[go: up one dir, main page]

WO2015140313A1 - Nécroptose induisant un fragment mlkl et ses inhibiteurs - Google Patents

Nécroptose induisant un fragment mlkl et ses inhibiteurs Download PDF

Info

Publication number
WO2015140313A1
WO2015140313A1 PCT/EP2015/055975 EP2015055975W WO2015140313A1 WO 2015140313 A1 WO2015140313 A1 WO 2015140313A1 EP 2015055975 W EP2015055975 W EP 2015055975W WO 2015140313 A1 WO2015140313 A1 WO 2015140313A1
Authority
WO
WIPO (PCT)
Prior art keywords
mlkl
necroptosis
inhibitor
pip
functional fragment
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/EP2015/055975
Other languages
English (en)
Inventor
Peter Vandenabeele
Yves DONDELINGER
Mathieu Bertrand
Wim Declercq
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universiteit Gent
Vlaams Instituut voor Biotechnologie VIB
Original Assignee
Universiteit Gent
Vlaams Instituut voor Biotechnologie VIB
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.)
Filing date
Publication date
Application filed by Universiteit Gent, Vlaams Instituut voor Biotechnologie VIB filed Critical Universiteit Gent
Publication of WO2015140313A1 publication Critical patent/WO2015140313A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates to a 4-helical bundle domain fragment (4HBD) of mixed-lineage kinase domain-like (MLKL) that is necessary and sufficient to induce necroptosis in cells, by binding to phosphatidylinositol phosphates (PIPs).
  • 4HBD 4-helical bundle domain fragment
  • MLKL mixed-lineage kinase domain-like
  • PIPs phosphatidylinositol phosphates
  • necroptosis is a caspase-independent form of cell death that contributes to the pathogenesis of several human diseases, including ischemia-reperfusion injury, sepsis and viral infection (Duprez et al., 201 1 ; Linkermann et al., 2013; Mocarski et al., 2012). Understanding the molecular mechanisms underlying necroptosis is therefore a priority that may lead to the development of new therapies for these diseases. Signal transduction during necroptosis has been mostly studied in the context of TNF signaling.
  • complex I a large plasma membrane-associated complex, known as complex I, that promotes cell survival by driving activation of the canonical NF- ⁇ pathway (Vandenabeele et al., 2010b). Inhibition of the NF- ⁇ pathway at the level of complex I, for example by degradation of clAP1/2 or inhibition of TAK1 kinase activity, consequently switches the pro-survival signal to a RIPK1 kinase-dependent pro-death signal (Ofengeim and Yuan, 2013).
  • RIPK1 substrates have not been identified, it is clear that the kinase activity of RIPK1 critically regulates signal transduction from complex I to the downstream cell death pathways, either apoptotic via activation of caspase-8 or necroptotic via activation of RIPK3 and MLKL.
  • RIPK1 kinase activity regulates the formation of the necrosome, a necroptosis-inducing complex consisting of RIPK1 , RIPK3 (Cho et al., 2009; He et al., 2009; Zhang et al., 2009) and MLKL (Sun et al., 2012; Zhao et al., 2012).
  • RIPK1 and RIPK3 bind to each other via homotypic RHIM-domain interactions, which allows them to form amyloid-like fibrillar structures (Li et al., 2012).
  • MLKL is recruited to the necrosome by the binding of its kinase-like domain to the kinase domain of RIPK3 (Sun et al., 2012; Xie et al., 2013).
  • Human RIPK3 phosphorylates human MLKL on Thr357 and Ser358, which are located in the activation loop of its kinase-like domain (Sun et al., 2012). These phosphorylation events were suggested to induce a conformational change in MLKL leading to its activation, as evidenced by the fact that certain mutations in the ATP-binding pocket of the mouse kinase-like domain are sufficient to induce its auto-activation (Murphy et al., 2013).
  • Mitochondria and more precisely mitochondrial ROS production, have been extensively associated with the execution phase of necroptosis (Goossens et al., 1995; Schulze-Osthoff et al., 1992; Vandenabeele et al., 2010a). A link between MLKL and the mitochondria was also recently revealed.
  • Patent application CN 102516384 describes MLKL as target point for a cell necrosis inhibitor, but it is focused on the phosphorylation and is limited to the part of the sequence outside the 4HBD.
  • MLKL translocates to the plasma membrane upon induction of necroptosis, where it interacts with phosphatidyl inositol phosphates (PIPs) via a patch of positively charged amino acids at the surface of a 4-helical bundle domain (4HBD) located in its N-terminal region. Even more surprisingly, we found that this domain is sufficient to induce leakage of PIP-containing liposomes.
  • a first aspect of the invention is a functional fragment of MLKL, comprising at least amino acids 1 -125 of SEQ ID No. 1 (4HBD+BR of MLKL).
