in the body. For example, binding of these proteins to various receptor molecules can be predicted by various computer models. Upon discovering that such binding in fact takes place, knowledge of the protein structure then allows chemists to design and attempt to synthesize small molecules which mimic the functional binding of apopain to its receptor. This is the method of "rational" drug design.
  • another aspect of the invention is to provide material which is a starting material in the rational design of drugs which mimic the action of apopain.
  • ICE/CED- 3-like protease family which divide into two distinct phylogenic subfamilies, have now been identified.
  • ICErel- ⁇ TX, ICH-2
  • ICE re l-ITI ICE rel-ITI
  • CED-3 CED-3
  • ICH-1 mimerase-activated protein
  • CPP32 apopain, Yama
  • Mch2 Mch3
  • ICE-LAP3 ICE-LAP3
  • apopain/CPP32 is a key player in this important physiological process 1 ' -20
  • Apopain incapacitates key homeostatic and repair enzymes at the onset of apoptosis by separating essential functional domains at (P4)Asp-X-X-Asp(Pl) motifs.
  • Known substrates for apopain include poly(ADP-ribose) polymerase (an enzyme involved in genome surveillance and DNA repair in stressed cells), the U l -70 kDa small nuclear ribonucleoprotein (which is necessary for mRNA splicing) and the 460 kDa catalytic subunit of the DNA-dependent protein kinase (which is essential for DNA double-strand break repair) ⁇ ' 9, 21-23
  • Additional potential substrates for apopain and apopain-like proteases during cell suicide include structural proteins, such as fodrin (non-erythroid spectrin), nuclear lamins and growth arrest-specific protein 2 (Gas2), as well as protein kinase C d and several as-yet-unidentified lupus autoantigens22, 24-27 T e e proteases thus appear to participate in a cascade of cleavage events that contribute to the disabling of homeostatic and repair processes as well as the systematic structural
  • ICE/CED-3 proteases all appear to be synthesized as proenzymes each of which is proteolytically processed to form a heterodimeric catalytic domain.
  • two heterodimers associate to form a tetramer, an observation that has led to the suggestion that this species is the catalytically active form of the enzyme2"> 29 ⁇ j- , e mos t distinctive catalytic property of this family is a near absolute requirement for Asp at the substrate P] position ⁇ * 5, 28-30 Despite these and other similarities, however, the substrate specificity for ICE and apopain are markedly different.
  • ICE has a preference for hydrophobic residues in the P4 position, a critical determinant, while apopain appears to be specific for Asp in this position.
  • apopain in complex with the peptide-aldehyde inhibitor Ac-DEVD-CHO at a nominal resolution of 2.5 A .
  • the tertiary and quaternary structures of apopain are strikingly similar to those of ICE (Fig. 1 ).
  • the pi 7 and p 12 subunits of apopain are folded into a compact cylinder approximately 40 x 33 x 25 A in size, and the folding is dominated by a central 6-stranded ⁇ -sheet with five parallel and one antiparallel strands.
  • apopain When the apopain and ICE structures are aligned on 194 residues with similar secondary structure or conformation, the average difference between equivalent alpha carbon atoms is 1.11 A.
  • the most significant differences in the overall structures of the two enzymes are that apopain lacks two small surface loops that are present in ICE and contains one extra loop not present in the other enzyme. Missing from apopain are groups equivalent to residues 156-160 (bottom left of Fig. IB) and 248-258 (top center of Fig. IB) of ICE.
  • Apopain contains a ten-residue insert at a position equivalent to residue 381 of ICE (top right of Fig.
  • this extra loop forms an irregular reverse turn over the bound inhibitor and contributes several residues that make up part of the binding site; addition of this loop is one of the most significant differences between the two classes of ICE-related proteases exemplified by these two enzymes.
  • ICE ICE, ICE re l-II, ICE re l-III
  • CED-3-like subfamily including C. elegans CED-3 itself, are predicted to have an equivalent loop (e.g. ICH-1 , Mch2, Mch3).
  • the resulting tetrametic assembly is approximately 50 x 35 x 25 A in size with a 12-stranded ⁇ -structure running through its center, and contains two catalytic sites at widely- separated parts of the molecule (Fig. 2).
  • the equivalent 388 alpha carbons differ by an average of only 2.01 A.
  • the two chains at the left of Fig. 2 are derived from the same p32 precursor.
  • This model is supported by the notion that the relatively compact heterodimer at the left of Fig. 2 is derived from a single precursor molecule, but it requires a relatively large conformational change upon processing. In the apopain structure, such a change would have only local effects and would not affect the overall structure of the protein.
  • a surface loop connects the C-terminus of the blue subunit with the N-terminus of the red chain. Such a loop would extend from the end of the ⁇ -strand comprising residues 278-283 (fourth from the left in Fig.
  • the sidechains at Pi and P4 are contained in pronounced depressions in the binding groove where they make numerous interactions with the protein, while the S2 and S3 sidechains point away from the body of the protein.
  • the S j subsite is remarkably similar in the ICE and apopain complexes.
