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WO2025051878A1 - Traitement de maladies inflammatoires - Google Patents

Traitement de maladies inflammatoires Download PDF

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WO2025051878A1
WO2025051878A1 PCT/EP2024/074863 EP2024074863W WO2025051878A1 WO 2025051878 A1 WO2025051878 A1 WO 2025051878A1 EP 2024074863 W EP2024074863 W EP 2024074863W WO 2025051878 A1 WO2025051878 A1 WO 2025051878A1
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plk1
nlrp3
activity
expression
test compound
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Xuan Li
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Cambridge Enterprise Ltd
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5355—Non-condensed oxazines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)
    • 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)
    • 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
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to methods of treating inflammatory diseases and diseases characterised by enhanced level of pro-inflammatory cytokines IL-10 and/or IL-18 caused by excessive NLR family Pyrin Domain Containing 3 (NLRP3) Inflammasome activation.
  • inflammatory diseases and diseases characterised by enhanced level of pro-inflammatory cytokines IL-10 and/or IL-18 caused by excessive NLR family Pyrin Domain Containing 3 (NLRP3) Inflammasome activation.
  • NLR family Pyrin Domain Containing 3 (NLRP3) inflammasome activation is linked with pathogenesis of various disorders including gout, atherosclerosis, cardiomyopathy, atrial fibrillation, Kawasaki disease, diabetes, Alzheimer’s disease, cancer, infection including COVID-19, and others (Martinon, F. et al. (2006); Strowig, T. et al (2012); Duewell, P. et al. (2010); Bracey, N. et al. (2013); Anzai, F. et al. (2020); Heneka, M.T. et al. (2013); Jourdan, T. et al. (2013); Karan, D. et al.
  • NLRP3 inflammasome activation is tightly operated by Toll-like receptor (TLR) and NOD-like receptor (NLR) pathways to co-ordinate the final output.
  • TLR Toll-like receptor
  • NLR NOD-like receptor
  • Activation of the TLRs primes the NLRP3 pathway to enhance the basal level expression of NLRP3 and IL-10 at the transcriptional level (He, Y. et al (2016).
  • the cells are then ready to receive various stimuli to activate NLRP3 inflammasome to form the multiprotein complex, including NLRP3 itself, ASC adaptor, and Caspase-1 (Latz, E. et al (2013)).
  • the complex is formed with NLRP3 connected to Caspase-1 through an adaptor protein named apoptosis- associated Speck-like protein with a Caspase-recruitment domain (ASC, encoded by Pycard).
  • ASC Caspase-recruitment domain
  • Activation of inflammasome pathways triggers Caspase-1 dependent processing of immature interleukin 10 (pro-IL-10) and interleukin 18 (pro-IL-18) into their bioactive counterparts.
  • pro-IL-10 immature interleukin 10
  • pro-IL-18 interleukin 18
  • TGN trans-Golgi network
  • NLRP3 inflammasome a macromolecular structure in the cell responsible for sensing danger and triggering an inflammatory response, is linked to the pathogenesis of heart failure following myocardial infarction (Toldo, S. et al Nature Reviews Cardiology (2016)).
  • IHD Ischemic Heart Disease
  • Ml Myocardial infarction
  • CHF chronic heart failure
  • Compromised cardiac function after Ml leads to CHF with systemic health complications, a major impact on quality of life with an annual mortality rate over 25% (Murray et al., 2013).
  • PLK1 Polo-like kinase 1
  • NLRP3 Pyrin Domain Containing 3
  • IL-10 enhanced inflammatory cytokines
  • Agents that reduce the expression or activity of PLK1 may therefore be useful in the treatment of inflammatory disease, for example by suppressing inflammatory responses, including inflammatory diseases regulated by the NLRP3 inflammasome.
  • a first aspect of the invention provides a method of treating inflammation comprising administering an agent that reduces the expression and/or activity of PLK1 to an individual in need thereof.
  • an inflammatory disease treated by a method of the first aspect may be characterised by excessive or aberrant activation of the NLR family Pyrin Domain Containing 3 (NLRP3) inflammasome.
  • NLRP3 Pyrin Domain Containing 3
  • a second aspect of the invention provides a method of treating a disease characterised by excessive or aberrant activation of the NLR family Pyrin Domain Containing 3 (NLRP3) inflammasome, the method comprising administering an agent that reduces the expression and/or activity of PLK1 to an individual in need thereof.
  • NLRP3 Pyrin Domain Containing 3
  • inflammation and diseases characterised by excessive or aberrant NLRP3 inflammasome activation may result in enhanced levels of pro-inflammatory cytokines, such as IL-1 and/or IL-18.
  • diseases characterised by excessive NLRP3 inflammasome activation may include cardiac dysfunction.
  • a third aspect of the invention provides a method of treating cardiac dysfunction, the method comprising administering an agent that reduces the expression and/or activity of PLK1 to an individual in need thereof.
  • a fourth aspect of the invention provides an agent that reduces the expression and/or activity of PLK1 for use in a method of the first, second or third aspects.
  • a fifth aspect of the invention provides the use of an agent that reduces the expression and/or activity of PLK1 in the manufacture of a medicament for use in a method of the first, second or third aspects.
  • a sixth aspect of the invention provides a method of screening for a compound for the treatment of inflammation, or a disease characterised by excessive or aberrant NLRP3 inflammasome activation comprising determining the effect of a test compound on the expression, amount or activity of PLK1 in a cell.
  • a compound that reduces the expression or activity of PLK1 may be a candidate compound for the treatment of inflammation or a disease characterised by excessive or aberrant NLRP3 inflammasome activation.
  • a seventh aspect of the invention provides a method of screening for a compound for the treatment of cardiac dysfunction comprising determining the effect of a test compound on the expression, amount or activity of PLK1 in a cell.
  • a compound that reduces the expression or activity of PLK1 may be a candidate compound for the treatment of cardiac dysfunction.
  • Figure 1 shows an unbiased BiolD screen of PLK1 interactome upon NLRP3 inflammasome activation that reveals a proximal association of PLK1 with NLRP3.
  • A Schematic representation of the bio-engineered plasmid expressing the biotin ligase BASU connected to murine PLK1 with a (GGGS)3 linker.
  • B Schematic representation of the biotinylated proteins associated with PLK1 and BASU in this assay.
  • C Transduced iBMDM were treated with biotin (50pM, 2 hours), and cell lysates were run on western blot to show biotinylated proteins stained by streptavidin-HRP.
  • iBMDM cells transduced with BASU-GS3-PLK1 were treated for inflammasome activation and biotin labelling as indicated. Cells were lysed and biotinylated proteins were purified using magnetic beads. Trypsination was followed by peptide quantification, and 5pg of peptides per sample were submitted for TMT labelling and Mass Spectrometry analysis.
  • E Volcano plot for interactome with PLK1 after NLRP3 inflammasome activation (activated), compared to the interactome with PLK1 under primed condition (primed).
  • Red dots represent the enhanced protein interaction in the Activated group
  • blue dots represent the enhanced protein interaction in the Primed group
  • grey dots are non-significant with the selected threshold cut-off (the cut-off threshold for log2FoldChange is 0.4, equal to complete 1 .3fold change; the significance adjusted P value is less than 0.05, by Benjamini-Hochberg correction).
  • F Gene Ontology analysis shows the up (red)/down (blue) - regulated interacting proteins with PLK1 after NLRP3 inflammasome activation in protein subgroups with corresponding numbers.
  • FIG. 2 shows PLK1 interacts with NLRP3 (A-C)
  • A-C NLRP3
  • Reconstituted HEK 293T cells were used to perform coimmunoprecipitations to determine association between PLK1 and NLRP3, using full-length proteins (A), NLRP3 domains (Pyrin domain: PYD; Pyrin domain deletion: A-PYD; Leucine-rich repeat: LRR; LRR deletion: A-LRR) with full-length PLK1 (B), or PLK1 domains (kinase domain: KD; kinase domain deletion: A- KD; Polo-Box domain 2 deletion: A-PBD2) with full-length NLRP3 (C). Domain structures of NLRP3 or PLK1 are shown (B, C).
  • Figure 3 shows PLK1 regulates microtubule nucleation and affects NLRP3 inflammasome positioning.
  • A, B Rosa CreErt2/wt Plk1 fl/fl BMDMs were treated with 4-OH Tamoxifen (0.002mg/mL 4-OH Tam, 24 hours) before and during priming (100ng/mL LPS, 5 hours), and then were activated (5mM ATP, 30 minutes).
  • FIG. 4 shows that PLK1 inhibition suppresses inflammatory response in LPS-induced endotoxemia model.
  • C57BL/6 wild-type (WT) and NLRP3 knock-out (KO) mice were treated with BI6727 (5mg/kg, i.p.) or control vehicle, followed by LPS administration (20mg/kg, i.p.).
  • BI6727 5mg/kg, i.p.
  • control vehicle i.p.
  • LPS administration 20mg/kg, i.p.
  • D, E Representative histopathological images from lung tissues and quantification of lung parenchymal area (D), and representative immunofluorescence images of Gr1 positive cell staining in lung tissue and the quantification (E).
  • F, G Representative histopathological images of the liver and the quantitative results of immune cell infiltration (F), and representative immunofluorescence images of Gr1 positive cells in the liver and the quantitative results (G).
  • FIG. 5 shows that PLK1 kinase inhibition suppresses inflammatory response in MSU-induced peritonitis model.
  • C57BL/6 wild-type mice and NLRP3 knock-out (KO) mice were treated with BI6727 (1 mg/kg, i.v.) or control vehicle, followed by MSU (0.5mg/mouse, i.p.).
  • A Experimental scheme. Samples for cytokine measurement and flow cytometry were collected at the indicated time points.
  • B, C IL1 p
  • C TNFa
  • n 6/group.
  • D-H Cells collected from the peritoneal cavity were analyzed by flow cytometry.
  • Non-parametric test was used to analyze the data of (B).
  • Unpaired t-test was used to analyze the data of (C).
  • Two-way ANOVA with Sidak post hoc test was used for statistical analysis (D-H). All data are mean ⁇ SEM.
  • Figure 6 shows the effect of PLK1 inhibitor BI6727 on cardiac function.
  • Figure 6A shows serum cardiac troponin I (cTropI) expression from blood day 1 after surgery.
  • Figure 6B shows ejection fraction and
  • Figure 6C shows fractional shortening measured from echocardiology images at baseline (pre-surgery) and 24h post-surgery.
  • Figure 6D-F show (D) Pulse, (E) Systolic, and (F) Diastolic blood pressure readings taken at baseline (pre-surgery) and 3 days post-surgery using an automated tail-cuff.
  • Figure 6G shows Ejection fraction over 28 days. ANOVA test or unpaired t test were used for statistical analysis, where p ⁇ 0.05 is considered significant. Lines and bars represent mean ⁇ SD. *p ⁇ 0.05.
