WO2020094613A1 - Nod2 inhibitors for the treatment of hereditary periodic fevers - Google Patents

Nod2 inhibitors for the treatment of hereditary periodic fevers Download PDF

Info

Publication number
WO2020094613A1
WO2020094613A1 PCT/EP2019/080183 EP2019080183W WO2020094613A1 WO 2020094613 A1 WO2020094613 A1 WO 2020094613A1 EP 2019080183 W EP2019080183 W EP 2019080183W WO 2020094613 A1 WO2020094613 A1 WO 2020094613A1
Authority
WO
WIPO (PCT)
Prior art keywords
methyl
methyloxy
fluoro
nod2
phenyl
Prior art date
Application number
PCT/EP2019/080183
Other languages
French (fr)
Inventor
Mathias Chamaillard
Lionel POULIN
Sylvain NORMAND
Thomas Alexander KUFER
Original Assignee
INSERM (Institut National de la Santé et de la Recherche Médicale)
Université de Lille
Institut Pasteur De Lille
Centre National De La Recherche Scientifique (Cnrs)
University Of Hohenheim
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by INSERM (Institut National de la Santé et de la Recherche Médicale), Université de Lille, Institut Pasteur De Lille, Centre National De La Recherche Scientifique (Cnrs), University Of Hohenheim filed Critical INSERM (Institut National de la Santé et de la Recherche Médicale)
Publication of WO2020094613A1 publication Critical patent/WO2020094613A1/en

Links

Classifications

    • 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
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • 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 and pharmaceutical compositions for the treatment of hereditary periodic fevers.
  • Mutations of the nucleotide-binding oligomerization domain protein 12 are causing a familial cold-induced auto -inflammatory syndrome (referred as FCAS2; OMIM 611762) that belongs to the group of hereditary recurrent fevers 1 .
  • FCAS2 familial cold-induced auto -inflammatory syndrome
  • OMIM 611762 familial cold-induced auto -inflammatory syndrome
  • the aforementioned Mendelian disorders are primarily characterized by recurrent episodes of fever and serosal inflammation (including sterile peritonitis, arthritis and abdominal pains) that may coincide with myalgia and urticarial rash.
  • NLRP12 In contrast to most members of the nucleotide-binding domain leucine-rich repeat proteins (NLR) family, NLRP12 (also known as NALP12, RNO, MONARCH- 1, and PYPAF-7) is thought to play a suppressive role on inflammatory responses. Indeed, overexpression of FCAS2-causing NLRP12 mutations results in unrestrained NF-kB and caspase-l activation 1 2 . Furthermore, the pro-inflammatory cytokine interleukin- 1b (IL- 1 b) is spontaneously secreted by peripheral blood mononuclear cells (PBMCs) from patients carrying NLRP12 mutations in contrast to cells from healthy controls 2 .
  • PBMCs peripheral blood mononuclear cells
  • Enterohemorragic E. coli EHEC
  • enteropathogenic E. coli EHEC
  • Infection by EPEC and EHEC results in the effacement of the brush border microvilli followed by bacterial attachment to the apical plasma membrane of intestinal epithelial cells.
  • Citrobacter rodentium is an extracellular enteric bacterial pathogen that naturally colonizes the caecum and the colon of mice 11 . As is observed in humans, C.
  • rodentium attaches to and colonizes the intestinal epithelium by triggering the development of lesions and infiltration of phagocytic mononuclear cells.
  • protective immunity to C. rodentium involves several Crohn’s disease predisposing genes, among which are the nucleotide-binding oligomerization domain containing protein 2 (encoded by the NOD2 gene) 12 and the autophagy 16-like 1 (encoded by the ATG16L1 gene).
  • NOD2 is a member of the NLR family that is required for local production of the chemokine CCL2 through the recruitment of the serine-threonine kinase RIPK2 (also known as Cardiak) 13 .
  • ATG16L1 was found to interact with NOD2 14 and to interfere with poly-ubiquitination of the serine-threonine kinase RIPK2 15 that is required for activation of nuclear factor kappa-light- chain-enhancer of activated B cells (NF-kB) in response to bacterial muramyl dipeptide (MDP). Consequently, animals that are hypomorphic for ATG16L1 expression showed enhanced Nod2 -mediated protection against C. rodentium 16 .
  • the present invention relates to methods and pharmaceutical compositions for the treatment of hereditary periodic fevers.
  • the present invention is defined by the claims.
  • NLRP12 nucleotide-binding oligomerization domain protein 12
  • MDP bacterial muramyl dipeptide
  • the first object of the present invention relates to a method for treating hereditary periodic fevers in a subject in need thereof comprising administering the subject with a therapeutically effective amount of at least one inhibitor of NOD2-mediated signaling pathway.
  • hereditary periodic fever has its general meaning in the art and in particular refers to a syndrome that is caused by truncating and missense mutations in the gene encoding the Nucleotide-binding oligomerization domain protein 12 (Nlrpl2) as described in Jeru I, Duquesnoy P, Fernandes- Alnemri T, Cochet E, Yu JW, Lackmy-Port-Lis M, et al. Mutations in NALP12 cause hereditary periodic fever syndromes.
  • the hereditary periodic fevers include the NLRPl2-associated hereditary periodic fever and more particularly a familial cold-induced auto -inflammatory syndrome (referred as FCAS2; OMIM 611762).
  • the method of the present invention is particularly suitable for preventing enteric bacterial infections as well as for increasing the subject’s tolerance towards the gut microbiota.
  • NOD2 has its general meaning in the art and refers to the nucleotide-binding oligomerization domain containing 2 protein. NOD2 activates NF-KB upon CARDs (caspase recruitment domains) interaction with the serine-theonine kinase RIPK2 (Receptor interacting protein-2 kinase). NOD2 is a cytoplasmic receptor which play a key role in innate immune surveillance. It recognizes both gram positive and gram negative bacterial pathogens upon sensing of bacterial muramyl dipeptide (MDP).
  • MDP bacterial muramyl dipeptide
  • activity of Nod2 refers to any activity of wild type Nod2. The term is intended to encompass all activities of Nod2 (e.g., including, but not limited to, activating NF-KB, binding to RIP2, and enhancing apoptosis).
  • an“inhibitor of NOD2 mediated pathway” refers to any compound natural or not that is able to inhibit NOD2 activity.
  • the“inhibitor of NOD2 mediated signaling pathway” refers to any compound in the art that interferes with the NOD2 signaling pathway by inhibiting the expression and/or activities of NOD2 and/or expression, phosphorylation and/or kinase activity of RIP2.
  • the inhibitor of NOD2-mediated pathway is a RIPK2 inhibitor.
  • RIPK2 has its general meaning in the art and refers to the Receptor interacting protein-2 (RIPK2) kinase, which is also referred to as CARD3, RICK, CARDIAK, or RIP2.
  • RIPK2 is a TKL family serine/threonine protein kinase involved in innate and adaptive immune signaling.
  • RIPK2 kinase is composed of an N-terminal kinase domain and a C-terminal caspase-recruitment domain (CARD) linked via an intermediate (IM) region ((1998) J. Biol. Chem. 273, 12296-12300; (1998) Current Biology 8, 885-889; and (1998) J. Biol. Chem.
  • a“RIPK2 inhibitor” refers to any compound natural or not which is capable of inhibiting the activity of RIPK2, in particular RIPK2 kinase activity.
  • RIPK2 inhibitors are well known in the art.
  • the term encompasses any RIPK2 inhibitor that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition or down-regulation of a biological activity associated with activation of the RIPK2.
  • the term also encompasses inhibitor of expression.
  • the RIPK2 inhibitor is selective over the other kinases.
  • RIPK2 inhibition of the compounds may be determined using various methods well known in the art.
  • the skilled man may use any commercially available RIPK2 kinase assay (see for example the RIPK2 assay commercially available from Promega: ADP - GloTM Kinase Assay is a luminescent kinase assay that measures ADP formed from a kinase reaction.
  • Typical assays are also described in WO2011123609, WO2011120025, WO2011120026, WO2011140442, W02012021580, W02012122011, and WO2013025958.
  • the RIPK2 inhibitor is a small organic molecule.
  • the RIPK2 inhibitor is DCAM-253 (2-dialkylamino-9- indazolyl-purine) that is disclosed in Yun Zhao oo Hye Song, Alfred M Ajami, and Hans- Christian ReineckerA New RIPK2 Kinase Inhibitor for the Treatment of Intestinal Inflammation The Journal of Immunology, 2012, 188, 169.5
  • the RIPK2 inhibitor is selected from the group consisting of compounds described in the International Patent Publications: WO2011120025, WO2011120026, WO2011123609, WO2011140442, W02012021580, W02012122011, WO2013025958, and WO2014043437, WO2014043446.
  • the RIPK2 inhibitor is selected from the group consisting of 4- amino-quinolines as described in WO2011140442.
  • the RIPK2 inhibitor is selected from the group consisting of:
  • the RIPK2 inhibitor is selected from the group consisting of N- pyrazolyl, N-quinolyl amines as described in W02012021580.
  • the RIPK2 is selected from the group consisting of:
  • the RIPK2 inhibitor is selected from the group consisting of imidazolyl- imidazoles as described in WO 2011123609.
  • the RIPK2 inhibitor is selected from the group consisting of: 2-[2-(4-chlorophenyl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-N-[2-(4- morpholinyl)ethyl] - 1 H-benzimidazo le-5 -carboxamide,
  • the RIPK2 inhibitor is selected from the group consisting of indazolyl-pyrimidines as described in WO2011120025.
  • the RIPK2 inhibitor is selected from the group consisting of:
  • the RIPK2 inhibitor is selected from the group consisting of amino-quinolines as described in W02012122011.
  • the RIPK2 inhibitor is selected from the group consisting of:
  • the RIPK2 inhibitor is selected from the group consisting of amino quinazolines as described in WO 2013025958.
  • the RIPK2 inhibitor is selected from the group consisting of:
  • the RIPK2 inhibitor is selected from the group consisting of aminoquinolines as described in WO2014043437.
  • the RIPK2 inhibitor is selected from the group consisting of:
  • the RIPK2 inhibitor is selected from the group consisting of amino quinazo lines as described in WO2013025958.
  • the RIPK2 inhibitor is 2- ((4-(benzo [djthiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7-yl)oxy)ethano 1 and is having the following structure:
  • the RIPK2 inhibitor is an inhibitor of RIPK2 expression.
  • An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.
  • said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme.
  • anti- sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of RIPK2 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of RIPK2, and thus activity, in a cell.
  • RIPK2 can be synthesized, e.g., by conventional phosphodiester techniques.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
  • Small inhibitory RNAs siRNAs
  • siRNAs can also function as inhibitors of expression for use in the present invention.
  • RIPK2 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that RIPK2 gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference or RNAi
  • Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing RIPK2.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and R A virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus
  • adenovirus adeno-associated virus
  • the RIPK2 inhibitor is administered to the subject in a therapeutically effective amount.
  • a therapeutically effective amount is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the active ingredient of the present invention e.g. RIPK2 inhibitor
  • pharmaceutically acceptable excipients e.g. RIPK2 inhibitor
  • sustained-release matrices such as biodegradable polymers
  • pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • a further aspect of the invention relates to a method for screening a plurality of test substances useful for the treatment of hereditary periodic fevers in a subject in need thereof comprising the steps consisting of (a) testing each of the test substances for its ability to inhibit NOD2 mediated pathway and (b) and positively selecting the test substances capable of inhibiting the NOD2 mediated pathway.
  • the screening method of the present invention comprises the step of (i) providing a RIPK2 protein; (ii) contacting the RIPK2 protein with a test substance wherein the substance is expected to inhibit the phosphorylation or kinase activity of the RIPK2 protein; and (iii) selecting a test substance as a candidate that decreases the phosphorylation level or the kinase activity of RIPK2 in comparison to a negative control that is not contacted with a test substance.
  • the screening method of the present invention comprises the steps of i) bringing into contact the test substance to be tested with a mixture of a first RIPK2 protein (2) a second NOD2 protein, ii) determining the ability of said test substance to inhibit the binding between the RIPK2 protein and the NOD2 protein and iii) positively selected the test substance that is capable to inhibit the binding between the RIPK2 protein and the NOD2 protein.
  • RIPK2 proteins come from various sources and sequences in the art may be used for the present disclosure as long as it contains a kinases activity.
  • the sequence of RIPK2 is known in the art, for example as NCBI reference NO. NP— 003812.1. In one embodiment, a full or partial length of RIPK2 can be used.
  • RIPK2 proteins are provided as a cell that endogenously or exogenously expressing the protein.
  • mammalian cells are prepared to express the protein of interest such as RIPK2 through a transient or stable transfection or cells that endogenously express the protein of interest may be used.
  • Cells endogenously expressing RIPK2 may include but is not limited to, macrophages, dendritic cells, neutrophils and epithelial cells, which may be obtained from various organs for example such as peritoneal cavity of a mouse.
  • the cells obtained may be cultured in a cell culture dish and treated with a test substance for a certain period time in a suitable medium, from which the whole proteins are extracted and tested/detected for kinase activity of RIPK2 protein.
  • a suitable medium from which the whole proteins are extracted and tested/detected for kinase activity of RIPK2 protein.
  • established cell lines may be used, in which case the cells are transfected with a plasmid expressing RIPK2.
  • the example of such cells include but is not limited to 293, 293T or 293 A (Graham F L, Smiley J, Russell W C, Naim R (July 1977).“Characteristics of a human cell line transformed by DNA from human adenovirus type 5”. J. Gen. Virol.
  • test substance refers generally to a material that is expected to decrease, reduce, suppress or inhibit the kinase activity of RIPK2 or its phosphorylation or to interfere the interaction between RIPK2 and NOD2, which include small molecules, high molecular weight molecules, mixture of compounds such as natural extracts or cell or tissue culture products, biological material such as proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamer, RNAzymes and DNAzymes, or glucose and lipids, but is not limited thereto.
  • the test substances may be polypeptides having amino acid residues of below 20, particularly 6, 10, 12, 20 aa or above 20 such as 50aa.
  • synthetic chemical library may be obtained from Maybridge Chemical Co. (UK), Comgenex(USA), Brandon Asociates(USA), Microsource(USA) and Sigma-Aldrich(USA).
  • the chemical library of natural origin may be obtained from Pan Laboratories (USA) and MycoSearch(USA).
  • Further test substances may be obtained by various combinatorial library construction methods known in the art including for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries.
  • Test substance of a library may be composed of peptides, peptoides, circular or liner oligomeric compounds, template based compounds such as benzodiazepine, hydantoin, biaryls, carbocyclic and polycyclic compounds such as naphthalene, phenothiazine, acridine, steroids and the like, carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocyclic compounds such as triazine, indole, thiazolidine and the like, but does not limited thereto.
  • template based compounds such as benzodiazepine, hydantoin, biaryls
  • carbocyclic and polycyclic compounds such as naphthalene, phenothiazine, acridine, steroids and the like
  • carbohydrate and amino acid derivatives dihydropyridine
  • benzhydryl and heterocyclic compounds such as triazine, indole, thiazolidine and
  • FIGURE
  • Figure 1 Oral (but not systemic) route of administration of 2-((4- (benzo[d]thiazol-5-ylamino)-6-(tert-butylsulfonyl)quinazolin-7-yl)oxy)ethanol
  • mice protects mice against lethal endotoxin shock.
  • Figure 2 The compound 21 inhibits cytokine secretion in response to either NOD2 (eg. bacterial muramyl dipeptide) or NODI (eg. FK565) agonist.
  • NOD2 eg. bacterial muramyl dipeptide
  • NODI eg. FK565
  • A Interleukin- 1 beta secretion by peripheral blood monocytes in response to active (eg. MDP) and inactive (eg. MDP-DD) bacterial muramyl dipeptide and lipopolysaccharide (LPS).
  • B Interleukin-6 and
  • C Interleukin-8 secretion by Beas2B epithelial cells in response to FK565. Cytokine levels were determined by specific ELISA
  • mice Mr/U2-deficient mice (. Nlrpl2 ' ) mice were generated through homologous recombination by using the Lex-l ES cells that are derived from the l29SvEvBrd strain (not shown).
  • a gene-targeting vector with a neomycin-resistance cassette was constructed to replace the first two exons of Nlrpl2. The latter is required to encode the Pyrin domain of NLRP12, which is essential for recruiting ASC and subsequently for activating Caspase-l . Genotyping of positive ES clones was accomplished by Southern blotting analysis.
  • Nlrpl2 ' mice were produced at the expected Mendelian ratio by crossing heterozygous animals and were crossed with AGi/2-dcficicnt mice 12 for generating animals that are deficient for both Nod2 and Nlrpl2. Genotyping of mouse tail DNA was performed to confirm the presence of the wild-type and/or targeted alleles (data not shown). The absence of Nlrpl2 mRNA in Nlrpl2 ' animals was confirmed by quantitative reverse-transcriptase (RT)-PCR (data not shown). r/?72-deficient (. Nlrpl2 ' ) mice were backcrossed onto a C57BL6/J background.
  • RT reverse-transcriptase
  • mice Bacterial infection. Age and sex-matched mice were orally inoculated with ⁇ l x 10 9 CFU of either C. rodentium strain DBS 100 or kanamycin-resistant C. rodentium strain DBS 120 for CFU counting in feces (kindly provided by D. Schauer, Massachusetts Institute of Technology). Histological scoring of inflammatory cells infiltration and of crypt length damage was blindly performed on hematoxylin and eosin (H&E) stained sections by two investigators.
  • H&E hematoxylin and eosin
  • mice underwent a lethal total- body irradiation. Twenty- four hours post-irradiation, mice received intravenously 5 x 10 5 fresh bone marrow cells. Blood was collected in heparin-containing tubes 7-8 weeks after bone- marrow transplantation and reconstitution efficiency was checked by flow cytometry using a FACS Canto II (BD biosciences) after cell staining by PE- conjugated anti-CD45. l (A20) and FITC- conjugated anti-CD45.2 (104) from BD biosciences. Two months after bone-marrow transplantation, the colonization resistance towards C. rodentium and the waterfall-shaped flow cytometric distribution of monocyte descendants was analyzed within the colon of chimeric mice.
  • mice Male mice (8-10 weeks old) were injected intraperitoneally with a non- lethal dose of highly purified LPS (10 mg/kg of highly purified E. coli 0l l l :B4 purchased from Invivogen) 24 hours before a secondary challenge with murabutide at 10 mg/kg (Invivogen).
  • mice were challenged with a lethal dose of highly purified LPS as a model of acute endotoxin septic shock (54 mg/kg of highly purified E. coli 0l l l :B4 purchased from Invivogen). Mice were monitored twice daily over a 6-day period.
  • mice The morphology of recruited cells within the peritoneum of mice was determined by cytological examination after centrifugation on glass microscope slides (Cytospin; Shandon), fixation and staining according to the manufacturer's recommendations (Diflf; Dade Behring Inc.). For cytokine measurements, serum was taken at 90’, 180’, 360’ and 540’ after secondary MDP challenge.
  • HEK293T cells were transfected with the indicated constructs using Lipofectamine2000 (Invitrogen), as indicated in the manufacturers’ protocol.