  • the local concentration of the two positive charges of the arginyl side chains in Si is stabilized by the negatively-charged aspartyl side chain of the modulators and substrates of these enzymes. It is quite likely that the conformation observed in the three-dimensional structures of inhibited apopain and ICE would be unstable in the uninhibited enzymes, where such stabilization is absent. These observations suggest either that the free enzyme contains a counterion such as acetate or several water molecules in Si , or that there is a significant local conformational change on the binding or release of modulators and substrates.
  • apopain Differences between the apopain and ICE complexes at S2 and S3 primarily arise from amino acid sequence differences.
  • the side chains of the P3 glutamyl residue points away from the body of the protein, but makes a salt link to the side chain of Arg341 ? consistent with the specificity of the enzyme at this site.
  • the Arg341 side chain also interacts with the Pi aspartyl side chain (see above).
  • the side chain of the P2 valine lies against a hydrophobic wall of the groove centered on the side chain of Tyr338
  • the S4 subsites differ radically in both their overall geometry and their chemical nature.
  • the S4 subsite of ICE is a large and shallow hydrophobic depression that easily accommodates a tyrosyl side chain, while the corresponding site in apopain is a narrow socket that closely envelops the Pi Asp sidechain.
  • the carboxyl group of the inhibitor makes specific polar interactions with the amide nitrogen atom of Phe 81B and the N ⁇ l atom of Asn342
  • This network of hydrogen bonds is extended by an interaction between the Asn342
  • the site is complementary in size and shape to an aspartyl side chain, and it contains two hydrogen bond donors firmly positioned with the appropriate geometry to interact with a carboxyl group.
  • the orientation of the side chain of Asn342 j s fixed by the interactions of its 0°1 , a hydrogen bond acceptor, with amide nitrogens of Lys344 a nd Asp345 ? hydrogen bond donors.
  • the three-dimensional structure of apopain is generally similar in tertiary and quaternary structure to that of ICE.
  • these two proteins interact with analogous modulator peptides in very similar ways in the S l -S3 subsites.
  • S4 subsites consistent with the amino acid sequence variation and substrate specificity differences between these two enzymes.
  • These differences provide a foundation for understanding the structural and functional differences between the two subfamilies of the ICE-specific proteases that these enzymes exemplify.
  • comparison of these two structures provides a structural basis for the design of highly specific modulators of these proteins that will be of great value in the study of the disease states and physiological functions with which they are associated.
  • the present invention relates to crystalline apopain, the structure of apopain as determined by X-ray crystallography, the use of that structure to solve the structure of apopain homologues and other crystal forms of apopain, mutants and co-complexes of apopain, and the use of the apopain structure and that of its homologues, mutants, and co- complexes to design modulators of apopain.
  • the present invention is directed to the three - dimensional structure of an isolated and purified enzyme designated apopain and its structure coordinates. Another aspect of the invention is to use the structure coordinates of the apopain crystal to reveal the atomic details of the active site and one or more of the accessory binding sites of apopain. Another aspect of the invention is to use the structure coordinates of an apopain crystal to solve the structure of a different apopain crystal or crystal of a mutant, homologue or co- complex of apopain. A further aspect of the invention is to provide apopain mutants characterized by one or more different properties compared to wild-type apopain.
  • Another aspect of this invention is to use the structure coordinates and atomic details of apopains or mutants or homologues or co-complexes thereof to design, evaluate computationally, synthesize and use modulators of apopain that prevent or treat the undesirable physical and pharmacological properties of apopain.
  • modulators may be used as therapies that are beneficial in the treatment of immune, proliferative and degenerative diseases including, but not limited to, immune deficiency syndromes (such as AIDS), autoimmune diseases, pathogenic infections, cardiovascular and neurological injury, alopecia, aging, cancer, Parkinson's disease, Alzheimer's disease, Huntington's disease, neurodegenerative disorders and spinal cord injury.
  • this invention relates to compounds of formula I (designated L-761 ,191):
  • this invention relates to modulators of the pro-apoptotic proteolytic activity of thiol proteinases which cause apoptosis at least in part by disabling the normal biological function of poly(ADP-ribose)polymerase, U I -70 kDa small ribonucleoprotein and the catalytic subunit of DNA- dependent protein kinase.
  • Figure 1 (A) is a schematic representation of the apopain:Ac-DEVD-CHO structure viewed down the crystallographic two-fold axis, ⁇ -structures are arrows; ⁇ -helices, coils. The bound inhibitor is represented by the short arrow at the top of the figure. The extra loop (residues 180A, 181A-I) in apopain is indicated by the arrow. The pi 7 chain is dark; the pi 2 chain, light.
  • Figure 1 (B) shows the ICE:YVAD structure oriented to maximize alignment with (A).
  • the two extra loops in ICE are indicated by the white arrows at the bottom (residues 156- 160) and top left (residues 248-253) of the figure.
  • the p20 chain is dark; the plO chain, light.