  • Figure 7 shows the effect of Plk1 inhibitor BI6727 on serum inflammatory biomarkers following myocardial infarction.
  • Serum expression of (7 A) IL-10, (7B) IL-5, (7C) IL-6, (7D) KCGRO, and (7E) TNFa measured 3 days following surgery using V-PLEX Mouse Proinflammatory Panel 1.
  • Figure 8 shows the effect of Plk1 inhibitor BI6727 on infarct scar size 28d following myocardial infarction.
  • Figure 8A shows a representative Masson’s staining sham(left), Ml+control (centre), MI+BI6727(right),
  • Figure 8B shows scar area (%) left ventricular area,
  • Figure 8C shows scar area (%) left ventricular midline area. Treatments were compared using unpaired t-test, where p ⁇ 0.05 is considered significant. Lines represent mean ⁇ SD.
  • FIG. 9 shows Plk1 inhibitor BI6727 has no acute effect on cardiac function in the absence of myocardial infarction. Treatments were compared using unpaired t-test, where p ⁇ 0.05 is considered significant. Lines represent mean ⁇ SD.
  • Figure 10 shows the effect of BI6727 on immune cell recruitment to the heart 3 days post-MI .
  • Surface marker expression determined on day 3 post-MI by flow cytometry (10A) CD45+ cells, (10B) Neutrophils (CD11b+, Ly6G high), (10C) Macrophages (CD11 b+, F480+), (10D) CD4+ T cells (CD3+ CD4+), (10E) CD8+ T Cells (CD3+, CD8+), (10F) B220+ B Cells.
  • Data are representative of the frequency of single cells. Data were statistically analysed using Kruskal-Wallis non-parametric testing, where p ⁇ 0.05 is considered significant, and presented as Mean ⁇ SD.
  • Figure 11 shows the effect of PLK1 inhibition on cytokines in LPS-induced endotoxemia model.
  • C57BL/6 wild-type (WT) and NLRP3 knock-out (KO) mice were treated with BI6727 (5mg/kg, i.p.) or control vehicle, followed by LPS administration (20mg/kg, i.p.).
  • Samples for cytokine measurement were collected at 3 hours after LPS administration.
  • IL6 and IL12 were undetectable in NLRP3 KO samples
  • B Serum samples were collected, and multiple cytokine levels were measured.
  • This invention relates to the finding that Polo-like kinase 1 (PLK1) mediates inflammatory responses and regulates the activation of the NLR family Pyrin Domain Containing 3 (NLRP3) inflammasome. Inhibition of PLK1 may therefore be useful in treating inflammation and inflammatory diseases including those characterised by excessive NLRP3 activation. Inhibition of PLK1 is also shown herein to improve cardiac function. This may be useful in the treatment of cardiac dysfunction in a patient with cardiac inflammation, for example a patient following myocardial infarction (Ml) or other ischaemic heart disease.
  • Ml myocardial infarction
  • PLK1 activity may be reduced in a patient by administering a compound that reduces the expression or activity of Polo-like kinase 1 (PLK1).
  • PLK1 Polo-like kinase 1
  • Polo-like kinase 1 is a Ser/Thr protein kinase that is highly expressed during mitosis and is involved in recruiting y-tubulin and pericentriolar matrix proteins to regulate centrosome maturation.
  • PLK1 (Gene ID: 5347) may be human PLK1 and may have the reference amino acid sequence of NCBI database entry NP_005021 .2.
  • PLK1 may be encoded by the reference nucleotide sequence of NCBI database entry NP_005030.6.
  • PLK1 has a kinase domain at residues 45 to 309 and two polo-box domains (PBDs) at residues 407 to 494 and 509 to 590 of the reference sequence NP_005021 .2.
  • Reducing PLK1 activity as described herein may cause PLK1 to be completely inactivated (i.e. PLK1 activity may be reduced to zero or substantially zero), or reduced by 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 95% or more in target cells relative to cells in which PLK1 is not reduced.
  • PLK1 activity may be completely inactivated (i.e. PLK1 activity may be reduced to zero or substantially zero), or reduced by 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 95% or more in target cells relative to cells in which PLK1 is not reduced.
  • Suitable assays for measuring PLK1 inhibition are known in the art.
  • reductions in PLK1 activity in the setting of NLRP3 inflammasome activation may be determined by reductions in IL-10 and/or IL-18 or their downstream cytokines such as IL-6 and/or I FNy, reduction in cell death as measured by lactate dehydrogenase (LDH) activity, reduction in high sensitivity C-reactive protein (hsCRP), reduced levels of ASC speck formation, and/or reduced levels of interaction of PLK1 with NLRP3.
  • LDH lactate dehydrogenase
  • hsCRP high sensitivity C-reactive protein
  • ASC speck formation reduced levels of ASC speck formation
  • Suitable agents for reducing PLK1 activity may include PLK1 inhibitors.
  • PLK1 inhibitors may, for example, include small chemical molecules, for example non-polymeric organic compounds having a molecular weight of 900 Daltons or less.
  • Suitable PLK1 inhibitors are well known in the art and include volasertib (BI6727; Pubchem CID 10461408 ; IUPAC; N-((1S,4S)-4-(4-(cyclopropylmethyl)piperazin-1-yl)cyclohexyl)-4-(((R)-7-ethyl-8-isopropyl-5-methyl-6- oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzamide); rigosertib (ON01910; Pubchem CID 6918736; 2-[2-methoxy-5-[[(E)-2-(2,4,6-trimethoxyphenyl)ethenyl]sulfonylmethyl]anilino]acetic acid); onvansertib (Pubchem CID 49792852; 1-(2-hydroxyethyl)-8-[5-(4-methylpiperazin-1
  • PLK1 inhibitors are disclosed in Hu et al PLoS One. 2013; 8(10): e78832; Vitour J Biol Chem. (2009) 284(33): 21797-21809; US8003785, US7718801 , and US7517873.
  • the PLK1 inhibitor is volasertib.
  • PLK1 inhibitor as used herein, cover pharmaceutically acceptable salts and solvates of these compounds.
  • suitable agents for reducing PLK1 expression or activity may also include heterobifunctional molecules, such as proteolysis targeting chimeras (PROTACs), which induce selective intracellular proteolysis of PLK1 .
  • These agents may reduce PLK1 activity in a cell by increasing intracellular degradation of PLK1 protein and thereby reducing the amount of active PLK1 protein that is present in the cell.
  • heterobifunctional molecules, such as PROTACs to reduce the amount of target protein in a cell is well known in the art (see for example Cermakova K et al (2016) Molecules. 23 (8): 1958; Bondeson, D et al (2017) Ann Rev Pharmacol Toxicol. 57: 107-123; Chi KR (2016). Nature Reviews. Drug Discovery.
  • a suitable heterobifunctional molecule may consist of a binding moiety for an E3 ubiquitin ligase and a binding moiety for PLK1 , the two binding moieties being connected by a linker. Binding of the heterobifunctional molecule in a cell results in the formation of an intracellular ternary complex comprising PLK1 and E3 ligase. PLK1 is ubiquitinylated by the E3 ligase in the complex and the ubiquitinylated PLK1 is subsequently recognised by the proteasome of the cell and degraded. Techniques for the generation of PROTACs are well known in the art.
  • an agent that reduces PLK1 activity may be a suppressor nucleic acid, or a nucleic acid encoding a suppressor agent.
  • Nucleic acid encoding a suppressor nucleic acid may be contained in a vector.
  • Suitable expression vectors are well-known in the art and include viral vectors, such as retroviral, adenoviral, adeno-associated viral (AAV), lentiviral, vaccinia or herpes vectors.
  • a suitable vector containing a nucleic acid encoding a suppressor nucleic acid may target a tissue affected by an inflammatory disorder. For example a vector may selectively transduce cells of the affected tissue relative to cells of other tissue.
  • Suitable vectors include tissue-specific AAV vectors.
  • the expression of active PLK1 protein may be reduced by a suppressor nucleic acid compared to a control cell or may be absent i.e. the transcription of the PLK1 gene and/or translation of PLK1 mRNA may be reduced or absent, such that the cell treated with the suppressor nucleic acids lacks or has a reduced amount of active PLK1 protein compared to a control cell. Reducing the amount of active PLK1 protein to 20% of the amount in control cells or lower is shown to be sufficient to induce cell death. For example, a cell may express up to 5%, up to 10%, up to 15% or up to 20%, of the active PLK1 polypeptide that is expressed by control cells.
  • nucleic acid suppression may be used to reduce the expression of active PLK1 polypeptide.
  • nucleic acid suppression techniques such as anti-sense and RNAi suppression, to down-regulate expression of target genes is well-established in the art.
  • Cells may be transfected with a suppressor nucleic acid (i.e. a nucleic acid molecule which suppresses PLK1 expression), such as a siRNA or shRNA, or a heterologous nucleic acid encoding the suppressor nucleic acid.
  • a suppressor nucleic acid i.e. a nucleic acid molecule which suppresses PLK1 expression
  • siRNA or shRNA a nucleic acid molecule which suppresses PLK1 expression
  • heterologous nucleic acid encoding the suppressor nucleic acid.
  • the suppressor nucleic acid reduces the expression of active PLK1 polypeptide by interfering with transcription and/or translation, thereby reducing PLK1 activity in the cells.
  • RNAi involves the expression or introduction into a cell of an RNA molecule which comprises a sequence which is identical or highly similar to the PLK1 coding sequence.
  • the RNA molecule interacts with mRNA which is transcribed from the PLK1 gene, resulting in the sequence specific degradation or specific post- transcriptional gene silencing (PTGS) of the mRNA.
  • PTGS post- transcriptional gene silencing
  • RNA molecule is preferably double stranded RNA (dsRNA) (Fire A. et al Nature 391 , (1998)). Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologous genes in a wide range of mammalian cell lines (Elbashir SM. et al. Nature, 411 , 494-498, (2001)).
  • RNA molecules for use in RNAi suppression include short interfering RNA (siRNA).
  • siRNA are double stranded RNA molecules of 15 to 40 nucleotides in length, preferably 15 to 28 nucleotides or 19 to 25 nucleotides in length, for example 19, 20, 21 , 22, 23, 24 or 25 nucleotides in length.
  • two unmodified 21 mer oligonucleotides may be annealed together to form a siRNA.
  • a siRNA molecule may contain a 3' and/or 5' overhang on each strand having a length of about 0, 1 , 2, 3, 4, or 5 nucleotides. The overhang lengths of the strands are independent, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand.
  • RNA molecules for use in RNAi include small hairpin RNAs (shRNAs).
  • shRNA are singlechain RNA molecules which comprise or consist of a short (e.g. 19 to 25 nucleotides) antisense nucleotide sequence, followed by a nucleotide loop of 5 to 9 nucleotides, and the complementary sense nucleotide sequence (e.g. 19 to 25 nucleotides).
  • the sense sequence may precede the nucleotide loop structure and the antisense sequence may follow.
