Activators of caspase-8/c-FLIP.SUB.L dimerization and their use in cancer therapy

Abstract

The present invention relates to activators of caspase-8/c-FLIP.sub.L dimerization and their use in enhancing the pro-apoptotic activity of the heterodimer and cancer therapy.

Claims

1. A compound, wherein said compound is 2-[[4-(4-pyridyl)benzoyl]amino]ethyl 3-(3-pyridylmethylsulfamoyl)benzoate according to formula III ##STR00006## or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising an effective amount of the compound according to claim 1, together with a pharmaceutically acceptable carrier.

Description

(1) The present invention will now be further described in the examples with reference to the accompanying figures, nevertheless, without wanting to be limited thereto. For the purposes of the present invention, all references as cited are incorporated by reference in their entireties. In the Figures:

(2) FIG. 1 shows the heterodimer structure of c-FLIP.sub.L and caspase-8 used for virtual screening for the discovery of FLIPins. (a) Model of heterodimer structure of c-FLIP.sub.L (shown in dark gray) and processed caspase-8-p43/p10 (shown in light gray) with a putative binding site for the compound according to the present invention. The compound binding site is shown as a gray surface (b) Superimposition of the crystal structure of the processed caspase-8 [PDB ID 4PRZ] on the heterodimer complex of procaspase-8/c-FLIP.sub.L [PDB ID 3H11] (c) Interaction of FLIPinB with binding site residues of the heterodimer complex; amino acid residues of c-FLIP.sub.L and caspase-8 are denoted in black and dark gray colors, respectively. Hydrogen bonds are depicted as green dotted lines. Amino acid residues are shown in line representation; Protein subunits are shown in cartoon representation: processed caspase-8 (light gray color), c-FLIP.sub.L (dark gray color). The preferred compound FLIPinB is shown in stick representation.

(3) FIG. 2 shows that the co-treatment of FLIPinB/DL promotes the loss of cell viability in cancer cell lines. HeLa-CD95 cervical carcinoma cancer cells (a), T cell leukemia Jurkat cells (b), HeLa-CD95-FL (c-FLIP.sub.L-overexpressing HeLa-CD95) cells (c, d) and acute myeloid leukemia MV-4-11 (e) cells were treated with the indicated concentration of FLIPinB for two hours and subsequently stimulated with CD95L (60 ng/mL in a+b; 238 ng/mL in c+d, 1000 in ng/mL e) for 6 hours (b-e) or 22 hours (a). Cell viability was measured using the Cell Titer-Glo-Luminescent Cell Viability Assay estimating the ATP content. Mean and standard deviation are shown (n=3). (f) HeLa-CD95 cells were pretreated with 40 M FLIPinB for two hours and stimulated with TRAIL (100 ng/mL). Dying cells were visualized with live cell imaging. Cells were stained with Cytotox Red Reagent. Fluorescence of dying cells was detected by the IncuCyte live cell analysis system (Essen Bioscience). In the lower part representative images after 4 hours of TRAIL stimulation are shown (bf=brightfield, M=medium, T=100 ng/mL TRAIL, F+T=40 M FLIPinB+100 ng/mL TRAIL).

(4) FIG. 3 shows that FLIPinB/DL co-treatment enhances caspase activity. (a+b) Jurkat cells were pre-treated with 20 M FLIPinB for two hours and stimulated with CD95L (60 ng/mL) for four hours (a) or 100 ng/mL TRAIL for three hours (b). Caspase-3/-7-activity was determined by the Caspase-Glo3/7 Assay. Caspase activity is shown in RU. Mean and standard deviations are shown (n=3). (c) HeLa-CD95 cells were pre-treated with 40 M FLIPinB for two hours and stimulated with CD95L (50 ng/mL). Caspase activity was visualized with live cell imaging. Cells were stained with Caspase-3/7 Green Apoptosis Assay Reagent. Fluorescence of dying cells was detected by the IncuCyte live cell analysis system (Essen Bioscience). In the lower part representative images after two hours of CD95L stimulation are shown (bf=brightfield, M=medium, C=50 ng/mL CD95L, F+C=40 M FLIPinB+50 ng/mL CD95L).