  • Figure 1 (C) shows the amino acid sequences of apopain and ICE aligned by matching residues that have similar secondary structures or conformations in the aligned three-dimensional structures of the two proteins. Lower-case letters represent residues for which no electron density is evident.
  • the amino acid residues of apopain are identified by the sequence numbers of the homologous residues in ICE in this alignment. Apopain sequence numbers are omitted when no ICE-related residue is present in apopain, and apopain-specific insertions are indicated by the additions of letters to the ICE sequence numbers.
  • the method used for production of apopain involves folding of active enzyme from its constituent pl7 and pi 2 subunits which are expressed separately in E. coli.
  • Amino acid numbering for the purpose of the recombinant engineering described in this section refers to amino acid positions within the apopain proenzyme as determined from the open reading frame of GenBank accession number U 13738 (the initiator methionine being residue 1 and the final residue preceding the stop codon being residue 402). The beginning and end of each subunit was determined by mass spectroscopy analysis and amino- terminal microsequencing of non-recombinant, purified human apopain.
  • the apopain/CPP32 pl7 subunit (Serl 1-Asp297) and pl2 subunit (Ser310_His402) were engineered for expression as MetSer 1 1. Asp297 and MetSer310-His402 constructs, respectively, by PCR- directed template modification.
  • a cDNA encoding the pi 7 subunit was amplified with the sense (forward) synthetic oligonucleotide 5'- GCT CTA GAC TCG AGT CAT GAG TGG AAT ATC CCT GGA CAA CAG TTA TAA AAT GG -3' (SEQ ID NO: l) plus the antisense (reverse) oligonucleotide 5'- GCT CTA GAC TCG AGT CAT GAT TAG TCT GTC TCA ATG CCA CAG TCC AGT TCT G -3 ' (SEQ ID NO:2) using the full length CPP32 ⁇ cDNA (3 ng/ ⁇ l) as template (Pwo polymerase (0.025 U/ ⁇ l) Boehringer Mannheim; 25 cycles of 1 min at 94°C, 1 min at 60°C, 45 sec at 72°C).
  • a cDNA encoding the pi 2 subunit was amplified the same way except that the sense (forward) oligonucleotide was 5'- GCT CTA GAC TCG AGT CAT GAG TGG TGT TGA TGA TGA CAT GGC GTG TC -3' (SEQ ID NO:3) and the antisense (reverse) oligonucleotide was 5'- GCT CTA GAC TCG AGT CAT GAT TAG TGA TAA AAA TAG AGT TCT TTT GTG AGC -3' (SEQ ID NO:4).
  • PCR fragments were purified, trimmed with Xba I then ligated into the Xba I site of pBluescript II SK(+) (Stratagene). Following sequence verification, inserts were excised from nested Bsp HI sites and ligated into the Nco I site of pET- 11 d (Novagen). Appropriately oriented clones were then transformed into E. coli BL21 (DE3)pLysS cells. Optimal growth conditions for production cultures employed M9 medium at 37° with overnight induction using 1 mM IPTG for expression of the recombinant proteins. (Under these conditions, the individual subunits were expressed at approximately 50 mg/liter.
  • the protein was localized exclusively in the inclusion body fraction where they generally constituted > 99% of the total protein.
  • the E coli were harvested, washed, and broken in the presence of protease inhibitors.
  • the inclusion bodies were then isolated and solubilized in 6 M guanidine- hydrochloride.
  • the denatured pi 7 and pi 2 subunits were mixed and rapidly diluted to a concentration of 100 ⁇ g/ml in 100 mM HEPES/KOH (pH 7.5) 10%
  • Mass spectral analysis of recombinant apopain complex indicates that it comprises intact pi 7 chains, spanning Ser ⁇ 41 to Asp297 5 and a mixture of pl2 chains, consisting of predominantly MetSer 10-His402 w j m some Ser310- His402
  • the resulting enzyme is indistinguishable from native enzyme with regard to kinetic parameters for inhibition by Ac-DEVD-CHO.
  • Three-dimensional diffraction data extending to a resolution of 2.5 A were collected at room temperature using a Siemens area detector and CuKoc radiation from a Rigaku RU-200 rotating-anode X-ray generator. These data were processed with the SAINT software package3°. 20,801 observations of 8.929 unique reflections were merged with an R-factor of 5.55%.
  • the structure was solved by molecular replacement, using X-PLOR37 and a model based on PDB 3 ⁇ entry 1ICE, the structure of inhibited ICE2 .
  • the current model was constructed by interactive model-building39 and refinement using X-PLOR ' . In early stages of model-building, phase refinement using SQUASH significantly improved the quality of electron density maps.
  • the current model was inspected against 10% simulated-annealing omit maps4l and comprises residues 149-295 of the pi 7 chain, residues 317-402 of the pi 2 chain, the bound inhibitor, and 25 ordered water molecules. Residues 141 -148 at the N-terminus of the pi 7 chain, 296-297 at the C-terminus of the pi 7 chain, and 310-316 at the N-terminus of the pi 2 chain are not visible in the current electron density maps, presumably due to disorder.