  • the nucleotide loop forms a hairpin turn which allows the base pairing of the complementary sense and antisense sequences to form the shRNA.
  • a suppressor nucleic acid such as a siRNA or shRNA, may comprise or consist of a sequence which is identical or substantially identical (i.e. at least 90%, at least 95% or at least 98% identical) to all or part (for example, 15 to 40 nucleotides) of a reference PLK1 nucleotide coding sequence.
  • Suitable reference sequences coding PLK1 that may be used for the design of suppressor nucleic acids are publically available and include the NCBI database entries set out above.
  • PLK1 activity is suppressed in cells by downregulation of the production of active PLK1 polypeptide by the suppressor nucleic acid.
  • a siRNA to suppress the expression of human PLK1 may comprise 18 to 22 contiguous nucleotides from the reference sequence.
  • Suppressor nucleic acids such as siRNAs and shRNAs, for reducing PLK1 expression may be readily designed using reference PLK1 coding sequences and software tools which are widely available in the art and may be produced using routine techniques.
  • a suppressor nucleic acid may be chemically synthesized; produced recombinantly in vitro or cells (Elbashir, S. M. et al., Nature 411 :494-498 (2001); Elbashir, S. M., et al., Genes & Development 15:188-200 (2001)) or obtained from commercial sources (e.g. Cruachem (Glasgow, UK), Dharmacon Research (Lafayette, Colo., USA)).
  • two or more suppressor nucleic acids may be used to suppress the expression of PLK1.
  • a pool of siRNAs may be employed. Suitable siRNAs and siRNA pools may be produced using standard techniques.
  • Nucleic acid suppression may also be carried out using anti-sense techniques.
  • Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of the base excision repair pathway component so that its expression is reduced or completely or substantially completely prevented.
  • antisense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the anti-sense oligonucleotides can interfere with expression control sequences.
  • the construction of anti-sense sequences and their use is well known in the art (Peyman and Ulman, Chemical Reviews, 90:543-584, (1990); Crooke, Ann. Rev. Pharmacol. Toxicol. 32:329-376, (1992)).
  • Anti-sense oligonucleotides may be generated in vitro or ex vivo for administration or anti-sense RNA may be generated in vivo within the cardiac cells in which down-regulation of PLK1 is desired.
  • doublestranded DNA may be placed under the control of a promoter in a "reverse orientation" such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the sense strand of the target gene.
  • the complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein.
  • the complete sequence corresponding to the PLK1 coding sequence in reverse orientation need not be used.
  • fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon.
  • a suitable fragment may have about 14-23 nucleotides, e.g. about 15, 16 or 17.
  • an agent that reduces PLK1 activity may be administered alone, the therapeutic agent will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the active agent.
  • a therapeutic agent may be admixed with other reagents, such as buffers, carriers, diluents, preservatives and/or pharmaceutically acceptable excipients in order to produce a pharmaceutical composition for use in treating cardiac dysfunction.
  • reagents such as buffers, carriers, diluents, preservatives and/or pharmaceutically acceptable excipients in order to produce a pharmaceutical composition for use in treating cardiac dysfunction. Suitable reagents are described in more detail below.
  • aspects of the invention provide a pharmaceutical composition comprising an agent that reduces PLK1 activity, and a pharmaceutically acceptable excipient and (ii) a method of producing a pharmaceutical composition for use in treating cardiac dysfunction comprising admixing an agent that reduces PLK1 activity as described above with a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a subject e.g., human
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • compositions suitable for administration include aqueous and nonaqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer’s Solution, or Lactated Ringer’s Injection. Suitable vehicles can be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.
  • a therapeutic agent or the pharmaceutical composition comprising the therapeutic agent as described herein may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to; parenteral, for example, by infusion, including intravenous infusion, in particular intravenous bolus infusion.
  • parenteral for example, by infusion, including intravenous infusion, in particular intravenous bolus infusion.
  • infusion techniques are known in the art and commonly used in therapy (see, e.g., Rosenberg et al., New Eng. J. of Med., 319:1676, 1988).
  • appropriate dosages of the therapeutic agent, and compositions comprising the therapeutic agent can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention.
  • the selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular cells, the route of administration, the time of administration, the rate of loss or inactivation of the cells, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient.
  • the amount of the therapeutic agent and the route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.
  • a typical oral dosage of a small molecule inhibitor is in the range of from about 0.05 to about 1000 mg, preferably from about 0.1 to about 500 mg, and more preferred from about 1 .0 mg to about 200 mg administered in one or more dosages such as 1 to 3 dosages.
  • the exact dosage will depend upon the frequency and mode of administration, the sex, age, weight and general condition of the subject treated, the nature and severity of the condition treated and any concomitant diseases to be treated and other factors evident to those skilled in the art.
  • parenteral routes such as intravenous, intrathecal, intramuscular and similar administration, typically doses are in the order of about half the dose employed for oral administration.
  • Agents that reduce PLK1 expression or activity are shown herein to reduce inflammatory responses including inflammatory responses mediated by the NRLP3 inflammasome activation and/or inflammatory cytokines (IL-10, TNFa, IFNy). Agents may therefore be useful in the treatment of inflammatory diseases, including inflammatory diseases characterised by excessive NRLP3 inflammasome activation and/or increased levels of inflammatory cytokines relative to healthy non-pathological controls. Suitable agents are described in detail above.
  • aspects of the invention provide an agent that reduces PLK1 activity as described herein for use in a method of treating inflammation or an inflammatory disease in an individual; the use of an agent that reduces PLK1 activity in the manufacture of a medicament for treating inflammation or an inflammatory disease in an individual; and a method of treating inflammation or an inflammatory disease comprising administering an agent that reduces PLK1 activity to an individual in need thereof.
  • Inflammatory diseases may include diseases characterised by increased, excessive or aberrant levels of inflammatory response relative to healthy non-pathological controls, such as chronic inflammation, gout, lupus, endometriosis, type 1 diabetes, fatty liver disease, autoimmune diseases, such as rheumatoid arthritis, cardiovascular disorders, gastrointestinal disorders, such as inflammatory bowel disease, and lung diseases, such as asthma.
  • diseases characterised by increased, excessive or aberrant levels of inflammatory response relative to healthy non-pathological controls such as chronic inflammation, gout, lupus, endometriosis, type 1 diabetes, fatty liver disease, autoimmune diseases, such as rheumatoid arthritis, cardiovascular disorders, gastrointestinal disorders, such as inflammatory bowel disease, and lung diseases, such as asthma.
  • aspects of the invention provide an agent that reduces PLK1 activity as described herein for use in a method of treating a disease characterised by excessive NRLP3 inflammasome activation in an individual; the use of an agent that reduces PLK1 activity in the manufacture of a medicament for treating a disease characterised by excessive NRLP3 inflammasome activation in an individual; and a method of treating a disease characterised by excessive NRLP3 inflammasome activation comprising administering an agent that reduces PLK1 activity to an individual in need thereof.
  • a disease characterised by excessive NRLP3 inflammasome activation may display dysfunctional or altered activation of the NRLP3 inflammasome relative to healthy non-pathological controls. For example, the timing, location or extent of NRLP3 inflammasome activation may be altered relative to non-pathological controls.
  • excessive NRLP3 inflammasome activation may include increased NRLP3 inflammasome activation and over-activation of the NRLP3 inflammasome. The activation of the NRLP3 inflammasome leads to caspase-1 activation and the conversion of pro- IL-1 p into IL-1 p and pro-IL-18 into IL-18.
  • Increased or excessive NRLP3 inflammasome activation may increase levels of active caspase 1 , IL-1 p and/or IL-18.
  • a disease suitable for treatment by PLK1 inhibition as described herein may be characterised by increased, elevated, excessive, or aberrant levels of active caspase 1 , and/or pro-inflammatory cytokines, such as IL-1 p and/or IL-18.
  • the amount or level of active caspase 1 , IL-1 p and/or IL-18 may be increased relative to non-pathological controls.
  • a disease characterised by excessive NRLP3 inflammasome activation may be associated with inflammation or inflammatory disease, for example chronic inflammation, and/or increased cell death through pyroptosis.
  • Pyroptosis is characterised by the caspase-mediated cleavage of gasdermin D.
  • NLRP3 inflammasome activation may include cardiac dysfunction, cryopyrin-associated periodic syndromes (CAPS), gout, atherosclerosis, diabetes, Alzheimer's disease, dry eye disease, such as Sjogren’s syndrome dry eye (SSDE), cardiomyopathy, atrial fibrillation, Kawasaki disease, cancer, and pathogen infection, such as bacterial or viral infection.
  • CAPS cryopyrin-associated periodic syndromes
  • gout atherosclerosis
  • diabetes Alzheimer's disease
  • dry eye disease such as Sjogren’s syndrome dry eye (SSDE)
  • SSDE dry eye disease
  • cardiomyopathy atrial fibrillation
  • Kawasaki disease cancer
  • pathogen infection such as bacterial or viral infection.
  • agents that reduce PLK1 activity are shown herein to improve cardiac function in model systems. In some preferred embodiments, agents that reduce PLK1 expression or activity may therefore be useful in the treatment of cardiac dysfunction.
  • aspects of the invention provide an agent that reduces PLK1 activity as described herein for use in a method of treating cardiac dysfunction in an individual; the use of an agent that reduces PLK1 activity in the manufacture of a medicament for treating cardiac dysfunction in an individual; and a method of treating cardiac dysfunction comprising administering an agent that reduces PLK1 activity to an individual in need thereof.
  • An individual with cardiac dysfunction suitable for treatment as described herein may have reduced or impaired cardiac function relative to a healthy control individual, for example as a result of or following a myocardial infarction.
  • Cardiac function may be determined, for example, from the ejection fraction (EF). Suitable techniques for EF measurement are well known in the art. In humans, a normal heart ejection fraction may vary between 50 to 70 percent. An ejection fraction measurement under 40 percent may be indicative of cardiac dysfunction, such as heart failure or cardiomyopathy.
  • EF ejection fraction
  • Cardiac dysfunction may include cardiomyopathy, such as sepsis induced cardiomyopathy, Takotsubo syndrome, myocarditis, and heart failure, for example chronic heart failure (CHF).
  • Chronic heart failure CHF may be characterised by reduced or impaired functioning of the heart muscle, resulting reduced pumping efficiency and insufficient blood flow to meet the body’s requirements.
  • CHF may be caused by ischaemic heart disease (IHD), which is characterised by reduced blood supply to the heart muscle.
  • IHD ischaemic heart disease
  • the IHD may take the form of a myocardial infarction (Ml), in which the blood supply to the heart muscle is completely blocked, leading to the death of cardiomyocytes.
  • Ml myocardial infarction
  • a PLK1 inhibitor as described herein may be useful in the treatment of post-MI CHF in an individual.
  • chronic heart failure may be characterised by a preserved ejection fraction (e.g. heart failure with preserved ejection fraction; HFpEF).