  • “Functional” as used here means that the fragment, when expressed in a cell, is inducing necroptosis.
  • said fragment consists of amino acids 1 -125 of SEQ ID No. 1.
  • said fragment is binding PIP.
  • the use of the functional fragment of MLKL as a medicament. Further provided is the use of the functional fragment of MLKL in the treatment of cancer. Further provided is the use of the functional fragment of MLKL in the treatment of multiple myeloma.
  • MLKL and PIP interact intracellularly
  • the intracellular delivery could be achieved by pH-sensitive drug delivery systems, use of cell-penetrating peptides or by adeno-associated virus (AAV)-based delivery system.
  • AAV adeno-associated virus
  • Another aspect of the invention is the use of a functional fragment of MLKL according to the invention to screen inhibitors of the MLKL-PIP interaction. Said screening may be done using a cellular necroptosis assay as described above. Alternatively, an in vitro assay is used using purified functional fragment and PIP whereby the binding and the disruption of the binding is directly checked.
  • Another aspect of the invention is an inhibitor of the MLKL-PIP interaction, obtainable by the use of a functional fragment of MLKL to screen inhibitors of the MLKL-PIP interaction, according to the invention.
  • said inhibitors interfere with the positively charged amino acids of the region 22-35 of SEQ ID No. 1 .
  • said inhibitor can be an antibody binding on said region, or a strongly positively charged peptide, such as but not limited to posKRas (SEQ ID No. 2).
  • the own MLKL interaction domain SEQ ID No. 3 is used as an inhibitor.
  • said inhibitor is a cell penetrating peptide (CPP) preferably a positively charged CPP.
  • CPP cell penetrating peptide
  • CPPs are known to the person skilled in the art, an overview of CPPs can be found in Lindgren and Langel (201 1 ); as a non-limiting example, the CPP can be selected from the group consisiting of TAT 47"57 (GRKKRRQRRRP; Vives et al., 1997) and ECP 32"40 (NYRWRCKNQ; Fang et al, 2013).
  • the CPP, or other inhibiting peptides can be used as such, or they can be modified to increase the stability, e.g. by pegylation, or by incorporation of D amino acids.
  • necroptosis associated diseases are known to the person skilled in the art, and include, but are not limited to ischemia-reperfusion injury, necrotizing pancreatitis, necrotizing dermatitis, inflammatory bowel disease, systemic inflammatory response syndrome, ocular disease, cancer, brain injury, multiple sclerosis and stroke.
  • RIPK3 kinase activities 293T cells were transfected with 1 ⁇ g of empty vector (EV), pLenti6-strephRIPK1 -FLAG (RIPK1/R1 ), pLenti6-strep-hRIPK3-FLAG (RIPK3/R3) or pLenti6- strep-hMLKL-FLAG (MLKL/ML). After 24 h, cell death was analyzed by Sytox Green positivity (A) or cells were lysed in 1 x laemmli buffer and lysates were immunoblotted (B).
  • EV empty vector
  • pLenti6-strephRIPK1 -FLAG RIPK1/R1
  • pLenti6-strep-hRIPK3-FLAG pLenti6- strep-hMLKL-FLAG
  • MLKL/ML pLenti6- strep-hMLKL-FLAG
  • C and E 293T cells were transfected with 1 ⁇ g of empty vector or pLenti6-strep-hMLKL-FLAG in the presence or absence of zVAD-fmk (C), nec-1 (E) or R3i (E). After 24 h, cell death was analyzed by SytoxGreen positivity and cell lysates were analyzed by immunoblotting.
  • D 293T cells were transfected with 100 ng of pLenti6-strep-hMLKL-FLAG, followed by analysis of PI positivity and morphology by time-lapse confocal microscopy.
  • HT-29 cells were pretreated 1 h with TAK1 i+zVAD in the presence or absence of nec-1 or R3i, followed by hTNF stimulation. After 20 h, cell death was analyzed by SytoxGreen positivity. Cell death data are presented as mean ⁇ SEM of three independent experiments.
  • FIG. 3 The full 4-helical bundle domain of MLKL is sufficient and absolutely required for necroptosis induction.
  • A 293T cells were transfected with 1 ⁇ g of empty vector or the indicated pLenti6-strep-hMLKL-FLAG mutants. After 24 h, cell death was analyzed by Sytox Green positivity and cell lysates were analyzed by immunoblotting.
  • B 293T cells were transfected with 50 ng of the indicated pLenti6-strep-hMLKL-EGFP mutants. The next day, GFP fluorescence was analyzed by confocal microscopy. The upper panel shows the picture before deconvolution and the lower panel after deconvolution to minimize the influence of out- of-focus light.
  • Cell death data are presented as mean ⁇ SEM of three independent experiments.
  • C Homology model of the N-terminal domain of human MLKL consisting of the 4-helical bundle domain (dark grey) and the brace region (light grey).
  • D-E 293T cells were transfected with 1 ⁇ g of empty vector or the indicated pLenti6-strep-hMLKL-FLAG mutants. After 24 h, cell death was analyzed by Sytox Green positivity and cell lysates were analyzed by immunoblotting. Cell death data are presented as mean ⁇ SEM of three independent experiments.