(5) FIG. 4 shows that FLIPinB/CD95L co-treatment enhances caspase activation. (a) HeLa-CD95 cells were pretreated with 20 M FLIPinB for two hours, followed by stimulation with CD95L (60 ng/mL) for three hours. Total cellular lysates were analyzed by Western Blot using the indicated antibodies. One representative experiment out of three independent experiments is shown. (b) HeLa-CD95 cells were treated with 20 M FLIPinB for up to 16 hours. Total cellular lysates were analyzed by Western Blot using the indicated antibodies. Actin was used as loading control.

(6) FIG. 5 shows that FLIPinB increases caspase-8 activity at the DISC. (a) Scheme presenting the proposed mechanism of FLIPinB action: FLIPinB binds to the caspase-8/c-FLIP.sub.L complex after initial processing of procaspase-8 to p43 and p10, which leads to the enhancement of caspase-8 activity in the p43/p10/FLIPinB/c-FLIP.sub.L complex. (b) HeLa-CD95 cells were pre-incubated with or without 20 M FLIPinB for two hours, followed by stimulation with 60 ng/mL CD95L for three hours. CD95-DISC-IPs were analyzed by Caspase-Glo-8-Assay. (c) HeLa-CD95 cells were stimulated with 60 ng/mL CD95L for three hours. CD95-DISC-IPs were analyzed by Western Blot analysis and probed for the indicated proteins. Actin was used as loading control. One representative experiment out of three is shown. (BC=control IP without antibody).

(7) FIG. 6 shows modeling caspase-8 activation at the DISC/DED filament upon action of FLIPinB. (a) The topology of ODE model describing the activation of caspase-8 in the DED filament. kddimerization rate for procaspase-8 dimer; C.Math.kddimerization rate for c-FLIP.sub.L/procaspase-8 and caspase-8/caspase-8 dimers; kpprocessing rate; kcd and kc3 rates for cell death substrate cleavage by caspase-8 and caspase-3, respectively. kc3, kc3_p3 rates of procaspase-3 cleavage by caspase-8 and caspase-3 respectively. (b-e) Simulations of the model and experimental data used for the model training. Lanes present model simulations, while points experimentally measured values. Treatment with CD95L is marked in blue, while the co-treatment CD95L/FLIPinB is shown in red.

(8) FIG. 7 shows model predictions and validation of FLIPinB action. (a) Simulations for cell death. Stimulation with CD95L is marked in blue, while the cotreatment CD95L/FLIPinB is shown in red. (b). HeLa-CD95 cells were pretreated with 40 M FLIPinB for two hours and stimulated with CD95L (50 ng/mL). Dying cells were visualized with live cell imaging. Cells were stained with Cytotox Red Reagent. Fluorescence of dying cells was detected by the IncuCyte live cell analysis system (Essen Bioscience). In the lower part representative images after 4 hours of CD95L stimulation are shown (bf=brightfield, M=medium, C=50 ng/mL CD95L, C+F=40 M FLIPinB+50 ng/mL CD95L).

EXAMPLES

(9) Material and Methods

(10) Virtual ScreeningVirtual screening (VS) was carried out using the Glide molecular docking software (Friesner et al., 2004, 2006) from the Schrdinger Small Molecule Drug Discovery Suite 2015-1 (Schrdinger, Inc). Molecular docking was performed in the standard-precision (SP) and extra-precision (XP) modes. Prior to VS, protein structures were processed using the Protein preparation wizard module in the Schrdinger Suite 2015-1 (Madhavi Sastry et al., 2013). Protein minimization was carried out using MacroModel from the Schrdinger Suite 2015-1 and OPLS_2005 force field. VS was performed using the ZINC12 library of commercially available compounds prepared for molecular docking, which contains more than 16 million purchasable compounds (Irwin et al., 2012; Sterling and Irwin, 2015). At the first step VS was executed using Glide in the standard precision mode selecting about 100,000 compounds with the best Glide SP scoring function values. The next round of VS was performed in the XP mode resulting in a selection of compounds with the best Glide XP scoring function values for further visual inspection and selection of final hits for experimental validation. Additionally, the inventors applied the same virtual screening pipeline for 3770 active substances from Pubchem Bioassay Database (AID 624356), which contains the results of HTS directed on identification of small molecule compounds sensitizing TRAIL through caspase-8 pathway. This allowed the inventors to select two more compounds, FLIPinQ and FLIPinR.

