Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the invention relates to compounds influencing mitosis and meiosis in eukaryotic cells and methods for identifying such compounds.
• the invention largely relates to the treatment and prevention of human conditions by modulating sister chromatid segregation.
• a key prerequisite for the successful division of one cell into two is the duplication and subsequent segregation of the cellular genome into the two forming daughter cells.
• Duplication of the genome by DNA replication occurs during synthesis (S) phase of the cell cycle, whereas segregation of the duplicated DNA takes place much later during anaphase of mitosis.
• S phase replicated DNA molecules remain physically attached to each other, a phenomenon called cohesion, until they are separated in anaphase.
• the cellular DNA is condensed into chromosomes, in which each of the two replicated DNA molecules are microscopically visible as sister chromatids.
• the cohesion that is holding sisters together has to be dissolved.
• This process is mediated by a protease, called separin or separase, that is cleaving a complex of chromosomal cohesion proteins (the cohesin complex) that is required to hold sister chromatids together.
• This cleavage reaction liberates sisters from each other so that they can be pulled towards opposite poles of the diving cells by the spindle apparatus (reviewed by Nasmyth et al., 2000).
• WO 00/48627 suggests a method for identifying compounds which exert their effect by directly modulating, in particular by inhibiting separase's proteolytic activity, i.e. by being protease inhibitors specific for separase.
• separase stands for "human separase”.
• the screening method for identifying compounds that have the ability of modulating sister chromatid separation in plant or animal cells as described in WO00/48627 comprises incubating separase, in the presence of the substrate(s) for its proteolytic activity and optionally its co-factor(s), with test compounds and determining the modulating effect of the test compounds on the proteolytic activity of the separase.
• the identification of small molecule inhibitors of separase requires enzyme assays in which the protease activity of separase can be directly measured.
• such assays are adaptable to high throughput formats so that large libraries of chemical compounds can be tested for their ability to inhibit separase.
• the previously reported experiments demonstrating that separase is associated with a protease activity were performed with separase isolated in small scale by immunoprecipitation or affinity chromatography from either yeast or human cells (Uhlmann et al., 2000; Waizenegger et al., 2000).
• WO00/48627 suggests to use full-length separase, preferably in recombinant form, for performing protease assays.
• protease activity of separase is tightly regulated during the cell cycle, ensuring that the ability of separase to cleave cohesin and thereby to dissolve sister chromatid cohesion is not activated before the transition from metaphase to anaphase.
• Work in budding yeast and human cells has shown that prior to anaphase separase is inhibited by a protein called securin (Ciosk et al., 1998; Uhlmann et al., 1999; Waizenegger et al., 2000). Securin binds to separase until securin is ubiquitinated by the anaphase-promoting complex and subsequently degraded by the 26S proteasome shortly before the onset of anaphase.
• securin The destruction of securin is thought to activate separase.
• separase itself is cleaved at the same time as securin is destroyed, resulting in at least two C-terminal cleavage products called p55 and p60 (Waizenegger et al., 2000).
• p55 and p60 C-terminal cleavage products
• separase protease assay suitable for high throughput format it is essential to know which form of separase represents the active enzyme and how the active form of separase can be obtained.
• the present invention provides the first evidence that the cleaved forms of separase represent the active protease and that N- and C-terminal cleavage products of separase remain physically associated.
• the present invention further provides evidence that securin inhibits separase by directly binding to it. Securin binding to separase could either directly block the access of substrates to the active site of separase or it could keep separase in a conformation in which its active site is not accessible to substrates.
• the present invention further provides evidence that it is the autocatalytic reaction, i.e. the reaction in which separase cleaves itself, which is responsible for the cleavage of separase into its active form.
• the present invention shows that active forms of tagged recombinant full-length separase can be obtained and that fluorogenic peptide substrates are useful to measure the protease activity of separase.
• the antibody beads with bound separase are incubated in mitotic Xenopus laevis egg extracts which are able to ubiquitinate and degrade the securin protein which is bound to separase when it is immunoprecipitated from mitotic human cell lysates.
• mitotic Xenopus extracts the majority of securin is degraded, but in addition a major portion of human separase is also cleaved.