  • Cells were harvested in RIPA buffer 24h post transfection (150 mM NaCl, 50 mM Tris pH 7.4, 1% Triton X-100, 0.1% SDS, 0.5% Na-deoxycholate) containing phosphatase inhibitors (20 M b-glycerophosphate, 5 mM NaF, 100 mM Na3V04) and protease inhibitors (Complete protease inhibitor cocktail with EDTA; Roche).
  • the lysate was cleared 20 min at 14,000 g for 20 mins and FLAG-tagged NOD2 was subsequently precipitated for 3h at 4°C using anti- FLAG M2 agarose (Sigma-Aldrich). Proteins were separated by Laemmli SDS-PAGE and visualized using either anti- FLAG M2 antibody (Stratagene, 1 :1,000) or anti-MYC antibody (Roche, 1 :1,000).
  • NOD2 and NLRP12 knockout THP-1 cells lines using CRISPR/Cas9 gene editing were deeply examined; a common translational start for all the reported iso forms were selected and used to potential KO CRISPR guide RNA pairs.
  • the gRNAs were subsequently identified using the sanger centre CRISPR webtool (http://www.sanger.ac.uk/htgt/wge/find_crisprs).
  • the chosen guide RNA were designed to cut as far upstream as possible to generate indels in the region containing the ATG start codon; an additional G were added to the 5' end of the guides to maximize expression from the U6 promoter.
  • Complementary oligos were designed and annealed to yield dsDNA inserts with compatible overhangs to BsmBI-digested vectors (Shalem,Sanjana,et ah, Science, 2014), the sense guides were inserted into the puromycin selectable plasmid LentiCRISPR/Cas9v2 (Addgene #52961).
  • HEK-293FT cells were co -transfected with the appropriate Lentivirus plasmids.
  • THP-l cells were infected with the produced lentivirus harboring the gRNA and the Cas9 protein.
  • the cell pools were subsequently single cell sorted by FACS and clones analyzed for NLRP12 or NOD2 depletion by immunob lotting, when applicable, and sequencing. Briefly, genomic DNA was isolated from cell candidates and the region surrounding the ATG start codon of both NOD2 and NLRP12 were amplified by PCR using a forward and a reverse primer.
  • the resulting PCR products were subcloned into the holding vector pUCl9 and around 10 colonies were picked for each clonal line. Plasmid DNAs were isolated and sent for sequencing with primers M13F and M13R to finally select successful cell line. The absence of the NOD2 or NLRP12 protein was then confirmed, when applicable, by western-blotting.
  • the stable THP-l cell line was generated using a retroviral system.
  • the Myc-BirA*-NOD2 was constructed using the pLXN-retro viral vector (ClonTech,USA).
  • the retrovirus vector was transfected into HEK293-FT cell line for the production of viruses.
  • the viruses were harvested after 72h and the THP-l cells were infected and subsequently selected using 250pg/mL of Geneticin® (LifeTechnologies, USA). After 14 days, the surviving cells were cell sorted and individual clones were grown for 2 months. Usually, the individual clones were tested for the proper expression of Myc-BirA*-NOD2 using the ⁇ z-NOD2 (2D9) monoclonal anti-body (SantaCruz, USA).
  • Luciferase Reporter Assays The NLRP12 coding sequence was inserted into the pcDNA3.l vector and site-directed mutagenesis was performed to generate the plasmids expressing the Arg284X and Arg352Cys mutations. 3xl0 4 HEK293T cells were seeded in a 96-well format directly prior transfection with the indicated amounts of plasmid using XtremeGene9 (Roche) as indicated in the manufacturers’ protocol.
  • luciferase activity was measured using a standard plate luminometer (Berthold Instruments). Luciferase activity was normalized as a ratio to b-galactosidase activity and standard deviation (SD) was calculated from triplets.
  • cycloheximide (Sigma-Aldrich) for 2 hours was performed on 5 million PBMCs that were isolated from fresh blood of one of the affected patient using Pancoll gradient centrifugation (BioTech) and cultured in RPMI 1640 medium. Flow cytometry analysis. Cells were stained and analyzed using a FACS LSRFortessa system (BD Biosciences). Dead cells were excluded with the LIVE/DEAD Fixable Violet Dead Cell staining kit (Life technologies). Lineage positive cells were excluded using the PerCP5.5 -conjugated anti-CD3 (17A2), anti-NKl.l(PKl36), anti- CDl9(6D5), anti-Ly6G (1A8) (Biolegend).
  • PerCP-conjugated anti-CCR3 (83103) added to the lineage staining to exclude eosinophils was from R&D. Allophycocyanin-Cy7-conjugated anti-CDl lb (Ml/70), PerCP5.5-conjugated anti-Ly6G (1A8), Brilliant violet 5l0-conjugated anti-MHC Class II (I-A/I-E) (M5/114.15.2) and FITC-conjugated and Alexa Fluor 700- conjugated anti-Ly6C (AL21) were from BD Pharmingen.
  • Cytokine measurement Cytokine levels were determined by ELISA kits, according to protocols provided by R&D Systems.
  • RNA/a/er® RNA/a/er®
  • the quality of the extracted RNA was confirmed by Agilent 2100 Bioanalyzer using RNA Nano 6000 (Agilent Technologies).
  • the 4x44K Whole Mouse Genome Oligo Microarrays was used to determine the gene expression profile of two biological replicates.
  • RNA was reverse-transcribed with the High- Capacity cDNA Archive kit (Applied Biosystems), according to the manufacturer’s instructions.
  • the resulting cDNA (equivalent to 5ng of total RNA) was amplified using the SYBR Green real-time PCR kit and detected on a Stratagene Mx3005P (Agilent Technologies).
  • RT-PCR was performed with the forward and reverse primers (sequences available upon request) that were designed using Primer express software, version 1.0 (Applied Biosystems, Foster City, CA).
  • a DNA melting curve analysis was carried out in order to confirm the presence of a single and specific amplicon. Actb was used as an internal reference gene in order to normalize the transcript levels.
  • Relative mRNA levels (2 DDa ) were determined by comparing (a) the PCR cycle thresholds (Ct) for the gene of interest and Actb (ACt) and (b) ACt values for treated and control groups (AACt).
  • RNA extraction from whole peripheral blood from controls and FCAS2 patients that were collected in PAXgene tubes was performed using PAXgene Blood RNA Kit (QIAGEN) following the manufacturer’s instructions.
  • RNA extraction from PBMCs was performed using the RNeasy Mini Kit (Qiagen) including DNase treatment according to the manufacturer’s instructions.
  • One pg of RNA was reversed transcribed in the presence of 2.5 mM of oligo-dT using the Reverse Transcriptor kit (Roche) following the manufacturer’s instructions.
  • RNA samples 75 ng were amplified using Q5 High-Fidelity 2X Master Mix (New England BioLabs).
  • Forward and reverse primers used in PCR amplification are located in the 5’UTR and 3’UTR of NLRP12 cDNA respectively (Sequences available upon request), which can amplify the two alleles of NLRP12 in the patients and the healthy donor.
  • Exon 3 of NLRP12 cDNA was sequenced with the Big Dye Terminator sequencing kit (Applied Biosystems) using two different primers (Table 1), and run on an ABI 3730 c 1 automated sequencer. Sequences were analyzed with SeqScape software (Applied Biosystems).
  • N-ethylmaleimide (Thermofisher scientific, USA) was added to the RIPA buffer to lyse the MGl32-treated samples.
  • a SDS gel was run and membranes were blotted against the NOD2-monoclonal antibody (2D9) (SantaCruz, USA) and RIPK2 (Cell Signalling Technology, USA) and Actin or Tubulin as a controls.
  • NOD2 ubiquitination was assessed using the ⁇ z-poly-K48 antibody (Millipore, USA).
  • the THP-l Myc-BirA*-NOD2 stable cell line was grown and a total of lxlO 7 of cells were used for these experiments.
  • Cells were incubated for 2 hours with MDP ( 1 Oiig/mL). After the 2 hours incubation with and without proteasome inhibitor MG132 (12.5mM), the cells were placed on ice, washed twice with PBS, spun down and incubated in lmL of lysis buffer (50mM HEPES pH7.5; l50mM NaCl; lx complete-EDTA free protease inhibitor; IX phosphatase inhibitor; 1% iGPAL and lmM PMSF).
  • lysis buffer 50mM HEPES pH7.5; l50mM NaCl; lx complete-EDTA free protease inhibitor; IX phosphatase inhibitor; 1% iGPAL and lmM PMSF.
  • NLRP12 interacts with NOD2 through a linker-region proximal to the nucleotide-binding domain that is required for ATP binding.
  • NLRP12 may potentially interact with both Caspase-activating recruitment domains (CARDs) of NOD2 but not that of NOD1 17
  • CARDs Caspase-activating recruitment domains
  • NLRP12 may promote MDP tolerance by dissociating the NOD2-HSP90 complex, which is required for NF-KB activation in response to bacteria 16 .
  • THP-l cells stably expressing the fusion protein Myc-BirA*-NOD2.
  • yeast two-hybrid screen data 17 we confirmed the NOD2-NLRP12 interaction in monocytic cell line when specifically inhibiting the proteasome degradation of NOD2 that is induced in response to MDP (data not shown).
  • NOD2-NLRP12 complex is not observed in response to bacterial lipopolysaccharide (LPS) that is not sensed by either NOD2 nor NLRP12 (data not shown).
  • LPS bacterial lipopolysaccharide
  • HEK-293T cells were next transfected with plasmids transiently expressing FLAG-tagged NOD2 and Myc-tagged NLRP12 (data not shown).
  • Overexpression of full- length FLAG-tagged NOD2 together with Myc-tagged NFRP12 resulted in an interaction with RIPK2, which was underrepresented in the complex at high NFRP12 concentration as shown by immunoprecipitation using an anti-FFAG antibody (data not shown).
  • NLRP12 dominantly suppress MDP-induced NF-kB activation by promoting degradation of the NOD2/RIPK2 complex.
  • NFRP12 As a potential checkpoint blocker of NOD2 signaling in monocytes, we examined the influence of NFPR12 on the stability and the activity of the NOD2/RIPK2 complex. Co-immunoprecipitation experiments revealed that NFRP12 expression promotes poly-ubiquitination of the NOD2/RIPK2 complex in HEK- 293T cells (data not shown). In contrast, the ubiquitination status of NOD1 was not influenced by full-length NFRP12 (data not shown).
  • NLRP12 deficiency impairs MDP tolerance in mice.
  • NLRP12 gene expression is observed in patients with septic shock 20 , suggesting a potential feed-back regulatory loop on NLRP12 function during sepsis.
  • endotoxin treatment negatively regulates NLRP12 promoter activity in human monocytes through the PR domain-containing 1, with ZNF domain 21 .
  • knocking-down NLRP12 expression may enhance TLR4 signaling in vitro 3
  • Wild-type and Nlrpl2- deficient mice were primed with a non-lethal dose of highly purified LPS from E coli 0111 :B4. As a consequence of a failure to negatively regulate NOD2 signaling, LPS-primed Nlrp 12 -deficient mice were significantly more susceptible to secondary MDP challenge when compared to similarly treated control animals (data not shown).
  • NLRP12 is dispensable for protecting mice against endotoxemia, but rather function as a negative regulator of NOD2 signaling in mice. MDP Tolerance is lost in monocytes that are either deficient for NLRP12 or expressing the NLRP12 mutation that is causing FCAS2.
  • the R284X nonsense mutation failed to inhibit the activation of NF-kB in response to MDP (data not shown) and of the JAK/STAT pathway by the S/T kinase TANK-binding kinase 1 that is commonly referred as TBK-l (data not shown). It coincided with a barely detectable recruitment of HSP90 by such mutation (data not shown) even if this was not related to a failure of the R284X nonsense mutation to interact with NOD2 (data not shown). As a consequence, loss of NLRP12 expression by CRISPR/Cas9 system in human monocytic THP-l cells enhanced secretion of TNF-a in response to MDP when compared to parental cells (data not shown).
  • MDP induced a greater secretion of either tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL-6) by PBMCs from patients bearing the R284X nonsense mutation when compared to control cells (data not shown).
  • TNF-a tumor necrosis factor alpha
  • IL-6 interleukin-6
  • Such lack of MDP tolerance was the consequence of the activation of a surveillance pathway referred to as nonsense-mediated mRNA decay, which can be blocked by cycloheximide (data not shown). This provided a potential explanation for the loss of tolerance to MDP that account for the failure to detect the truncated protein in such mutant cells by western blotting (data not shown).
  • Loss of tolerance in the intestine of 7Wfy /2-deficient mice is caused by type I and/or III interferon downstream of NOD2 signaling.
  • IFI44 interferon-induced protein 44
  • IFIT2 interferon-induced protein with tetratricopeptide repeats 2
  • APOL9a/b Apo lipoprotein L9
  • OF2 2’-5’-Oligoadenylate synthetase 2
  • ISRE Interferon Stimulated Response Elements
  • the transcript level of the above-mentioned differentially expressed genes was primarily enriched in primary IECs isolated from both the colon and the caecum of Nlrp 12 -deficient mice (data not shown).
  • immunohistochemical analysis revealed an enhanced production of either IFIT2 or OAS2 that is essentially restricted to the epithelium of the caecum and proximal colon from Nlrpl2- deficient mice (data not shown). This is in line with the idea that NOD2-mediated inflammasome activation is enhanced by IL-32 25 , which subsequently triggers type I/III IFNs activation 2627 .
  • IFIT2 primarily functions as a downstream effector of the IFN-l receptor 28 that may subsequently influence the outcome of Nlrp 12 -deficient mice in response to endotoxin shock 29 . While IFIT2 is thought to regulate cell death and inflammation 29 , epithelial proliferation is also orchestrated by several additional ISG that were upregulated within the epithelium of Nlrp 12 -deficient mice 30 , such as Apol9a/b 31 .
  • NLRP12 deficiency contributes to a greater colonization resistance against attaching-and-effacing bacterial pathogen through activation of NOD2 signaling in monocytes.
  • C. rodentium 33 34 which is a mouse-restricted model for attaching and effacing (A/E) enteric bacterial-induced diarrhea such as those caused by EPEC and EHEC.
  • C. rodentium colonizes the caecum and the colon of mice through attachment to the epithelium, effacement of microvilli-covered surface and the formation of pedestal-like structure 9 .
  • NLRP12 negatively MDP tolerance by regulating the stability of NOD2/RIPK2 complex
  • rodentium from the gut lumen resulted in a greater bacterial dissemination in the spleen (data not shown). This was associated with spenomegaly (data not shown) and tissue pathology as evidenced respectively by about 47 percent increase in spleen weight (data not shown) and by the enhanced histological score (data not shown) and a 30 percent increase in the crypt length (data not shown).
  • R A was extracted from the caecum of infected mice at day 0 and day 7 post-infection and a genome-wide analysis of the acute transcriptional response to the pathogen was performed (data not shown). As expected, C.
  • rodentium may exploit NLRP12 signaling for limiting the accumulation of monocytes when being recruited at the site of the infection.
  • NLRP12 signaling for limiting the accumulation of monocytes when being recruited at the site of the infection.
  • no change in autophagy induction was observed at either day 7 or 14 post-infection (data not shown) and the monocytes isolated from intestine of ///72-dcficicnt mice showed a similar MHCII expression with a progressive loss of Ly6C marker when compared to that in control mice (data not shown).
  • NOD2 signaling promotes accumulation of phagocytes to the site of infection in A7/y /2-dcficicnt mice, which may subsequently contribute to the improved clearance of C. rodentium 9 .
  • NLRP12 as a checkpoint blocker of NOD2 signaling in monocytes that provides a potential explanation for the recurrent episodes of serosal inflammation (including peritonitis and abdominal pains) in patients bearing non sense mutations in the NLRP12-encoding gene 1 .
  • Such paradigm indicates that disease manifestation in patients with NLRP12 mutations is likely initiated by the influence of some specific interactions with the gut microbiota that are regulated by the Crohn’s disease predisposing NOD2 gene 41 . In mice, this may subsequently account for the colitis-prone changes in the composition of the gut microbiota that are caused by NLRP12 deficiency 40 .
  • the exact role of NLRP12 signaling on NOD2-mediated resilience of the gut microbiota now deserves further experimental studies with littermate controls and co-housed mice.
  • CATERPILLER protein monarch- 1 is an antagonist of toll-like receptor-, tumor necrosis factor alpha-, and Mycobacterium tuberculosis-induced pro-inflammatory signals. The Journal of biological chemistry 280, 39914-39924 (2005).
  • Heat shock protein 90 associates with monarch- 1 and regulates its ability to promote degradation of NF-kappaB- inducing kinase. Journal of immunology 179, 6291-6296 (2007).
  • IL-32 synergizes with nucleotide oligomerization domain (NOD) 1 and NOD2 ligands for IL-lbeta and IL-6 production through a caspase 1 -dependent mechanism. Proceedings of the National Academy of Sciences of the United States of America 102, 16309-16314 (2005).
  • IFIT2 is an effector protein of type I IFN-mediated amplification of lipopolysaccharide (LPS)-induced TNF-alpha secretion and LPS-induced endotoxin shock. Journal of immunology 191, 3913-3921 (2013).
  • LPS lipopolysaccharide
  • IFN-lambda IFN- lambda

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of hereditary periodic fevers. In particular, the present invention relates to a method for treating hereditary periodic fevers in a subject in need thereof comprising administering the subject with a therapeutically effective amount of at least one inhibitor of NOD2-mediated signaling pathway.

Description

NOD2 INHIBITORS FOR THE TREATMENT OF HEREDITARY PERIODIC FEVERS
FIELD OF THE INVENTION:
The present invention relates to methods and pharmaceutical compositions for the treatment of hereditary periodic fevers.