(11) Cell linesHuman cervical cancer HeLa-CD95 cells (Neumann et al., 2010) (CD95-overexpressing cells) and HeLa-CD95-FL cells (CD95/c-FLIP.sub.L-overexpressing cells) were maintained in DMEM/Ham's (Merck Millipore, Germany) supplemented with 10% heat-inactivated fetal calf serum, 1% Penicillin-Streptomycin and 0.0001% Puromycin in 5% CO.sub.2. Human acute myeloid leukemia MV-4-11 cells and T lymphoma Jurkat cells were maintained in RPMI 1640 (Thermo Fisher Scientific Inc., USA) supplemented with 10% heat-inactivated fetal calf serum and 1% Penicillin-Streptomycin in 5% CO.sub.2.

(12) Antibodies and ReagentsThe following antibodies were used for Western Blot analysis: polyclonal anti-caspase-3 antibody (#9662), polyclonal anti-PARP antibody (#9542), monoclonal anti-RIPK1 XP antibody (#3493), polyclonal anti-actin antibody (A2103, Sigma-Aldrich, Germany), polyclonal anti-CD95 antibody (sc-715), polyclonal anti-mCherry antibody (ab183628), monoclonal anti-caspase-10 antibody (MO59-3), monoclonal anti-FADD antibody (clone 1C4), monoclonal anti-caspase-8 antibody (clone C15) and monoclonal c-FLIP antibody (clone NF6). Horseradish peroxidase-conjugated goat anti-mouse IgG1, -2a, -2b, goat anti-rabbit and rabbit anti-goat were from Santa Cruz (California, USA). Recombinant TRAIL (KillerTRAIL) and recombinant CD95L (SuperFasLigand) were from Enzo Life Sciences, Germany. The monoclonal anti-APO-1 antibody (mouse-IgG3) was used for immunoprecipitations (IPs). All chemicals were of analytical grade and purchased from Merck (Darmstadt, Germany) or Sigma (Germany). The anti-APO-1, C15, NF6 and 1C4 antibodies were a kind gift of Prof. P. H. Krammer (DKFZ, Heidelberg). Recombinant LZ-CD95L was produced as described (Fricker et al., 2010).

(13) Analysis of total cell lysates by Western Blot analysisThe Western Blot analysis of total cellular lysates was performed in accordance to the inventors' previous reports (Schmidt et al., 2015).

(14) CD95 DISC-IPThe CD95 DISC-IP (Immunoprecipitation) from 510.sup.6 HeLa-CD95 cells were done as described before (Pietkiewicz et al., 2015). In addition, DISC-IPs were washed four times with PBS, which was followed by Western Blot analysis or caspase-8 activity assays.

(15) Caspase-8 activity assayEach of protein A beads samples with CD95 DISC-IPs were resuspended in 95 L of CHAPS-Buffer (50 mM HEPES pH=7.2; 50 mM NaCl; 10 mM EDTA; 5% Glycerin; 10 mM DTT, 0.1% CHAPS) and transferred into a 96-well plate. Caspase-8 activity was measured according to manufacturer's instructions (Caspase-Glo 8 Assay, Promega, Germany). Every condition was performed in duplicate. The luminescence intensity was analyzed by a microplate reader Infinite M200pro (Tecan, Switzerland).

(16) Cell viability quantification by ATP assay50 L of the CellTiter-Glo solution was added to each well and measurements were performed according to manufacturer's instructions (CellTiter-Glo Luminescent Cell Viability Assay, Promega, Germany). The luminescence intensity was analyzed by a microplate reader Infinite M200pro (Tecan, Switzerland). The viability of untreated cells was taken as one relative unit (RU). Every condition was performed in duplicate.