• the antibody beads with bound separase are incubated with either purified cohesin complexes or with recombinant SCC1 , which is the subunit of the cohesin complex that is cleaved by separase.
• the activity of separase is then monitored by analyzing the cleavage of SCC1 , which can be analyzed by SDS gel electrophoresis and subsequent immunoblotting with antibodies to SCC1. If recombinant radioactively labeled SCC1 is used the gels can also be analyzed by autoradiography or Phosphorimaging. In the experiments of the invention (Example 1 ), it was found that securin is able to bind to separase.
• securin can bind to the cleaved form of separase as well as to the full-length separase. It was found that securin binding to separase inhibits the protease activity of separase (Fig. 1).
• the immunoprecipitates were incubated in mitotic Xenopus egg extracts to allow securin destruction and separase cleavage.
• Recombinant forms of the p55 and p60 cleavage products are produced in suitable expression systems, purified and tested for their ability to cleave SCC1 , or a fragment thereof that contains the separase cleavage site, in vitro.
• Standard expression systems such as E. coli, budding yeast, Baculovirus infected Sf9 and Hi5 insect cells and transfected mammalian, e.g. human cells can be used.
• standard biochemical protocols can be used, e.g. those described in WO00/48627 for obtaining separase.
• the respective fragment can be employed in the protease assay (as described in WO00/48627) as a substitute for the full-length separase molecule.
• N- and C-terminal cleavage products of separase remain associated with each other (see above, Figure 6) it is also possible that both the N- and the corresponding C-terminal fragments will be required to obtain active recombinant separase.
• the two or more fragments can either be expressed individually, purified and then mixed together, or they can be co-expressed in expression systems as listed above, and the obtained complexes containing both N- and C-terminal fragments are purified. All of these forms, e.g. all combinations of C-terminal and corresponding N-terminal fragments, will then be tested for their ability to cleave SCC1 in vitro. If any of the complexes described above yields human separase activity, the respective complex of separase fragments can be employed in the proteolytic assay in the same manner as a C-terminal fragment by itself, as described above.
• full-length human separase is expressed in expression systems as listed above, the recombinant protein is isolated and activated, e.g. by incubation in mitotic Xenopus egg extracts, to induce its activation by cleavage.
• the assay is performed by employing the respective form of separase (fragment(s)) in combination with securin.
• the separase (fragment) is activated in the presence of securin in cell extracts, e.g. Xenopus laevis cell extracts.
• securin, or a fragment thereof that proves to be sufficient for activation of separase is employed in recombinant form, based on the cDNA sequence (Lee et al., 1999; Zhang et al, 1999).
• the separase (fragment) and securin can either be expressed and purified separately and then combined or they can be co-expressed and co-purified; as described above.
• synthetic peptide substrates for separase are designed and synthesized that allow the simple detection of protease activity in high throughput format, e.g. by fluorogenic methods.
• the proteolytic assays suitable for this purpose have been described in WOOO/48627.
• substrate peptides containing the separase recognition sequence see WOOO/48627
• AMC 7-amino-4-methyl-coumarin group
• the present invention relates to methods for identifying a compound that has the ability of modulating sister chromatid separation by inhibiting the proteolytic
• proteolytic screening assay method of the invention e.g. carried out with one or more separase fragments, in the presence or absence of securin, can be carried out according to standard methods, in particular as described in WO 00/48627:
• a further example for a suitable assay is based on the phenomenonon of fluorescence resonance energy transfer (FRET), as described by Gershkovich et al., 1996 or by Matayoshi et al., 1990. Additional examples for assays that may be used in the present invention for a high-throughput screening method to identify inhibitors of separase activity were described by Gray et al., 1994, Murray et al., 1993, Sarubbi et al., 1991.
• FRET fluorescence resonance energy transfer
• Fluorescent or radioactive labels and the other reagents for carrying out the enzymatic reaction on a high-throughput scale are commercially available and can be employed according to supplier's instructions (e.g. Molecular Probes, Wallac).
• supplier's instructions e.g. Molecular Probes, Wallac.