  • Figure 2 is a schematic representation of the apopain tetramer oriented as in Figure 1.
  • the heterodimer at the left of the figure is identical to Fig IA.
  • the pi 7 chain is colored dark; the pi 2 chain light.
  • the colors are reversed in the heterodimer at the right.
  • the bound inhibitor molecules are the short arrows at the top of the dimer on the left side of the figure and the bottom of the dimer on the right.
  • Figure 3 shows hydrogen bonds and other polar interactions between the bound N-acetyl tetrapeptide aldehyde inhibitor and (A) ICE and (B) apopain.
  • Figure 4(A) shows the S4 subsite of the apopain: Ac-DEVD- CHO complex.
  • the acetyl group and S4 aspartyl residue of the bound inhibitor are depicted as ball-and-stick models; the protein as rods. Hydrogen bonds are drawn as dashed white lines.
  • Figure 4(B) shows the S4 subsite of the ICE:YVAD complex. (A) and (B) are oriented to maximize global alignment of the two proteins.
  • the present invention is directed to the three- dimensional structure of an isolated and purified enzyme designated apopain and its structure coordinates. Another aspect of the invention is to use the structure coordinates of the apopain crystal to reveal the atomic details of the active site and one or more of the accessory binding sites of apopain. Another aspect of the invention is to use the structure coordinates of an apopain crystal to solve the structure of a different apopain crystal or crystal of a mutant, homologue or co- complex of apopain. A further aspect of the invention is to provide apopain mutants characterized by one or more different properties compared to wild-type apopain.
  • Another aspect of this invention is to use the structure coordinates and atomic details of apopains or mutants or homologues or co-complexes thereof to design, evaluate computationally, synthesize and use modulators of apopain that prevent or treat the undesirable physical and pharmacological properties of apopain.
  • modulators may be used as therapies that are beneficial in the treatment of immune, proliferative and degenerative diseases including, but not limited to, immune deficiency syndromes (such as AIDS), autoimmune diseases, pathogenic infections, cardiovascular and neurological injury, alopecia, aging, cancer, Parkinson's disease, Alzheimer's disease, Huntington's disease, neurodegenerative disorders and spinal cord injury.
  • this invention relates to compounds of formula I (designated L-761,191):
  • this invention relates to modulators of the pro-apoptotic proteolytic activity of thiol proteinases which cause apoptosis at least in part by disabling the normal biological function of poly(ADP-ribose)polymerase.
  • Apoptosis constitutes a systematic means of cell suicide within an organism during normal morphogenesis, tissue remodeling as well as in response to pathogenic infections or other irreparable cell damage. Inappropriate apoptosis may underlie the etiology of human diseases such as Alzheimer's, Parkinson's and Huntington's diseases, immune deficiency and autoimmune disorders, ischemic cardiovascular and neurological injury, alopecia, leukemias, lymphomas and other cancers, which therefore makes the control of apoptosis an important potential target for therapeutic intervention.
  • PARP poly(ADP-ribose) polymerase
  • PARP is proteolytically cleaved at the onset of apoptosis by a hitherto- unidentified protease with properties that resemble those of ICE.
  • the cleavage site within PARP (DEVD216-G217) resembles one of the two sites in proIL-l ⁇ (FEAD27-G28) that are recognized and cleaved by ICE.
  • Proteolytic cleavage of PARP at this site results in the separation of the two zinc-finger DNA-binding motifs in the amino-terminus of PARP from the automodification and poly(ADP-ribos)ylating catalytic domains located in the carboxy-terminus of the polypeptide.
  • This cleavage precludes the catalytic domain of PARP from being recruited to sites of DNA damage and presumably disables the ability of PARP to coordinate subsequent repair and genome maintenance events.
  • the Ca2+/Mg2+-dependent endonuclease implicated in the internucleosomal DNA cleavage that is a hallmark of apoptosis is negatively regulated by poly(ADP-ribos)ylation20-22.
  • the seven known members of the ICE/CED-3 family of cysteine proteases which are of human origin are ICE, ICE rel -II, ICE rel - ffl, ICH-1 , CPP32, Mch2 and Mch3. Each is capable of initiating an apoptotic response when transfected into host cells; however, it is possible that overexpression of any protease may cause non-specific induction of cell death. Cytoplasmic expression of other proteases, such as trypsin, chymotrypsin, proteinase K or granzyme B, for example, have also been shown to induce apoptosis27,28.
  • ced-3 In the nematode C. elegans, deletion or mutation of a single gene, ced-3, abolishes apoptotic death.
  • ced-3 was found to be homologous to the gene for mammalian interleukin- 1 ⁇ converting enzyme (ICE), which encodes a protease whose only known function is the cleavage of the inactive 31 kDa proIL-l ⁇ cytokine precursor to the active 17 kDa form.
  • ICE mammalian interleukin- 1 ⁇ converting enzyme
  • apopain/CPP32 is the specific ICE/CED-3-like cysteine protease that cleaves PARP and other substrates at the onset of apoptosis in mammalian cells.