(17) Caspase-3/7 activity assayCaspase activity was measured by adding 50 L of the Caspase-Glo3/7 solution to each well. The luminescence intensity was analyzed by a microplate reader Infinite M200pro (Tecan, Switzerland) according to manufacturer's instructions (Caspase-Glo 3/7 Assay, Promega, Germany). The caspase activity of non-treated cells was taken as one relative unit (RU). Every condition was performed in duplicate.

(18) Cell death detection using IncuCyte live cell imagingHeLa-CD95 cells in a 96 well plate (510.sup.3 cells/well) were pre-incubated with FLIPinB for 2 hours. Subsequently cells were stimulated with CD95L (50 ng/mL). To monitor cell death, cells were treated with IncuCyte Cytotox Red Reagent (1:4000) and Caspase-3/7 Green Apoptosis Assay Reagent (1:5000). The cells were imaged every hour by IncuCyte ZOOM (Essen BioScience, Michigan) with a lox objective for a period of 6 hours. Every condition was performed in triplicate. Data were analyzed by the IncuCyte ZOOM Software (2016A).

(19) ModelingOrdinary differential equations (ODEs) were used to model caspase-8 activation at the DISC with or without FLIPinB. In brief, the model was fitted to cell viability data, caspase-3 activity assays and in vitro caspase-8 activity after immunoprecipitation of DISC complex. In vitro caspase-8 activity was estimated from the rate of caspase-8 substrate cleavage, assuming that the caspase-8 substrate is present in excess.

(20) Parameter fitting was done using the differential evolution method implemented in scipy.optimize.differential_evolution (Storn and Price, 1997). The Chi-square objective function based on deviation between model data and experimental data points was minimized to adjust parameter values.

(21) Identifability analysis of parameters was carried out by implementing the profile likelihood method (Raue et al., 2009). Profile likelihoods for each parameter were evaluated by fixing corresponding parameters in a wide range of values, while re-optimizing all other parameters. 95% finite sample confidence intervals for the parameters were estimated.

(22) Virtual Screening Identified Compounds Targeting c-FLIP (FLIPins)

(23) A closed conformation of the unprocessed L2 loop of procaspase-8 in the procaspase-8/c-FLIP.sub.L heterodimer was suggested to be crucial for its pro-apoptotic function, due to its stabilizing effect on the active center of caspase-8. In the closed conformation, the side chains of amino acid residues of the unprocessed L2 loop occupy a groove on the c-FLIP.sub.L protein located between the 6 and 3 regions (6/3 groove) (Yu, Jeffrey and Shi, 2009). The switch of the L2 loop from a closed to an open conformation due to the L2 loop cleavage is expected to abolish the interactions within the 6/3 groove of c-FLIP.sub.L and subsequently diminished the activity of the heterodimer. In this regard, the L2 loop cleavage leads to the generation of caspase-8-p43 and p10 cleavage products resulting in a complex comprising caspase-8-p43/p10 and c-FLIP.sub.L (denoted thereafter p43/p10/c-FLIP.sub.L). Accordingly, the inventors assumed that if the 6/3 groove will be occupied with a rationally designed small molecule mimicking the stabilizing effect of the L2 loop in a closed conformation, it would rescue the activity of the p43/p10/c-FLIP.sub.L complex (FIG. 1a). In order to identify such compounds a virtual screening was performed.

(24) To carry out the virtual screening, the structure of the p43/p10/c-FLIP.sub.L complex with the L2 loop in an open conformation had to be computed. The structure of the p43/p10/c-FLIP.sub.L complex was derived from two previously obtained structures: the crystal structure of procaspase-8/c-FLIP.sub.L heterodimer of [PDB ID 3H11] (Yu, Jeffrey and Shi, 2009) and the structure of the p18 subunit of caspase-8 [PDB ID 4PRZ] (Vickers et al., 2014). The latter was used to obtain information on the structure of caspase-8 with the L2 loop in an open conformation. The structure of the p18 and p10 parts of caspase-8 in p43/p10/c-FLIP.sub.L complex was predicted based on the structural superimposition of p18 structure [PDB ID .sub.4PRZ] on the structure of procaspase-8 [PDB ID 3H11] followed by structural minimization (FIG. 1b). During the structure preparation water molecules from the crystal structure [PDB ID 3H11] located on the interface between procaspase-8 and c-FLIP.sub.L were kept, assuming that they play a role in complex stabilization. The generated 3D structure of the p43/p10/c-FLIP.sub.L complex was used for virtual screening (FIG. 1b).