BACKGROUND OF THE INVENTION:
Mutations of the nucleotide-binding oligomerization domain protein 12 (NLRP12) are causing a familial cold-induced auto -inflammatory syndrome (referred as FCAS2; OMIM 611762) that belongs to the group of hereditary recurrent fevers1. The aforementioned Mendelian disorders are primarily characterized by recurrent episodes of fever and serosal inflammation (including sterile peritonitis, arthritis and abdominal pains) that may coincide with myalgia and urticarial rash. In contrast to most members of the nucleotide-binding domain leucine-rich repeat proteins (NLR) family, NLRP12 (also known as NALP12, RNO, MONARCH- 1, and PYPAF-7) is thought to play a suppressive role on inflammatory responses. Indeed, overexpression of FCAS2-causing NLRP12 mutations results in unrestrained NF-kB and caspase-l activation1 2. Furthermore, the pro-inflammatory cytokine interleukin- 1b (IL- 1 b) is spontaneously secreted by peripheral blood mononuclear cells (PBMCs) from patients carrying NLRP12 mutations in contrast to cells from healthy controls2. Besides its interaction with ASC to recruit caspase-l and to form an inflammasome with proteolytic activity against pro-ILI b and pro-ILl83, biochemical studies revealed that NLRP12 also interacts with NF-KB-inducing kinase (NIK) and IL-l receptor-associated kinase 1 (IRAK-l) in human monocytes4 5. However, it is worth noting that neutralizing I LI b signaling by the ILI b-rcccptor antagonist (namely Anakinra or Kineret®) was inefficient in maintaining a clinical response in FCAS2 patients 2. Foremost, genetic ablation of Nlrpl2 renders mice highly susceptible to colitis and colitis-associated colorectal cancer6 7, while being resistant to Salmonellosis8. However, the function of NLRP12 on bacterial tolerance and host defense remains largely unappreciated as mutant animals were also found highly susceptible to infection by a vaccinated strain of Yersinia pestis9. Collectively, these paradigms argue for the need to better understand the complex regulatory mechanisms by which NLRP12 signaling could contribute to bacterial tolerance while being exploited by some bacterial pathogens9. Escherichia coli is a versatile Gram negative commensal that typically colonizes the gastrointestinal tract within a few hours after birth. E. coli successfully exploits several ways to efficiently compete with other microorganisms allowing it to occupy specific niches in the gut. Enterohemorragic E. coli (EHEC) and enteropathogenic E. coli (EPEC) remains an important cause of diarrhea or hemorrhagic colitis in humans worldwide10. Infection by EPEC and EHEC results in the effacement of the brush border microvilli followed by bacterial attachment to the apical plasma membrane of intestinal epithelial cells. Similar to the related EPEC and EHEC, Citrobacter rodentium is an extracellular enteric bacterial pathogen that naturally colonizes the caecum and the colon of mice11. As is observed in humans, C. rodentium attaches to and colonizes the intestinal epithelium by triggering the development of lesions and infiltration of phagocytic mononuclear cells. Of note, protective immunity to C. rodentium involves several Crohn’s disease predisposing genes, among which are the nucleotide-binding oligomerization domain containing protein 2 (encoded by the NOD2 gene)12 and the autophagy 16-like 1 (encoded by the ATG16L1 gene). In contrast to NLRP12, NOD2 is a member of the NLR family that is required for local production of the chemokine CCL2 through the recruitment of the serine-threonine kinase RIPK2 (also known as Cardiak)13. Indeed, a prolonged bacterial shedding in infected .V i/2-dcficicnt mice results at least partially from an impaired recruitment of monocytes12. It is also worth noting that ATG16L1 was found to interact with NOD2 14 and to interfere with poly-ubiquitination of the serine-threonine kinase RIPK215 that is required for activation of nuclear factor kappa-light- chain-enhancer of activated B cells (NF-kB) in response to bacterial muramyl dipeptide (MDP). Consequently, animals that are hypomorphic for ATG16L1 expression showed enhanced Nod2 -mediated protection against C. rodentium 16.
SUMMARY OF THE INVENTION:
The present invention relates to methods and pharmaceutical compositions for the treatment of hereditary periodic fevers. In particular, the present invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION:
Mutations in the nucleotide-binding oligomerization domain protein 12 (NLRP12) are causing recurrent episodes of serosal inflammation through still poorly understood mechanisms. Herein, the inventors show that NLRP12 efficiently sequesters HSP90 and promotes K48-linked ubiquitination and degradation of NOD2 in response to bacterial muramyl dipeptide (MDP). NOD2 directly interacts with a linker-region proximal to the nucleotide-binding domain of NLRP12, whereas blocking proteasome pathway strengthens its endogenous interaction with NOD2 in monocytes. Consequently, the disease-causing NLRP12 R284X mutation fails to repress MDP-induced NF-kappaB activation by escaping from the NOD2/HSP90 complex. While Nlrpl2 deficiency renders septic mice highly susceptible to overt lethal reactions towards MDP, a sustained Nod2-dependent responsiveness to MDP is observed among monocytes lacking Nlrpl2. This loss of tolerance results in greater colonization resistance in the gut as a consequence of Nod2-dependent accumulation of inflammatory mononuclear cells and epithelial induction of several interferon-stimulated genes. The studies unveil an unexpected diverging effect of Nod2 and Nlrpl2 deficiency, suggesting that inhibition of Nod2 signaling represents a target for the treatment of FCAS2 and more generally hereditary periodic fevers.
Thus the first object of the present invention relates to a method for treating hereditary periodic fevers in a subject in need thereof comprising administering the subject with a therapeutically effective amount of at least one inhibitor of NOD2-mediated signaling pathway.
As used herein the term“hereditary periodic fever” has its general meaning in the art and in particular refers to a syndrome that is caused by truncating and missense mutations in the gene encoding the Nucleotide-binding oligomerization domain protein 12 (Nlrpl2) as described in Jeru I, Duquesnoy P, Fernandes- Alnemri T, Cochet E, Yu JW, Lackmy-Port-Lis M, et al. Mutations in NALP12 cause hereditary periodic fever syndromes. Proc Natl Acad Sci U S A 2008, 105(5): 1614-1619; Jeru I, Le Borgne G, Cochet E, Hayrapetyan H, Duquesnoy P, Grateau G, et al. Identification and functional consequences of a recurrent NLRP12 missense mutation in periodic fever syndromes. Arthritis and rheumatism 2011, 63(5): 1459-1464. And Borghini S, Tassi S, Chiesa S, Caroli F, Carta S, Caorsi R, et al. Clinical presentation and pathogenesis of cold-induced autoinflammatory disease in a family with recurrence of an NLRP12 mutation. Arthritis and rheumatism 2011, 63(3): 830-839. Accordingly, the hereditary periodic fevers include the NLRPl2-associated hereditary periodic fever and more particularly a familial cold-induced auto -inflammatory syndrome (referred as FCAS2; OMIM 611762).
In particular, the method of the present invention is particularly suitable for preventing enteric bacterial infections as well as for increasing the subject’s tolerance towards the gut microbiota.
As used herein the term“NOD2” has its general meaning in the art and refers to the nucleotide-binding oligomerization domain containing 2 protein. NOD2 activates NF-KB upon CARDs (caspase recruitment domains) interaction with the serine-theonine kinase RIPK2 (Receptor interacting protein-2 kinase). NOD2 is a cytoplasmic receptor which play a key role in innate immune surveillance. It recognizes both gram positive and gram negative bacterial pathogens upon sensing of bacterial muramyl dipeptide (MDP). As used herein, the term“activity of Nod2” refers to any activity of wild type Nod2. The term is intended to encompass all activities of Nod2 (e.g., including, but not limited to, activating NF-KB, binding to RIP2, and enhancing apoptosis).
Accordingly an“inhibitor of NOD2 mediated pathway” refers to any compound natural or not that is able to inhibit NOD2 activity. In particular, the“inhibitor of NOD2 mediated signaling pathway” refers to any compound in the art that interferes with the NOD2 signaling pathway by inhibiting the expression and/or activities of NOD2 and/or expression, phosphorylation and/or kinase activity of RIP2. Thus, in some embodiments, the inhibitor of NOD2-mediated pathway is a RIPK2 inhibitor.
As used herein, the term“RIPK2” has its general meaning in the art and refers to the Receptor interacting protein-2 (RIPK2) kinase, which is also referred to as CARD3, RICK, CARDIAK, or RIP2. RIPK2 is a TKL family serine/threonine protein kinase involved in innate and adaptive immune signaling. RIPK2 kinase is composed of an N-terminal kinase domain and a C-terminal caspase-recruitment domain (CARD) linked via an intermediate (IM) region ((1998) J. Biol. Chem. 273, 12296-12300; (1998) Current Biology 8, 885-889; and (1998) J. Biol. Chem. 273, 16968-16975). Upon exposure to bacterial muramyl dipeptide, NOD2 oligomerize and the downstream serine-threonine kinase RIPK2 is recruited through homophilic CARD-CARD interactions. Once activated the RIPK2 signaling pathways leads to the activation of the NF-kB transcription factor through the ubiquitination of the IKKg/NEMO subunit of the signalosome (Inohara et al. 2000; Ogura et al. 200lb; Kobayashi et al. 2002; Abbott et al. 2004). Consistently, genetic ablation of either Nod2 or RIPK2 renders mice unresponsive to bacterial muramyl dipeptide.
As used herein, a“RIPK2 inhibitor” refers to any compound natural or not which is capable of inhibiting the activity of RIPK2, in particular RIPK2 kinase activity. RIPK2 inhibitors are well known in the art. The term encompasses any RIPK2 inhibitor that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition or down-regulation of a biological activity associated with activation of the RIPK2. The term also encompasses inhibitor of expression. In some embodiments, the RIPK2 inhibitor is selective over the other kinases. By“selective” it is meant that the inhibition of the selected compound is at least 10- fold, preferably 25-fold, more preferably lOO-fold, and still preferably 300-fold higher than the inhibition of the other kinases. The RIPK2 inhibition of the compounds may be determined using various methods well known in the art. In particular, the skilled man may use any commercially available RIPK2 kinase assay (see for example the RIPK2 assay commercially available from Promega: ADP - Glo™ Kinase Assay is a luminescent kinase assay that measures ADP formed from a kinase reaction. Typical assays are also described in WO2011123609, WO2011120025, WO2011120026, WO2011140442, W02012021580, W02012122011, and WO2013025958. Typically, the RIPK2 inhibitor is a small organic molecule.
In some embodiments, the RIPK2 inhibitor is DCAM-253 (2-dialkylamino-9- indazolyl-purine) that is disclosed in Yun Zhao oo Hye Song, Alfred M Ajami, and Hans- Christian ReineckerA New RIPK2 Kinase Inhibitor for the Treatment of Intestinal Inflammation The Journal of Immunology, 2012, 188, 169.5
In some embodiments, the RIPK2 inhibitor is selected from the group consisting of compounds described in the International Patent Publications: WO2011120025, WO2011120026, WO2011123609, WO2011140442, W02012021580, W02012122011, WO2013025958, and WO2014043437, WO2014043446.
In some embodiments, the RIPK2 inhibitor is selected from the group consisting of 4- amino-quinolines as described in WO2011140442. In particular, the RIPK2 inhibitor is selected from the group consisting of:
- N- 1 ,3-Benzothiazol-5-yl-6-(methylsulfonyl)-4-quinolinamine,
- N-(2-methylphenyl)-6-(methylsulfonyl)-4-quinolinamine,
4-chloro-2- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenol,
4-fluoro-2- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenol,
- N- 1H- 1 ,2,3-benzotriazol-5-yl-6-(methylsulfonyl)-4-quinolinamine,
3-fluoro-4- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenol,
2- chloro-4- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenol,
3- methyl-2- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenol,
4- chloro-3- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenol,
3- chloro-4- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenol,
4- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenol,
2-fluoro-4- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenol,
- N-lH-indol-6-yl-6-(methylsulfonyl)-4-quinolinamine,
5- fluoro-2- {[6-(methylsulfonyl)-4-quinolinyl]amino} phenol
2-chloro-6-methyl-3- {[6-(methylsulfonyl)-4-quinolinyl]amino} phenol, 6- (methylsulfonyl)-N-phenyl-4-quinolinamine,
- N-(2-fluorophenyl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(2,5-difluorophenyl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(2,6-difluorophenyl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(2,4-dichlorophenyl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(2,4-dimethylphenyl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(3,5-difluorophenyl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(5-fluoro-2-methylphenyl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(3-chlorophenyl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(4-fluorophenyl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(4-chlorophenyl)-6-(methylsulfonyl)-4-quinolinamine,
(4-methyl-3- {[6-(methylsulfonyl)-4-quinolinyl]amino} phenyl)methanol,
- N- [4-methyl-3 -(methyloxy)phenyl] -6-(methylsulfonyl)-4-quinolinamine,
(3- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenyl)methanol,
- N- 1 ,3-benzodioxol-4-yl-6-(methylsulfonyl)-4-quinolinamine,
- N-(4- {[(2-chlorophenyl)methyl] oxy}phenyl)-6-(methylsulfonyl)-4-quinolinamine,
- 2-fluoro-5- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenol, 4-fluoro-3- {[6- (methylsulfonyl)-4-quino linyljamino } pheno 1,
2,4-dimethyl-5- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenol,
3-methyl-5- {[6-(methylsulfonyl)-4-quinolinyl]amino} phenol,
- N- 1H- 1 ,2,3-benzotriazol-4-yl-6-(methylsulfonyl)-4-quinolinamine,
5- {[6-(methylsulfonyl)-4-quino linyljamino} - 1 ,3-benzoxathiol-2-one,
- N-(4-methyl-lH-l,2,3-benzotriazol-6-yl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(5-fluoro-lH-l,2,3-benzotriazol-6-yl)-6-(methylsulfonyl)-4-quinolinamine
- N-(5-methyl-lH-l,2,3-benzotriazol-6-yl)-6-(methylsulfonyl)-4-quinolinamine,
- N-l,2,3-benzothiadiazol-5-yl-6-(methylsulfonyl)-4-quinolinamine,
- N- 1 ,2-benzisoxazol-6-yl-6-(methylsulfonyl)-4-quinolinamine,
- N- [2,4-dimethyl-5 -(methyloxy)phenyl] -6-(methylsulfonyl)-4-quino linamine,
6-(methylsulfonyl)-N-[4-(phenyloxy)phenyl]-4-quinolinamine,
- N- [3 -(methyloxy)phenyl] -6-(methylsulfonyl)-4-quino linamine,
2- methyl-3- {[6-(methylsulfonyl)-4-quino linyljamino} phenol,
2-methyl-5-{[6-(methylsulfonyl)-4-quino linyljamino} phenol,
- N- [2-chloro-5 -(methyloxy)phenylj -6-(methylsulfonyl)-4-quino linamine,
- N- [4-fluoro-3 -(methyloxy)phenylj -6-(methylsulfonyl)-4-quino linamine, 3- methyl-4- {[6-(methylsulfonyl)-4-quinolinyl] amino} phenol,
6-(methylsulfonyl)-N-phenyl-4-quinolinamine,
- N- 1 -benzofuran-4-yl-6-(methylsulfonyl)-4-quinolinamine,
- N-[4-bromo-3-(methyloxy)phenyl]-6-(methylsulfonyl)-4-quinolinamine,
2-chloro-5-{[6-(methylsulfonyl)-4-quinolinyl] amino} phenol,
- N- 1 ,3-benzothiazol-6-yl-6-(methylsulfonyl)-4-quinolinamine,
- N-2H-indazo 1-3 -yl-6-(methylsulfonyl)-4-quino linamine,
- N- 1 ,3-benzothiazol-5-yl-6-[(l -methylethyl)sulfonyl]-4-quino linamine,
- N-lH-indazo l-6-yl-6- [(l-methylethyl)sulfonyl] -4-quino linamine,
- N- 1 ,3-benzothiazol-5-yl-6-(ethylsulfonyl)-4-quinolinamine,
6-(ethylsulfonyl)-N- 1 H-indazol-6-yl-4-quino linamine,
6-( (6-[( 1 -methylethyl)sulfonyl]-4-quinolinyl} amino)- 1 H-indazole-5 -carboxamide, 6- [(l-methylethyl)sulfonyl] -N-(7 -methyl- lH-indazol-6-yl)-4-quino linamine,
- N- [4-chloro-3 -(methyloxy)phenyl] -6- [(l-methylethyl) sulfonyl] -4-quino linamine,
6-( (6-[( 1 -methylethyl)sulfonyl]-4-quinolinyl} amino)- 1 H-indazole-5 -carbonitrile,
- N-lH-indazo 1-3 -yl-6- [(l-methylethyl) sulfonyl] -4-quino linamine,
6- [(l-methylethyl)sulfonyl] -N- [5 -(methyloxy)-lH-indazo 1-3 -yl] -4-quino linamine,
- N-(5 -fluoro-lH-indazo 1-3 -yl)-6- [(l-methylethyl)sulfonyl] -4-quino linamine,
- N-(3 -methyl- lH-indazol-6-yl)-6-(methylsulfonyl)-4-quinolinamine,
- N- [4-chloro-3 -(methyloxy)phenyl] -6-(metliylsulfonyl)-4-quino linamine,