• the specific assay design depends on various parameters, e.g. on the size of the substrate used. In the case of using a short peptide, the fluorescence quenching or the fluorescence resonance energy transfer methods are preferred examples for suitable assay technologies.
• the fluorescence quenching (Resonance Energy Transferong RET) assay relies on synthetic substrates which are capable of direct, continuous signal generation that is proportional to the extent of substrate hydrolysis.
• the substrate peptide carries a fluorescent donor near one end and an acceptor near the other end.
• the fluorescence of the substrate is initially quenched by intramolecular RET between donor and acceptor.
• RET Resonance Energy Transferong RET
• Example 9 this type of assay is exemplified by use of AMC; which serves as a donor fluorophore and in the case of the separase-specific peptide substrates the amino acid bonds of the peptides function as acceptor chromophores.
• AMC which serves as a donor fluorophore and in the case of the separase-specific peptide substrates the amino acid bonds of the peptides function as acceptor chromophores.
• An assay of this type may be also carried out as follows: the solution of the labeled substrate (e.g.
• labeling pairs that are suitable for the method of the invention are commercially availabe, e.g. Europium (Eu) and Allophycocyanin (APC), Eu and Cy5, Eu and PE (Wallac, Turku, Finland).
• the compounds identified in the above methods have the ability to interfere with sister chromatid separation by modulating the proteolytic activity of separase.
• the present invention also relates to compounds which act as inhibitors of separase for use in human therapy, in particular cancer therapy.
• the invention relates to a pharmaceutical composition which contains, as the active ingredient, one or more compounds which interfere with or modulate sister chromatid separation by inhibiting separase activity.
• the invention comprises pharmaceutically active compounds and their use in therapy, which are small chemical molecules that have been identified as separase inhibitors in the screening method of the invention.
• separase inhibitors can be obtained starting from the recombinant active separase.
• synthetic peptide derivatives exemplified by derivatives of SVEQGR, DREIMR, SFEILR or EWELLR (e.g. Bio-SVEQGR- amk) can be used as the structural basis to develop peptidomimetic molecules that inhibit separase.
• the cleavage the cleavage sequence of human SCC1 or human separase can preferably be used.
• the assays described above using recombinant active separase and peptide substrates, e.g. fluorogenic peptides, can be used to optimize such compounds.
• Inhibitors of human separase activity identified in the screening methods of the invention or based on rational inhibitor design can be used as cytotoxic therapeutics for the treatment of diseases that are caused by uncontrolled cell proliferation, such as cancers, leukaemias, or cardiac restenosis.
• Species specific inhibitors of separase from eukaryotic pathogenic microorganisms can be used to treat infectious diseases caused by such microorganisms, for example infections caused by pathogenic fungi or diseases caused by parasites such as Leishmania species.
• RNA interference experiments can be used to knock out separase expression in human cultured cells, according to known methods, as described, e.g. by Elbashir et al, 2001.
• Influencing the process of sister chromatid separation may be also beneficial in preventing birth defects caused by missegration of chromosomes in human meioses. For example, since cases of human aneuploidy such as Down's syndrome may be caused by premature separation of sister chromatids (Griffin, 1996), the use of a drug that inhibits separase activity might be able to reduce precocious sister separation and thereby the incidence of aneuploidy in human fetuses.
• the invention relates to separase inhibitors for the prevention of birth defects caused by missegration of chromosomes in human meioses.
• the efficacy of compounds identified as separase inhibitors in the method of the invention can be tested for in vivo efficacy either on yeast cells or in mammalian cells.
• Effective compounds should block (or at least in some way interfere with) sister chromatid separation, which can be measured, e.g. by using CenV-GFP in yeast, as described by Ciosk et al., 1998, or standard cytological techniques in mammalian cells.
• Compounds effective in tumor therapy should be either cytostatic or cytotoxic. Substances whose potential for therapeutic use has been confirmed in such secondary screens can be further tested for their effect on tumor cells.
• tumor cell proliferation To test the inhibition of tumor cell proliferation, primary human tumor cells are incubated with the compound identified in the screen and the inhibition of tumor cell proliferation is tested by conventional methods, e.g. bromo-desoxy- uridine or 3 H incorporation. Compounds that exhibit an anti-proliferative effect in these assays may be further tested in tumor animal models and used for the therapy of tumors.