  • the central role played by apopain/CPP32 in mammalian cell death is further substantiated by potent and selective modulators which prevent apoptosis from occurring in vitro.
  • the cloned apopain cDNA may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant apopain.
  • Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, yeast, bluegreen algae, plant cells, insect cells and animal cells.
  • An appropriately constructed expression vector may contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
  • a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
  • a strong promoter is one which causes mRNAs to be initiated at high frequency.
  • Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
  • a variety of mammalian expression vectors may be used to express recombinant apopain in mammalian cells.
  • mammalian expression vectors which may be suitable for recombinant apopain expression, include but are not limited to, pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO- pSV2-neo (ATCC 37593) pBPV- 1 (8-2) (ATCC 371 10), pdBPV-
  • MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).
  • DNA encoding apopain may also be cloned into an expression vector for expression in a recombinant host cell.
  • Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria, yeast, mammalian cells and insect cells.
  • Cell lines derived from mammalian species include but are not limited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651 ), CHO- Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
  • the expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, infection, protoplast fusion, and electroporation.
  • the expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce apopain protein. Identification of apopain expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-apopain antibodies, and the presence of host cell-associated apopain activity.
  • Expression of apopain cDNA may also be performed using in vitro produced synthetic mRNA.
  • Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes.
  • modified apopain cDNA molecules are constructed. Host cells are transformed with the cDNA molecules and the levels of apopain RNA and protein are measured.
  • apopain protein in host cells are quantitated by a variety of methods such as immunoaffinity and/or ligand affinity techniques, apopain-specific affinity beads or apopain-specific antibodies are used to isolate 35s-methionine labeled or unlabelled apopain protein. Labeled apopain protein is analyzed by SDS-PAGE. Unlabelled apopain protein is detected by Western blotting, ELISA or RIA employing apopain specific antibodies.
  • apopain protein may be recovered to provide apopain in active form.
  • apopain purification procedures are available and suitable for use.
  • Recombinant apopain may be purified from cell lysates or from conditioned culture media, by various combinations of, or individual application of fractionation, or chromatography steps that are known in the art.
  • recombinant apopain can be separated from other cellular proteins by use of an immuno-affinity column made with monoclonal or polyclonal antibodies specific for full length nascent apopain or polypeptide fragments of apopain
  • the recombinant protein may be used to generate antibodies.
  • antibody as used herein includes both polyclonal and monoclonal antibodies, as well as fragments thereof, such as, Fv, Fab and F(ab)2 fragments that are capable of binding antigen or hapten.
  • Monospecific antibodies to apopain are purified from mammalian antisera containing antibodies reactive against apopain or are prepared as monoclonal antibodies reactive with apopain using standard techniques.
  • Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for apopain.
  • Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the apopain, as described above.
  • Enzyme-specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with rabbits being preferred, with an appropriate concentration of apopain either with or without an immune adjuvant.
  • Monoclonal antibodies (mAb) reactive with apopain may be prepared by conventional methods, such as by immunizing inbred mice with apopain.
  • the mice are immunized with about 0.1 mg to about 10 mg, preferably about 1 mg, of apopain in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant. Freund's complete adjuvant is preferred.
  • the mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 0.1 to about 10 mg of apopain in a buffer solution such as phosphate buffered saline (PBS) by the intravenous (rV) route.
  • PBS phosphate buffered saline
  • Lymphocytes from antibody-positive mice are obtained by removing spleens from immunized mice by standard procedures known in the art.
  • Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner under conditions which will allow the formation of stable hybridomas.
  • Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.
  • DMEM Dulbecco's Modified Eagles Medium
  • Supernatant fluids are collected form growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using apopain as the antigen.
  • SPIRA solid phase immunoradioassay
  • Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.
  • In vitro production of anti-apopain is carried out by growing the hydridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb.
  • the mAb are purified by techniques known in the art.
  • Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of apopain in body fluids or tissue and cell extracts.
  • serological or immunological assays include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of apopain in body fluids or tissue and cell extracts.
  • Methods such as those described above may be used to produce monospecific antibodies may be utilized to produce antibodies specific for apopain polypeptide fragments or full-length nascent apopain polypeptide.
  • Apopain antibody affinity columns are made by adding the antibodies to a gel support, such as Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support.
  • the antibodies are then coupled to the gel via amide bonds with the spacer arm.
  • the remaining activated esters are then quenched with 1 M ethanolamine HCl (pH 8).
  • the column is washed with water followed by 0.23 M glycine HCl (pH 2.6) to remove any non-conjugated antibody or extraneous protein.
  • the column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing apopain or apopain fragments are slowly passed through the column.
  • the column is then washed, and the protein is eluted.
  • the purified apopain protein is then dialyzed against phosphate buffered saline.
  • Kits containing apopain cDNA, antibodies to apopain or apopain protein may be prepared. Such kits are used to detect DNA which hybridizes to apopain DNA or to detect the presence of apopain protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic analyses and epidemiological studies.
  • the DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of apopain DNA, apopain RNA or apopain protein.