- N-(3-fluoro-lH-indazol-6-yl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(4-chloro-lH-indazol-3-yl)-6-(methylsulfonyl)-4-quinolinamine,
- N- [5 -(methyloxy)-lH-indazo 1-3 -yl] -6-(methylsulfonyl)-4-quino linamine,
- N-(5-fluoro-lH-indazol-3-yl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(5-chloro-lH-indazol-3-yl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(6-chloro-lH-indazol-3-yl)-6-(methylsulfonyl)-4-quinolinamine,
- N-lH-indazo l-6-yl-6-(methylsulfonyl)-4-quino linamine,
- N-(4-methyl-l,3-benzothiazol-5-yl)-6-(methylsulfonyl)-4-quino linamine,
- N-(4-bromo- 1 ,3-benzothiazol-5-yl)-6-(methylsulfonyl)-4-quinolinamine,
- N-(4-chloro-l,3-benzothiazol-5-yl)-6-(methylsulfonyl)-4-quino linamine,
- 6- [(1, l-dimethylethyl)sulfonyl] -N-(5 -fluoroA6-[(l, l-dimethylethyl)sulfonyl] -N-(5 - fluoro-6-methyl-lH-pyrazolo[3,4-/7]pyridin-3-yl)-4- quino linamine,
6- [( 1 -methylethyl)sulfonyl]-N-( 1 -methyl- 1 H-indazol-3 -yl)-4-quino linamine,
6-[( 1 , 1 -dimethylethyl)sulfonyl]-N- 1 H-indazol-6-yl-4-quinolinamine, 6- [(1, l-dimethylethyl)sulfonyl] -N-(3 -fluoro-lH-indazol-6-yl)-4-quino linamine,
6- [(1, l-dimethylethyl)sulfonyl] -N-(3 -methyl- lH-indazol-6-yl)-4-quino linamine,
6- [(1, l-dimethylethyl)sulfonyl] -N-lH-indazo 1-3 -yl-4-quino linamine,
6- [(1, l-dimethylethyl)sulfonyl] -N-(5 -fluoro-lH-indazo 1-3 -yl)-4-quino linamine,
- N- [4-chloro-3 -(methyloxy)phenyl] -6- [(1, l-dimethylethyl)sulfonyl] -4-quino linamine,
- N-(3 -fluoro-lH-indazo l-6-yl)-6- [(l-methylethyl)sulfonyl] -4-quino linamine,
6- [(l-methylethyl)sulfonyl] -N-(3 -methyl- lH-indazol-6-yl)-4-quino linamine,
6- [(l-methylethyl)sulfonyl] -N- [7-(trifluoromethyl)-lH-indazol-3 -yl] -4-quino linamine,
- N-(5 -fluoro-lH-indazo 1-3 -yl)-6- [(trifluoromethyl)sulfonyl] -4-quino linamine,
- N-(6-(((tetrahydrofuran-2-yl)methyl)sulfonyl)quinolin-4-yl)benzo[d]thiazol-5-amine, 4-(l,3 -benzothiazo 1-5 -ylamino)-6- [(trifluoromethyl)sulfonyl]quino line,
- N-(6-(tert-butylsulfonyl)quino lin-4-yl)thiazo lo [5 ,4-b]pyridin-6-amine,
- N-2,l ,3-Benzoxadiazol-5-yl-6-(methylsulfonyl)-4-quino linamine,
3- {[4-(l ,3-Benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl} -3-methyl- 1 -butanol, 6-(methylsulfonyl)-N-(2-phenylethyl)-4-quino linamine,
6- [(4-aminophenyl)sulfonyl] -N- 1 ,3 -benzothiazol-5 -yl-4-quino linamine,
- N- 1 ,3 -benzothiazol-5 -yl-6-(4-pyridinylsulfonyl)-4-quinolinamine,
3- {[4-(l ,3-benzothiazol-5-ylamino)-6-quinolinyl]thio} -3-methyl- 1 -butanol,
- N- 1 ,3 -benzothiazol-5 -yl-6-(ethylsulfonyl)-4-quinolinamine,
- 3- {[4-(l ,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}-l-propanol,
4- {[4-(l ,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}-l -butanol,
2- {[4-(l ,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}ethanol,
- N- 1 ,3 -benzothiazol-5 -yl-6- [( 1 , 1 -dimethyl ethyl)sulfonyl] -4-quino linamine,
- N- 1 ,3 -benzothiazol-5 -yl-6- [( 1 , 1 -dimethyl ethyl)thio]-4-quinolinamine,
- N- 1 ,3 -benzothiazol-5 -yl-6- [( 1 -methylpropyl)thio] -4-quino linamine,
- N- 1 ,3 -benzothiazol-5 -yl-6-(ethylthio)-4-quinolinamine,
6-[(2-aminoethyl)thio]-N- 1 ,3 -benzothiazol-5 -yl-4-quinolinamine,
- N- 1 ,3-benzothiazol-5-yl-6-(cyclopentylthio)-4-quinolinamine,
- N- 1 ,3 -benzothiazol-5 -yl-6-(cyclopentyl sulfonyl)-4-quino linamine,
3- {[4-(lH-indazol-6-ylamino)-6-quinolinyl]thio} -3 -methyl- 1 -butanol,
3- {[4-(lH-indazol-6-ylamino)-6-quinolinyl]sulfonyl} -3-methyl- 1 -butanol,
- N-l,3-benzothiazol-5-yl-6-[(l-methyl-4-piperidinyl)thio]-4-quino linamine,
- N- 1,3 -benzothiazol-5 -yl-6-(2-pyrimidinylthio)-4-quinolinamine,
2- {[4-(l ,3-benzothiazol-5-ylamino)-6-quinolinyl]thio}-l-propanol, 2- {[4-(l ,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}-l-propanol,
2- [l-( {[4-( 1 ,3 -benzothiazol-5 -ylamino)-6-quinolinyl]thio} methy l)cy clopropy 1] ethano 1,
2- [l-( { [4-(l,3 -benzothiazo 1-5 -ylamino)-6-quino linyljsulfbnyl [methyl )cyclopropyl] ethanol,
2- {[4-(l ,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}-2-methyl-l-propanol,
2- {[4-(l ,3-benzothiazol-5-ylamino)-6-quinolinyl]thio}-2-methyl-l-propanol,
2- {[4-(l ,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfmyl}-2-methyl-l-propanol 2- { [4-( 1 H-indazol-6-ylamino)-6-quinolinyl]sulfonyl} -2-methyl- 1 -propanol, methyl 2- { [4-(l, 3 -benzothiazo 1-5 -ylamino)-6-quino linyljthio } -2-methylpropanoate,
- methyl 2-{[4-(l,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}-2- methylpropanoate, N- 1 ,3-benzothiazol-5-yl-6- {[ 1 , 1 -dimethyl-3- (methyloxy)propyl]thio) -4-quinolinamine,
- N-(5-fluoro-lH-indazol-3-yl)-6-(tetrahydro-2H-pyran-4-ylsulfonyl)-4-quinolinamine, 2-( {4-[(5-fluoro-lH-indazol-3-yl)amino]-6-quinolinyl} sulfonyl)ethanol,
- N-l,3-benzothiazol-5-yl-6-(lH-l,2,4-triazol-3-ylsulfonyl)-4-quinolinamine,
- N-(5-fluoro-lH-indazol-3-yl)-6-(lH-imidazol-4-ylsulfonyl)-4-quinolinamine,
- N-(5-fluoro-lH-indazol-3-yl)-6-(lH-l,2,4-triazol-3-ylsulfonyl)-4-quinolinamine,
- N-(5 -fluoro-lH-indazo 1-3 -yl)-6-(tetrahydro-3 -furanylsulfonyl)-4-quino linamine,
- N-l, 3 -benzothiazol-5-yl-6-[(2-methyltetrahydro-3-furanyl)sulfonyl] -4- quino linamine,
- N-l,3-benzothiazol-5-yl-6- {[2-(methyloxy)ethyl]sulfonyl} -4-quinolinamine,
- N- 1 ,3-benzothiazol-5-yl-6-(methylthio)-4-quinolinamine,
2- { [4-(l, 3 -benzothiazo 1-5 -ylamino)-6-quino linyljthio } ethano 1,
- N-l ,3 -benzothiazol-5 -yl-6- {[2-(diethylamino)ethyl]thio} -4-quinolinamine,
- N-l,3-benzothiazol-5-yl-6- {[2-(diethylamino)ethyl]sulfonyl} -4-quinolinamine,
- N-l, 3 -benzothiazol-5 -yl-6-(tetrahydro-3-furanylthio)-4-quinolinamine,
- N-l, 3 -benzothiazol-5 -yl-6-(tetrahydro-3-furanylsulfonyl)-4-quinolinamine,
- N-l, 3 -benzothiazol-5 -yl-6-(tetrahydro-2H-pyran-4-ylthio)-4-quinolinamine,
- N- 1 ,3 -benzothiazol-5 -yl-6-(tetrahydro-2H-pyran-4-ylsulfonyl)-4-quinolinamine,
- N- 1 ,3 -benzothiazol-5 -yl-6-(2 -propen- 1 -ylsulfonyl)-4-quino linamine,
- N-l, 3 -benzothiazol-5 -yl-6- {[2-(4-morpholinyl)ethyl]thio} -4-quinolinamine,
- N-l,3-benzothiazol-5-yl-6- {[2-(3,5-dimethyl-4-isoxazolyl)ethyl]thio} -4- quino linamine, - N-l, 3 -benzothiazol-5 -yl-6- {[2-(3, 5 -dimethyl-4-isoxazolyl)ethyl]sulfonyl} -4- quinolinamine,
- N-(6-((l-methylpiperidin-4-yl)sulfonyl)quino lin-4-yl)benzo [djthiazo 1-5 -amine,
1 , 1 -dimethyl ethyl 4- {[4-(l ,3 -benzothiazol-5 -ylamino)-6-quinolinyl]thio} - 1 - piperidinecarboxylate,
1 , 1 -dimethyl ethyl 4- {[4-(l ,3 -benzothiazol-5 -ylamino)-6-quinolinyl]sulfonyl} - 1 - piperidinecarboxylate,
- N-l,3-benzothiazol-5-yl-6-[(2,2,2-trifluoroethyl)thio]-4-quinolinamine,
- N-l,3-benzothiazol-5-yl-6-[(2,2,2-trifluoroethyl)sulfonyl]-4-quinolinamine,
- N-l, 3 -benzothiazol-5 -yl-6-(tetrahydro-2H-thiopyran-4-ylthio)-4-quinolinamine,
2- {[4-( 1,3 -benzothiazol-5 -ylamino)-6-quinolinyl]sulfinyl}ethanol,
- N-(6-(((35)-tetrahydro-2H-pyran-3 -yl)sulfonyl)quino lin-4-yl)benzo [djthiazo 1-5 -amine, 2- { [4-(l,3 -benzothiazo 1-5 -ylamino)-6-quino linyljthio } etliano 1,
- N- 1 ,3 -benzothiazol-5 -yl-6- [( 1 -methylethyl)tliio] -4-quinolinamine,
2- { [4-(l, 3 -benzothiazo 1-5 -ylamino)-6-quino linyljthio } -2-metliylpropanoic acid, 2-{[4-(l,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}-2-methylpropanoic acid,
2- {[4-( 1,3 -benzothiazol-5 -ylamino)-6-quinolinyl]sulfinyl}ethanol,
4- [(5 -Hydroxy-2-methylphenyl)amino] -N-(phenylmethyl)-6-quino linecarboxamide,
- N-cyclohexyl-4-[(5-hydroxy-2-methylphenyl)amino]-6-quino linecarboxamide,
4- [(5 -hydroxy-2-methylphenyl)amino] -N- [3 -(4-morpholinyl)propylJ -6- quino linecarboxamide,
4- [(5 -hydroxy-2-methylphenyl)amino] -N-(tetrahydro-2H-pyran-4-ylmethyl)-6- quino linecarboxamide,
4- [(5 -hydroxy-2-methylphenyl)amino] -N-(3 -phenylpropyl)-6-quino linecarboxamide, 4-[(5-hydroxy-2-methylphenyl)amino]-N-[(3R)- 1 -(phenylmethyl)-3-pyrrolidinyl]-6- quino linecarboxamide,
4-[(5-hydroxy-2-methylphenyl)amino]-N- (3-[methyl(phenyl) aminojpropyl} -6- quino linecarboxamide,
4- [(5 -hydroxy-2-methylphenyl)amino] -N- [3 -(lH-imidazo l-l-yl)propyl] -6- quino linecarboxamide,
4- [(5 -hydroxy-2-methylphenyl)amino] -N- [2-(lH-imidazo 1-4 -yl) ethyl] - 6 - quino linecarboxamide,
- N- [( 1 S)-2-hydroxy- 1 -( 1 H-imidazol-4-ylmethyl)ethyl] -4- [(5 -hydroxy-2- methylphenyl)amino] - 6-quino linecarboxamide, - N-(lH-benzimidazo l-2-ylmethyl)-4- [(5 -hydroxy-2-methylphenyl)amino] -6- quino linecarboxamide,
4- [(5 -hydroxy-2-methylphenyl)amino]-N-[2-(l-pyrrolidinyl)ethyl] -6- quino linecarboxamide,
4-[(5-hydroxy-2-methylphenyl)amino]-N-[2-(methylsulfonyl) ethyl] -6- quino linecarboxamide,
4- [(5 -hydroxy-2-methylphenyl)amino] -N- [2-(lH-indo 1- 3 -y 1) ethyl] - 6 - quino linecarboxamide,
4- [(5 -hydroxy-2-methylphenyl)amino] -N- [(6-methyl-2-pyridinyl)methyl]-6- quino linecarboxamide,
- N-(4,5 -dimethyl- 1 ,3 -thiazol-2-yl)-4- [(5 -hydroxy-2-methylphenyl)amino]-6- quino linecarboxamide,
4-(l,3-benzothiazol-5-ylamino)-N-(phenylmetliyl)-6-quinolinecarboxamide,
- N-l,3 -Benzothiazo 1-5 -yl-6-(4-morpho linylsulfonyl)-4-quino linamine,
4-(l,3 -Benzothiazo 1-5 -ylamino)-N-(3 -methyl-3 -oxetanyl)-6-quino linesulfonamide, 4-(l,3-benzothiazol-5-ylamino)-N-(l-methyletliyl)-6-quinoline sulfonamide,
- N- 1 ,3-benzothiazol-5-yl-6-(l -piperidinylsulfonyl)-4-quinolinamine,
4-(l, 3 -benzothiazo 1-5 -ylamino)-N,N-dimethyl-6-quino linesulfonamide,
4-(l,3-benzothiazol-5-ylamino)-N-methyl-N-(methyloxy)-6-quino linesulfonamide, 4-(l,3-benzothiazol-5-ylamino)-N-(2-hydroxyethyl)-N-metliyl-6- quino linesulfonamide,
- N-(2-hydroxyethyl)-4-(lH-indazol-6-ylamino)-N-methyl-6-quino linesulfonamide, 4-{[4-chloro-3-(methyloxy)phenyl]amino}-N-(2-liydroxyetliyl)-N-metliyl-6- quino linesulfonamide,
4-{[4-chloro-3-(methyloxy)phenyl]amino}-6-quinolinesulfonamide,
4-( 1 H-indazol-6-ylamino)-6-quino linesulfonamide,
4-(l,3-benzothiazol-5-ylamino)-N-4-piperidinyl-6-quino linesulfonamide,
4-(l,3-benzothiazol-5-ylamino)-N-(4-piperidinylmethyl)-6-quino linesulfonamide,
- N-l,3-benzothiazol-5-yl-6-(4-morpholinylsulfonyl)-4-quino linamine,
4-(l, 3 -benzothiazo 1-5 -ylamino)-N- [(6-methyl-2-pyridinyl)metliyl] -6- quino linesulfonamide,
4-(l, 3 -benzothiazo 1-5 -ylamino)-N- [2-(dimethylamino)ethyl] -6-quino linesulfonamide, 4-(l, 3 -benzothiazo 1-5 -ylamino)-N- [2-(methyloxy)ethyl] -6-quino linesulfonamide,
4-(l, 3 -benzothiazo 1-5 -ylamino)-N- [3 -(4-morpho linyl)propyl] -6-quino linesulfonamide, 4-(l,3-benzothiazol-5-ylamino)-N-(tetrahydro-2H-pyran-4-ylmethyl)-6- quino linesulfonamide,
4-(l,3-benzothiazol-5-ylamino)-N-(tetrahydro-2H-pyran-4-yl)-6-quinolinesulfonamide, 4-(l,3-benzothiazol-5-ylamino)-N-cyclohexyl-6-quino linesulfonamide,
4-(l,3 -benzothiazo 1-5 -ylamino)-N- [2-(methylsulfonyl)ethyl] -6-quino linesulfonamide, 4-(l,3-benzothiazol-5-ylamino)-N-(phenylmethyl)-6-quinolinesulfonamide,
- N~2- {[4-(l,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}glycinamide,
- N~3- {[4-(l,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}-beta-alaninamide,
4-(l, 3 -benzothiazo 1-5 -ylamino)-N-(2-hydroxyethyl)-6-quino linesulfonamide,
4-(l, 3 -benzothiazo 1-5 -ylamino)-N-3 -oxetanyl-6-quino linesulfonamide,
4-(l,3-benzothiazol-5-ylamino)-N-(2-{[2-(methyloxy)etliyl]oxy}etliyl)-6- quino linesulfonamide,
4-(l,3-benzothiazol-5-ylamino)-N-methyl-N-(l-methylethyl)-6-quino linesulfonamide, 4-( 1 H-indazol-6-ylamino)-N,N-dimethyl-6-quinolinesulfonamide,
4- { [4-chloro-3 -(methyloxy)phenyl] amino } -N,N-dimethyl-6-quinolinesulfonamide,
- N-lH-indazol-6-yl-6-(4-morpholinylsulfonyl)-4-quinolinamine,
- N-[4-chloro-3-(methyloxy)phenyl]-6-(4-morpliolinylsulfonyl)-4-quinolinamine,
4-( 1 H-indazol-6-ylamino)-N-( 1 -methylethyl)-6-quinolinesulfonamide,
4- {[4-chloro-3-(methyloxy)phenyl]amino}-N-(l-methylethyl)-6-quino linesulfonamide,
- N- 1 ,3-benzothiazol-5-yl-6-(l -pyrrolidinylsulfonyl)-4-quinolinamine,
- methyl 1- { [4-(l, 3 -benzothiazo 1-5 -ylamino)-6-quinolinyl] sulfonyl} -L-prolinate,
- methyl (3S,6R)-l-{[4-(l,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}-6-methyl-3- piperidinecarboxylate,
l-{[4-(l,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}-3-pyrrolidinol,
- N-l, 3 -benzothiazo 1-5 -yl-6- [(3 -methyl-4-morpho linyl)sulfonyl] -4-quino linamine,
- N-l, 3 -benzothiazo 1-5 -yl-6- [(4-methyl- l-piperazinyl)sulfonyl] -4-quino linamine,
- N-l, 3 -benzothiazo 1-5 -yl-6-(4-thiomorpholinylsulfonyl)-4-quino linamine,
- N- 1 ,3-benzothiazol-5-yl-6-[(l , 1 -dioxido-4-thiomorpholinyl)sulfonyl]-4- quino linamine,
- N-l, 3 -benzothiazo 1-5 -yl-6- {[(3 R)-3-methyl-4-morpholinyl]sulfonyl} -4-quino linamine,
- ((2S,5R)-4-{[4-(l,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}-5-ethyl-2- morpholinyl)methano 1,
- N-l, 3-benzothiazol-5-yl-6-{[(3S)-3-methyl-4-morpholinyl]sulfonyl} -4-quino linamine,
- N- 1 ,3-benzothiazol-5-yl-6-(l -piperazinylsulfonyl)-4-quinolinamine, ((2S,5R)-4-{[4-(l,3-benzothiazol-5-ylamino)-6-quinolinyl]sulfonyl}-5-methyl-2- morpholinyl)methano 1,
(4- { [4-(l,3 -benzothiazo 1-5 -ylamino)-6-quinolinyl] sulfonyl} -2-morpho linyl)methano 1,
4-(l,3-benzothiazol-5-ylamino)-N-methyl-N-[2-(methyloxy)ethyl]-6- quino linesulfonamide,
4-(l, 3 -benzothiazo 1-5 -ylamino)-N- [2-(methyloxy)ethyl] -6-quino linesulfonamide,
- N-(5-fluoro-lH-indazol-3-yl)-6-(4-morpholinylsulfonyl)-4-quinolinamine,
- N-l, 3 -benzothiazo 1-5 -yl-6- [(2,2-dimethyl-4-morpholinyl)sulfonyl] -4-quino linamine,
- N-l, 3 -benzothiazo 1-5 -yl-6- [(2-methyl-4-morpho linyl)sulfonyl] -4-quino linamine,
4- [(7-chloro-lH-indazo 1-3 -yl)amino] -N- [2-(methyloxy)ethyl] -6-quino linesulfonamide,
- N-l,3-Benzothiazol-5-yl-N-[6-(methylsulfonyl)-4-quinolinyl]acetamide,
- N-l,3-benzothiazol-5-yl-N-[6-(methylsulfonyl)-4-quinolinyl]methanesulfonamide,
- N- 1 ,3-Benzoxazol-5-yl-6-(methylsulfonyl)-4-quinolinamine,
- N-[4-chloro-3-(methyloxy)phenyl]-6-(tetrahydro-2H-pyran-4-ylsulfonyl)-4- quino linamine,
- N-[4-chloro-3-(methyloxy)phenyl]-6-(tetrahydro-2H-pyran-4-ylsulfinyl)-4- quino linamine
and pharmaceutically acceptable salts thereof
In some embodiments, the RIPK2 inhibitor is selected from the group consisting of N- pyrazolyl, N-quinolyl amines as described in W02012021580. In particular, the RIPK2 is selected from the group consisting of:
6-[(l , 1 -dimethylethyl)sulfonyl]-N-(4, 5-dimethyl- 1 H-pyrazo 1-3 -yl)-4-quino linamine,
- N-(4,5-dimethyl-lH-pyrazol-3-yl)-6-(isopropylsulfonyl) quinolin-4-amine,
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-6-((tetrahydro-2H-pyran-4-yl)sulfonyl)quino lin-4- amine,
- N-(3, 4-dimethyl- lH-pyrazol-5-yl)-6-(methylsulfonyl)-4-quino linamine,
- N-(4,5-dimethyl-lH-pyrazol-3-yl)-6-[(trifluoromethyl) sulfonyl] -4-quino linamine,
6- [( 1 , 1 -dimethylethyl)sulfonyl] -N- [4-methyl-5 -(trifluoromethyl)- 1 H-pyrazo 1-3 -yl] -4- quino linamine,
6- [( 1 , 1 -dimethylethyl)sulfonyl] -N-( 1 ,3 ,4-trimethyl- 1 H-pyrazo 1-5 -yl)-4-quino linamine, 6- [( 1 -methylethyl)sulfonyl] -N- [4-methyl-5 -(trifluoromethyl)- 1 H-pyrazol-3 -yl] -4- quino linamine, - N-(4-methyl-5-(trifluoromethyl)-lH-pyrazol-3-yl)-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quino lin-4-amine,
- N-(3, 4-dimethyl- lH-pyrazol-5-yl)-6-(((tetrahydrofuran-2-yl)methyl)sulfonyl)
quino lin-4-amine,
- N-(3,4-dimethyl-lH-pyrazol-5-yl)-6-[(2,2,2-trifluoroethyl)sulfonyl]-4-quinolinamine, 6-(tert-butylsulfonyl)-N-(4, 5-dimethyl- lH-pyrazol-3-yl)-N-methylquinolin-4-amine, (R)-6-(tert-butylsulfinyl)-N-(4, 5-dimethyl- lH-pyrazol-3-yl)quinolin-4-amine,
- (S)-6-(tert-butylsulfinyl)-N-(4,5-dimethyl-lH-pyrazol-3-yl)quinolin-4-amine,
- 6-(tent-butylsulfonyl)-N-(4,5-dimethyl-lH-pyrazol-3-yl)-3-dueteroquinolin-4-amine,
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-6-((4-fluorotetrahydro-2H-pyran-4- yl)sulfonyl)quino lin-4-amine,
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-6-((4-methyltetrahydro-2H-pyran-4- yl)sulfonyl)quino lin-4-amine,
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-6-(tetrahydro-3-furanylsulfonyl)-4-quinolinamine,
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-6-[(2-methyltetrahydro-3-furanyl)sulfonyl]-4- quinolinamine,
- N-(3, 4-dimethyl- lH-pyrazol-5-yl)-6-(tetrahydro-2H-pyran-4-ylthio)-4-quinolinamine,
2-({4-[(4, 5-dimethyl- lH-pyrazol-3-yl)amino]-6-quinolinyl}sulfonyl)ethanol, ethyl 3-({6-[(l,l -dimethylethyl)sulfonyl] -4-quino linyl} amino)-4-methyl- 1 H-pyrazo le- 5-carboxylate,
3-({6-[(l,l -dimethylethyl)sulfonyl] -4-quino linyl} amino)-4-methyl- 1 H-pyrazo 1-5 - yljmethanol,
[3-( (6-[(l , 1 -Dimethylethyl)sulfonyl]-4-quino linyl} amino)-5 -methyl- 1 H-pyrazo 1-4- yljmethanol,
3-({6-[(l,l -Dimethylethyl)sulfonyl] -4-quino linyl} amino)-5 -methyl- 1 H-pyrazo le-4- carboxylic acid,
(R)-N-(3 ,4-dimethyl- 1 H-pyrazo 1-5 -yl)-6-((tetrahydro-2H-pyran-4-yl)sulfinyl)quino lin-
4-amine, and
(S)-N-(3 ,4-dimethyl- 1 H-pyrazo 1-5 -yl)-6-((tetrahydro-2H-pyran-4-yl)sulfinyl)quino lin- 4-amine,
and pharmaceutically acceptable salts thereof.