  • FIG. 4 The full 4-helical bundle domain of MLKL is sufficient and absolutely required for necroptosis induction.
  • A 293T cells were transfected with 100 ng of pLenti6- strephMLKL 1 -180-FLAG, followed by analysis of PI positivity and morphology by time-lapse confocal microscopy.
  • B 293T cells were transfected with 50 ng of the indicated pLenti6-strep- hMLKL-EGFP mutants. The next day, GFP fluorescence was analyzed by confocal microscopy. Only few cells were detected due to cytotoxicity of the pLenti6-strep-hMLKL- EGFP construct.
  • FIG. 5 Cell death correlates with the formation of high molecular weight MLKL oligomers that are not inhibited by NSA.
  • A 293T cells were transfected with 1 ⁇ g of empty vector or the indicated pLenti6-strep-hMLKL-FLAG mutants. After 24 h, cells were lysed in 1x laemmli buffer with 50 mM DTT (reducing) or without (non-reducing). Cell lysates were analyzed by immunoblotting.
  • B HT-29 cells were pretreated for 1 h with TAK1 i+zVAD in the presence or absence of NSA, followed by hTNF stimulation. After 20 h, cell death was analyzed by SytoxGreen positivity.
  • (C) 293T cells were transfected with 1 ⁇ g of empty vector or pLenti6-strep-hMLKL-FLAG in the presence or absence of NSA. After 24 h, cell death was analyzed by SytoxGreen positivity and cell lysates were analyzed by immunoblotting.
  • (D) 293T cells were transfected with 1 ⁇ g of empty vector or the indicated pLenti6-strep-hMLKL-FLAG mutants. After 24 h, cells were lysed in 1 x laemmli buffer with 50 mM DTT (reducing) or without (non-reducing). Cell lysates were analyzed by immunoblotting.
  • 293T cells were transfected with 1 ⁇ g of empty vector or the indicated pLenti6-strep-hMLKL-FLAG mutants in the presence or absence of NSA. After 24 h, cells were lysed in 1 x laemmli buffer with 50 mM DTT (reducing) or without (nonreducing). Cell lysates were analyzed by immunoblotting. Cell death data are presented as mean ⁇ SEM of three independent experiments. * indicates a nonspecific band.
  • FIG. 7 Positive charges in the 4-helical bundle of MLKL are required for recruitment of MLKL to the plasma membrane and induction of necroptosis.
  • MLKL green
  • B 293T cells were transfected with 1 ⁇ g of empty vector or the indicated pLenti6-strep-hMLKL-FLAG mutants. After 24 h, cell death was analyzed by Sytox Green positivity and cell lysates were analyzed by immunoblotting.
  • (C) 293T cells were transfected with 50 ng of pLenti6-strep- hMLKL-EGFP mutants. The next day, GFP fluorescence was analyzed by confocal microscopy. The upper panel shows the picture before deconvolution and the lower panel after deconvolution to minimize the influence of out-of-focus light.
  • (D) 293T cells were transfected with 1 ⁇ g of empty vector or the indicated pLenti6-strep-hMLKL-FLAG mutants. After 24 h, cells were lysed in 1 x laemmli buffer with 50 mM DTT (reducing) or without (nonreducing). Cell lysates were analyzed by immunoblotting.
  • FIG. 8 Positive charges in the 4-helical bundle of MLKL are required for recruitment of MLKL to the plasma membrane and induction of necroptosis.
  • A 293T cells were transfected with 50 ng of the indicated pLenti6-strep-hMLKL-EGFP mutants. The next day, GFP fluorescence was analyzed by confocal microscopy. Only few cells were detected due to cytotoxicity of the pLenti6-strep-hMLKL-EGFP construct.
  • B 293T cells were transfected with 1 ⁇ g of empty vector or the indicated pLenti6-strep-hMLKL-FLAG mutants. After 24 h, cell death was analyzed by Sytox Green positivity and cell lysates were analyzed by immunoblotting. Cell death data are presented as mean ⁇ SEM of three independent experiments.
  • FIG. 9 MLKL interacts with phosphatidyl inositol phosphates by positive charges in its N-terminal 4-helical bundle.
  • Recombinant GST-hMLKL 1 -210 or GST-hMLKL 1 -210 9posE were first incubated with PreScission protease to remove the GST tag and were then incubated individually with either a more general lipid strip (upper panel) or a PIP strip (lower panel). Binding was revealed by infrared fluorescence detection using the Odyssey system. The "Blank” is spotted with xylene cyanol and this interfered with detection in the red channel.
  • B-C 293T cells were transfected with 333 ng of empty vector or the indicated pLenti6-strep- hMLKL-FLAG in the presence of increasing concentrations of either PH-BTK or PH-PLC5 plasmids or the combination of both. After 24 h, cell death was analyzed by Sytox Green positivity and cell lysates were analyzed by immunoblotting. Cell death data are presented as mean ⁇ SEM of three independent experiments.
  • MLKL interacts with phosphatidyl inositol phosphates by positive charges in its N-terminal 4-helical bundle.