• Toxicity and therapeutic efficacy of the compounds identified as drug candidates by the method of the invention can be determined by standard pharmaceutical procedures, which include conducting cell culture and animal experiments to determine the IC 50 , LD50, the ED50 The data obtained are used for determining the human dose range, which will also depend on the dosage form (tablets, capsules, aerosol sprays, ampules, etc.) and the administration route (oral, buccal, nasal, paterental or rectal).
• a pharmaceutical composition containing the compound as the active ingredient can be formulated in conventional manner using one or more physologically active carriers and excipients. Methods for making such formulations can be found in manuals, e.g. "Remington Pharmaceutical Sciences".
• Separase inhibitors may also be useful in applications which aim at the deliberate polyploidisation of plant cells for crop development.
• yeast it has been shown that inhibition of separase activity prevents chromosome separation without blocking cell cycle progression and therefore gives rise to cells with increased ploidy.
• Inhibitors that block separase's protease activity could therefore be used to increase the ploidy of any eukaryotic cell, including all plant cells.
• Increasing the ploidy of plant cells is useful for 1 ) producing larger plants, 2) for increasing the ploidy of breeding stocks, and 3) for generating fertile hybrids.
• the present invention relates, in a further aspect, to separase inhibitors for the treatment of plant cells for increasing their ploidy.
• the screening method of the invention can be easily adapted by employing plant components, i.e. a plant separase and a plant homolog of SCC1. Sequence homologs of plant separase and SCC1 are present in databases, e.g. of the Arabidopsis thaliana genome.
• Separase inhibitors which impair sister chromatid separation may also be used in cytological analyses of chromosomes, for example, in medical diagnoses of chromosome structure.
• Fig. 1 Securin acts as an inhibitor for separase
• Fig. 2 Yeast peptides inhibit proteolytic activity of human separase in similar concentration as they inhibit yeast separase and influence the processing of human separase
• Fig.3 Human peptides inhibit proteolytic activity of human separase in a similar concentration as the yeast peptides do
• Fig. 4 Addition of yeast peptides at different stages during the activation of separase suggests that the cleaved forms are the active forms of separase and securin binding blocks access of peptide substrates to the active site of separase
• Fig. 5 Mitotically activated separase has autocatalytic activity
• Fig. 6 N- and C-terminal cleavage products of human separase stay associated after mitotic cleavage and ectopically expressed separase is able to cleave human SCC1
• Fig. 7 Working model for the activation of human separase
• Fig. 8 Mapping of the cleavage sites in human separase
• Fig. 9 A Principle of fluorogenic separase inhibitor screening assay
• Fig. 10 In vitro assay using activated separase bound to microbeads
• Fig. 11 Transcleavage separase screening assay showing inhibiting effect of small molecular compounds
• Inactive human separase (separase) immunoprecipitates were activated in mitotic Xenopus egg extracts (for details see Waizenegger et al., 2000). 30 microliter beads were incubated with 40 ⁇ g bacterially expressed wildtype securin, destruction box deleted form of securin (Gmachl et al., 2000) or BSA diluted in XB + 1 mM DTT for 40 min at RT. Subsequently the beads were washed with XB + 1 mM DTT and with TBS + 0.5 M NaCI + 1 mM DTT + 0.5% TWEEN20, followed by one wash with XB + 1m DTT. To control the rebinding of securin an aliquot of 5 ⁇ l was taken per assay and subsequently analysed by immunoblotting with antibodies against separase and securin.
• the beads were used for the SCC1 in vitro cleavage assay: 20 ⁇ l beads were mixed with 30 ⁇ l of the following SCC1 in vitro translation mix ( SCCImyc IVT + 1 ⁇ l PLK-GST + 0.3 ⁇ l 1 M MgCI 2 , 0.3 ⁇ l 100 mM ATP, 0.12 ⁇ l 250 mM EGTA, 13.78 ⁇ l XB + 1mM DTT) and incubated at 22° C and 1200 rpm. 5 ⁇ l were taken per time point, the reaction was stopped by addition of SDS loading buffer. Samples were analyzed by immunoblotting with mouse monoclonal antibodies against myc (9E10).