In some embodiments, the RIPK2 inhibitor is selected from the group consisting of imidazolyl- imidazoles as described in WO 2011123609. In particular, the RIPK2 inhibitor is selected from the group consisting of: 2-[2-(4-chlorophenyl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-N-[2-(4- morpholinyl)ethyl] - 1 H-benzimidazo le-5 -carboxamide,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl]-/V-methyl- 1 -(2-pyridinylmethyl)- 1
H- benzimidazole-5 -carboxamide,
2-[2-(4-chlorophenyl)-l H-imidazol-4-yl]-/V-methyl-l -(tetrahydro-2- furanylmethyl)- 1 H-benzimidazo le-5 -carboxamide,
2-[2-(4-chlorophenyl)-l H-imidazol-4-yl]-/V-methyl-l -(tetrahydro-3-furanyl)- 1 H- benzimidazole-5 -carboxamide,
2-[2-(4-chlorophenyl)-l H-imidazol-4-yl]-/v-methyl-l -[2-(2-pyridinyl)ethyl]-l H- benzimidazole-5 -carboxamide,
2-{2-[2-fluoro-5-(methyloxy)phenyl]-l H-imidazol-4-yl}-N-methyl-l -[2- (methyloxy)ethyl]- 1 H-benzimidazo le-5 -carboxamide,
- N-methyl-l -[2-(methyloxy)ethyl]-2-{2-[3-(methyloxy)phenyl]-l H-imidazol- 4-yl} - 1 H- benzimidazole-5 -carboxamide,
2-{2-[2,5-bis(methyloxy)phenyl]-l H-imidazol-4-yl} -N-methyl-l -[2- (methyloxy)ethyl]- 1 H-benzimidazole-5 -carboxamide,
- N-methyl-l -[2-(methyloxy)ethyl]-2-{2-[2-(methyloxy)phenyl]-l H-imidazol- 4-yl} - 1 H- benzimidazole-5 -carboxamide,
W-methyl-l -[2-(methyloxy)ethyl]-2-{2-[4-(methyloxy)phenyl]-l H-imidazol- 4-yl} - 1 H- benzimidazole-5 -carboxamide,
2- {2-[4-chloro-3-(methyloxy)phenyl]- 1 H-imidazol-4-yl} -N,N-dimethyl- 1-[2- (methyloxy)ethyl]- 1 H-benzimidazo le-5 -carboxamide,
2-[2-(4-chloro-3-hydroxyphenyl)-l H-imidazol-4-yl]-N,N-dimethyl-l -[2- (methyloxy)ethyl]- 1 H-benzimidazo le-5 -carboxamide,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-N-methyl-l-[2-
(methyloxy)ethyl]-l H- benzimidazole-5 -carboxamide,
2-{2-[4-chloro-3-(methyloxy)phenyl]-l H-imidazol-4-yl} -N-methyl-l -[2- (methyloxy)ethyl]- 1 H-benzimidazo le-5 -carboxamide,
- N-methyl-l -[2-(methyloxy)ethyl]-2-{2-[4-(methylsulfonyl)phenyl]-l H- imidazol-4-yl}- 1 H-benzimidazo le-5 -carboxamide,
2- {2-[4-(aminosulfonyl)phenyl]- 1 H-imidazol-4-yl} -N-methyl- 1-[2-
(methyloxy)ethyl]- 1 H-benzimidazole-5 -carboxamide,
2-[2-(l H-indol-5-yl)-l H-imidazol-4-yl]-N-methyl-l-[2-(methyloxy)ethyl]-l H- benzimidazole-5 -carboxamide, 2-[2-(l H-indol-6-yl)-l H-imidazol-4-yl]-N-methyl-l-[2-(methyloxy)ethyl]-l H- benzimidazole-5 -carboxamide,
2- {2-[3-(aminosulfonyl)phenyl]- 1 H-imidazol-4-yl} -N-methyl- 1-[2-
(methyloxy)ethyl]- 1 H-benzimidazole-5 -carboxamide,
2- {2-[3-(aminocarbonyl)phenyl]- 1 H-imidazol-4-yl} -N-methyl- 1-[2-
(methyloxy)ethyl]- 1 H-benzimidazole-5 -carboxamide,
2-[2-(l H-indazol-5-yl)-l H-imidazol-4-yl]-N-methyl-l-[2-(methyloxy)ethyl]-l H- benzimidazole-5 -carboxamide,
2-[2-(l H-indazol-6-yl)-l H-imidazol-4-yl]-N-methyl-l-[2-(methyloxy)ethyl]-l H- benzimidazole-5 -carboxamide,
2-[2-(2, 1 ,3-benzoxadiazol-5-yl)-l H-imidazol-4-yl]-N-methyl-l-[2-
(methyloxy)ethyl]- 1 H-benzimidazole-5 -carboxamide,
2-[2-(2, 1 ,3-benzoxadiazol-5-yl)-l H-imidazol-4-yl]-N,N-dimethyl-l -[2- (methyloxy)ethyl]- 1 H-benzimidazole-5 -carboxamide,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-N,N-dimethyl-l -[2- (methyloxy)ethyl]- 1 H-benzimidazole-5 -carboxamide,
5-(4- {6-methyl- l-[2-(methyloxy)ethyl]-l H-benzimidazol-2-yl}-l H-imidazol- 2-yl)- 2, 1 ,3-benzoxadiazole,
5-(4- {6-methyl- l-[2-(methyloxy)ethyl]-l H-benzimidazol-2-yl}-l H-imidazol- 2-yl)-l ,3- benzothiazole,
2- {2-[4-chloro-3-(methyloxy)phenyl]- 1 H-imidazol-4-yl} -6-methyl- 1-[2- (methyloxy)ethyl]- 1 H-benzimidazole,
2-chloro-5-(4- {6-methyl- 1 -[2-(methyloxy)ethyl]-l H-benzimidazol-2-yl}-l H- imidazo l-2-yl)pheno 1,
2-[2-(4-chlorophenyl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-l H- benzimidazole,
- methyl 2- [2-(4-chlorophenyl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-l H- benzimidazole-5-carboxylate,
2-[2-(4-chlorophenyl)-l H-imidazol-4-yl]-N,l -bis[2-(methyloxy)ethyl]-l H- benzimidazole-5 -carboxamide,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl]-N-methyl- 1 -[2-(methyloxy)ethyl]- 1 H- benzimidazole-5 -carboxamide,
2-[2-(4-chlorophenyl)-l H-imidazol-4-yl]-N,N-dimethyl-l -[2-
(methyloxy)ethyl] -L benzimidazo le-5 -carboxamide, 2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl]- 1 [2-(methyloxy)ethyl]-5
(trifluoromethyl)- 1 H-benzimidazole,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl]-6-(methyloxy)- 1 [2-
(methyloxy)ethyl]-l H- benzimidazole,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl] -5 -methyl- 1 -[2-(methyloxy)ethyl]- 1 H- benzimidazole,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl] -6-methyl- 1 -[2-(methyloxy)ethyl]- 1 H- benzimidazole,
N, 1 -bis [2-(methyloxy)ethyl] -2-(2 -phenyl- 1 /-/-imidazol-4-yl)-l /-/- benzimidazole-5- carboxamide,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl]- 1 -(2-hydroxyethyl)-N-[2-
(methyloxy)ethyl]- 1 H-benzimidazole-5 -carboxamide,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl]-N-methyl- 1 -[2-(methyloxy)ethyl]- 1 H- benzimidazole-6-carboxamide,
2-[2-(4-chlorophenyl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-l H- benzimidazole-6- carboxylic acid,
2-[2-(3-chlorophenyl)- 1 H-imidazol-4-yl]-N-methyl- 1 -[2-(methyloxy)ethyl]- 1 H- benzimidazole-5 -carboxamide,
2-[2-(4-chlorophenyl)-l H-imidazol-4-yl]-N-methyl-l -propyl- 1 H- benzimidazole-5- carboxamide,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl]- 1 -[2-(ethyloxy)ethyl]-N-methyl- 1 H- benzimidazole-5 -carboxamide,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl] -7-methyl- 1 -[2-(methyloxy)ethyl]- 1 H- benzimidazole,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl]- 1 - [2-(methyloxy)ethyl] -N-4- pyridinyl-l H- benzimidazole-5 -carboxamide,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl]- 1 - [2-(methyloxy)ethyl] -N-2- pyridinyl-l H- benzimidazole-6-carboxamide,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl]- 1 - [2-(methyloxy)ethyl] -N-3 - pyridinyl-l H- benzimidazole-6-carboxamide,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl]- 1 - [2-(methyloxy)ethyl] -N-4- pyridinyl-l H- benzimidazole-6-carboxamide,
2-[2-(4-chlorophenyl)- 1 H-imidazol-4-yl]- 1 - [2-(methyloxy)ethyl] -N-2- pyridinyl-l H- benzimidazole-5 -carboxamide, 2-[2-(4-chlorophenyl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-l H- imidazo[4,5- cjpyridine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l-[2-(methyloxy)ethyl]-l H- benzimidazo le-6-carbonitrile,
5 - {4- [6-methyl- 1 - [2-(methyloxy)ethyl] -5 -(methylsulfonyl)- 1 H-benzimidazo 1- 2-yl]-l H- imidazol-2-yl}-l ,3-benzothiazole,
5-(4-{6-fluoro-l -[2-(methyloxy)ethyl]-l H-benzimidazol-2-yl}-l H-imidazol- 2-yl)-l ,3- benzothiazole,
5-(4- (7-chloro- 1 -[2-(methyloxy)ethyl]- 1 H-benzimidazo l-2-yl} - 1 H-imidazol- 2-yl)-l ,3- benzothiazole,
5-(4- (4-methyl- 1 -[2-(methyloxy)ethyl]- 1 H-benzimidazo l-2-yl} - 1 H-imidazol- 2-yl)-l ,3- benzothiazole,
5-(4- (6-chloro- 1 -[2-(methyloxy)ethyl]- 1 H-benzimidazo l-2-yl} - 1 H-imidazol- 2-yl)-l ,3- benzothiazole,
5-(4- (6-bromo- 1 -[2-(methyloxy)ethyl]- 1 H-benzimidazo l-2-yl} - 1 H-imidazol- 2-yl)-l ,3- benzothiazole,
5-(4-{6-(ethyloxy)-l -[2-(methyloxy)ethyl]-l H-benzimidazol-2-yl}-l H- imidazol-2-yl)- 1 ,3-benzothiazole,
5 - (4- [ 1 - [2-(methyloxy)ethyl] -6-(trifluoromethyl)- 1 H-benzimidazo l-2-yl] - 1 H- imidazol- 2-yl} - 1 ,3-benzothiazole,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-/V,/V,6-trimethyl-l -[2- (methyloxy)ethyl]- 1 H-benzimidazo le-5 -carboxamide,
5- (4- [6-methyl- 1 -[2-(methyloxy)ethyl]-5-(4-morpholinylcarbonyl)- 1 -N- benzimidazol- 2-yl]- 1 H-imidazol-2-yl}-l ,3-benzothiazole,
5-(4- (6-methyl-5- {[(3R)-3-methyl-4-morpholinyl]carbonyl} - 1 -[2-
(methyloxy)ethyl]- 1 H-benzimidazol-2-yl}-l H-imidazol-2-yl)-l ,3- benzothiazole,
5-(4-{6-methyl-5-([(3S)-3-methyl-4-morpholinyl]carbonyl}-l-[2- (methyloxy)ethyl]- 1 H-benzimidazol-2-yl}-l H-imidazol-2-yl)-l ,3- benzothiazole,
5-(4- (6-methyl- 1 -[2-(methyloxy)ethyl]-5-[(4-methyl- 1 -piperazinyl)carbonyl]- l-N-benzimidazol-2-yl}-l H-imidazol-2-yl)-l ,3-benzothiazole,
methyl 2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l -[2- (methyloxy)ethyl]-l H- benzimidazole-6-carboxylate, 2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-l H- benzimidazole-6-carboxylic acid,
- methyl 2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l-[2- (methyloxy)ethyl]-l H- benzimidazole-5 -carboxy late,
2-{2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-l H- benzimidazol-5-yl} -2-propanol,
2-{2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-l H- benzimidazol-5-yl} -2-propanol,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-3-[2-(methyloxy)ethyl]-3H- imidazo [4 , 5 -6]pyridine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazo l-4-yl] -5 -methyl-3 -[2-
(methyloxy)ethyl]-3H- imidazo[4,5-6]pyridine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazo l-4-yl]- 1 -[2-(methyloxy)ethyl]-l H- imidazo [4 , 5 -6]pyridine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazo l-4-yl]-6-methyl-3- [2-
(methyloxy)ethyl]-3H- imidazo[4,5-6]pyridine,
{2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l-[2-(methyloxy)ethyl]-l H- benzimidazol-5-yl}methanol,
5-{4-[l -[2-(methyloxy)ethyl]-5-(trifluoromethyl)-l H-benzimidazol-2-yl]-l H- imidazol- 2-yl} - 1 ,3-benzothiazole,
5-{4-[l -[2-(methyloxy)ethyl]-5-(methylsulfonyl)-l H-benzimidazol-2-yl]-l H- imidazol- 2-yl} - 1 ,3-benzothiazole,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazo l-4-yl]- 1 -[2-(methyloxy)ethyl]-l H- benzimidazole-5 -sulfonamide,
5-(4-{5-bromo-l-[2-(methyloxy)ethyl]-l H-benzimidazol-2-yl}-l H-imidazo 1- 2-yl)-l ,3- benzothiazole,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazo l-4-yl]- 1 -[2-(methyloxy)ethyl]-l H- benzimidazo le-5 -carbonitrile,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-6-chloro-l -[2- (methyloxy)ethyl]-l H- benzimidazole-5 -carboxylic acid,
5-(4-{l-[2-(methyloxy)ethyl]-l H-benzimidazol-2-yl}-l H-imidazo l-2-yl)- 1 ,3- benzothiazole,
5-{4-[l -[2-(methyloxy)ethyl]-4-(methylsulfonyl)-l H-benzimidazol-2-yl]-l H- imidazol- 2-yl} - 1 ,3-benzothiazole, 5 -(4- { 1 - [2-(methyloxy)ethyl] -4- [(phenylmethyl)sulfonyl] - 1 H-benzimidazo 1- 2-yl} - 1 H- imidazol-2-yl)-l ,3-benzothiazole,
5-{4-[l -[2-(methyloxy)ethyl]-4-(4-morpholinyl)-l H-benzimidazo l-2-yl]- 1 H- imidazol-2- yl} - 1 ,3-benzothiazole,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-4-(4- morpholinyl)- 1 H-imidazo[4,5-c]pyridine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-4-[(3S)-3-methyl-4- morpholinyl]-l-[2- (methyloxy)ethyl]-l H-imidazo[4,5-c]pyridine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-4-[(3R)-3-methyl-4- morpholinyl]-l -[2- (methyloxy)ethyl]-l H-imidazo[4,5-c]pyridine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-4-[(3R)-3-ethyl-4- morpholinyl]-l -[2- (methyloxy)ethyl]-l H-imidazo[4,5-c]pyridine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazo l-4-yl]-4-(2-methyl-4-morpholinyl)- l-[2- (methyloxy)ethyl]-l H-imidazo[4,5-c]pyridine,
((2S,5R)-4-{2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l-[2-
(methy lo xy)ethy 1] - 1 H-imidazo[4,5-c]pyridin-4-yl}-5-ethyl-2- morpholinyl)methano 1,
((2S,5S)-4-{2-[2-(l 3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l -[2- (methy lo xy)ethy 1] - 1 H-imidazo[4,5-c]pyridin-4-yl}-5-methyl-2- morpholinyl)methano 1,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-4-(l- piperidinyl)- 1 H-imidazo[4,5-c]pyridine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-4-(l- pyrrolidinyl)-l H-imidazo[4,5-c]pyridine, 4- (l-azetidinyl)-2-[2-(l ,3- benzothiazol-5-yl)-l H-imidazol-4-yl]-l -[2- (methyloxy)ethyl]-l H- imidazo[4,5-c]pyridine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-4-(4- methyl-l - piperazinyl)-l H-imidazo[4,5-c]pyridine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazo l-4-yl] -4- [(3 S)-3, 4-dimethyl- 1- piperazinyl]-l- [2-(methyloxy)ethyl]-l H-imidazo[4,5-c]pyridine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-N-methyl-N, 1 -bis [2-
(methyloxy)ethyl]- 1 H-imidazo[4,5-c]pyridin-4-amine,
2-[{2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazo l-4-yl]- 1 -[2-(methyloxy)ethyl]-l H- imidazo[4,5-c]pyridin-4-yl} (methyl)amino]ethanol, 2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-N-ethyl-N, l-bis [2- (methyloxy)ethyl]- 1 H-imidazo[4,5-c]pyridin-4-amine,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl] -6-methyl- 1 -[2-