  • the recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of apopain.
  • kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container.
  • the carrier would further comprise reagents such as recombinant apopain protein or anti-apopain antibodies suitable for detecting apopain.
  • the carrier may also contain means for detection such as labeled antigen or enzyme substrates or the like.
  • Nucleotide sequences that are complementary to the apopain encoding cDNA sequence can be synthesized for antisense therapy.
  • These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2'-0-alkylRNA, or other apopain antisense oligonucleotide mimetics.
  • Apopain antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harbouring the antisense sequence, apopain antisense therapy may be particularly useful for the treatment of diseases where it is beneficial to reduce apopain activity.
  • Apopain gene therapy may be used to introduce apopain into the cells of target organs.
  • the apopain gene can be ligated into viral vectors which mediate transfer of the apopain DNA by infection of recipient host cells.
  • Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus and the like.
  • apopain DNA can be transferred into cells for gene therapy by non-viral techniques including receptor- mediated targeted DNA transfer using ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection membrane fusion or direct microinjection. These procedures and variations of them are suitable for ex vivo as well as in vivo apopain gene therapy.
  • Apopain gene therapy may be particularly useful for the treatment of diseases where it is beneficial to elevate apopain activity.
  • compositions comprising apopain DNA or apopain protein may be formulated as described elsewhere in this application or according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences.
  • a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of the protein or DNA.
  • Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose apopain related disorders. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
  • the pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
  • this invention is also directed to those DNA sequences which contain alternative codons which code for the eventual translation of the identical amino acid.
  • a sequence bearing one or more replaced codons will be defined as a degenerate variation.
  • mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
  • DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally-occurring peptide.
  • Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate.
  • a "functional derivative" of apopain is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of apopain.
  • the term “functional derivatives” is intended to include the "fragments,” “variants,” “degenerate variants,” “analogs” and “homologues” or to "chemical derivatives" of apopain.
  • the term “functional derivatives" is intended to include the "fragments,” “variants,” “degenerate variants,” “analogs” and “homologues” or to "chemical derivatives" of apopain.
  • fragment is meant to refer to any polypeptide subset of apopain.
  • variant is meant to refer to a molecule substantially similar in structure and function to either the entire apopain molecule or to a fragment thereof.
  • a molecule is "substantially similar” to apopain if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical.
  • analog refers to a molecule substantially similar in function to either the entire apopain molecule or to a fragment thereof.
  • the term "chemical derivative" describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
  • the present invention is also directed to methods for screening for compounds which modulate that expression of DNA or RNA encoding apopain as well as the function of apopain protein in vivo. Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.
  • Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding apopain or the function of apopain protein.
  • Compounds that modulate the expression of DNA or RNA encoding apopain or the function of apopain protein may be detected by a variety of assays.
  • the assay may be a simple "yes/no" assay to determine whether there is a change in expression or function.
  • the assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample.
  • the invention encompasses compounds of formula I (L-761 ,191):
  • compositions of the present invention comprise a compound of Formula I as an active ingredient or a pharmaceutically acceptable salt, thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, ammonium, potassium, sodium, zinc and the like. Particularly preferred are the calcium, magnesium, potassium, and sodium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
  • basic ion exchange resins such as
  • salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.
  • acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like.
  • Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.
  • the magnitude of therapeutic dose of a compound of Formula I will, of course, vary with the nature of the severity of the condition to be treated and with the particular compound of Formula VI and its route of administration and vary upon the clinician's judgment. It will also vary according to the age, weight and response of the individual patient. An effective dosage amount of the active component can thus be determined by the clinician after a consideration of all the criteria and using is best judgment on the patient's behalf.
  • An ophthalmic preparations for ocular administration comprising 0.001-1 % by weight solutions or suspensions of the compounds of Formula I in an acceptable ophthalmic formulation may be used.
  • Any suitable route of administration may be employed for providing a mammal, especially a human with an effective dosage of a compound of the present invention.
  • oral, parenteral and topical may be employed.
  • Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
  • compositions of the present invention comprise a compound of Formula I as an active ingredient or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic bases or acids and organic bases or acids.
  • compositions include compositions suitable for oral, parenteral and ocular (ophthalmic). They may be conveniently presented in unit dosage form and prepared by any of the methods well- known in the art of pharmacy.
  • the compounds of Formula I can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, alcohols, oils, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case or oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaque
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil emulsion.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into active ingredient with the carrier which constitutes one or more necessary ingredients.
  • the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
  • each tablet contains from about 1 mg to about 500 mg of the active ingredient and each cachet or capsule contains from about 1 to about 500 mg of the active ingredient.
  • Compounds of the instant invention are conveniently prepared using procedures known in the art.
  • Proteolytic cleavage of poly(ADP-ribose)polymerase may be measured as followed.
  • the cDNA encoding human poly(ADP-ribose)polymerase (clone pCD-12; GenBank accession no. M32721 ; NCBI gi 190266) was excised from its cloning vector by Xho I restriction digestion then ligated into Xho I-cut, CIP-treated pBluescript U SK+ (Stratagene). Following transformation into competent Escherichia coli cells, colony purification and propagation of the resulting transformed cells in liquid culture, the plasmid DNA was purified and the orientation of the PARP cDNA was determined by restriction enzyme analysis.