  • Recombinant GST, GST-hMLKL 1 -210, GST-hMLKL 1 -210 9posE, and the PH domain of PLC5, PIP(4,5)2 GRIP were incubated with a PIP strip and binding was revealed by infrared fluorescence detection using the Odyssey system.
  • FIG. 11 Interfering with the formation of PI(5)P or PI(4,5)P2 inhibits TNF-induced necrosis.
  • L929sAhFAS cells (A) and FADD-/- Jurkat cells (B) were pretreated for 30 min with the indicated compounds and subsequently stimulated by hTNF. Cell death was analyzed by
  • A-D Liposomes consisting of 95% phosphatidylcholine (PC) supplemented with either (D) 5% phosphatidylinositol (3,4,5)-triphosphate (PIP3), (C) 5% phosphatidylinositol (4,5)-diphosphate (PIP2), (B) 5% phosphatidylinositol (5)-phosphate (PIP) or (A) 5% phosphatidylinositol (PI) were incubated with 500 nM of the indicated recombinant proteins of which GST was clipped of. GST was included to control for any residual GST still present in the recombinant protein samples. After 30 min carboxyfluorescein release was measured. The data are presented as mean ⁇ SD of one representative experiment.
  • sequences encoding wild-type RIPK1 , RIPK3 and MLKL and the mutated and truncated versions of MLKL were cloned into pENTR3C using the cloneEZ PCR cloning kit (GenScript, Piscataway, NJ, USA). Next, these sequences were transferred into home-made modified pLenti6 vectors, i.e. pLenti6-FLAG-puromycin or pLenti6-EFGP-V5-BLAST destination vector, using the LR gateway recombination system (Life Technologies, Carlsbad, CA, USA).
  • the plasmid encoding a modified version of the positively charged C-terminal part of kRas has been described elsewhere (Yeung et al., 2006).
  • the plasmids containing the pleckstrin homology domains of BTK and PLC5 were a kind gift from Prof. J. Gettemans (Nanobody Lab, Department of Biochemistry, Ghent University, Ghent, Belgium).
  • 293T and L929sAhFAS cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, L-glutamine (200 mM) and sodium pyruvate (400 mM).
  • HT-29 cells were cultured in EMEM supplemented with 10% FCS and 1x MEM nonessential amino acids solution (Sigma).
  • FADD-/- Jurkat cells were cultured in RPMI media supplemented with 10% FCS, Lglutamine, sodium pyruvate, and ⁇ -mercaptoethanol.
  • Anti-FLAG Sigma-Aldrich
  • anti-FLAG-HRP Sigma- Aldrich
  • anti-actin MP Biomedicals
  • anti-caspase-3 Cell Signaling #9662
  • anti-EGFP BD Biosciences
  • Recombinant human TNF-a produced and purified to at least 99% homogeneity in our laboratory, has a specific biological activity of 3x107 lU/mg and was used at 600 lU/ml (20 ng/ml) to stimulate HT-29 cells.
  • the caspase peptide inhibitor zVAD-fmk (Bachem) was used at 20 ⁇ .
  • Necrostatin-1 and PITenin-7 were acquired from Calbiochem and were used at 10 ⁇ and 33 ⁇ , respectively.
  • the specific RIPK3 inhibitor (R3i) GSK'840 was a kind gift from GSK and was used at 10 ⁇ .
  • Necrosulfonamide (Toronto Research Chemicals) was used at 10 ⁇ .
  • the TAK1 kinase inhibitor NP-009245 (AnalytiCon Discovery GmbH), a derivative of (5Z)-7-Oxozeaenol, was used at 1 ⁇ .
  • the PIKfyve inhibitor YM201636 (Cayman Chemical) was used at 10 ⁇ .
  • 293T were seeded the day before transfection at 300 000 per well in a 6-well plate. The next day, 293T cells were transfected with 1 ⁇ g DNA by using jetPEI transfection reagent according to the manufacturer's instructions.
  • SytoxGreen (Invitrogen) was added 24 h after transfection at a final concentration of 5 ⁇ .
  • SytoxGreen intensity was measured by a Fluostar Omega fluorescence plate reader (BMG Labtech) using an excitation filter of 485 nm, an emission filter of 520 nm, gains set at 1 100, 40 flashes per well and orbital averaging with a diameter of 7 mm.
  • all cells were lysed by adding Triton x-100 at a final concentration of 0.1 % and SytoxGreen intensity was measured again.
  • L929sAhFAS, FADD-/- Jurkat cells or HT-29 cells were seeded at 10 000, 50 000 or 40 000 cells per well, respectively, in triplicates in a 96-well plate. The next day, cells were pre-treated with the indicated compounds for 30 min and then stimulated with hTNF (600 lU/ml) in the presence of 5 ⁇ SytoxGreen (Invitrogen). SytoxGreen intensity was measured at intervals of one hour by using a Fluostar Omega fluorescence plate reader, with an excitation filter of 485 nm, emission filter of 520 nm, gains set at 1 100, 20 flashes per well and orbital averaging with a diameter of 3 mm.
  • cell death was calculated by subtracting the induced SytoxGreen fluorescence from the background fluorescence and then dividing the difference by the maximal fluorescence (minus the background fluorescence) obtained by full permeabilization of the cells by using Triton x-100 at a final concentration of 0.1 %. All cell death data are presented as mean ⁇ SEM of three independent experiments.