(25) Structural rearrangements in the course of the transformation from the closed to the open conformation of the L2 loop might lead to formation of a cavity on the binding interface of p43/p10/c-FLIP.sub.L complex according to in silico model (FIG. 1a). Therefore, the inventors expected that virtual screening would allow to identify compounds binding within this cavity to c-FLIP.sub.L, thereby imitating the stabilizing effect of the L2 loop in a closed conformation and leading to an increase in caspase-8 activity. As a result of the inventors' virtual screening, 5000 compounds with the best scoring function values were selected for further visual inspection. Visual verification was aimed at filtering of possible docking artifacts as well as at selection of compounds able to mimic key interactions observed in the L2 loop in the procaspase-8/c-FLIP.sub.L heterodimer structure. In this regard, the key criterion was the possibility of a compound to form a hydrogen bond with the carbonyl oxygen of Ser318 of c-FLIP.sub.L, that was supposed to mimic the interaction between R376 of procaspase-8 with c-FLIP.sub.L in the procaspase-8/c-FLIP.sub.L heterodimer. Finally, the inventors selected suitable compounds termed FLIPins (FLIP inhibitors) for in vitro experimental tests.

(26) FLIPinB/FLIPinB Enhances CD95L-/TRAIL-Mediated Loss of Cell Viability

(27) As an initial experimental screening, the effect of FLIPins was examined by measuring the viability of Jurkat cells upon stimulation with FLIPin/CD95L in comparison with CD95L-only treatment. FLIPins that showed significant loss of viability upon their administration to Jurkat cells were not further considered. The compound designated as FLIPinB showed the best properties in the initial selection. Hence, its effects on CD95L- or TRAIL-induced cell death were further exploited in Jurkat, HeLa-CD95 (HeLa cells overexpressing CD95) (Neumann et al., 2010) and HeLa-CD95-FL (HeLa-CD95 cells overexpressing c-FLIP.sub.L) cells. Administration of FLIPinB in Jurkat and HeLa-CD95 cells enhanced CD95L- and TRAIL-induced loss of cell viability and increased caspase activity. High levels of c-FLIP.sub.L are associated with resistance to DR-mediated apoptosis (Fricker et al. 2010). Accordingly, FLIPinB administration allowed to overcome the resistance of HeLa-CD95-FL cells to CD95L. These data provide the first evidence that the compound FLIPinB promotes CD95L- and TRAIL-induced loss of cell viability and caspase activation.

(28) Subsequently, the structural properties of FLIPinB were further optimized using computer design methods. In order to construct a water soluble compound and improve its binding affinity to the caspase-8/c-FLIP.sub.L heterodimer, several optimization steps were introduced. This included the substitution of benzene in biphenyl and benzylsulfamic groups by pyridine expecting an increased binding to Arg337 and Trp466 in the side chains of c-FLIP.sub.L (FIG. 1c). The modification of the FLIPinB structure led to the small molecule FLIPinB, which contains the core element of FLIPinB. Introduction of side chains did not perturb the binding of FLIPinB to c-FLIP.sub.L. The key interactions observed in the predicted FLIPinB/c-FLIP.sub.L/p43/p10-caspase-8 complex are shown in the FIG. 1c. Importantly, the phenyl-pyrimidine ring occupies a cavity formed between the 6-sheet strands and the 3-helix of c-FLIP.sub.L. FLIPinB forms two hydrogen bonds with the carbonyl group of Ser318 and a water molecule from the structure [PDB ID 3H11], which was kept for the virtual screening.