(methyloxy)ethyl]-4-(4- morpholinyl)- 1 /-/-imidazo[4,5-c]pyridine,
5-{4-[l -[2-(methyloxy)ethyl]-5-(4-morpholinylsulfonyl)-l H-benzimidazol-2- yl]-l-N- imidazol-2-yl}-l ,3-benzothiazole,
5- {4-[l -[2-(methyloxy)ethyl]-5-(l -piperazinylsulfonyl)-l /-/-benzimidazol-2- yl]-l H- imidazol-2-yl}-l ,3-benzothiazole,
5-{4-[l -[2-(methyloxy)ethyl]-5-(l-pyrrolidinylsulfonyl)-l H-benzimidazol-2- yl]-l H- imidazol-2-yl}-l ,3-benzothiazole,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-N,l -bis[2-(methyloxy)ethyl]- 1 H- benzimidazole-5 -sulfonamide,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-6-methyl-N, 1 -bis[2-
(methyloxy)ethyl]- 1 H-benzimidazole-5 -sulfonamide,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl] -6-methyl- 1 -[2-
(methyloxy)ethyl]-l H- benzimidazole-5 -carboxylic acid,
5-(4- {6-methyl- 1 -[2-(methyloxy)ethyl]-5-[(4-methyl- 1 -piperazinyl)sulfonyl]- 1 /-/- benzimidazol-2-yl}-l /-/-imidazol-2-yl)-l ,3-benzothiazole,
5- {4- [6-methyl- 1 -[2-(methyloxy)ethyl]-5-(4-morpholinylsulfonyl)-l /-/- benzimidazol-2- yl]-l /-/-imidazol-2-yl}-l ,3-benzothiazole,
2-[2-(l ,3-benzothiazol-5-yl)-l H-imidazol-4-yl]-l -[2-(methyloxy)ethyl]-l H- benzimidazole-5 -carboxylic acid,
5-(l-methyl-4-{6-methyl-l -[2-(methyloxy)ethyl]-l /-/-benzimidazol-2-yl}-l /- /- imidazol- 2-yl)-l ,3-benzothiazole,
2'-(l ,3-benzothiazol-5-yl)-l -[2-(methyloxy)ethyl]-l H, 1 'H-2,4'-biimidazole, 2'-(l ,3-benzothiazol-5-yl)-4,5-dimethyl-l-[2-(methyloxy)ethyl]-l H, 1 Ή-2,4'- biimidazole,
2'-(l ,3-benzothiazol-5-yl)-4,5-diethyl-l-[2-(methyloxy)ethyl]-l H, 1 Ή-2,4'- biimidazole,
- 5-(4-{l-[2-(methyloxy)ethyl]-4,5,6,7-tetrahydro-l /-/-benzimidazol-2-yl}-l /-/- imidazol- 2-yl)-l ,3-benzothiazole,
and pharmaceutically acceptable salts thereof In some embodiments, the RIPK2 inhibitor is selected from the group consisting of indazolyl-pyrimidines as described in WO2011120025. In particular, the RIPK2 inhibitor is selected from the group consisting of:
- N4-(5-fluoro-l H-indazol-3-yl)-N4-methyl- N2-[3,4,5-tris(methyloxy)phenyl]-
2.4-pyrimidinediamine,
- N2-[3,4-bis(methyloxy)phenyl]-N4-l H-pyrazolo[3,4-b]pyridin-3-yl-2,4- pyrimidinediamine,
3-( {4-[(5-fluoro- 1 H-indazol-3-yl)amino]-2-pyrimidinyl} amino)-N,N- dimethyl- benzenesulfonamide,
- N4-(5-fluoro- 1 H-indazol-3-yl)-N2-[3-(methoxy)-5-(methylsulfonyl)phenyl]-
2.4- pyrimidinediamine,
- N4-(5-fluoro-6-methyl-l H-pyrazolo[3,4-b]pyridin-3-yl)-N4-methyl-N2-[3- methyl-5- (methylsulfonyl)phenyl]pyrimidine-2, 4-diamine,
- N4-(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N -methyl-N2-[3-(methyloxy)- 5- (methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
2- (ethyloxy)-5-({4-[(5-fluoro-l H-indazol-3-yl)amino]-2-pyrimidinyl}amino)- N,N- dimethylbenzenesulfonamide,
- N2-(l ,l-dioxido-l -benzothien-4-yl)-N4-(5-fluoro-l H-indazol-3-yl)-2,4- pyrimidinediamine,
3- ((4-((5-fluoro-6-methyl-l H-indazol-3-yl)(methyl)amino)pyrimidin-2- yl)amino)-5 - methylbenzenesulfonamide,
- N -[2,3-dimethyl-5-(methylsulfonyl)phenyl)-N4-(5-fluoro-l H-indazol-3-yl)-
2,4- pyrimidinediamine,
2-{[3-({4-[(5-fluoro-l H-indazol-3-yl)amino]-2- pyrimidinyl} amino)phenyl]sulfinyl} ethanol,
- N2-[3,4-bis(methyloxy)phenyl]-N4-[4-(methyloxy)-l H-indazol-3-yl]-2,4- pyrimidinediamine,
- N2-[3,4-bis(methyloxy)phenyl]-N4-(6-methyl-lH-indazol-3-yl)-2,4- pyrimidinediamine,
- N2-[3,4-bis(methyloxy)phenyl]-N -{6-[(phenylmethyl)oxy]-l H-indazol-3-yl}-
2,4- pyrimidinediamine,
- N2-[3,4-bis(methyloxy)phenyl]-N4-l H-indazol-3-yl-2,4-pyrimidinediamine,
- N4 5-(methyloxy)-l H-indazol-3-yl]-N2-[3,4,5-tris(methyloxy)phenyl]-2,4- pyrimidinediamine, - N2-[3,4-bis(methyloxy)phenyl]-N -(6-chloro N2-[3,4-bis(methyloxy)phenyl]- N4-(5-chloro- 1 H-indazol-3-yl)-2,4-pyrimidinediamine,
- N2-[3,4-bis(methyloxy)phenyl]-N4-[5-(methyloxy)-l H-indazo l-3-yl]-2, 4- pyrimidinediamine,
- N2-[3,4-bis(methyloxy)phenyl]-N4-(7-fluoro-l H-indazo l-3-yl)-2, 4- pyrimidinediamine,
- N -1 H-indazo l-3-yl-N2-[4-(4-methyl-l-piperazinyl)phenyl]-2, 4- pyrimidinediamine,
- N2-[3,4-bis(methyloxy)phenyl]-N4-(4-fluoro-l H-indazo l-3-yl)-2, 4- pyrimidinediamine,
- N2-[3,4-bis(methyloxy)phenyl]-N4-[6-(methyloxy)-l H-indazo l-3-yl]-2, 4- pyrimidinediamine,
- N2-[3,4-bis(methyloxy)phenyl]-N4-(4,5-dichloro-l H-indazo l-3-yl)-2, 4- pyrimidinediamine,
- N -[3,4-bis(methyloxy)phenyl]-N4-(6-chloro-l-methyl-l H-indazol-3-yl)-2,4- pyrimidinediamine,
3-[(4-{[5-(methyloxy)-l H-indazo 1-3 -yl] amino} -2- pyrimidinyl)amino]benzenesulfonamide,
- N2-[3,4-bis(methyloxy)phenyl]-N4-(5-fluoro-l H-indazo l-3-yl)-2, 4- pyrimidinediamine,
- N2-[3,4-bis(methyloxy)phenyl]-N -(5,7-difluoro-lH-indazol-3-yl)-2,4- pyrimidinediamine,
- N2-[3,4-bis(methyloxy)phenyl]-N4-(6,7-difluoro-l H-indazo l-3-yl)-2, 4- pyrimidinediamine,
- N4-(5-fluoro-l H-indazol-3-yl)-N2-[3-(l -pyrrolidinylsulfonyl)phenyl]-2,4- pyrimidinediamine,
- N2-[4-fluoro-3-(methylsulfonyl)phenyl]-N -[5-(methyloxy)-l H-indazo 1-3 -yl]- 2,4- pyrimidinediamine,
- N4-(5-f)uoro-lH-indazol-3-yl)-N2-[4-fluoro-3-(methylsulfonyl)phenyl]-2,4- pyrimidinediamine,
- N4-(5 -fluoro- 1 H-indazo 1-3 -yl)-N4-methyl-N2- [3 ,4,5 -tris(methyloxy)phenyl] - 2,4- pyrimidinediamine,
- N4-(5-fluoro-l H-indazo 1-3 -yl)-N -methyl-N2-[3-(methylsulfonyl)phenyl]-2,4- pyrimidinediamine, - N2-[3,4-bis(methyloxy)phenyl]-N4-[ 1 -methyl-5-(methyloxy)- 1 H-indazol-3- yl]-2,4- pyrimidinediamine,
- N -[5-(methyloxy)-l H-indazol-3-yl]-N2-[3,4,5-tris(met yloxy)phenyl]-2,4- pyrimidinediamine,
- N2-[4-fluoro-3-(methyloxy)phenyl]-N4-[5-(methyloxy)- 1 H-indazol-3-yl]-2,4- pyrimidinediamine,
- N 2-(dimethylamino)ethyl]-N-met yl-3-[(4-{[5-(methyloxy)-l H-indazol-3- yljamino} - 2-pyrimidinyl)amino]benzenesulfonamide,
- N,N-dimethyl-3-[(4-{[5-(methyloxy)-l H-indazol-3-yl]amino}-2- pyrimidinyl)amino]benzenesulfonamide,
- N4-[5-(methyloxy)-l H-indazol-3-yl]-N2-[3-(l-pyrrolidinylsulfonyl)phenyl]- 2,4- pyrimidinediamine,
- N -{3-[l -(dimethylamino)-2,2,2-trifluoroet yljpheny!}- N -(5-fluoro-l H- indazo l-3-yl)- 2,4-pyrimidinediamine,
2-[3-( {4-[(5-fluoro- 1 H-indazol-3-yl)amino]-2-pyrimidinyl} amino)phenyl]-2- methylpropanenitrile,
- N4-(5-fluoro-l H-indazol-3-yl)-N2-[3-(methylsulfonyl)phenyl]-
2,4pyrimidinediamine,
- N2-(2, 3-dihydro- 1 ,4-benzodioxin-6-yl)-N -(5-fluoro-l H-indazol-3-yl)-2,4- pyrimidinediamine,
- N -(5-fluoro-l H-indazol-3-yl)-N2-[4-methyl-3-(met ylsulfonyl)phenyl]-2,4- pyrimidinediamine,
2-{[3-({4-[(5-fluoro-l H-indazol-3-yl)amino]-2- pyrimidinyl} amino)phenyl]sulfonyl} -2- methyl- 1 -propanol,
4-( (4-[(5-fluoro- 1 H-indazol-3-yl)amino]-2-pyrimidinyl} amino)-2,6- bis(methylo xy)pheno 1,
- N4-(5-fluoro-l H-indazol-3-yl)-N2-l H-indol-6-yl-2,4-pyrimidinediamine,
- N4-(5-fluoro-l H-indazol-3-yl)-N2-l H-indol-4-yl-2,4-pyrimidinediamine,
- N4-(5-fluoro-l H-indazol-3-yl)-N2-[l -(methylsulfbnyl)-l H-indol-6-yl]-2,4- pyrimidinediamine,
- N -(5-fluoro-l H-indazol-3-yl)-N2-[2-methyl-3-(methylsulfonyl)phenyl]-2,4- pyrimidinediamine,
- N -(5-fluoro-l H-indazol-3-yl)-N2-[l -(methylsulfonyl)-l H-indol-5-y IJ-2,4- pyrimidinediamine, - N -(7-chloro-l H-indazol-3-yl)-N2-[4-methyl-3-(methylsulfbnyl)phenyl]-2,4- pyrimidinediamine, N2- [4-methyl-3 -(methylsulfbnyl)pheApyrimidinediamine,
- N4-(5-fluoro- 1 H-indazol-3-yl)-N2-[3-methyl-5-(methylsulfbnyl)phenyl]-2,4- pyrimidinediamine,
- N -(5-fluoro-l H-indazol-3-yl)-N2-[4-(methylsulfbnyl)phenyl]-2,4- pyrimidinediamine, N4-(5-fluoro-l H-indazol-3-yl)-N2-[3-(4-morp olinylsulfonyl)p enyl]-2,4- pyrimidinediamine,
3-( {4-[(5-fluoro- 1 H-indazol-3-yl)amino]-2-pyrimidinyl} amino)-N-
(methyloxy)benzenesulfonamide,
3-({4-[(5-fluoro-l H-indazol-3-yl)amino]-2-py rimidinyl}amino)-N-methyl-N- (methyloxy)benzenesulfonamide,
5-({4-[(5-fluoro-l H-indazol-3-yl)amino]-2-pyrimidinyl}amino)-N,2-Dlmet yl- N- (methyloxy)benzenesulfonamide,
- N2-[3-(ethyloxy)-5-(methylsulfonyl)p enyl]-N4-(5-fluoro-l H-indazol-3-yl)-
2,4- pyrimidinediamine,
- N4-(5-fluoro- 1 H-indazol-3-yl)-N2- {3-[(l -methylethyl)sulfonyl]phenyl} -2,4- pyrimidinediamine,
- N2-(3,5-dimethylphenyl)-N4-(5-fluoro-l H-indazol-3-yl)-2,4- pyrimidinediamine, N -(5-fluoro-l H-indazol-3-yl)-N2-{4-methyl-3-[(l- methylethyl)sulfonyl]phenyl} -2,4- pyrimidinediamine,
- N4-(5-fluoro- 1 H-indazol-3-yl)-N2-[4-methyl-3-(methylsulfonyl)phenyl]-2,4- pyrimidinediamine,
2-(ethyloxy)-5-( (4-[(5-fluoro- 1 H-indazol-3-yl)amino]-2-pyrimidinyl} amino)- N,N- dimethylbenzenesulfonamide,
- N4-(5-fluoro- 1 H-indazol-3-yl)-N2-[3-(methyloxy)-5-(methylsulfonyl)phenyl]-
2,4- pyrimidinediamine,
- N2-[3-(ethylsulfonyl)-5-(methyloxy)phenyl]-N4-(5-fluoro- 1 H-indazol-3-yl)-
2,4- pyrimidinediamine,
- N2- (4-(ethyloxy)-3-[(l -methylethyl)sulfonyl]phenyl} -N4-(5-fluoro- 1 H- indazo l-3-yl)- 2,4-pyrimidinediamine,
- N4-(5-fluoro-l H-indazol-3-yl)-N -methyl-N2-[4-methyl-3-
(methylsulfonyl)phenyl]- 2,4-pyrimidinediamine,
- N4-(5-fluoro-l H-indazol-3-yl)-N4-methyl-N2-[3-methyl-5-
(methylsulfonyl)phenyl]- 2,4-pyrimidinediamine, - N4-ethyl-N4-(5-fluoro-l H-indazol-3-yl)-N2- [3 -methyl-5 -
(methylsulfonyl)phenylJ-2,4- pyrimidinediamine,
- N -(5-fluoro-l H-indazol-3-yl)-N2-(l -methyl-l H-imidazol-2-yl)-2,4- pyrimidinediamine,
- N4-(5-fluoro-l H-indazo!-3-yl)-N2-[3-(met yloxy)-5-(methylsulfonyl)phenyl]- 2,4- pyrimidinediamine,
2- {[5-({4-[(5-fluoro-l H-indazol-3-yl)amino]-2-pyrimidinyl}amino)-2- methylp enyl]sulfonyl}-2-met yl-l -propanol,
- N4-(5-fluoro-l H-indazol-3-yl)-N2-{4-fluoro-3-[(l
methylethyl)sulfonyl]phenyl} -2,4- pyrimidinediamine,
- N -(5-fluoro-l H-indazol-3-yl)-N4-methyl-N2-[3-(methyloxy)-5-
(methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N-[3-( (4-[(5-fluoro- 1 H-indazol-3-yl)amino]-2-pyrimidinyl} amino)phenyl]-N- methylmethanesulfonamide,
- N2 3-(dimethylamino)-5-(methylsulfonyl)phenyl]-N -(5-fluoro-l H-indazol-3- yl)-2,4- pyrimidinediamine,
3- ({4-[(5-fluoro-l H-indazol-3-yl)amino]-2-pyrimidinyl}amino)benzarriide,
- N4-(5-fluoro- 1 H-indazol-3-yl)-N2-[2-fluoro-4-(methylsulfonyl)phenyl]-2,4- pyrimidinediamine,
- N4-(5-fluoro-l H-indazol-3-yl)-N2-[3-(methylsulfonyl)-5-(l
pyrrolidinyl)phenyl]-2,4- pyrimidinediamine,
- N2-[3-(ethyloxy)-5-(ethylsulfonyl)phenyl]-N -(5-fluoro-l H-indazol-3-yl)-2,4- pyrimidinediamine,
- N4-(5-fluoro- 1 H-indazol-3-yl)-N2-[4-(methylsulfonyl)-2, 3-dihydro- 1 - benzo furan-6- yl] -2,4-pyrimidinediamine,
- N -(5-fluoro-l H-indazol-3-yl)-N -[4-fluoro-3-(methylsulfonyl)phenyl]-N - methyl-2,4- pyrimidinediamine,
- N4-(5-fluoro-l H-indazol-3-yl)-N -methyl-N2-[2-methyl-3-
(methylsulfonyl)phenyl]- 2,4-pyrimidinediamine,
- N2-[3,4-Dlmethyl-5-(methylsulfonyl)phenyl]-N -(5-fluoro-l H-indazol-3-yl)- N4- methyl-2, 4-pyrimidinediamine,
- N -(5-fluoro-l H-indazol-3-yl)-N2-[2-(methyloxy)-5-(methylsulfonyl)phenyl]- 2,4- pyrimidinediamine, 3-({4-[(5-fluoro-l H-indazo l-3-yl)amino]-2-pyrimidinyl}amino)-4-
(methyloxy)benzenesulfonamide,
- N -(5-fluoro-l H-indazol-3-yl)-N2-{3-[(trifluoromethyl)sulfonyl]p enyl}-2,4- pyrimidinediamine,
- N2- 1 -benzothienA-yl-N4-(5-fluoro- 1 H-indazol-3-yl)-2,4-pyrimidinediamine,
- N2-[2,4-dimet yl-5-(met ylsulfonyl)phenyl]-N4-(5-fluoro-l H-indazo 1-3 -yl)-
2,4- pyrimidinediamine,
7-( {4-[(5-fluoro- 1 H-indazol-3-yl)amino]-2-pyrimidinyl} amino)-2,3-d'ihydro- 1- benzofuran-5 -sulfonamide,
- N4-(5-fluoro- 1 H-indazo l-3-yl)-N2-[2-fluoro-3-(methylsulfonyl)phenyl]-2, 4- pyrimidinediamine,
- N2- [4-methyl-3 -(methylsulfonyl)phenyl] -N4-[6-(trifluoromcthyl)- 1 H-indazo 1- 3 -yl] -2, pyrimidinediamine,
- N -(6,7-Dlfluoro-l H-indazo 1-3 -yl)-N2- [4-methyl-3 -(methylsulfonyl)phenyl] -
2,4- pyrimidinediamine,
- N -(6-methyl- 1 H-indazo l-3-yl)-N2-[4-methyl-3-(methylsulfonyl)phenyl]-2, 4- pyrimidinediamine,
- N4-(5-chloro-l H-indazo 1-3 -yl)-N2-[4-methyl-3-(methylsulfonyl)p enyl]-2,4- pyrimidinediamine,
- N4-(4-chloro- 1 H-indazo l-3-yl)-N2-[4-methyl-3-(methylsulfonyl)phenyl]-2, 4- pyrimidinediamine,
- N4-(7-fluoro- 1 H-indazo l-3-yl)-N2-[4-methyl-3-(methylsulfonyl)phenyl]-2, 4- pyrimidinediamine,
- N4-(5,7-Dlfluoro-l H-indazo ! -3 -yl)-N2- [4-methyl-3 -(methylsulfonyl)phenyl] -
2,4- pyrimidinediamine,
- N4-(5,6-difluoro-l H-indazo 1-3 -yl)-N2- [4-methyl-3 -(methylsulfonyl)phenyl] -
2,4- pyrimidinediamine,
- N4-(5-fluoro- 1 H-indazo l-3-yl)-N2-[2-methyl-5-(methylsulfonyl)phenyl]-2, 4- pyrimidinediamine,
- N4-(5-fluoro-l H-indazo l-3-yl)-N2-[3, 4, 5-tris(methyloxy)phenyl]-2, 4- pyrimidinediamine,
- N2-[4-methyl-3-(met ylsulfonyl)phenyl]-N4-[5-(methyloxy)-l H-indazo 1-3 -yl]-