  • Treatment using PLK1 inhibition as described herein may be useful in limiting infarct size, reducing cardiac overload, improving cardiac function, reducing inflammatory responses and/or improving cardiac remodelling.
  • an individual suitable for treatment as described herein may have heart failure following a myocardial infarction (Ml).
  • Administration of an agent that reduces PLK1 activity as described herein after myocardial infarction may improve cardiac function in the individual. This may be useful for example, in reducing the extent of the myocardial infarction in the individual and/or in preventing or reducing the risk of additional myocardial infarction in the individual.
  • An individual with an inflammatory disease for example, a cardiac dysfunction, such as CHF
  • a cardiac dysfunction such as CHF
  • Treatment may be any treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, delaying, abating or arresting one or more symptoms and/or signs of the condition or prolonging survival of a subject or patient beyond that expected in the absence of treatment.
  • some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, delaying, abating or arresting one or more symptoms and/or signs of the condition or prolonging survival of a subject or patient beyond that expected in the absence of treatment.
  • Treatment as a prophylactic measure is also included.
  • a prophylactic measure i.e. prophylaxis
  • an individual susceptible to or at risk of the occurrence or re-occurrence of inflammatory disease or a disease characterised by excessive NRLP3 inflammasome activation for example a cardiac dysfunction, chronic heart disease, ischemic heart disease or myocardial infarction, may be treated as described herein.
  • Such treatment may prevent, delay or reduce the risk of the occurrence or re-occurrence of the condition in the individual.
  • Treatment may include therapeutic and prophylactic or preventative treatment (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset.
  • treatment may reduce the risk of myocardial infarction occurring or recurring in the individual; delay the onset of myocardial infarction or recurrent myocardial infarction; or reduce the severity of myocardial infarction or recurrent myocardial infarction after onset.
  • An individual suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), or a human.
  • a rodent e.g. a guinea pig, a hamster, a rat, a mouse
  • murine e.g. a mouse
  • canine e.g. a dog
  • feline e.g. a cat
  • equine e.g. a horse
  • the individual is a human.
  • non-human mammals especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or rabbit animals) may be employed.
  • Administration of an agent that reduces PLK1 activity is normally in a "therapeutically effective amount” or “prophylactically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, A method according to administration, the scheduling of administration and other factors known to medical practitioners.
  • An agent that reduces PLK1 activity may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the circumstances of the individual to be treated.
  • an agent that reduces PLK1 activity as described herein may be administered in combination with one or more other therapies.
  • ACE angiotensin-converting enzyme
  • other therapies suitable for the treatment of cardiac dysfunction may include angiotensin-converting enzyme (ACE) inhibitors, such as ramipril, perindopril, enalapril, lisinopril and captopril, angiotensin II receptor blockers, such as candesartan, telmisartan, losartan and valsartan, phosphodiesterase inhibitors or beta-adrenergic receptor agonists; beta blockers, such as carvedilol, metoprolol and bisoprolol, diuretics, such as furosemide and bumetanide, mineralocorticoid receptor antagonists, such as spironolactone and eplerenone, inotropes, ivabradine, saculbitril valsartan, hydralazine with nitrate, and digoxin
  • ACE angiotensin-converting enzyme
  • Other therapies that may be administered in combination with an agent that reduces PLK1 activity may include treatments for infection, such as antibiotics and anti-viral therapies.
  • the compounds When the therapeutic agents are used in combination with additional therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route.
  • a therapeutic agent When a therapeutic agent is used in combination with an additional therapeutic agent active against the same disease, the dose of each agent in the combination may differ from that when the therapeutic agents are used alone.
  • Prescription of treatment is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated.
  • Appropriate doses of therapeutic polypeptides are well known in the art (Ledermann J.A. et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922).
  • Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used.
  • a therapeutically effective amount or suitable dose of an agent that reduces PLK1 activity may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the agent that reduces PLK1 activity is for prevention or for treatment, the size and location of the area to be treated, the precise nature of the agent and the nature of any detectable label or other molecule attached to the agent.
  • a typical dose of a therapeutic agent as described herein will be in the range of 0.1 mg/kg to 100mg/kg, for example 1 to 80mg/kg.
  • a dose in the range 100 pg to 1 g may be used for systemic applications, and 1 pg to 1 mg for topical applications.
  • An initial higher loading dose, followed by one or more lower doses, may be administered.
  • the treatment schedule for an individual may be dependent on the pharmacokinetic and pharmacodynamic properties of the composition, the route of administration and the nature of the condition being treated.
  • an agent that reduces PLK1 activity as described herein may be administered by the intravenous route, although other routes (topical, enteral, and parenteral), such as oral, subcutaneous, inhalation, ocular, intraperitoneal, enema, intrathecal, intracardiac, intrathoracic, guided delivery and other administration may also be appropriate, depending on the context.
  • routes topical, enteral, and parenteral
  • oral, subcutaneous, inhalation, ocular, intraperitoneal, enema, intrathecal, intracardiac, intrathoracic, guided delivery and other administration may also be appropriate, depending on the context.
  • an agent that reduces PLK1 activity as described herein may be administered locally to an affected tissue.
  • the agent may be delivered to the heart through nanoparticles, or ultrasound-guided transthoracic injection to treat a cardiac dysfunction or disorder as described herein.
  • Administration of therapeutic agent as described herein can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment.
  • Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
  • Treatment may be periodic, and the period between administrations may be about one week or more, e.g. about two weeks or more, about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more.
  • treatment may be every two to four weeks or every four to eight weeks.
  • a method of screening for a compound useful in the treatment of an inflammatory disease, a disease characterised by excessive NRLP3 inflammasome activation and/or improving cardiac function may comprise; determining the activity of PLK1 in the presence and absence of a test compound.
  • a decrease in PLK1 activity in the presence relative to the absence of the test compound may be indicative that the test compound is a candidate compound for use in treating an inflammatory disease or a disease characterised by excessive NRLP3 inflammasome activation and/or improving cardiac function in the patient.
  • PLK1 activity may be determined in the context of NLRP3 inflammasome activation by measuring IL-10 and/or IL- 18 or their downstream cytokines such as IL-6 and/or IFNy, hsCRP, cell death through lactate dehydrogenase (LDH) activity, levels of ASC speck formation, and/or the amount of interaction of PLK1 with NLRP3.
  • IL-10 and/or IL- 18 or their downstream cytokines such as IL-6 and/or IFNy, hsCRP, cell death through lactate dehydrogenase (LDH) activity, levels of ASC speck formation, and/or the amount of interaction of PLK1 with NLRP3.
  • a method of screening for a compound useful in treating an inflammatory disease, a disease characterised by excessive NRLP3 inflammasome activation and/or improving cardiac function in a patient may comprise; determining the binding of a test compound to PLK1 or a fragment thereof.
  • Suitable PLK1 fragments may comprise one or both polo-box domains (PBDs).
  • PBDs polo-box domains
  • Binding of the test compound to one or both polo-box domains of PLK1 may be indicative that the test compound is a candidate compound for use in treating a disease characterised by excessive NRLP3 inflammasome activation and/or improving cardiac function.
  • the polo-box domains (PBDs) are located in PLK1 at positions corresponding to residues 407 to 494 and 509 to 590 of the reference sequence NP_005021.2.
  • method of screening for a compound useful in treating an inflammatory disease, a disease characterised by excessive NRLP3 inflammasome activation and/or improving cardiac function in a patient may comprise; determining the binding of PLK1 to NRLP3 in the presence and absence of a test compound.
  • a decrease in binding in the presence relative to the absence of the test compound may be indicative that the test compound is a candidate compound for use in treating an inflammatory disease, a disease characterised by excessive NRLP3 inflammasome activation and/or improving cardiac function.
  • a method of screening for a compound useful in the treatment of an inflammatory disease may comprise; determining the amount of PLK1 protein in a mammalian cell in the presence and absence of a test compound.
  • a decrease in the amount of PLK1 protein in the cell in the presence relative to the absence of the test compound may be indicative that the test compound is a candidate compound for use in treating a disease characterised by excessive NRLP3 inflammasome activation and/or improving cardiac function.
  • a method of screening for a compound useful in the treatment of an inflammatory disease may comprise; determining the expression of PLK1 in a mammalian cell in the presence and absence of a test compound.
  • a decrease in expression of PLK1 in the cell in the presence relative to the absence of the test compound may be indicative that the test compound is a candidate compound for use in treating a disease characterised by increased NRLP3 inflammasome and/or improving cardiac function.
  • Suitable methods for determining PLK1 activity; the binding of PLK1 to NLRP3; the amount of PLK1 protein in a cell; and/or PLK1 expression are well known in the art.
  • PLK1 for use in screening methods may be an isolated polypeptide comprising the full-length PLK1 sequence, for example a PLK1 reference sequence, as set out herein, or a fragment thereof. Suitable fragments may include at least 50, at least 100 or at least 150 contiguous amino acids from a PLK1 reference sequence. Isolated PLK1 polypeptides may be produced using standard recombinant techniques.
  • test compound may be an isolated molecule or may be comprised in a sample, mixture or extract, for example, a biological sample.
  • Compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programmes and may include, for example, small organic molecules, polypeptides and nucleic acids, such as aptamers. Extracts of plants, microbes or other organisms, which contain several characterised or uncharacterised components may also be used.
  • Combinatorial library technology provides an efficient way of testing a potentially vast number of different compounds for ability to modulate PLK1 activity.
  • Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others. The use of peptide libraries may be preferred in certain circumstances.
  • libraries of biological molecules such as aptamers or antibody molecules.
  • test compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.001 nM to 1 mM or more concentrations of putative inhibitor compound may be used, for example from 0.01 nM to 100pM, e.g. 0.1 to 50 pM, such as about 10 pM. Even a compound which has a weak effect may be a useful lead compound for further investigation and development.
  • Test compounds may include peptides derived from PLK1 or binding partners thereof, such as NLRP3, as described above.
  • Membrane permeable peptide fragments of from 5 to 40 amino acids, for example, from 6 to 10 amino acids may be tested for their ability to bind to PLK1 or inhibit its activity.
  • the modulatory properties of a peptide above may be increased by the addition of one of the following groups to the C terminal: chloromethyl ketone, aldehyde and boronic acid. These groups are transition state analogues for serine, cysteine and threonine proteases.
  • the N terminus of a peptide fragment may be blocked with carbobenzyl to inhibit aminopeptidases and improve stability (Proteolytic Enzymes 2nd Ed, Edited by R. Beynon and J. Bond, Oxford University Press, 2001).
  • Test compounds may include antibodies, antibody fragments and antibody derivatives and nonimmunoglobulin binding molecules, such as aptamers, trinectins, anticalins, kunitz domains, transferrins, nurse shark antigen receptors and sea lamprey leucine-rich repeat proteins.
  • Candidate modulatory antibody molecules may be characterised, and their binding regions determined to provide single chain antibodies and fragments thereof which are responsible for inhibiting activity or blocking interactions with binding partners.