• Interphase Xenopus egg extracts were driven into mitosis in the presence of 1 mM Biotin-SVEQGR-amk or DMSO. Those mitotic Xenopus egg extracts were used to activate a batch of separase immunoprecipitates.
• Biotin labeled peptides were detected via immunoblotting according to Faleiro et al., 1997.
• Securin acts as an inhibitor for separase
• the amount of securin that bound to separase was at least as high as the amount of securin that had been bound to separase originally before securin was degraded by incubating the separase immunoprecipitates in mitotic Xenopus extracts, although the majority of separase had been cleaved during the incubation in the Xenopus extract. This result shows that securin can bind to the cleaved form of separase as well as to the full-length separase.
• Fig. 1A Separase immunoprecipitates (separase IP) obtained from Nocodazole arrested HeLa cells and bound by antibodies against the C-terminus of separase coupled to beads were activated in mitotic Xenopus egg extracts (separase
• Fig.l B Activated separase immunprecipitates which were either incubated with buffer, wildtype securin, destruction box deleted securin or BSA were incubated SCC1-myc reaction mix. Aliquots were taken at indicated timepoints and analysed by immunoblotting with antibodies against myc. Cleaved SCC1 is marked by arrows.
• Yeast peptides inhibit proteolytic activity of human separase in similar concentration as they inhibit yeast separase and influence the processing of human separase
• the two inhibitors are therefore called Bio- SVEQGR-cmk and Bio-SVEQGR-amk.
• These inhibitors were added to human separase that had been isolated by immunoprecipitation and had been activated in mitotic Xenopus extracts as above, it was observed that both inhibitors are able to block the ability of separase to cleave SCC1 ( Figure 2A).
• the concentration of these peptide derivatives required to inhibit human separase was similar to the concentration needed to inhibit separase from budding yeast (compare Fig. 2, upper panel and WO 00/48627, Uhlmann et al., 2000).
• Fig. 2A The structure of the yeast peptides
• Fig. 2B Separase immunoprecipitates obtained from nocodazole arrested HeLa cells bound by antibodies against the C-terminus of separase coupled to beads were activated in mitotic Xenopus egg extracts. Subsequently samples were incubated with indicated concentrations of yeast peptides (Biotin- SVEQGR-cmk or Biotin-SVEQGR-amk). After a short wash samples were mixed with SCC1-myc reaction mix for 1 hour. Samples were analysed by immunoblotting with antibodies against myc. Cleaved SCC1 is marked by an arrow.
• Fig. 2C The samples (see B) were immunblotted with antibodies against separase.
• Human peptides inhibit proteolytic activity of human separase in a similar concentration as the yeast peptides inhibit yeast and human separase
• Separase immunoprecipitates obtained from nocodazole-arrested HeLa cells bound by antibodies against the C-terminus of separase coupled to beads were activted in mitotic Xenopus egg extracts. Subsequently samples were incubated with the indicated concentrations of human peptides (DREIMR-amk or Biotin-DREIMR-amk). After washing samples were mixed with SCC1-myc reaction mix for 1 hour. Samples were analysed by immunoblotting with antibodies against myc. For control a sample was treated with the same concentration of DMSO which was used for the solubilization of the peptides (DMSO). The SCC1-myc reaction mix was loaded as an input control (SCCImyc input).
• peptide inhibitors developed on the basis of the cleavage recognition site of human SCC1 , "DREIMR", were able to inhibit separase at a similar concentration as the peptide inhibitors derived from yeast (Fig. 3).
• Fig. 3A shows the structure of the human peptide
• Fig. 3B shows that human peptide derivatives inhibit the SCC1 cleavage activity of separase.
• Full length SCC1 is indicated by an arrowhead, cleaved SCC1 is marked by an arrow.
• Fig. 4A Separase immunoprecipitates (separase IP) obtained from Nocodazole arrested HeLa cells bound by antibodies against the C-terminus of separase coupled to beads were activated in mitotic Xenopus egg extracts (separase ip 1 TM* 0 ' 10 ). Aliquots were analysed by immunoblotting with antibodies against separase and securin.