2,4- pyrimidinediamine, - N'-[3-( {4-[(5-fluoro- 1 H-indazol-3-yl)amino]-2-pyrimidinyl} amino)phenyl]-
N,N- dimethylsulfamide,
3-({4-[(5-fluoro-l H-indazol-3-yl)amino]-
2Ayrimidinyl}amino)benzenesulfonamide,
5-({4-[(5-fluoro-l H-indazol-3-yl)amino]-2-pyrimidinyl}amino)-2- methylbenzenesulfonamide,
- N4-(6 -difluoro-l H-indazol-3-yl)-N4-methyl-N2-[4-methyl-3-(met ylsulfonyl)phenyl]- 2,4-pyrimidinediamine,
- N4-(6,7-difluoro-l H-indazol-3-yl)-N4-met yl-N2-[3-met yl-5-(met ylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N -(6J-difluoro-l H-indazol-3-yl)-N4-methyl-N2-[3,4,5- tris(methyloxy)phenyl]-2,4- pyrimidinediamine,
- N4-(7-fluoro-l H-indazol-3-yl)-N -methyl-N2-[4-methyl-3-(methylsulfonyl)p enyl]- 2,4-pyrimidinediamine,
- N4-(7-fluoro-l H-indazol-3-yl)-N -methyl-N2- [3 -methyl-5 -
(methylsulfonyl)phenyl]- 2,4-pyrimidinediamine,
- N -(7-fluoro-l H-indazol-3-yl)-N4-methyl-N2-[3-(methyloxy)-5-
(methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N -(7-fluoro-l H-indazol-3-yl)-N -methyl-N2-[3,4,5-tris(methyloxy)phenyl]- 2,4- pyrimidinediamine,
- N4-(5,6-difluoro-l H-indazol-3-yl)-N4-methyl-N2-[3,4,5- tris(methyloxy)phenyl]-2,4- pyrimidinediamine,
- N4-(5,6-difluoro-l H-indazol-3-yl)-N -methyl-N2-[3-(methyloxy)-5- (met ylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N4-(5,6-difluoro-l H-indazol-3-yl)-N4-met yl-N2-[4-methyl-3-(met ylsulfonyl)phenyl]- 2,4-pyrimidinediamine,
- N4-(5,6-difluoro-l H-indazol-3-yl)-N4-methyl-N2-[3-met yl-5-
(methylsulfonyl)phenyl]- 2,4-pyrimidinediamine,
- N4-(6,7-difluoro-l H-indazol-3-yl)-N -methyl-N -[3-(methyloxy)-5- (methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N -(1 H-indazol-3-yl)-N2-(3-methyl-5-(methylsulfonyl)phenyl)pyrimidine-2,4- diamine,
- N4-( 1 H-indazo 1-3 -yl)-N2-(4-methyl-3 -(methylsulfonyl)phenyl)pyrimidine-
2,4- diamine, - N4-(7-fluoro-l H-indazol-3-yl)-N2-(3 -methyl-5 -
(methylsulfonyl)phenyl)pyrimidine- 2,4-diamine,
- N -(4-fluoro-l H-indazol-3-yl)-N -methyl-N2-(4-methyl-3- (methylsulfonyl)phenyl)pyrimidine-2, 4-diamine,
- N4-(6J-Dlfluoro-l H-indazol-3-yl)-N2-(3 -methyl-5 -
(methylsulfonyl)phenyl)pyrimidine-2, 4-diamine,
- N4-(4-fluoro-l H-indazol-3-yl)-N -methyl-N2-(3 -methyl-5 -
(methylsulfonyl)phenyl)pyrimidine-2, 4-diamine,
- N -(5-fluoro-6-methyl-l H-indazo 1-3 -yl)-N4-methyl-N2- [3 -methyl-5- (met ylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N4-(7-fIuoro-6-methyl-l H-indazol-3-yl)-N4-methyl-N2-[3-methyl-5-
(methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N4-(5,6-difluoro-l H-indazo 1-3 -yl)-N2- [3 -methyl-5 -(methylsulfonyl)phenyl] - 2,4- pyrimidinediamine,
- N4-(5,6-difluoro-l H-indazo 1-3 -yl)-N2- [3 -(methyloxy)-5-
(methylsulfonyl)phenyl]-2,4- pyrimidinediamine,
- N4-(5,6-difluoro-l H-indazo l-3-yl)-N2-[3, 4, 5-tris(methyloxy)phenyl]-2, 4- pyrimidinediamine,
- N4-(6-fluoro-5-methyl-l H-indazo 1-3 -yl)-N4-methyl-N2- [3 -methyl-5- (met ylsulfonyl)phenyl]-2,4-pyrimidinediamine,
2- chloro-5-({4-[(5-fluoro-l H-indazo 1-3 -yl)amino] -2- pyrimidinyl}amino)benzenesulfonamide,
- N4-(5-fluoro-6-methyl-l H-indazo 1-3 -yl)-N -methyl-N2- [3 -methyl-5- (met ylsulfonyl)phenyl]-2,4-pyrimidinediamine,
3-( (4-[(7-fluoro- 1 H-indaz0l-3-yl)(methyl)amino]-2-pyrimidinyl} amino)-5- met ylbenzenesulfonamide,
- N4-(7-fluoro- 1 H-indazo 1-3 -yl)-N4-methyl-N2-(3-(methylsulfonyl)-5-
(pyrrolidin-l - yl)phenyl)pyrimidine-2, 4-diamine,
3- ({4-[(6J-difluoro-l H-indazo l-3-yl)(methyl)amino]-2-pyrimidinyl}amino)-5- methylbenzenesulfonamide,
- N -(7-fluoro-l H-indazo l-3-yl)-N4-methyl-N2-(3-methyl-5-(pyrrolidin- 1- yl)phenyl)pyrimidine-2, 4-diamine,
- N4-(7-fluoro-l H-indazo 1-3 -yl)-N -methyl-N2-(3 -methyl-5 - morpholinophenyl)pyrimidine-2, 4-diamine, N4-(7-fluoro-6-methyl- 1 H- indazo 1-3 -yl)-N4-methyl-N2- [3 -(methyloxy)-5 - (methylsulfonyl)phenyl] -2,4- pyrimidinediamine,
- N -(5-fluoro-6-methyl-l H-indazol-3-yl)-N -methyl-N2-[3-(methyloxy)-5- (methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N -(6,7-difluoro-l H-indazol-3-yl)-N2-(l ,l-dioxido-l -benzothien-6-yl)-N4- methyl-2,4- pyrimidinediamine,
- N4-methyl-N4-(5 -methyl- 1 H-indazol-3-yl)-N2-[3-(methyloxy)-5-
(methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N4-methyl-N -(5 -methyl- 1 H-indazol-3-yl)-N2 3 -methyl-5 -
(methylsulfonyl)phenyl]- 2,4-pyrimidinediamine,
3 -( (4- [(7-fluoroA-methyl- 1 H-indazo 1-3 -yl)(methyl)amino] -
2Ayrimidinyl} amino)-5- methylbenzenesulfonamide,
- N4-(7-fluoro-l H-indazo 1-3 -yl)-N2-(3-methoxy-5-
(methylsulfonyl)phenyl)pyrimidine- 2,4-diamine,
5-((4-((7-fluoro-l H-indazo 1-3 -yl)(met yl)amino)pyrimidin-2-yl)amino)-2- methoxybenzenesulfonamide,
- N -(6-methyl- 1 H-indazo l-3-yl)-N2-[3-methyl-5-(methylsulfonyl)phenyl]-2, 4- pyrimidinediamine,
- N4-(6,7-difluoro-l H-indazo 1-3 -yl)-N2- [3 -(methyloxy)-5-
(methylsulfonyl)phenyl]-2,4- pyrimidinediamine,
3 -((4-((5 ,6-difluoro- 1 H-indazo 1-3 -yl)(methyl)amino)pyrimidin-2-yl)amino)-5 - methylbenzenesulfonamide,
- N4-(5,6-difluoro-l H-indazo 1-3 -yl)-N2-(4-methoxy-3-(methylsulfonyl)phenyl)- N4- methylpyrimidine-2, 4-diamine,
- N -(7-fluoro-l H-indazo 1-3 -yl)-N2-(4-methoxy-3 -(methylsulfo nyl)phenyl)-N4- methylpyrimidine-2, 4-diamine,
- N -(7-fluoro-l H-indazo l-3-yl)-N4-methyl-N2-(3-methyl-5-((tetrahydro-2H- pyran-4- yl)sulfonyl)p enyl)pyrimidine-2, 4-diamine,
3-( (4-[(6J-difluoro- 1 H-indazo l-3-yl)(methyl)amino]-2-pyrimidinyl} amino)-5- (methyloxy)benzenesulfonamide,
3 -((4-((5 ,6-difluoro- 1 H-indazo 1-3 -yl)(methyl)amino)pyrimidin-2-yl)amino)-5 - methoxybenzenesulfonamide,
3 -( (4- [(5 -fluoro-6-methyl- 1 H-indazo 1-3 -yl)(methyl)amino] -2- pyrimidinyl} amino)-5- (methyloxy)benzenesulfonamide. 3-( (4-[(7-fluoro-6- methyl- 1 H-indazol-3-yl)(methyl)amino]-2-pyrimidinyl} amino)-5A
(methyloxy)benzenesulfonamide,
3-( {4-[(6,7-difluoro- 1 H-indazol-3-yl)amino]-2-pyrimidinyl} amino)-5- methylbenzenesulfonamide,
3-((4-((7-fluoro- 1 H-indazo 1-3 -yl)amino)pyrimidin-2-yl)amino)-5- methoxybenzenesulfonamide,
3-((4-((7-fluoro- 1 H-indazo 1-3 -yl)(methyl)amino)pyrimidin-2-yl)amino)-5- methoxybenzenesulfonamide,
- N2-[4-(ethyloxy)-3-(methylsulfonyl)phenyl]-N4-(7-fluoro-lH-indazol-3-yl)- N4-methyl- 2,4-pyrimidinediamine,
3 -methoxy-5 -((4-(methyl(7-(trifluoromethyl)- 1 H-indazo 1-3 - yl)amino)pyrimidin-2- yl)amino)benzenesulfonamide,
3 -methyl-5 -[(4- (methyl[6-(trifluoromethyl)- 1 H-indazo l-3-yl]amino} -2- pyrimidinyl)amino]benzenesulfonamide,
3-(methyloxy)-5-[(4- (methyl[6-(trifluoromethyl)- 1 H-indazo l-3-yl]amino} -2- pyrimidinyl)amino]benzenesulfonamide,
3 -methyl-5 -((4-(methyl(7-(trifluoromet yl)-l H-indazo 1-3 -yl)amino)pyrimidi yl)amino)benzenesulfonamide,
- N,N-dimethyl-3-{[4-(l H-pyrazolo[3,4-b]pyridin-3-ylamino)-2- pyrimidinyl] amino } benzenesulfonamide,
- N4-(5 -methyl- 1 H-pyrazolo[3,4-b]pyridin-3-yl)-N2-[3-
(methylsulfonyl)phenyl]-2,4- pyrimidinediamine,
- N2-[4-methyl-3-(methylsu!fonyl)phenyl]-N4-(5-methyl-l H-pyrazolo[3,4- b]pyridin-3- yl)-2,4-pyrimidinediamine,
- N2-[4-fluoro-3-(methylsulfonyl)phenyl]-N -(5-methyl-l H-pyrazolo[3,4- b]pyridin-3- yl)-2,4-pyrimidinediamine,
- N -(5-fiuoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N2-[3-(methylsuifonyl)phenyl]- 2,4- pyrimidinediamine,
- N2-[4-fluoro-3-(methylsulfonyl)phenyl]-N -(5-fluoro-l H-pyrazolo[3,4- b]pyridin-3-yl)- 2,4-pyrimidinediamine,
- N4-(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N2-[4-methyl-3-
(methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N4-(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N2-{4-methyl-3-[(l- methylethyl)sulfonyl]phenyl} -2,4-pyrimidinediamine, - N4-(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N4-methyl-N2-[3-
(methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N4-(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N2-{4-methyl-3-[(l- methylethyl)sulfonyl]phenyl}-2,4-pyrimidinediamine,
- N4-(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N2-[3-(methyloxy)-5-
(methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N4-(5-fluoro-lH-pyrazolo[3,4-b]pyridin-3-yl)-N4-methyl-N2-[3-methyl-5- (methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N -(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N -met yl-N2-[4-methyl-3- (methylsulfonyl)phenyl]-2,4-pynmidinediamine,
- N4-(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N2-[3-(metylsulfonyl)-5-(4- morpholinyl)phenyl]-2,4-pyrimidinediamine,
- N4-(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N2-[4-methyl-3-
(methylsulfonyl)p enyl]-2,4-pyrimidinediamine,
- N2-[3,4-Dlmethyl-5-(methylsulfonyl)phenyl]-N4-(5-fluoro-l H-pyrazolo[3,4- b]pyridin-3-yl)-2,4-pyrimidinediamine,
- N4-(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N4-methyl-N2-[4-methyl-3- (methylsulfonyl)-5-(l-pyrrolidinyl)phenyl]-2,4-pyrimidinediamine,
- N4-(6-methyl-l H-pyrazolo[3,4-b]pyridin-3-yl)-N2-[3-
(iTiethylsulfonyl)phenyl]-2,4- pyrimidinediamine,
- N2-[3-[(difluoromethyl)oxy]-5-(met ylsulfonyl)phenyl]-N -(5-fluoro-l H- pyrazolo[3,4- b]pyridin-3-yl)-N4-methyl-2,4-pyrimidinediamine,
- N2-[3-(3,3-difluoro-l-pyrrolidinyl)-5-(methylsulfonyl)p enyl]-N4-(5-fluoro-l H- pyrazolo[3,4-b]pyridin-3-yl)-N4-methyl-2,4-pyrimidinediamine,
- N -(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N4-methyl-N2-[4-
(methylsulfonyl)-2,3- dihydro- 1 -benzofuran-6-yl]-2,4-pyrimidinediamine,
- N4-(5-fluoro-l H-pyrazoio[3,4-b]pyridin-3-yl)-N2-[3,4,5- tris(methyloxy)phenyl]-2,4- pyrimidinediamine,
- N4-(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)-N4-methyl-N2-[3,4,5- tris(methyloxy)phenyl]-2,4-pyrimidinediamine,
- N2-[4-met yl-3-(methylsulfonyl)phenyl]-N4-(6-methyl-l H-pyrazolo[3,4- b]pyridin-3- yl)-2,4-pyrimidinediamine,
- N2-{3-[(l ,l-Dlmethylethyl)sulfonyl]-5-methylphenyl}-N4-(5-fluoro-l H- pyrazolo[3,4- b]pyridin-3-yl)-2,4-pyrimidinediamine, - N2-[3-[(l , 1 -Dlmethylethyl)sulfonyl]-5-(trifluoromethyl)phenyl]-N4-(5-fluoro- 1 H- pyrazolo[3,4-b]pyridin-3-yl)-2,4-pyrimidinediamine,
- N 5-fluoro-6-methyl-l HAyrazolo[3,4-b]pyridin-3-yl)-N -methyl-Nz-[3- (methyloxy)- 5-(methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N4-(5-fluoro-6-methyl-l H-pyrazolo[3,4-b]pyridin-3-yl)-N -methyl-N2-[3- methyl-5- (methylsulfonyl)phenyl]pyrimidine-2, 4-diamine,
- N4-(5-fluoro-6-methyl-l H-pyrazolo[3,4-b]pyridin-3-yl)-N2-[3-(methyloxy)-5- (methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N -(5-fluoro-6-methyl-l H-pyrazolo[3,4-b]pyridin-3-yl)-N -methyl-Nz-[3- (methylsulfonyl)-5-(l-pyrrolidinyl)phenyl]-2,4-pyrimidinediamine,
- N4-(5-fluoro-6-methyl-l H-pyrazolo[3,4-b]pyridin-3-yl)-N -methyl-N2-[4- methyl-3- (methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N4-(5-fluoro-6-methyl-l H-pyrazolo[3,4-b]pyridin-3-yl)-N4-methyl-N2-[3- (methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
- N4-(5-fluoro-6-methyl-l H-pyrazolo[3,4-b]pyridin-3-yl)-N4-methyl-N2-[3,4,5- tris(methyloxy)phenyl]-2,4-pyrimidinediamine,
- N2 3-[(l -dimethylethyl)sulfonyl]-5-methylphenyl}-N4-(5-fluoro-6-methyl-l H- pyrazolo[3,4-b]pyridin-3-yl)-N4-methyl-2,4-pyrimidinediamine,
- N -(5-fluoro-l ,6-dimethyl-l H-pyrazolo[3,4-b]pyridin-3-yl)-N4-methyl-N2- [3 -methyl-5 - (methylsulfonyl)phenyl] -2,4-pyrimidinediamine,
3-({4-[(5-fluoro-6-methyl-l H-pyrazolo[3,4-b]pyridin-3-yl)(methyl)amino]-2- pyrimidinyl}amino)benzenesulfonamide,
- 3-({4-[(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)amino]-2- pyrimidinyl}amino)benzenesulfonamide,
- N4-(5-fluoro-6-methyl-l H-pyrazolo[3,4-b]pyridin-3-yl)-N2-[3-fiuoro-5- (methylsulfonyl)phenyl]-N -methyl-2, 4-pyrimidinediamine,
- 3-({4-[(5-fluoro-l/-/-pyrazolo[3,4-b]pyridin-3-yl)(methyl)amino]-2- pyrimidinyl}amino)benzenesulfonamide,
- 2-c loro-5-({4-[(5-fluoro-lH-pyrazolo[3,4-b]pyridin-3-yl)amino]-2- pyrimidinyl}amino)benzenesulfonamide,
2-chloro-5-({4-[(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)(methyl)amino]-2- pyrimidinyl}amino)benzenesulfonamide,
- 5-({4-[(5-fluoro-l H-pyrazolo[3,4-b]pyridin-3-yl)(methyl)amino]-2- pyrimidinyl}amino)-2-methylbenzenesulfonamide, - N4-(5-fluoro-7-oxido-l HAyrazolo[3,4-b]pyridin-3-yl)-N -methyl-N 3-methyl-
5- (methylsulfonyl)phenyl]-2,4-pyrimidinediamine,
5 -fluoro-6-methyl-3 -(methyl(2-((3 -methyl-5 -
(methylsulfonyl)phenyl)amino)pyrimidin-4-yl)amino)-l H-pyrazolo[3,4- b] pyridi nc 7-oxide,
- N4-(5-fluoro-6-methyl-l H-pyrazolo[3,4-b]pyridin-3-yl)-N4-methyl-N2-(3- methyl-5- ((tetrahydro-2H-pyranA-yl)sulfonyl)phenyl)pyrimidine-2, 4-diamine, and pharmaceutically acceptable salts thereof.