(29) Importantly, the same interaction features are observed for the L2 loop in the procaspase-8/c-FLIP.sub.L complex. Additionally, the sulfonamide group is able to form hydrogen bonds with the hydroxyl group of Thr407 of c-FLIP.sub.L and caspase-8 carbonyl oxygen of E396 (FIG. 1c), replacing corresponding water molecule observed in the c-FLIP.sub.L heterodimer structure [PDB ID 3H11]. Remarkably, FLIPinB occupies Dithiane Diol binding cavity, according to reference crystal structures [PDB ID 4PRZ], what is likely beneficial for its binding free energy.

(30) The designed small compound FLIPinB efficiently promoted a loss of cell viability in a dose- and time-dependent manner induced by CD95L in several cell lines including HeLa-CD95, Jurkat, HeLa-CD95-FL and MV-4-11 cells (FIG. 2a-e). These observations were supported by measuring cell death in HeLa-CD95 cells via live cell imaging using the IncuCyte technology (FIG. 20. In particular, cell death was detected using Cytotox Red, a dye which penetrates into dying cells with a compromised plasma membrane, providing the possibility to detect dying cells (FIG. 2f). Importantly, FLIPinB enhanced TRAIL-induced cell death, which was most prominent in the first hours after TRAIL treatment (FIG. 2f). Taken together, these experiments have demonstrated that the optimized compound FLIPinB promotes CD95L- and TRAIL-mediated loss of cell viability.

(31) FLIPinB Enhances CD95L-/TRAIL-Mediated Caspase Activity

(32) To ensure that FLIPinB promotes DL-mediated cell death via the apoptotic branch of the pathway the inventors analyzed its effects on caspase activity. Both FLIPinB/CD95L and FLIPinB/TRAIL co-treatment led to an increase of caspase-3/7 activity in Jurkat cells, compared to CD95L-treatment only (FIG. 3a, b). This was consistent with the analysis of caspase-3/7 activity with live cell imaging, which showed an increase in caspase-3/7 activity in single cells upon FLIPinB/CD95L and FLIPinB/TRAIL co-administration in Hela-CD95 cells (FIG. 3c). These data were further supported by Western Blot analysis, demonstrating that FLIPinB increases CD95L-induced cleavage of procaspase-8 to p18, RIPK1 and PARP (FIG. 4a). In this regard, a slight increase of RIPK1 cleavage indicated that FLIPinB might act via increasing caspase-8 activity, because RIPK1 isone of the caspase-8 substrates. Collectively, these data demonstrate that FLIPinB enhances caspase activation induced by DLs and that it acts on the apoptotic branch of the pathway.

(33) c-FLIP proteins are characterized by a short half-life, which plays a central role in the regulation of their inhibitory action via fine-tuning of their intracellular concentration. The binding of FLIPinB to c-FLIP.sub.L might cause a conformational change leading to recruitment of putative DUB and subsequent c-FLIP.sub.L proteosomal degradation. In order to check if FLIPinB triggers a decrease of c-FLIP.sub.L levels in the cell and thereby induces apoptosis, the inventors analyzed whether the addition of FLIPinB to HeLa-CD95 cells changes the level of c-FLIP.sub.L (FIG. 4b). Time-dependent analysis of c-FLIP.sub.L levels by Western Blot demonstrated no influence of FLIPinB on c-FLIP.sub.L expression in the first hours after its administration (FIG. 4b). Thus, it was concluded that FLIPinB does not act via modulating the protein level of c-FLIP.sub.L.

(34) FLIPinB Acts Via Enhancement of Caspase-8 Activity at the DISC

(35) DISC is a central platform for procaspase-8 activation. The predicted in silico mechanism of the FLIPinB effect involves the increased activity of procaspase-8/c-FLIP.sub.L heterodimer directly at the DISC. The suggested mechanism of FLIPinB action assumes that it binds to the procaspase-8/c-FLIP.sub.L complex after initial processing of procaspase-8 to p43 and p10 proteolytic fragments (FIG. 5a). The initial processing is expected to lead to a structural rearrangement of L2 loop from a closed to an open conformation, leading to emergence of a cavity at the complex interface and subsequent destabilization of the complex. The inventors assume that FLIPinB occupies the position of the L2 loop of procaspase-8 in the closed conformation, which leads to the stabilization of the complex and rescue of caspase-8 activity in the p43/p10/c-FLIP.sub.L complex (FIG. 5a).