• Fig. 4B Separase IPs (see A) were either preincubated with Biotin-SVEQGR- amk (preinc. with inh. peptide) or with DMSO (preinc. with DMSO), subsequently washed and aliquots were taken. Thereafter they were incubated in mitotic Xenopus egg extracts and washed again. Samples were taken for analysis (1 i, 1c). Separase IPs were activated either in mitotic Xenopus egg extracts which were driven into mitosis in the presence of Biotin-SVEQGR-amk or DMSO, subsequently washed and aliquots were taken for analysis (2i, 2c).
• Fig. 4C Samples 1 i, 1c, 2i, 2c, 3i and 3c (see B) were mixed with SCC1-myc reaction mix for 1 hour and analysed by immunoblotting with antibodies against myc. Arrows indicate the first and second SCC1 cleavage product.
• Fig. 4D Activated separase immunoprecipitates bound by antibodies against the C-terminus of separase to beads were first incubated with recombinant securin or, for control, with recombinant truncated cyclinB. After washing the precipitates were incubated with the human peptide inhibitor. Thereafter, the immunoprecipitates were again washed and analyzed by immunoblotting.
• the resulting DNA fragment was inserted via EcoRV and Spel into the existing truncated form of human separase (KIAA 0165).
• the complete coding sequence of human separase is shown in SEQ ID NO: 1
• the amino acid sequence is shown in SEQ ID NO:2.
• Human separase was N-terminally tagged with a Flag epitope and transiently transfected in HeLa cells either in a single transfection or in a cotransfection with human securin which was C-terminally tagged with a myc epitope.
• HeLa cells were also transiently transfected with securin-myc. After 24 hours transfection 330 nM nocodazole was added for 18 hours. Cells were harvested, washed with PBS and cell extracts were generated as described in Waizenegger et al. 2000. These cell extracts were used to immunoprecipitate exogenous Flag-separase with mouse anti-Flag antibodies bound to sepharose.
• Mitotically activated separase has autocatalytic activity
• a myc-tagged form of separase obtained by in vitro transcription and translation was used as a substrate.
• the transcription-translation reaction was carried out in the presence of 35 S-labeled methionine and cysteine, resulting in radiolabeled translation products.
• Figure 5 shows that only upon mitotic activation of separase separase-myc is autocatalytically cleaved.
• Fig. 5A Separase immunoprecipitates (separase IP) obtained from Nocodazole arrested HeLa cells bound by antibodies against the C-terminus of separase coupled to beads were either incubated in mitotic (separase
• Fig. 5B Separase immunoprecipitates were incubated either in a mitotic or interphase Xenopus egg extracts and then mixed with recombinant separase- myc reaction mix. At indicated time points samples were taken and analysed by immunoblotting with antibodies against myc.
• N-and C-terminally cleavage products of separase remain physically associated and tagged recombinant human separase is active
• tagged forms of separase were generated.
• N-terminally FLAG-tagged full-length separase was transiently expressed in HeLa cells as described in Example 5B and isolated by immunoprecipitation with an anti-FLAG antibody.
• the immunoprecipitates were incubated in mitotic Xenopus egg extracts to allow securin destruction and separase cleavage ( Figure 6A). After re-isolation the immunoprecipitates were immunoblotted with anti-separase antibodies, anti-securin antibodies or anti-FLAG antibodies. Both C-and N-terminal separase fragments could be detected in the immunoprecipitates, suggesting that the N- and C-terminal separase fragments remain associated after cleavage ( Figure 6A).
• Fig. 6A HeLa cells were transiently transfected with FLAG-separase, FLAG- separase and securin-myc or only with securin-myc. Mitotic extracts were performed from these cells and used for immunoprecipitation with anti-FLAG antibodies bound to sepharose (IP). The immunoprecipitates were activated in mitotic Xenopus egg extracts (IP m ). The immunoprecipitates were analysed by immunoblotting with antibodies against securin, separase (7A6) and FLAG.
• Fig. 6B Aliquots of above described immunoprecipitates were incubated with SCC1-myc reaction mix. At indicated time points samples were withdrawn and analysed by immunoblotting with antibodies against myc. Arrows indicate SCC1-myc cleavage products.