  • 293T cells were seeded at 10 000 cells per well in an 8-well Ibiditreat ⁇ -slide from Ibidi. The next day, cells were transfected with 50 ng of the indicated pLenti6-strephMLKL-EGFP mutants using jetPEI transfection reagent according to the company's instructions. After 24 hours, cells were fixed with 4% PFA for 15 min at room temperature. Images were acquired using a Leica TCS SP5 confocal system (Leica, Mannheim, Germany) using a 63X HCX PL Apo 1 .4 oil immersion objective, with a format of 1024x1024, a line average of 4 at 400 Hz, and a zoom of 2.5.
  • Stacks were imaged at a z-step of 83.9.
  • the brightfield images were acquired with the 633 laser line, with a gain of 300.
  • the GFP fluorescence was imaged with the 488 argon laser line at 28%, bandwidth of 498-589, gain of 893.
  • Deconvolution of the GFP signal was performed on Volocity software (Perkin Elmer), and image reconstruction was performed using ImageJ.
  • Images were acquired on a format of 1024x1024, with a line average of 2, scan speed of 400 Hz, and pinhole of 2AU. Excitation for both propidium iodide and brightfield was done with the 543 laser line at 3%, with a detection ban of 553-727. Images were acquired every 15 minutes for 16 hours. Image reconstruction was performed on ImageJ software.
  • MLKL (PDB: 4BTF) using SWISS-MODEL (Arnold et al., 2006).
  • the model was evaluated by uploading the PDB coordinate file to RAMPAGE (Lovell et al., 2003) and ProSA-web server
  • the coding sequences of MLKL or the 9posE mutant were cloned into the pGEX- 6P-2 backbone by using cloneEZ. Both plasmids were transformed in E. coli strain BL21 (DE3) and exponentially growing cultures of the WT (37°C) were induced with 1 .0 mM isopropyl- ⁇ - Dthiogalactopyranoside (IPTG) and incubated for 4 h at 37°C. The cultures of the 9 posE mutant was grown at 28°C, induced with 0.5 mM IPTG and incubated overnight at 20°C.
  • Elution fractions containing GST-hMLKL were pooled and dialyzed against buffer D (50 mM Tris-HCI pH 7.0, 150 mM NaCI, 1 mM EDTA, 1 mM DTT).
  • the GST-hMLKL fusion protein was digested with the PreScission Protease (GE Healthcare) to clip off GST.
  • the digested sample was run on a glutathione sepharose 4B column (GE Healthcare) preequilibrated with buffer B for removal of the GST-tag and the PreScission Protease.
  • Flow-through fractions containing hMLKL were pooled.
  • the purified recombinant hMLKL protein was dialyzed against buffer B. The purity of the fractions was checked by SDS-PAGE.
  • PIP and lipid strips were purchased from Echelon Biosciences. Both strips were blocked overnight at 4°C in buffer A (PBS pH 7.4, 3% (w/v) fatty acid free BSA).
  • Recombinant GST- hMLKL (AA 1 -210) WT and mutant were incubated on PIP and lipid strips for 1 h at room temperature in buffer B (PBS pH 7.4, 0.1 % (v/v) Tween-20, 3% (w/v) fatty acid free BSA).
  • GST-hMLKL proteins were revealed with goat anti-GST antibody (GE Healthcare) or rabbit anti-MLKL antibody (Sigma) in buffer B and visualized by infrared fluorescence detection using the Odyssey system (Li-Cor Biosciences).
  • Carboxyfluorescein (CF)-containing liposomes were prepared as described before (Antonsson et al., 1997), but with a modified lipid composition. Briefly, 1 mg lipid containing 95% (mol %) phosphatidylcholine and 5% (mol %) phosphatidylinositol, phosphatidyl (5)-phosphate, phosphatidyl (4,5)-diphosphate or phosphatidylinositol (3,4,5)-triphosphate were dried under nitrogen and solubilized in 1 ml PBS, pH 7.4, containing 20 mM CF (purity > 99%) and 30 mg of octyl glucoside/ml.
  • MLKL is the most downstream protein in the RIPK1 -RIPK3-MLKL axis and it makes this cellular system ideal for studying necroptotic signaling downstream of MLKL in detail.
  • Example 2 The full 4-helical bundle domain is sufficient and absolutely required for necroptosis induction
  • MLKL consists of an N-terminal 4-helical bundle domain (4HBD) fused by a brace region (BR) to a C-terminal inactive kinase-like domain (KLD) (Murphy et al., 2013). It had been shown that RIPK3 binds MLKL by kinase-kinase-like domain interactions, and that the subsequent phosphorylation of MLKL within its activation loop by RIPK3 is required for necroptosis induction (Murphy et al., 2013; Sun et al., 2012; Xie et al., 2013).
  • MLKL and MLKL-containing complexes have been reported in various subcellular compartments, such as the cytosol (Sun et al., 2012), in association with the mitochondria (Wang et al., 2012), in the mitochondria-associated membrane fraction (Chen et al., 2013), and very recently in the plasma membrane (Cai et al., 2013; Chen et al., 2014).
  • cytosol Un et al., 2012
  • mitochondria-associated membrane fraction Choen et al., 2013