  • Suitable antibodies may be obtained using techniques which are standard in the art, including, for example immunising a mammal with a suitable peptide, such as a fragment of the pro-inflammatory polypeptide, or isolating a specific antibody from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see W092/01047.
  • a suitable peptide such as a fragment of the pro-inflammatory polypeptide
  • isolating a specific antibody from a recombinantly produced library of expressed immunoglobulin variable domains e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see W092/01047.
  • Aptamers directed to PLK1 are also putative agents for modulating PLK1 , respectively.
  • Aptamers are nucleic acids that specifically bind to a target molecule.
  • aptamers are small nucleic acids ranging from 15- 50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets.
  • Aptamers can bind very tightly with kd for the target molecule of less than 10- 12 M.
  • Aptamers may bind PLK1 with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10000-fold difference in binding affinities between a target molecule and another molecule that differ at only a single position on the molecule.
  • An aptamer may have a kd with PLK1 of at least 10, 100, 1000, 10,000, or 100,000-fold lower than the kd with a control polypeptide.
  • the production and use of aptamers is well known in the art (see for example Bunka et al Curr Opin Pharmacol 2010 10 (5) 557-562).
  • a test compound identified as inhibiting PLK1 activity may be investigated further using one or more secondary screens.
  • a secondary screen may involve testing for a biological function or activity in vitro and/or in vivo, e.g. in an animal model. For example, the ability of a test compound to improve cardiac function may be determined.
  • a secondary screen may involve determining the selectivity of a compound for PLK1 by screening against a panel of isolated enzymes.
  • the effect of a test compound identified as a PLK1 inhibitor may be determined in vitro on mammalian cells or in model systems. For example, the effect of the identified test compound on NLRP3 inflammasome activation may be determined. In some embodiments, the effect of the test compound on the contractility of cardiomyocytes may be determined.
  • a method may further comprise modifying the compound to optimise its pharmaceutical properties. Suitable methods of optimisation, for example by structural modelling, are well known in the art. Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing.
  • test compound identified as a PLK1 inhibitor may be isolated and/or purified or alternatively, it may be synthesised using conventional techniques of recombinant expression or chemical synthesis. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. Methods described herein may thus comprise formulating the test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier for therapeutic application.
  • mice All in vivo experiments using mice were approved by the Home Office, UK, and were performed under PPL PA4BDF775.
  • mice Wild-type C57BL/6J and Nlrp3-/- 1 mice were described. All mice were fully backcrossed to a C57BL/6 background. Prof Clare Bryant (Veterinary Medicine, University of Cambridge) and Prof Bernard Ryffel (CNRS, INEM UMR7355) provided some of Nlrp3-/- mice. Dr David Adams (Wellcome Sanger Institute, Cambridge, UK) provided RosaCreErt2/CreErt2 mice. Dr Guillermo de career and Dr Marcos Malumbres provided Plk1 flox mice (Spanish National Cancer Research Centre, Spain). LyMcre mice were from the Jackson laboratory.
  • mice Age (8-14 weeks), and sex and genetic background (C57BL/6J)-matched male wild-type mice and NLRP3 knock-out mice were used.
  • Mice fed with a chow diet were injected i.p. with 5mg/kg BI6727 (Selleckem, S2235) resuspended in vehicle control.
  • Vehicle control was 4% DMSO (Sigma, D5879) in corn oil (Sigma, C8267).
  • One hour after BI6727 administration the mice were injected i.p. with 20mg/kg LPS (Sigma, O111 :B4, L4391) resuspended in PBS.
  • 3 hours after LPS injection animals were sacrificed, and peritoneal exudates and blood were collected and measured for cytokine measurement. 6 hours after the LPS injection, another batch of mice was sacrificed and then lung and liver tissue were harvested for analysis.
  • mice Age (8-12 weeks), sex and genetic background (C57BL/6J)-matched male wild-type mice and NLRP3 knockout mice were used. Mice fed with a chow diet were injected i.v. with 1 mg/kg BI6727 (Selleckem, S2235) resuspended in vehicle control as previously described (Rudolph et al Clin Cancer Res. 2009;15(9):3094- 102). Vehicle control was 0.01 % DMSO in 0.9% NaCI with 0.1 N HCI. One hour after BI6727 administration, the mice were injected i.p. with MSU crystals (0.5mg/mouse, resuspended in 200uL PBS). 5 hours after MSU challenge, animals were sacrificed, and peritoneal exudates were collected for cytokine measurement. 6 hours after MSU challenge, another batch of mice was sacrificed and then cells in the peritoneal cavity were collected for flow cytometry analysis.
  • HE Hematoxylin & Eosin staining was performed based on the procedures previously described (Feldman et al Methods Mol Biol. 2014;1180:31-43.).
  • the buffers and reagents used are listed below: Harris’ Hematoxylin (Sigma, HHS128), Scott’s Solution (20g Magnesium Sulphate and 2g Sodium Bicarbonate were dissolved in one liter of distilled water), Eosin (Sigma, HT1 10280), destaining Solution (250mL Methanol, 250mL filtered H2O and 5mL concentrated HCI), and DPX (Sigma, 06522).
  • the severity of lung injury was assessed by the area of parenchyma.
  • the lung parenchymal area assessed combined the area of the alveolar septum, the area of alveolar exudation, the area of inflammatory cell infiltration, and the area of bleeding. The area was determined based on the measurement using Image J software. For each mouse, the lung parenchymal area measurement was averaged upon 4-6 fields per section, one section per level, and 3 different levels in total. For each mouse, the infiltrated immune cells in the liver sinusoid were averaged upon 4-6 fields per section, one section per level, and 3 different levels in total.
  • cryosectioned tissues were used for immunofluorescence as previously described (Zhou et al Bio Protoc. 2017;7(10)).
  • the images were taken with a Leica microscope (DM6000 B). All analysis was performed using 4-6 fields per section at one level (comparable levels among different mice) for each mouse.
  • EB1 comets were quantified manually in FIJI (Image J) as previously described (Gavilan et al EMBO Rep. 2018;19(1 1)).
  • Single-plane images focused on the brightest pericentrin signal were marked with a point of interest corresponding to the pericentrin focus.
  • a circular selection of diameter 3pm was centred around the point of interest.
  • EB1 fluorescence intensity was measured within the circular selection. For each randomly selected field of view, all focused centrosomes were analysed.
  • peritoneal lavage samples were collected and centrifuged, and the total number of cells was counted using a cell counter (NucleoCounter, Chemometec). Collected cell samples were then stained with a viability dye (Zombie Aqua, Biolegend, 423102) for 10 minutes at room temperature in the dark. Then samples were stained with desired antibodies as previously described (Lu et al Immunity. 2020;52(5):782-93 e5; Clement M, et al. Circ Res. 2018; 122(6):813-20). Data were analysed by FlowJo software (v10.8.1 , Becton, Dickinson & Company).
  • Total immune cells are defined as cells with CD45 surface marker, excluding debris and gating on singlets to identify live cells. From CD45 positive cells, the cells within CD45+B220-CD4-CD8-Ly-6C-Ly- 6G-CD11 b+F4/80+ gate represent macrophage. B cells are defined as cells with B220+ surface marker and T cells are defined as cells with CD4/CD8+ surface marker. The cells within CD45+B220-CD4-CD8- Ly- 6C+Ly-6G+gate represent neutrophils.
  • ATP A7699-1 G
  • Ultrapure LPS Ultrapure LPS (0111 :B4) were obtained from Sigma.
  • Imject Alum was obtained from Thermo Fisher.
  • Nigericin BML-CA421-0005
  • Flagellin and poly were obtained from Invivogen.
  • MSU crystals and cholesterol crystals were made as described 48 49 .
  • DOTAP Roche
  • TranslT/LT1 MirusBio
  • TransIT/TKO MirusBio
  • pCMV- 3Tag-1 Full-length and truncated human NLRP3 (94-979; 1-220; 1-389; 1-574; 575-979) were subcloned into pCMV- 3Tag-1 as described.
  • pCMV3-HA-human PLK1 was acquired from Sino Biological (HG10676-NY). Truncations of PLK1 (1-361 , 1-498, 362-603) were then cloned into a pCMV5-HA vector.
  • the BASU sequence (Addgene, 107250) was subcloned into pLenti-C-Myc-DDK-P2A-Puro (OriGene, PS100092V5).
  • Antibodies against NLRP3 (Adipogen, Cryo2; Sigma, HPA012878), IL-1 p (R&D, AF-401-NA), Caspase-1 (Santa Cruz, SC-154), HA tag (Santa Cruz, y-11), Flag tag (Sigma, F1804), PLK1 (Abeam, ab17056), p- Actin (Cell Signalling Technology 3700S), ASC (Enzo Life Sciences, AD1 -905-173-100) were used for western blots.
  • Antibodies against PLK1 Proteintech, 10305-1 -AP
  • NLRP3 (Abeam, ab4207) were used for in situ proximity ligation assay.
  • Antibodies against PLK1 (Proteintech, 10305-1 -AP), PhosphohistoneH3 (Biolegend, 650806), ASC (Biolegend, 653904), rabbit IgG isotype control (Thermo Fisher, 02-6102) and AF488 Donkey anti rabbit (Thermo Fisher, A-21206) were used for flow cytometry, permeabilizing and fixing cells with the Foxp3 transcription factor staining buffer set (Thermo Fisher).
  • Antibodies against PLK1 (Abeam, ab17056), ASC (Santa Cruz, sc-22514-R), y-tubulin (Sigma, T5326), EB1 (BD, 610534) and pericentrin (Abeam, ab4448) were used for immunofluorescence, fixing cells with methanol for 5 minutes at - 20°C. Nuclei were stained with DAPI (Sigma, D9542). The pericentrin antibody (Abeam, ab4448) was also used for Imagestream experiments.
  • Wild-type and RosaCreErt2/wt Plk1 fl/fl bone marrow cells were obtained from C57BL/6 mice. Bone marrow cells were differentiated into macrophages as previously described (Guarda 2009 Nature. 2009;460(7252):269-73). The differentiation of BMDM was confirmed by flow cytometry with markers CD11 b and F4/80. PLK1 depletion in the RosaCreErt2/wt Plk1 fl/fl system was carried out by incubation of 0.002mg/mL 4OH-Tamoxifen (H7904-5MG, Sigma) for 24h.
  • tRPE cells were cultured in DMEM/F12 media with HEPES and L- Glutamine (Thermo Fisher, 11320033) supplemented with 10% FBS and antibiotics. Incubation of tRPE PLKIas cells overnight with 20 pM of the purine analogue 3MP-PP1 (Cayman Chemicals, 17860) was used to induce cell cycle arrest, and cells in cell cycle arrest were counted over three fields of view. Cells were cultured at 37°C, 5% CO2 in the humidified incubator. Isolation of Plk1 knock-out peritoneal macrophages
  • Peritoneal fluids were obtained by peritoneal lavage from LysM cre/wt Plk1 fl/fl , LysM cre/wt Plk1 fl/Wt and LysM cre/wt Plk1wt/wt ma
  • Peritoneal macrophages were purified with the AutoMACS Pro Separator system (Miltenyi, 130-092-545) and purity was confirmed by flow cytometry with markers CD11 b and F4/80.