• the cleavage sites in human separase were mapped by a method that has been previously used to map cleavage sites in SCC1 (WOOO/48627). Briefly, truncated versions of the human separase cDNA were generated by polymerase chain reactions, and the resulting cDNAs were used directly for coupled in vitro transcription-translation reactions by using rabbit reticulocyte lysates. The transcription-translation reactions were carried out in the presence of 35 S-labeled methionine and cysteine, resulting in radiolabeled translation products. These were then separated by SDS gel electrophoresis side by side with the in vitro cleavage products of mitotically activated human separase immunoprecipitates which were detected by immunoblotting.
• N-terminally truncated separase cDNA was generated by polymerase chain reaction.
• the 5'primers contain a sequence with the T7 polymerase binding site
• the PCR products were in vitro transcribed and translated with the TNT system (Promega).
• the recombinant products, which start with an exogenous methionine at the indicated amino acids obtained, were loaded side by side with in vitro cleaved separase on a SDS-PAGE.
• For immunoblotting the mouse anti-separase antibody (7A6) was used. The results are shown in Fig. 8.
• Assay for identifying separase inhibitors using a fluorogenic peptid substrate In order to establish a robust screening assay (based on liquid phase fluorescence energy transfer) for identifying inhibitors of recombinant human separase, four peptide substrates (1 : SFEILR-AMC, 2: SFEILRG-AMC, 3:EWELLR-AMC and 4: DREIMR-AMC) were synthesized. These peptides are linked to AMC (7-Amido-4-methylcoumarin), a fluorogenic group, which has been described for proteolytic assays, such as for trypsin (Zimmerman et al., 1977) and cathepsin B (Barrett and Kirschke, 1981 ).
• AMC serves as a donor fluorophore and in the case of the separase-specific peptide substrates the amino acid bonds of the peptides function as acceptor chromophores (Fig. 9A).
• the peptide substrates are cleaved at the P1 ' -AMC junction; by processing the peptide-AMC bond the unquenched AMC is set free and can be monitored as increasing fluorescence.
• the designed peptide substrates represent the intramolecular cis-cleavage site in separase itself (peptides 1 - 3) and the intermolecular trans-cleavage site in cohesin respectively (peptide 4).
• Trypsin solution (Gibco 043-90317 FU) was diluted 1 :1000 in Hepes buffer containing 20mM Herpes (pH: 7.7), 100mM KCI, 1mM MgCI 2 , 0.1 mM CaCI 2 and 1 mM DTT (freshly added). 1 ⁇ l of peptide 1 (4mg/ml in DMSO) was added, mixed and measured in a Hitachi f-2000 fluorescence spectrophotometer (Ex: 355 and Em: 460nm). A typical kinetics is shown in Fig. 9B.
• separase assay was performed as follows: ⁇ 500 ⁇ l of separase bead suspension was diluted with 1800 ⁇ l Hepes buffer. 30 ⁇ l of the diluted suspension was applied per well of a 96 "Packard OptiPlate black" plate. Additionally, 70 ⁇ l Hepes buffer and 1 ⁇ l of a LMW-compound stock solution (5mg/ml in DMSO) were added per well.
• the reaction was initialized by adding 1 ⁇ l of the peptide 1 (4mg/ml in DMSO).
• the reaction was monitored in a Fluoroskan II 96-well reader (Ex: 355nm, Em: 460nm) at room temperature (Fig. 10).
• As a control measurement was performed in the presence of 2 ⁇ l DMSO per 100 ⁇ l reaction volume whereas for inhibition a cleavage peptide linked to an AMK (acyl-oxymethyl ketone) residue, which was described in WO 00/48627, was used.
• the AMK residue serves as a broad-spectrum inhibitor for many cysteine and serine proteases (for details see Beynon and Bond, 1989).
• Ciosk R Zaariae W, Michaelis C, Shevchenko A, Mann M, Nasmyth K; Cell 1998 Jun 12;93(6):1067-76
• Waizenegger IC Hauf S, Meinke A, Peters; JM Cell 2000 Oct 27;103(3):399- 410

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