  • Cai et al., 2013; Chen et al., 2014 we fused full length MLKL, N-terminal 4HBD+BR and C-terminal KLD to GFP and analyzed the subcellular localization of the different fusion proteins by confocal microscopy.
  • Example 3 Cell death correlates with the formation of high molecular weight MLKL oligomers that are not inhibited by NSA
  • HMW oligomers Figure 6A
  • MLKL is able to form HMW oligomers, which is in contrast to the recently reported MLKL trimers or tetramers (Cai et al., 2013; Chen et al., 2014).
  • Our results also highlight a clear correlation between the ability to form HMW oligomers and necroptosis induction. Moreover, we found that the formation of these oligomers is possible only when the full 4HBD is present.
  • Necrosulfonamide is a recently discovered potent inhibitor of necroptosis that covalently binds to cysteine 86 in a loop between helices a3 and a4 in the 4HBD of human MLKL (Sun et al., 2012). As the full 4HBD of MLKL is required and sufficient for the formation of HMW oligomers and the induction of necroptosis, we evaluated the effect of NSA on both MLK- induced oligomerization and necroptosis. We pretreated 293T cells with NSA before transiently overexpressing MLKL.
  • Example 4 Positive charges in the 4-helical bundle of MLKL are required for recruitment of MLKL to the plasma membrane and induction of necroptosis
  • MLKL directly binds to plasma membrane components to mediate its cytotoxicity.
  • Protein-membrane interactions can be mediated by a broad spectrum of protein domains, including C1 , C2, PH, FYVE, PX, ENTH, ANTH, BAR and FERM domains (Cho and Stahelin, 2005).
  • a universal theme is that protein-membrane interaction is regulated by electrostatic interactions between the negatively charged phospholipids of the plasma membrane and the positively charged amino acids in a domain of the membrane-binding protein. Because the 4HBD is sufficient to induce necroptosis after ectopic expression, we investigated whether the 4HBD contains a patch rich in positively charged amino acids.
  • Example 5 MLKL interacts with phosphatidyl inositol phosphates by positive charges in its N-terminal 4-helical bundle
  • PH domains have different specificities for distinct phosphatidyl inositol phosphates.
  • the PH domain of PLC5 mainly binds to PI(4,5)P2 ( Figure 10A; PI(4,5)P2 GRIP), whereas the PH domain of BTK has a specificity towards PI(3,4,5)P3 (Garcia et al., 1995; Rameh et al., 1997; Salim et al., 1996).
  • Example 7 The interaction between MLKL and PIPs permeabilizes liposomes
  • the 4HBD of MLKL is structurally similar to a-pore forming toxins. These bacterial toxins consist of helical bundle domains that can oligomerize into cytolytic pores in the plasma membrane (Parker and Feil, 2005). As translocation of MLKL to the plasma membrane is required for its function and oligomerization correlates with its killing potential, we investigated whether MLKL itself has pore forming capacities. To do so, we incubated the recombinant N- terminal domain of MLKL (4HBD + BR) with phosphatidylcholine liposomes containing 5% PI, PI(5)P, PI(4,5)P2 or PI(3,4,5)P3 ( Figure 1 1A-D).
  • SWISS-MODEL workspace a webbased environment for protein structure homology modelling. Bioinformatics 22, 195- 201 .
  • Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nature cell biology 16, 55-65.
  • RIPK3 contributes to TNFR1 -mediated RIPK1 kinase dependent apoptosis in conditions of clAP1/2 depletion or TAK1 kinase inhibition. Cell death and differentiation 20, 1381 -1392.
  • Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.
  • Pore-forming protein toxins from structure to function.
  • the mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell 148, 228-243.
  • RIP3 an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325, 332-336. Zhao, J., Jitkaew, S., Cai, Z., Choksi, S., Li, Q., Luo, J., and Liu, Z.G. (2012).
  • Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF- induced necrosis. Proceedings of the National Academy of Sciences of the United States of America 109, 5322-5327.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un fragment de domaine de faisceau à 4 hélices (4HBD) de type domaine de la kinase de lignage mixte (MLKL) qui est nécessaire et suffisant pour induire une nécroptose dans des cellules, par liaison aux phosphatidylinositol phosphates (PIP). L'invention concerne en outre l'utilisation de ce domaine pour cribler des inhibiteurs de la liaison MLKL-PIP, et l'utilisation de ces inhibiteurs pour empêcher une nécroptose et pour traiter des maladies associées à une nécroptose.
PCT/EP2015/055975 2014-03-20 2015-03-20 Nécroptose induisant un fragment mlkl et ses inhibiteurs Ceased WO2015140313A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP14160920 2014-03-20
EP14160920.6 2014-03-20