  • Cells were cultured in RPMI 1640 Glutmax (Thermo Fisher) supplemented with 10% FBS and antibiotics for 2 hours prior to priming with LPS and activation with ATP.
  • Lentivirus transduction pLenti-BASU or pLenti-BASU-Plk1 were packaged and produced by transient transfection of HEK293/T17 cells using the TranslT-LT1 (MirusBio, MIR 2300). iBMDMs were transduced with packaged lentivirus as previously described. 24-48 hours after transduction, cells were placed in media without virus, and puromycin selection was performed to establish the stable iBMDM cells expressing BASU or BASU-PLK1 .
  • Stable iBMDMs expressing BASU or BASU-PLK1 were primed with 100 ng/ml of LPS (Sigma) for 5-6 hours, and/or activated 10pM nigericin for 2 hours. 30 minutes prior to nigericin activation, cells were treated with 100 n M of Ac-YVAD-cmk (Sigma, SML0429) and 5mM of glycine. During nigericin treatment, cells were incubated with 50 n M of Biotin. After that, both floating and adherent cells were collected in PBS with 1 mM EDTA.
  • the resulting cell pellets were lysed in the lysis buffer (50mM Tris pH 7.4, 500 mM NaCI, 0.4%SDS, 5mM EDTA, 1 %Triton) freshly supplemented with 1 mM DTT and 1 tablet of completeTM Mini EDTA-free Protease Inhibitor Cocktail (Roche, 1 1836170001) per 10mL of buffer.
  • the lysates were then passed through a 25g syringe 10 times for complete lysis. Then the samples were sonicated on a Bioruptor Pico (Diagenode) (Sonication cycle: 30s On, 30s Off; 5 Cycles, 4°C).
  • wash buffer 1 2%SDS
  • wash buffer 2 50mM HEPES pH 7.4, 1 mM EDTA, 500mM NaCI, 1 %Triton X-100, 0.1 %Nadeoxycholate
  • wash buffer 3 50mM Tris pH 7.4, 1 mM EDTA, 250mM LiCI, 0.5% P-40, 0.5%Na-deoxycholate
  • wash buffer 4 50mM Tris pH 7.4, 50mM NaCI, 0.1 %NP-40).
  • reducing solution 10mM DTT in 100mM triethylammonium bicarbonate (TEAB)
  • TEAB buffer for 15 minutes on a rotating wheel.
  • Trypsin digest was performed overnight at 37°C with 20ng/pL MS grade Trypsin (Therm
  • the Dionex Ultimate 3000 RSLC nanoUHPLC (Thermo Fisher) system and a Lumos Orbitrap mass spectrometer (Thermo Fisher) were used.
  • the peptides were loaded onto a pre-column (Thermo Fisher PepMapTM 100 C18, 5 pm particlesize, 100A pore size, 0.3 mm diameter x 5mm length) with 0.1 %formic acid for 3 minutes at a flow rate of 10pl/minute.
  • the peptides were eluted onto the analytical column (Easyspray TM column; Thermo Fisher PepMapTM C18, 2pm particle size, 100A pore size, 75pm diameter x 50cm length) for separation of peptides via reverse-phase chromatography at a flow rate of 300nl/minute.
  • the eluted peptides were introduced into the mass spectrometer with an Easy-Spray source (Thermo Fisher), m/z values were measured with an Orbitrap mass analyser with a resolution of 120,000 and were scanned between m/z 380-1500Da.
  • Precursor ions were isolated and fragmented by collision-induced dissociation (CID, Normalised Collision Energy (NCE): 35%), which were analysed in the linear ion trap.
  • CID collision-induced dissociation
  • NCE Normalised Collision Energy
  • the orbitrap analyser measured all m/z values and the relative abundance of each reporter ion and all fragment in each MS step with a resolution at 60,000.
  • Raw data processing was performed using Proteome Discoverer v.2.4 (Thermo Scientific). The data was searched against the Uniprot Mouse database and a database of common contaminants by the Mascot search algorithm (Matrix Science).
  • the spectra identification was performed with the following parameters: MS accuracy, 10 ppm; MS/MS accuracy, 0.8 Da; up to two trypsin missed cleavage sites allowed; carbamidomethylation of cysteine and TMT tagging of lysine and peptide N-terminus as fixed modifications; and oxidation of methionine and deamidated asparagine and glutamine as variable modifications.
  • the Percolator node was used for FDR estimation and only peptide identifications of high confidence (FDR ⁇ 1%) were accepted.
  • a total of 6856 proteins were identified from three TMT batches and 12 samples. However, only 3063 of these proteins were consistently identified in all 12 samples. Missing protein values across different samples in one TMT batch are relatively low, and no protein with missing values was exclusively present in the primed or activated samples. Missing values were removed from subsequent analysis. Differential expression (DE) analysis was performed to identify interacting proteins which are upregulated and downregulated in activated versus primed cells. There were 60 proteins with low FDR confidence, which we removed to keep high-quality proteins in the DE analysis. A total of 3063 proteins remained after removing the missing value and low FDR.
  • DE Differential expression
  • the cells were cultured on collagen (Sigma) coated 8-well chamber slide (Nunc).
  • PLA assay was performed according to manufacturer’s instruction (Duolink). Living cells were fixed by 4% PFA, and then subject to PLA assay. Nucleus was revealed in blue (DAPI) in the images. PLA signal (green) per cytoplasm of a cell were acquired using Duolink analysis software (Duolink). Co-immunoprecipitation assay
  • 1 mM Na3VO4 and 50 mM NaF 1 tablet of completeTM, Mini, EDTA-free Protease Inhibitor Cocktail per 10ml buffer (Roche).
  • biolayer interferometry experiments were performed using the Octet RED96 (ForteBio) instrument at 25oC, 1000 rpm.
  • Recombinant full-length NLRP3 tagged with GST (Caltag Medsystems), diluted in assay buffer (100 mM HEPES, 100 mM NaCI, pH 7.4, 0.01% BSA, 0.01% Tween) to 6.9 nM, was immobilised on anti-GST-coated fibre-optic biosensors (ForteBio).
  • a blank anti-GST coated biosensor was used as a control reference.
  • Immobilised biosensors were then suspended in buffer containing recombinant full-length Plk1 (tagged with His; MRC-PPU, UK) at 500-15.125 nM for 300 s to measure the association phase and transferred to buffer for 300 s to measure the dissociation phase.
  • Biosensors were regenerated between each reading using 10 mM glycine (pH 2.0) for 5 s and neutralised in assay buffer for 5 s; this was repeated 3 times for each cycle.
  • Subtraction of reference sensor data and Savitsky-Golay filtering were conducted in ForteBio Data Analysis software (version 8.0) and fitted using the Kinetics Binding function in Prism software (GraphPad).
  • Delta centroid distances between ASC-mCerulean and PE staining for pericentrin were calculated with the following function: Delta Centroid XY_M03_Ch03 - Pericentrin_lntensityWeighted_M07_Ch07 - ASCJntensity Weighted. ELISA and Meso Scale Discovery (MSD)
  • ELISA kits of mouse IL-1 p ELISA were obtained from BD biosciences and Thermo Fisher Scientific, and Mouse TNFa was obtained from R&D. ELISA assays were performed according to manufacturer’s instructions. MSD assay of mouse IL-1 p was performed by core biochemical assay laboratory of Cambridge University Hospitals, and the detection limit of mouse IL-1 p by MSD is 1 .2 pg/mL.
  • mice were initially anesthetised (ketamine 100 mg/kg + xylazine 10 mg/kg by injection into the intraperitoneal cavity for induction and 1-2% isoflurane inhalation for maintenance) and then intubated endotracheally and ventilated at a rate of 130 breaths/min.
  • a left thoracotomy was performed through the fourth intercostal space and the main branch of LAD was ligated permanently using an 7-0 prolene suture.
  • Significant colour changes and elevation of ST segment at the ischemic area was considered indicative of successful coronary occlusion.
  • Mice in the sham group underwent the same surgical procedure, except that the LAD was not ligated.
  • BI6727 (5 mg/kg) or equivalent concentration of DMSO in sterile saline solution was injected into the intraperitoneal cavity. Mice in the chronic (28 day) study received further injections on days 2 and 7 post-ML One mouse was culled on day 4 due to repeated suture
  • Cardiac function was determined by echocardiography (VisualSonics, Vevo 3100,40 MHz, MX55D transducer). After alignment with the papillary muscles using the long and short axis B-mode, M-mode images were recorded, and measurements were made retrospectively using VevoLAB software (Version 3.2, VisualSonics).
  • Hearts and spleens were collected on ice in PBS. Tissues were blotted and weighed. Heart tissues were minced and incubated in collagenase solution (collagenase II, elastase, DNase I) at 37°C with shaking for 45 min and rinsed through a 40 pm strainer. Spleens were minced over a 100 pm strainer and rinsed through with PBS. Erythrocytes were lysed using ACK buffer (150 mM NH4CI, 10 mM KHCO3, 0.1 mM EDTA, pH 7.6). Isolated cells were counted and resuspended in a PBS solution containing 2.5% FBS, 1 mM EDTA.
  • collagenase solution collagenase solution
  • DNase I elastase
  • Arterial blood pressure was measured using an automated tail-cuff BP-2000 Blood Pressure Analysis System (Visitech Systems). Mice were trained for measurement conditions on consecutive days for 1 week. After training, BP was measured once prior to surgery. Mice were placed in tail-cuff restraints over a warmed surface (39 °C) and fifteen consecutive BP measurements were taken, with the final ten readings per mouse recorded and averaged.
  • BP Blood Pressure Analysis System
  • PLK1 data were expressed as Mean ⁇ SEM. Comparisons of the two different PLK1 groups were analysed by unpaired t test. For more than two groups, ANOVA test was used. P ⁇ 0.05 (*), P ⁇ 0.01 (**), P ⁇ 0.001 (***), or P ⁇ 0.0001 (****) was considered statistically significant.
  • Interphase PLK 1 inhibition reduces IL 1(3 levels upon NLRP3 inflammasome activation
  • BMDMs murine bone marrow-derived macrophages
  • iBMDM immortalized BMDM
  • Proximity proteomics reveals PLK1 interactome upon NLRP3 inflammasome activation
  • Bio-ID proximity-dependent biotin identification
  • PLA In situ proximity ligation assay
  • Microtubule associated transport is the key intracellular machinery that is involved in regulating protein subcellular localization (Bauer et al Traffic. 2015; 16(10): 1039-61 ).