  • a fluorogenic derivative of the tetrapeptide recognized by apopain and corresponding to the Pi to P4 amino acids of the PARP cleavage site, Ac-DEVD-AMC (AMC, amino-4-methylcoumarin) was prepared as follows: i) synthesis of N-Ac-Asp(OBn)-Glu(OBn)-Val- CO2H, ii) coupling with Asp(oBn)-7-amino-4-methylcoumarin, iii) removal of benzyl groups.
  • Standard reaction mixtures 300 ⁇ l final volume, contained Ac-DEVD-AMC and purified or crude PARP-cleavage apopain/CPP32 enzyme in 100 mM Hepes/KOH (pH 7.5), 10% (w/v) sucrose, 0.1 % (w/v) CHAPS, 10 mM dithiothreitol, and were incubated at 25°C. Reactions were monitored continuously in a spectrofluorometer at an excitation wavelength of 380 nm and an emission wavelength of 460 nm.
  • structure coordinates refers to mathematical coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of an apopain molecule in crystal form.
  • the diffraction data are used to calculate an electron density map of the repeating unit of the crystal.
  • the electron density maps are used to establish the positions of the individual atoms within the unit cell of the crystal.
  • the term "heavy atom derivatization” refers to the method of producing a chemically modified form of the crystal of apopain.
  • a crystal is soaked in a solution containing heavy metal atom salts or organometallic compounds, which can diffuse through the crystal and bind to the surface of the protein.
  • the location(s) of the bound heavy metal atom(s) are determined by X-ray diffraction analysis of the soaked crystal. This information is used to generate the phase information used to construct three-dimensional structure of the protein.
  • unit cell refers to the basic parallelipiped shaped block.
  • the entire volume of a crystal may be constructed by regular assembly of such blocks.
  • space group refers to the arrangement of symmetry elements of a crystal.
  • molecular replacement refers to a method that involves generating a preliminary model of an apopain crystal whose structure coordinates are not known, by orienting and positioning a molecule whose structure coordinates are known. Phases are then calculated from this model and combined with observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are known.
  • the structure coordinates of apopain may be used to design compounds that bind to the enzyme and alter its physical properties in a variety of ways.
  • the structure coordinates of the enzyme may also be used to screen computationally small molecule data bases for compounds that bind to the enzyme.
  • the structure coordinates of apopain mutants may also facilitate the identification of related proteins or enzymes , thereby further leading to novel therapeutic modes for treating or preventing apopain-mediated conditions.
  • the method used for production of apopain involves folding of active enzyme from its constituent pl7 and pi 2 subunits which are expressed separately in E. coli.
  • the apopain/CPP32 pi 7 subunit (Ser I 41 _Asp297) and pi 2 subunit (Ser310_His402) were engineered for expression as MetSer ⁇ 41-Asp297 nd MetSer I0-His402 constructs, respectively, by PCR-directed template modification.
  • a cDNA encoding the pl7 subunit was amplified with the sense (forward) synthetic oligonucleotide 5'- GCT CTA GAC TCG AGT CAT GAG TGG AAT ATC CCT GGA CAA CAG TTA TAA AAT GG -3' (SEQ ID NO:5) plus the antisense (reverse) oligonucleotide 5'- GCT CTA GAC TCG AGT CAT GAT TAG TCT GTC TCA ATG CCA CAG TCC AGT TCT G -3' (SEQ ID NO:6) using the full length CPP32 ⁇ cDNA (3 ng/ ⁇ l) as template (Pwo polymerase (0.025 U/ ⁇ l) Boehringer Mannheim; 25 cycles of 1 min at 94°C, 1 min at 60°C, 45 sec at 72°C).
  • a cDNA encoding the pl2 subunit was amplified the same way except that the sense (forward) oligonucleotide was 5'- GCT CTA GAC TCG AGT CAT GAG TGG TGT TGA TGA TGA CAT GGC GTG TC -3 ' (SEQ ID NO:7) and the antisense (reverse) oligonucleotide was 5'- GCT CTA GAC TCG AGT CAT GAT TAG TGA TAA AAA TAG AGT TCT TTT GTG AGC -3' (SEQ ID NO:8) .
  • PCR fragments were purified, trimmed with Xba I then ligated into the Xba I site of pBluescript II SK(+) (Stratagene). Following sequence verification, inserts were excised from nested Bsp HI sites and ligated into the Nco I site of pET-1 Id (Novagen). Appropriately oriented clones were then transformed into E. coli BL21 (DE3)pLysS cells. Optimal growth conditions for production cultures employed M9 medium at 37° with overnight induction using 1 mM EPTG for expression of the recombinant proteins. (Under these conditions, the individual subunits were expressed at approximately 50 mg/liter.
  • the protein was localized exclusively in the inclusion body fraction where they generally constituted > 99% of the total protein.