(36) To test this hypothesis, CD95 DISC was immunoprecipitated from HeLa-CD95 cells with or without pre-treatment with FLIPinB using anti-CD95 (anti-APO-1) antibodies. Subsequently, the caspase-8 activity in the immunoprecipitates (IPs) was analysed using Caspase-8-Glo assay (FIG. 5b-c). DISC-IPs contained all core components of the DISC: CD95, FADD, procaspases-8 and 10 and their cleavage products (FIG. 5b). Co-treatment with FLIPinB increased caspase-8 activity at the DISC indicating that FLIPinB acts directly at the DISC by specifically increasing the caspase-8 activity (FIG. 5c). This experimental analysis further supports the computational predictions on the mechanism of FLIPinB.

(37) Validation of the Cell Response to FLIPinB Treatment with a Computational Model

(38) In order to obtain detailed quantitative insights into the mechanism of FLIPinB function at the DED chain, the inventors used the cutting edge technology of mathematical modeling. To this end, a biochemical model of procaspase-8 activation at the DED filament has been created based on ODEs. The topology of the model included the formation of the homo-(procaspase-8/procaspase-8) and heterodimers (procaspase-8/c-FLIP.sub.L) at the DED chains/filaments (FIG. 6a). After homo- and heterodimerization procaspase-8 undergoes autocatalytic activation in the DED filament which is followed by intra- and intermolecular processing of the dimers into the p43 and p10. This is accompanied by the caspase-8-mediated cleavage of procaspase-3 to caspase-3 and its subsequent activation leading to cell death. The ratios between procaspase-8 and c-FLIP at the DISC were based on quantitative proteomics analysis performed in the inventors' previous work and indicating substoichiometric amounts of c-FLIP in the DED filament with the ratio of 1 to 10 (c-FLIP to procaspase-8) (Schleich et al., 2012). This subsequently results in the decreased ratios of procaspase-8/c-FLIP.sub.L heterodimers to procaspase-8/procaspase-8 homodimers. The initial binding constants and related parametrization were based on the inventors' previous models of the DISC generated in HeLa-CD95 cells (Fricker et al., 2010).

(39) The generated model was trained and validated with experimental data for caspase-8 activity at the DISC and caspase-3/7 activity in the total cellular lysates (FIG. 6b-e). The modeling predicted that FLIPinB would stabilize p43/p10/c-FLIP.sub.L complex up to four hours after CD95L stimulation resulting in its sustained activity over this period of time (FIG. 6c). Indeed, the in silico simulations have shown that the concentration of the p43/p10/c-FLIP.sub.L complex increases from 1 to 3 nM within the first hours after addition of CD95L (FIG. 6c). This in turn results in the subsequent increase of the concentration of active caspase-8 upon addition of FLIPinB for 4 h after stimulation (FIG. 6c). This prediction was fitting well to the experimental data on caspase activation and finally provided the explanation why the effects of FLIPinB on caspase activity are only detectable in the first hours after DL stimulation.

(40) To test how the model would compute the timing of cell death upon addition of FLIPinB the simulations for the apoptosis induction with 60 ng/ml of CD95L with or without FLIPinB were performed. The model has predicted the increase in a rate of cell death within the first four hours after stimulation, which disappeared at later time points (FIG. 7a). Modeling predictions were validated by measuring cell death in HeLa-CD95 cells via live cell imaging using the IncuCyte technology as described previously (FIG. 7b). In accordance with in silico predictions FLIPinB enhanced CD95L-induced cell death in vitro in the first hours after CD95L co-treatment (FIG. 7b).

(41) Taken together, the inventors' modeling approach fully support the mechanism of FLIPinB action manifesting in the FLIPinB-mediated increase of the stability of heterodimer and thereby prolonging its catalytic life at the DED filament.

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