  • PLK1 could influence NLRP3 subcellular localization
  • biochemical fractionation assay As shown, there were comparable levels of NLRP3 expression in these cells (Fig. 3E). However, in comparison with cells with no PLK1 depletion upon NLRP3 inflammasome activation, there was a reduced presence of NLRP3 in both membrane and insoluble cytoskeleton fractions of the PLK1 depleted cells, despite no apparent changes in the cytosolic fractions (Fig. 3F). These results suggest an altered NLRP3 trafficking between different cellular organelles.
  • PLK1 kinase inhibition suppresses ll_1[3 levels in inflammatory models in vivo
  • PLK1 is essential for cell proliferation and survival during development, and the generic Plk1 knock-out mice display an embryonic lethality phenotype (Wachowicz et al Bioessays. 2016;38 Suppl 1 :S96-S106). Therefore, we applied a widely used pharmacological PLK1 kinase inhibitor BI6727, at doses of 5 mg/kg intraperitoneal (i.p.) in LPS-induced endotoxemia model (Fig.4A), or 1 mg/kg intravenous (i.v.) in MSU- induced peritonitis model (Fig.5A). The doses are lower than the doses at which BI6727 is normally used in cancer studies (Rudolh D Clin Cancer Res.
  • IL1 p production is largely or totally dependent on NLRP3 (Martinon F et al. Nature. 2006;440(7081):237-41 ; He Y et al Nature. 2016;530(7590):354-7; Sutterwala et al Immunity. 2006;24(3):317-27).
  • IL1 p level was largely dependent on NLRP3 activation in the LPS-induced endotoxemia model as IL1 p was substantially reduced in NLRP3 knock-out (KO) control mice, regardless of pharmacological treatment (Fig. 4B, 4C).
  • BI6727 treatment there was only a limited effect of BI6727 on reducing IL1 p level in the peritoneal fluids of NLRP3 KO mice (Fig.
  • LPS administration leads to systematic inflammatory responses including inflammation in the lung (Okamoto et al Am J Pathol. 2021 ;191 (9): 1526-36) and liver (Maehara et al Faseb j. 2020;34(11):15197-207), and the degree of inflammatory response could largely depend on NLRP3 (Grailer et al J Immunol. 2014; 192 (12): 5974-83).
  • BI6727 significantly alleviated LPS-induced inflammatory response in the lung (Fig. 4D, 4E) and the liver (Fig. 4F, 4G), evidenced by reduced levels of alveolar wall thickness (Fig.
  • BI6727 did not further limit the inflammatory response in NLRP3 KO mice (Fig. 4D-4G), further confirming that the effect of BI6727 on dampening inflammation depends on NLRP3.
  • BI6727 in vivo, we also used an MSU-induced peritonitis model (Fig. 5A), in which IL10 level is completely dependent on NLRP3 activation and the increased level of IL1 p upon MSU treatment leads to neutrophil infiltration into the peritoneal cavity.
  • Fig. 5A MSU-induced peritonitis model
  • IL10 level in peritoneal lavage was substantially reduced under BI6727 treatment without significantly altering TNFa
  • BI6727 reduced the number of infiltrated neutrophils (not macrophage, B cells, and T cells) in the peritoneal cavity, and this reduction was dependent on NLRP3 (Fig. 5D-5H).
  • PLK1 takes a moonlighting role in interphase to control inflammation. This work expands our current understanding of PLK1 and NLRP3 functions, as well as our knowledge of how the subcellular compartments are possibly modulated by microtubule organization to deliver the inflammatory output.
  • PLK1 inhibitor BI6727 has an acute protective effect on cardiac function following myocardial infarction.
  • BI6727 volasertib
  • LAD myocardial infarction
  • BI6727 (5 mg/kg) or control (DMSO) in saline solution was injected into the intraperitoneal space one hour following surgery and echocardiography images were acquired at multiple time-points pre- and post- Ml (pre-surgery, 1 day, 7 days, 14 days, and 28 days).
  • BP Arterial blood pressure
  • a multiplex panel of 10 pro- and anti-inflammatory cytokines was used to determine the expression levels of serum cytokines 3 days post-MI (Figure 8).
  • KCGRO neutrophil-recruitment factor

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Abstract

La présente invention concerne le traitement d'une inflammation, ou de maladies caractérisées par une activation excessive ou aberrante de l'inflammasome NLRP3 ("NLR family Pyrin Domain Containing 3"), par exemple un dysfonctionnement cardiaque, par réduction de l'expression et/ou de l'activité de la kinase 1 de type Polo (PLK1). Dans certains modes de réalisation, le traitement peut comprendre l'administration d'un inhibiteur de PLK1 à un individu en ayant besoin. L'invention concerne également des méthodes de criblage de composés candidats appropriés pour une utilisation dans de tels traitements.
PCT/EP2024/074863 2023-09-05 2024-09-05 Traitement de maladies inflammatoires Pending WO2025051878A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001047A1 (fr) 1990-07-10 1992-01-23 Cambridge Antibody Technology Limited Procede de production de chainon de paires a liaison specifique
US7517873B2 (en) 2007-03-22 2009-04-14 Hoffman-La Roche Inc. Substituted pyrimidodiazepines
US7718801B2 (en) 2004-08-31 2010-05-18 Banyu Pharmaceutical Co., Ltd. Substituted imidazole derivative
US8003785B2 (en) 2008-06-18 2011-08-23 Takeda Pharmaceutical Company Limited Halo-substituted pyrimidodiazepines

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001047A1 (fr) 1990-07-10 1992-01-23 Cambridge Antibody Technology Limited Procede de production de chainon de paires a liaison specifique
US7718801B2 (en) 2004-08-31 2010-05-18 Banyu Pharmaceutical Co., Ltd. Substituted imidazole derivative
US7517873B2 (en) 2007-03-22 2009-04-14 Hoffman-La Roche Inc. Substituted pyrimidodiazepines
US8003785B2 (en) 2008-06-18 2011-08-23 Takeda Pharmaceutical Company Limited Halo-substituted pyrimidodiazepines

Non-Patent Citations (140)

* Cited by examiner, † Cited by third party
Title
"Harrison's Principles of Internal Medicine", 2001, OXFORD UNIVERSITY PRESS
"Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING COMPANY
ADAMO, L. ET AL., NATURE REVIEWS CARDIOLOGY, 2020
ANDERSON, J. L.MORROW, D. A., NEW ENGLAND JOURNAL OF MEDICINE, 2017
ANGELLBAULCOMBE, EMBO JOURNAL, vol. 16, no. 12, 1997, pages 3675 - 3684
ARCHAMBAULT V ET AL., NATURE REVIEWS MOLECULAR CELL BIOLOGY., vol. 10, no. 4, 2009, pages 265 - 75
ARCHAMBAULT, V. ET AL., ONCOGENE, vol. 34, no. 37, 2015, pages 4799 - 4807
ARCHAMBAULT, V.GLOVER, D.M., NATURE REVIEWS. MOLECULAR CELL BIOLOGY, vol. 10, 2009, pages 265 - 275
BAGSHAWE K.D. ET AL., ANTIBODY, IMMUNOCONJUGATES AND RADIOPHARMACEUTICALS, vol. 4, 1991, pages 915 - 922
BALDRIGHI MARTA ET AL: "PLK1 inhibition dampens NLRP3 inflammasome-elicited response in inflammatory disease models", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 133, no. 21, 1 November 2023 (2023-11-01), US, XP093214243, ISSN: 1558-8238, DOI: 10.1172/JCI162129 *
BALDRIGHI, M.MALLAT, Z.LI, X, ATHEROSCLEROSIS, 2017
BARRETTA ML ET AL., NAT COMMUN., vol. 7, 2016, pages 11727
BARRETTA, M.L. ET AL., NAT COMMUN, vol. 7, 2016, pages 11727
BAUER ET AL., TRAFFIC., vol. 16, no. 10, 2015, pages 1039 - 61
BLOOM, M. W. ET AL., NAT. REV. DIS. PRIM., 2017
BONDESON, D ET AL., ANN REV PHARMACOL TOXICOL., vol. 57, 2017, pages 107 - 123
BUNKA ET AL., CURR OPIN PHARMACOL, vol. 10, no. 5, 2010, pages 557 - 562
BURKARD ME ET AL., PROC NATL ACAD SCI U S A., vol. 104, no. 11, 2007, pages 4383 - 8
CAO, Y. ET AL., SCI REP, vol. 8, 2018, pages 1055
CERMAKOVA K ET AL., MOLECULES, vol. 23, no. 8, 2018, pages 1958
CHEN ET AL., ONCOTARGET, vol. 8, no. 1, 2017, pages 1234 - 1246
CHEN S ET AL., BIOORG MED CHEM LETT., vol. 22, no. 2, 2012, pages 1247 - 50
CHEN, G.CHELU, M. G.DOBREV, D.LI, N.., FRONTIERS IN PHYSIOLOGY, 2018
CHEVRIER, N. ET AL., CELL, vol. 147, no. 4, 2011, pages 853 - 867
CHI KR, NATURE REVIEWS. DRUG DISCOVERY., vol. 15, no. 5, 2016, pages 295 - 7