  • the E coli were harvested, washed, and broken in the presence of protease inhibitors.
  • the inclusion bodies were then isolated and solubilized in 6 M guanidine-hydrochloride.
  • the denatured pl7 and pi 2 subunits were mixed and rapidly diluted to a concentration of 100 ⁇ g/ml in 100 mM HEPES/KOH (pH 7.5) 10% (w/v) sucrose, 0.1 % (w/v) CHAPS, 0.5 M NaCl, 10 mM DTT.
  • EXAMPLE 2 Purification of Apopain The active enzyme was subsequently separated from incorrectly folded protein by anion-exchange chromatography using a 1 ml HiTrap Q column (Pharmacia). Mass spectral analysis of recombinant apopain complex indicates that it comprises intact p 17 chains, spanning Serl41 to Asp297 ? and a mixture of pi 2 chains, consisting of predominantly MetSer3 0-His402 w ith some Ser310-
  • the apopain:Ac-DEVD-CHO complex was crystallized by hanging-drop vapor diffusion.
  • 1.5 ⁇ l drops of protein-inhibitor solution (8.7 mg/ml in 10 mM Tris-HCl pH 8.5, 10 mM DTT, 3 mM NaN3) were mixed with an equal volume of reservoir buffer (7% PEG- 6000 (w/w), 0.10 M sodium citrate pH 5.0, 10 mM DTT, 3 mM NaN3) and incubated at room temperature.
  • Three-dimensional diffraction data extending to a resolution of 2.5 A were collected at room temperature using a Siemens area detector and CuKoc radiation from a Rigaku RU-200 rotating-anode X-ray generator. These data were processed with the SAINT software package3 « . 20,801 observations of 8,929 unique reflections were merged with an R-factor of 5.55%.
  • the structure was solved by molecular replacement, using X-PLOR3 ' and a model based on PD ⁇ ° entry 1ICE, the structure of inhibited ICE ⁇ °.
  • the current model was constructed by interactive model-building39 and refinement using X- PLOR 7.
  • the current model was inspected against 10% simulated-annealing omit maps41 and comprises residues 149-295 of the pi 7 chain, residues 317- 402 of the pi 2 chain, the bound inhibitor, and 25 ordered water molecules. Residues 141-148 at the N-terminus of the pl7 chain, 296- 297 at the C-terminus of the p 17 chain, and 310-316 at the N-terminus of the pi 2 chain are not visible in the current electron density maps, presumably due to disorder.
  • ADDRESSEE CHRISTINE E. CARTY - MERCK & CO. , INC.

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Abstract

L'invention a trait à de l'apopaïne cristallisée, à l'utilisation qui est faite de cette structure pour dissoudre la structure d'homologues d'apopaïne ainsi que celle d'autres formes cristallines de l'apopaïne, de mutants et de co-complexes de celle-ci. Elle concerne également l'emploi de la structure d'apopaïne et de celle de ses homologues, mutants et co-complexes pour élaborer des effecteurs de l'apopaïne.
PCT/US1997/002578 1996-02-23 1997-02-19 Structure cristalline d'apopaine et son utilisation WO1997031018A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006833A3 (fr) * 1996-08-12 1998-03-19 Mittl Peter Structure cristalline de la cpp32
WO2000077184A1 (fr) * 1999-06-10 2000-12-21 Pharmacia & Upjohn Company Cristaux de caspase-8 et modeles et procedes correspondants

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996033268A1 (fr) * 1995-04-21 1996-10-24 Merck & Co., Inc. Apopaïne

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US5856116A (en) * 1994-06-17 1999-01-05 Vertex Pharmaceuticals, Incorporated Crystal structure and mutants of interleukin-1 beta converting enzyme

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1996033268A1 (fr) * 1995-04-21 1996-10-24 Merck & Co., Inc. Apopaïne

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF EXPERIMENTAL MEDICINE, May 1996, Volume 183, CASCIOLA-ROSEN et al., "Apopain/CPP32 Cleaves Proteins That are Essential for Cellular Repair; A Fundamental Principle of Apoptotic Death", pages 1957-1964. *
NATURE STRUCTURAL, BIOLOGY, July 1996, Vol. 3, No. 7, ROTONDA et al., "The Three-Dimensional Structure of Apopain/CPP32, a Key Mediator of Apoptosis", pages 620-625. *
NATURE, 1995, Volume 377, 05 October 1995, DARMON et al., "Activation of the Apoptotic Protease CPP32 by Cytotoxic T-Cell-Derived Granzyme B", pages 446-448. *
See also references of EP0885243A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006833A3 (fr) * 1996-08-12 1998-03-19 Mittl Peter Structure cristalline de la cpp32
US7052851B2 (en) 1996-08-12 2006-05-30 Novartis International Ag Crystal structure of cysteine protease
WO2000077184A1 (fr) * 1999-06-10 2000-12-21 Pharmacia & Upjohn Company Cristaux de caspase-8 et modeles et procedes correspondants

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