Publications (1)

Publication Number Publication Date
WO2015140313A1 true WO2015140313A1 (fr) 2015-09-24

Family

ID=50289591

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2015/055975 Ceased WO2015140313A1 (fr) 2014-03-20 2015-03-20 Nécroptose induisant un fragment mlkl et ses inhibiteurs

Country Status (1)

Country Link
WO (1) WO2015140313A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019048639A1 (fr) * 2017-09-07 2019-03-14 Vib Vzw Protéine de type domaine kinase à lignée mixte dans le traitement immunothérapeutique du cancer
IL279559A (en) * 2020-12-17 2022-07-01 Yeda Res & Dev Controlling ubiquitination of mlkl for treatment of disease

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010022444A1 (fr) * 2008-08-25 2010-03-04 The Walter And Eliza Hall Institute Of Medical Research Procédés et agents pour moduler des voies de signalisation de kinase par modulation de protéine de type domaine kinase de lignage mixte (mlkl)
EP2243828A1 (fr) * 2009-04-24 2010-10-27 DKFZ Deutsches Krebsforschungszentrum Utilisation de polypeptides "mixed lineage kinase like" (polypeptides MLKL) pour le traitement contre le cancer
US20110097303A1 (en) * 2009-10-27 2011-04-28 Michael Zasloff Methods and compositions for treating and preventing viral infections

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010022444A1 (fr) * 2008-08-25 2010-03-04 The Walter And Eliza Hall Institute Of Medical Research Procédés et agents pour moduler des voies de signalisation de kinase par modulation de protéine de type domaine kinase de lignage mixte (mlkl)
EP2243828A1 (fr) * 2009-04-24 2010-10-27 DKFZ Deutsches Krebsforschungszentrum Utilisation de polypeptides "mixed lineage kinase like" (polypeptides MLKL) pour le traitement contre le cancer
US20110097303A1 (en) * 2009-10-27 2011-04-28 Michael Zasloff Methods and compositions for treating and preventing viral infections