CHIAPPA MICHELA ET AL: "Present and Future Perspective on PLK1 Inhibition in Cancer Treatment", FRONTIERS IN ONCOLOGY, vol. 12, 2 June 2022 (2022-06-02), XP093214468, ISSN: 2234-943X, DOI: 10.3389/fonc.2022.903016 *
CLAUSEN BE ET AL., TRANSGENIC RES., vol. 8, no. 4, 1999, pages 265 - 77
CLAUSEN, B.E. ET AL., TRANSGENIC RES, vol. 8, 1999, pages 265 - 277
CLÉMENT M ET AL., CIRC RES., vol. 122, no. 6, 2018, pages 813 - 20
COLICINO EG ET AL., CYTOSKELETON (HOBOKEN)., vol. 75, no. 11, 2018, pages 481 - 94
CONDUIT PT ET AL., DEV CELL., vol. 28, no. 6, 2014, pages 659 - 69
CONDUIT, P.T. ET AL., DEV CELL, vol. 28, 2014, pages 659 - 669
CROOKE, ANN. REV. PHARMACOL. TOXICOL., vol. 32, 1992, pages 329 - 376
DE CARCER, G. ET AL., NAT. MED., vol. 23, 2017, pages 964 - 974
DICK, S. A.EPELMAN, S, CIRCULATION RESEARCH, 2016
DORETH CHRISTIAN: "The PLK1 and NLRP3 interactome in NLRP3 inflammasome activation", 1 June 2021 (2021-06-01), University of Cambridge, XP093214296, Retrieved from the Internet <URL:https://www.repository.cam.ac.uk/items/49adc894-a034-47fe-976f-331490d330fc> [retrieved on 20241011], DOI: https://doi.org/10.17863/CAM.78122 *
DUEWELL,P. ET AL., NATURE, vol. 464, 2010, pages 1357 - 1361
EL MAADIDI, S. ET AL., S. SCI. REP., 2019
ELBASHIR, S. M. ET AL., GENES & DEVELOPMENT, vol. 15, 2001, pages 188 - 200
ELBASHIR, S. M. ET AL., NATURE, vol. 411, 2001, pages 494 - 498
EUR J CANCER, vol. 69, 2016, pages S117
EVERETT, B. M. ET AL., CIRCULATION, 2019
FELDMAN ET AL., METHODS MOL BIOL., vol. 1180, 2014, pages 31 - 43
FIRAT-KARALAR EN ET AL., METHODS CELL BIOL., vol. 129, 2015, pages 153 - 70
FIRE A. ET AL., NATURE, vol. 391, 1998
GAVILAN ET AL., EMBO REP., vol. 19, no. 11, 2018
GRAILER ET AL., J IMMUNOL., vol. 192, no. 12, 2014, pages 5974 - 83
GUARDA, G. ET AL., NATURE, vol. 460, no. 7245, 2009, pages 269 - 273
HAREN ET AL., PIOS ONE, vol. 4, no. 6, 2009, pages 5976
HAREN, L. ET AL., PLOS ONE, vol. 4, 2009, pages 5976
HARTUPEE, J.MANN, D. L., NATURE REVIEWS CARDIOLOGY, 2016
HE, Y. ET AL., NATURE, vol. 530, no. 7590, 2016, pages 354 - 357
HE, Y. ET AL., TRENDS BIOCHEM SCI, vol. 41, 2016, pages 1012 - 1021
HENEKA, M.T. ET AL., NATURE, vol. 502, 2013, pages 674 - 678
HU ET AL., PLOS ONE., vol. 8, no. 10, 2013, pages 78832
HU, J. ET AL., PLOS ONE, vol. 8, 2013, pages 78832
HU, J. ET AL., PLOS ONE, vol. 8, no. 10, 2013, pages 78832
ITO M ET AL., NAT COMMUN., vol. 6, 2015, pages 7360
JOURDAN, T. ET AL., NAT MED, vol. 19, 2013, pages 1132 - 1140
KEPPNER S ET AL., CHEMMEDCHEM., vol. 4, no. 11, 2009, pages 1806 - 9
KHARE, S. ET AL., IMMUNITY, vol. 36, no. 3, 2012, pages 464 - 476
KIM, J. ET AL., NAT COMMUN, vol. 6, 2015, pages 10076
KISHI, K. ET AL., MOL CELL BIOL, vol. 29, 2009, pages 3134 - 3150
KONG D ET AL., J CELL BIOL., vol. 206, no. 7, 2014, pages 855 - 65
KONG, D. ET AL., J CELL BIOL, vol. 206, 2014, pages 855 - 865
LATZ, E. ET AL., NAT REV IMMUNOL, vol. 13, 2013, pages 397 - 411
LEDERMANN J.A. ET AL., INT. J. CANCER, vol. 47, 1991, pages 659 - 664
LENS, S.M. ET AL., NAT REV CANCER, vol. 10, 2010, pages 825 - 841
LI, X. ET AL., NAT COMMUN, vol. 8, 2017, pages 15986
LI, X. ET AL., NAT. COMMUN., 2017
LIN YC ET AL., J LEUKOC BIOL., vol. 97, no. 5, 2015, pages 825 - 35
LIU DONGLING ET AL: "Role of NLRP3 inflammasome in the pathogenesis of cardiovascular diseases", BASIC RESEARCH IN CARDIOLOGY, SPRINGER BERLIN HEIDELBERG, BERLIN/HEIDELBERG, vol. 113, no. 1, 9 December 2017 (2017-12-09), pages 1 - 14, XP036391685, ISSN: 0300-8428, [retrieved on 20171209], DOI: 10.1007/S00395-017-0663-9 *
LIU X ET AL., MBIO, vol. 13, no. 3, 2022, pages 0068322
LIU, J. ET AL., CLIN. MED. INSIGHTS CARDIOL., 2016
LU ET AL., IMMUNITY., vol. 52, no. 5, 2020, pages 782 - 93
MAEHARA ET AL., FASEB J., vol. 34, no. 11, 2020, pages 15197 - 207
MAGUPALLI ET AL., SCIENCE, vol. 2020, no. 6510, pages 369
MAHEN R ET AL., PROC NATL ACAD SCI U S A., vol. 108, no. 22, 2011, pages 9310 - 5
MAN, S.M. ET AL., PNAS, vol. 111, 2014, pages 7403 - 7408
MANN, D, L CIRCULATION RESEARCH, 2015
MARTINON, F. ET AL., NATURE, vol. 440, no. 7081, 2006, pages 237 - 241
MCINNES C ET AL., NAT CHEM BIOL., vol. 2, no. 11, 2006, pages 608 - 17
MISAWA, T. ET AL., NAT IMMUNOL, vol. 14, 2013, pages 454 - 460
MOREAU ET AL., CANCER RES., vol. 77, no. 13, pages S4178
MURAKAMI ET AL., ARTHRITIS RHEUM., vol. 54, no. 2, 2006, pages 455 - 62
MURAKAMI, Y. ET AL., ARTHRITIS AND RHEUMATISM, vol. 54, 2006, pages 455 - 462
MURUGAN ET AL., MOL CELLS, vol. 32, no. 3, 2011, pages 209 - 220
NASCIMENTO, D. S. ET AL., PLOS ONE, 2011
NORMANDIN ET AL., SCI REP, vol. 6, 2016, pages 37581
OAKLEY, B.R. ET AL., MOL BIOL CELL, vol. 26, 2015, pages 2957 - 2962
OKAMOTO ET AL., AM J PATHOL., vol. 191, no. 9, 2021, pages 1526 - 36
OZAKI, Y. ET AL., MOL CELL, vol. 47, no. 5, 2012, pages 694 - 706
PEYMANULMAN, CHEMICAL REVIEWS, vol. 90, 1990, pages 543 - 584
RAJAMAKI ET AL., PLOS ONE, vol. 5, no. 7, 2010, pages 11765
RAJAMAKI, K. ET AL., PLOS ONE, vol. 5, 2010, pages 11765
RAMANATHAN M ET AL., NAT METHODS., vol. 15, no. 3, 2018, pages 207 - 12
RAMANI A ET AL., CELL REP., vol. 25, no. 13, 2018, pages 3618 - 30
RAMANI, A. ET AL., CELL REP, vol. 25, 2018, pages 3618 - 3630
ROSENBERG ET AL., NEW ENG. J. OF MED., vol. 319, 1988, pages 1676
ROUX KJ ET AL., J CELL BIOL., vol. 196, no. 6, 2012, pages 801 - 10
RUDOLPH D ET AL., CLIN CANCER RES., vol. 15, no. 9, 2009, pages 3094 - 102
RUDOLPH, D. ET AL., CLIN CANCER RES, vol. 15, 2009, pages 3094 - 3102
SAMIR P ET AL., NATURE, vol. 573, no. 7775, 2019, pages 590 - 4
SCHNELLBAECHER A ET AL., BIOTECHNOL BIOENG., vol. 116, no. 6, 2019, pages 1537 - 55
SCHOPP ET AL., NAT COMMUN., vol. 8, 2017, pages 15690
SDELCI, S. ET AL., CURR BIOL, vol. 22, 2012, pages 1516 - 1523
SEIDI ET AL., ELIFE., 2018, pages 7
SESTER DP ET AL., CURR PROTOC IMMUNOL., vol. 114, 2016
SESTER DP ET AL., J IMMUNOL., vol. 194, no. 1, 2015, pages 455 - 62
SESTER, D.P. ET AL., CURR PROTOC IMMUNOL, vol. 114, 2016
SESTER, D.P. ET AL., J IMMUNOL, vol. 194, 2015, pages 455 - 462
SHENOY AR ET AL., SCIENCE, vol. 336, no. 6080, 2012, pages 481 - 5
SHI, H. ET AL., NAT IMMUNOL, vol. 17, 2016, pages 250 - 258
SODERBERG, O. ET AL., METHODS, vol. 45, no. 3, 2008, pages 227 - 232
STREBHARDT K., NAT REV DRUG DISCOV., vol. 9, no. 8, 2010, pages 643 - 60
STREBHARDT, K., NAT REV DRUG DISCOV, vol. 9, 2010, pages 643 - 660
STROWIG, T. ET AL., NATURE, vol. 481, no. 7413, 2012, pages 278 - 286
STUTZ A ET AL., METHODS MOL BIOL., vol. 1040, 2013, pages 91 - 101
STUTZ, A. ET AL., METHODS MOL BIOL, vol. 1040, 2013, pages 91 - 101
SUTTERWALA, F.S. ET AL., IMMUNITY, vol. 24, no. 3, 2006, pages 317 - 327
SWANSON, K. V., NATURE REVIEWS IMMUNOLOGY, 2019
TOLDO, S.ABBATE, A., NATURE REVIEWS CARDIOLOGY, 2018
VAN VUGT, M. A. T. M.MEDEMA, R. H., ONCOGENE, 2005
VENTURA, A. ET AL., NATURE, vol. 445, no. 7128, 2007, pages 661 - 665
VITOUR ET AL., J BIOL CHEM., vol. 284, no. 33, 2009, pages 21797 - 809
VITOUR, D. ET AL., J BIOL CHEM, vol. 284, 2009, pages 21797 - 21809
VITOUR, D. ET AL., J. BIOL. CHEM., 2009
VOINNETBAULCOMBE, NATURE, vol. 389, 1997, pages 553
WACHOWICZ, P. ET AL., BIOESSAYS, vol. 38, 2016, pages S96 - S106
WEIBRECHT I ET AL., EXPERT REV PROTEOMICS., vol. 7, no. 3, 2010, pages 401 - 9
WEIBRECHT, I. ET AL., EXPERT REV PROTEOMICS, vol. 7, 2010, pages 401 - 409
WHITFIELD, M.L. ET AL., MOL BIOL CELL, vol. 13, 2002, pages 1977 - 2000
WOODRUFF, J.B. ET AL., PHILOS TRANS R SOC LOND B BIOL SCI, vol. 369, 2014
YANG, X. ET AL., EMBO J., vol. 39, no. 2, 2020, pages 102201
YAO, C. ET AL., CIRCULATION, vol. 138, 2018, pages 2227 - 2242
ZENG, J. ET AL., EBIOMEDICINE, vol. 75, 2021, pages 103803
ZHONG, Z. ET AL., NATURE, vol. 560, 2018, pages 198 - 203
ZHOU ET AL., BIO PROTOC., vol. 7, no. 10, 2017
ZITOUNI, S. ET AL., NATURE REVIEWS MOLECULAR CELL BIOLOGY, vol. 15, 2014, pages 433 - 452
ZUBA-SURMA E.K. ET AL., FOLIA HISTOCHEM CYTOBIOL, vol. 45, 2007, pages 279 - 290

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