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JAMES M. MURPHY ET AL: "The Pseudokinase MLKL Mediates Necroptosis via a Molecular Switch Mechanism", IMMUNITY, vol. 39, no. 3, 1 September 2013 (2013-09-01), pages 443 - 453, XP055188506, ISSN: 1074-7613, DOI: 10.1016/j.immuni.2013.06.018 *
Q REMIJSEN ET AL: "Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis", CELL DEATH AND DISEASE, vol. 5, no. 1, 16 January 2014 (2014-01-16), pages e1004, XP055189354, DOI: 10.1038/cddis.2013.531 *
WANG HUAYI ET AL: "Mixed Lineage Kinase Domain-like Protein MLKL Causes Necrotic Membrane Disruption upon Phosphorylation by RIP3", MOLECULAR CELL, vol. 54, no. 1, 10 April 2014 (2014-04-10), pages 133 - 146, XP028847296, ISSN: 1097-2765, DOI: 10.1016/J.MOLCEL.2014.03.003 *
YVES DONDELINGER ET AL: "MLKL Compromises Plasma Membrane Integrity by Binding to Phosphatidylinositol Phosphates", CELL REPORTS, vol. 7, no. 4, 1 May 2014 (2014-05-01), pages 971 - 981, XP055189352, ISSN: 2211-1247, DOI: 10.1016/j.celrep.2014.04.026 *
ZHENYU CAI ET AL: "Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis", NATURE CELL BIOLOGY, vol. 16, no. 1, 8 December 2013 (2013-12-08), pages 55 - 65, XP055188505, ISSN: 1465-7392, DOI: 10.1038/ncb2883 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019048639A1 (fr) * 2017-09-07 2019-03-14 Vib Vzw Protéine de type domaine kinase à lignée mixte dans le traitement immunothérapeutique du cancer
CN111655278A (zh) * 2017-09-07 2020-09-11 维伯Vzw公司 免疫治疗性癌症控制中的混合谱系激酶结构域样蛋白
IL279559A (en) * 2020-12-17 2022-07-01 Yeda Res & Dev Controlling ubiquitination of mlkl for treatment of disease

Similar Documents

Publication Publication Date Title
Dondelinger et al. MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates
Peraro et al. Diversity-oriented stapling yields intrinsically cell-penetrant inducers of autophagy
Weber et al. Recent insights into the regulatory networks of NLRP3 inflammasome activation
Gerke et al. Annexins—a family of proteins with distinctive tastes for cell signaling and membrane dynamics
Teasdale et al. Insights into the PX (phox-homology) domain and SNX (sorting nexin) protein families: structures, functions and roles in disease
Vafiadaki et al. Phospholamban interacts with HAX-1, a mitochondrial protein with anti-apoptotic function
Atkins et al. The role of Drp1 adaptor proteins MiD49 and MiD51 in mitochondrial fission: implications for human disease
Zhou et al. Targeting Hippo pathway by specific interruption of YAP‐TEAD interaction using cyclic YAP‐like peptides
Wang et al. Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display
Dietrich et al. TRPC6: physiological function and pathophysiological relevance
Du et al. The tight junction protein, occludin, regulates the directional migration of epithelial cells
d'Angelo et al. Interaction of ezrin with the novel guanine nucleotide exchange factor PLEKHG6 promotes RhoG-dependent apical cytoskeleton rearrangements in epithelial cells
Chen et al. Striatins contain a noncanonical coiled coil that binds protein phosphatase 2A A subunit to form a 2: 2 heterotetrameric core of striatin-interacting phosphatase and kinase (STRIPAK) complex
EP3065759B1 (fr) Agents immunosuppresseurs et leur utilisation en thérapie
Silver et al. Apical polarity proteins recruit the RhoGEF Cysts to promote junctional myosin assembly
Senatore et al. The TBC1D31/praja2 complex controls primary ciliogenesis through PKA‐directed OFD1 ubiquitylation
Ivanova et al. The trans-membrane domain of Bcl-2α, but not its hydrophobic cleft, is a critical determinant for efficient IP3 receptor inhibition
Poissonnier et al. Disrupting the CD95–PLCγ1 interaction prevents Th17-driven inflammation
Qian et al. UVRAG cooperates with cargo receptors to assemble the ER‐phagy site
Swanson et al. The Skap-hom dimerization and PH domains comprise a 3′-phosphoinositide-gated molecular switch
Chandrakumar et al. Tankyrase regulates epithelial lumen formation via suppression of Rab11 GEFs
Feldman et al. Peptide inhibition of the SETD6 methyltransferase catalytic activity
WO2015140313A1 (fr) Nécroptose induisant un fragment mlkl et ses inhibiteurs
Lall et al. Structural and functional analysis of FIP2 binding to the endosome-localised Rab25 GTPase
Huang et al. The NTE domain of PTENα/β promotes cancer progression by interacting with WDR5 via its SSSRRSS motif

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15711184

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15711184

Country of ref document: EP

Kind code of ref document: A1