In some embodiments, the RIPK2 inhibitor is selected from the group consisting of amino-quinolines as described in W02012122011. In particular, the RIPK2 inhibitor is selected from the group consisting of:
6-(tert-butylsulfonyl)-N-(4, 5-dimethyl- lH-pyrazo l-3-yl)-7-methoxyquinolin-4- amine;
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-7-(methyloxy)-6-(tetrahydro-2H-pyran-4- ylsulfmyl)-4- quinolinamine;
- 6- [(1, l-dimethylethyl)sulfmyl] -N-(4, 5 -dimethyl- lH-pyrazo 1-3 -yl)-7- (methyloxy)-4- quinolinamine;
2-((4-((4, 5 -dimethyl- lH-pyrazo 1-3 -yl)amino)-7-methoxyquino lin-6- yl)sulfonyl)-2-methylpropan- l-o 1;
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-7-methoxy-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4- amine;
2-((4-((4, 5 -dimethyl- lH-pyrazo 1-3 -yl)amino)-7-methylquino lin-6- yl)sulfonyl)ethano 1;
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-6-((2,2-dimethyltetrahydro-2H-pyran-4- yl)sulfonyl)-7- methoxyquino lin-4-amine;
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-7-methoxy-6-((4-methyltetrahydro-2H- pyran-4- yl)sulfonyl)quinolin-4-amine;
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-7-methoxy-6-((2- methoxyethyl)sulfonyl)quinolin-4-amine;
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-7-methoxy-6-(((3R,4R)-3-methyltetrahydro- 2H-pyran-4- yl)sulfonyl)quino lin-4-amine;
- N-(4,5-dimethyl-lH-pyrazol-3-yl)-6-(((2R,6S)-2,6-dimethyltetrahydro-2H- pyran-4-yl)sulfonyl)-7- methoxyquino lin-4-amine; - 6-(tert-butylsulfonyl)-N-(5-fluoro-lH-pyrazolo[3,4-b]pyridin-3-yl)-7- methoxyquino lin-4-amine;
- N-[4-chloro-3-(methyloxy)phenyl]-6-[(l , 1 -dimethyl ethyl)sulfonyl]-7-
(methyloxy)-4- quinolinamine;
- N- [4-chloro-3 -(methyloxy)phenyl] -7-(methyloxy)-6-(tetrahydro-2H-pyran-4- ylsulfonyl)-4- quinolinamine;
- N- 1 ,3 -benzothiazol-5 -yl-7-(methyloxy)-6-(tetrahydro-2H-pyran-4- ylsulfonyl)-4-quino linamine;
2- { [4- { [4-chloro-3 -(methyloxy)phenyl] amino } -7-(methyloxy)-6- quinolinyl] sulfonyl} ethanol;
- N-(5-fluoro-lH-indazol-3-yl)-7-(methyloxy)-6-(tetrahydro-2H-pyran-4- ylsulfonyl)-4- quinolinamine;
2- {[4-[(4,5-dimethyl-lH-pyrazol-3-yl)amino]-7-(methyloxy)-6- quinolinyljsulfonyl} ethanol;
- N- [4-chloro-3 -(methyloxy)phenyl]-6- [( 1 -methylethyl) sulfonyl] -7- (methyloxy)-4-quino linamine;
- N- 1 ,3 -benzothiazol-5 -yl-6-[(l -methylethyl) sulfonyl] -7-(methyloxy)-4- quino linamine;
- N-(4,5-dimethyl-lH-pyrazol-3-yl)-6-[(l -methylethyl) sulfonyl]-7-(methyloxy)- 4-quinolinamine;
- N-(5-fluoro-lH-indazol-3-yl)-6-[(l -methylethyl) sulfonyl] -7-(methyloxy)-4- quino linamine;
2- {[4-(l,3-benzothiazol-5-ylamino)-7-(methyloxy)-6-quinolinyl]sulfonyl} ethanol;
6-(isopropylsulfonyl)-7-methoxy-N-(4-methyl-5-(trifluoromethyl)-lH-pyrazol-
3-yl)quinolin-4- amine;
6-(tert-butylsulfonyl)-7-methoxy-N-(4-methyl-5-(trifluoromethyl)-lH-pyrazol- 3-yl)quinolin-4- amine;
6-(tert-butylsulfonyl)-N-(4, 5-dimethyl- lH-pyrazol-3-yl)-7-ethoxyquinolin-4- amine;
6- (tert-butylsulfonyl)-7-ethoxy-N-(5 -fluoro-lH-indazol-3 -yl)quino lin-4-amine;
7- chloro-N-(4, 5-dimethyl- lH-pyrazol-3-yl)-6-((tetrahydro-2H-pyran-4-yl) sulfonyl)quinolin-4- amine; - N-(4, 5-dimethyl- lH-pyrazol-3-yl)-7-methyl-6-((tetrahydro-2H-pyran-4-yl) sulfonyl)quinolin-4- amine;
7-chloro-N-(5-fluoro-lH-indazol-3-yl)-6-((tetrahydro-2H-pyran-4-yl) sulfonyl)quino lin-4-amine;
- N-(5 -fluoro-lH-indazo 1-3 -yl)-7-methyl-6-((tetrahydro-2H-pyran-4-yl)
sulfonyl)quino lin-4-amine;
- N-(5-fluoro-lH-indazol-3-yl)-6-((tetrahydro-2H-pyran-4-yl)sulfbnyl)-7- (trifluoromethyl) quinolin- 4-amine;
6-(tert-butylsulfonyl)-N-(5 -fluoro-lH-indazo 1-3 -yl)-7-(trifluoromethyl) quino lin-4-amine;
6-(tert-butylsulfonyl)-N-(4, 5-dimethyl- lH-pyrazol-3-yl)-7-methylquinolin-4- amine;
6-(tert-butylsulfonyl)-N-(5 -fluoro-lH-indazo 1-3 -yl)-7-methylquino lin-4-amine; 6-(tert-butylsulfonyl)-N-(5 -fluoro-lH-indazo 1-3 -yl)-7-methoxy quino lin-4- amine;
6-(tert-butylsulfonyl)-7-chloro-N-(5-fluoro-lH-indazol-3-yl)quinolin-4-amine; 6-(tert-butylsulfonyl)-7-ethyl-N-(5 -fluoro-lH-indazo 1-3 -yl)quino lin-4-amine;
- N-(5 -fluoro-lH-indazo 1-3 -yl)-6-(isopropylsulfonyl)-7-methylquino lin-4-amine;
- N-(4,5-dimethyl-lH-pyrazol-3-yl)-6-(isopropylsulfonyl)-7-methylquinolin-4- amine;
6- (tert-butylsulfonyl)-N-(4, 5-dimethyl- lH-pyrazol-3-yl)-7-ethylquinolin-4- amine;
7- ethyl-N-(5-fluoro-lH-indazol-3-yl)-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quino lin-4-amine;
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-7-ethyl-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4- amine;
(3 -((6-(tert-butylsulfonyl)-7-methoxy quino lin-4-yl)amino)-4- methy lpheny l)methano 1;
- 7-ethoxy-N-(5-fluoro-lH-indazol-3-yl)-6-(isopropylsulfonyl)quinolin-4-amine;
- N-(7-chloro-lH-indazol-3-yl)-7-methoxy-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4- amine;
6-(tert-butylsulfonyl)-N-(7-fluoro-lH-indazo 1-3 -yl)-7-methoxy quino lin-4- amine; - 6-(tert-butylsulfonyl)-N-(5 ,7-difluoro- lH-indazol-3 -yl)-7-methoxyquinolin-4- amine;
- 6-(tert-butylsulfonyl)-N-(6,7-difluoro-lH-indazol-3-yl)-7-methoxyquinolin-4- amine;
6-(tert-butylsulfonyl)-N-(7-chloro-lH-indazo 1-3 -yl)-7-methoxyquino lin-4- amine;
6-(tert-butylsulfonyl)-7-methoxy-N-(5 -methoxy-lH-indazo 1-3 -yl)quino lin-4- amine;
6-(tert-butylsulfonyl)-N-(7-fluoro-lH-indazo 1-3 -yl)-7-methylquino lin-4-amine;
- 6-(tert-butylsulfonyl)-N-(5 ,7-difluoro-lH-indazo 1-3 -yl)-7-methylquino lin-4- amine;
6-(tert-butylsulfonyl)-N-(5 -methoxy-lH-indazo 1-3 -yl)-7-methylquino lin-4- amine;
- 6-(tert-butylsulfonyl)-N-(6,7-difluoro-lH-indazol-3-yl)-7-methylquinolin-4- amine;
6- (tert-butylsulfonyl)-N-(7-chloro-lH-indazol-3 -yl)-7-methylquino lin-4- amine;
7- methoxy-N-(4-methyl-5-(trifluoromethyl)-lH-pyrazol-3-yl)-6-((tetrahydro- 2H-pyran-4- yl)sulfonyl)quino lin-4-amine;
- N-(5 ,7-difluoro- lH-indazol-3-yl)-7-methoxy-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4- amine;
- N-(4-chloro-lH-indazol-3-yl)-7-methoxy-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4- amine;
- N-(6-chloro-lH-indazol-3-yl)-7-methoxy-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4- amine;
- N-(6,7-difluoro-lH-indazol-3-yl)-7-methoxy-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4- amine;
7-methoxy-N-(5-methoxy-lH-indazol-3-yl)-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4- amine;
- N-(5-chloro-lH-indazol-3-yl)-7-methoxy-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4- amine;
- N-(7-chloro-lH-indazol-3-yl)-7-methoxy-6-((4-methyltetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4-amine; N- 1 ,3-benzothiazol-5-yl-6-(methylsulfonyl)-4- quinolinamine; 7 -bromo-N-(4, 5-dimethyl- lH-pyrazo l-3-yl)-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4- amine;
7-bromo-6-(tert-butylsulfonyl)-N-(4, 5-dimethyl- lH-pyrazo l-3-yl)quinolin-4- amine;
7-bromo-N-(4-methyl-5-(trifluoromethyl)-lH-pyrazol-3-yl)-6-((tetrahydro-2H- pyran-4- yl)sulfonyl)quinolin-4-amine;
- 7-bromo-N-(4,5-dimethyl-lH-pyrazol-3-yl)-6-(isopropylsulfonyl)quinolin-4- amine;
- 7-bromo-N-(5-fluoro-lH-indazol-3-yl)-6-(isopropylsulfonyl)quinolin-4-amine;
7-bromo-N-(5-fluoro-lH-indazol-3-yl)-6-((tetrahydro-2H-pyran-4- yl)sulfonyl)quino lin-4-amine;
- N- 1 ,3-benzothiazol-5-yl-6-[(l , 1 -dimethylethyl)sulfonyl]-7-(methyloxy)-4- quinolinamine;
- 6-(tert-butylsulfonyl)-4-((4,5-dimethyl-lH-pyrazol-3-yl)amino)quinolin-7-ol;
2-((6-(tert-butylsulfonyl)-4-((4, 5-dimethyl- lH-pyrazol-3-yl)amino)quinolin-7- yl)oxy)ethanol;
- 6- (tert-butylsulfonyl)-7-(difluoromethoxy)-N-(4,5-dimethyl-lH-pyrazol-3-yl) quino lin-4-amine;
7- (difluoromethoxy)-N-(4, 5-dimethyl- lH-pyrazol-3-yl)-6-((tetrahydro-2H- pyran-4- yl)sulfonyl)quinolin-4-amine;
2-((4-(benzo[d]thiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quino lin-7- yl)oxy)ethanol;
(3-((6-(tert-butylsulfonyl)-7-methoxyquinolin-4-yl)amino)-4-methyl- lH- pyrazo 1-5 -yl)meth N-(4, 5-dimethyl- lH-pyrazo l-3-yl)-7-methyl-6-((4- methyltetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4-amine;
- N-(5-fluoro-lH-indazol-3-yl)-7-methyl-6-((4-methyltetrahydro-2H-pyran-4- yl)sulfonyl)quino lin-4-amine;
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-7-ethyl-6-((4-methyltetrahydro-2H-pyran-4- yl)sulfonyl)quino lin-4-amine;
7-ethyl-N-(5-fluoro-lH-indazol-3-yl)-6-((4-methyltetrahydro-2H-pyran-4- yl)sulfonyl)quinolin-4- amine;
- N-(7-chloro-lH-indazol-3-yl)-7-methyl-6-((4-methyltetrahydro-2H-pyran-4- yl)sulfonyl)quinolin- 4-amine;
and pharmaceutically acceptable salts thereof. In some embodiments, the RIPK2 inhibitor is selected from the group consisting of amino quinazolines as described in WO 2013025958. In particular, the RIPK2 inhibitor is selected from the group consisting of:
4-methy 1-3 - { [6-(methylthio)-4-quinazolinyl] amino } phenol,
4-methyl-3 - { [6-(methylsulfonyl)-4-quinazolinyl] amino } phenol,
- N- 1 ,3-benzothiazol-5-yl-6-[(l , 1 -dimethylethyl)thio]-4-quinazolinamine,
- N- 1 ,3-benzothiazol-5-yl-6-[(l , 1 -dimethylethyl)sulfonyl]-4-quinazolinamine, 6-(tert-butylsulfonyl)-N-(5 -fluoro-lH-indazo 1-3 -yl)quinazo lin-4-amine,
- N-l ,3 -benzothiazo 1-5 -yl-6- [(l-methylethyl)sulfonyl] -4-quinazo linamine,
2- {[4-(l ,3-benzothiazol-5-ylamino)-6-quinazolinyl]sulfonyl}ethanol,
- N- 1 ,3 -benzothiazo 1-5 -yl-6-(tetrahydro-2H-pyran-4-ylsulfonyl)-4- quinazo linamine,
- N- 1 ,3 -benzothiazo 1-5 -yl-6-(tetrahydro-2H-pyran-4-ylsulfonyl)-4- quinazo linamine,
2-((4-((4, 5 -dimethyl- lH-pyrazo 1-3 -yl)amino)-7-methoxyquinazo lin-6- yl)sulfonyl)ethano 1,
- N-(5 -fluoro-lH-indazo 1-3 -yl)-6- [(l-methylethyl)sulfonyl] -4-quinazo linamine,
- N-(4, 5-dimethyl- lH-pyrazol-3-yl)-6-[(l-methylethyl)sulfonyl]-4- quinazo linamine,
6-(tert-butylsulfonyl)-N-(5 -(trifluoromethyl)-lH-pyrazo 1-3 -yl)quinazo lin-4- amine,
6-(tert-butylsulfonyl)-N-(l ,3, 4-trimethyl- lH-pyrazol-5-yl)quinazolin-4-amine,
- N-(6-(tert-butylthio)-7-methoxyquinazolin-4-yl)benzo [d]thiazol-5 -amine,
- N-(6-(tert-butylsulfonyl)-7-methoxyquinazolin-4-yl)benzo [(ijthiazo 1-5 -amine,
- N-(6-(isopropylsulfonyl)-7-methoxyquinazolin-4-yl)benzo[(i]thiazol-5-amine,
4-(benzo[<i]thiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7-o 1,
- N-(6-(tert-butylsulfonyl)-7-ethoxyquinazo lin-4-yl)benzo [(ijthiazo 1-5 -amine,
- N-(6-(tert-butylsulfonyl)-7-ethoxyquinazolin-4-yl)-N-ethylbenzo[(i]thiazol-5 - amine,
2- ((4-(benzo[<i]thiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7- yl)oxy)ethanol,
- N-(6-(tert-butylsulfonyl)-7-(difluoromethoxy)quinazolin-4-yl)benzo[(i]thiazol-
5 -amine, - N-(6-(tert-butylsulfonyl)-7-(2,2,2-trifluoroethoxy)quinazolin-4- yl)benzo[(i]thiazo 1-5 -amine,
- N-(6-(tert-butylsulfonyl)-7-(methoxymethoxy)quinazolin-4- yl)benzo[(i]thiazo 1-5 -amine,
- N-(6-(tert-butylsulfonyl)-7-(cyclohexylmethoxy)quinazolin-4- yl)benzo[(i]thiazo 1-5 -amine,
3 - ((4-(benzo[<i]thiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7- y l)oxy)prop an- l-o 1,
- N-(6-(tert-butylsulfonyl)-7-((tetrahydro-2H-pyran-4-yl)oxy)quinazolin-4- yl)benzo[(i]thiazo 1-5 -amine,
- N-(6-(tert-butylsulfonyl)-7-(2-chloroethoxy)quinazolin-4-yl)benzo[(i]thiazol-5- amine,
(R)-l-((4-(benzo [<i]thiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7- yl)oxy)propan-2-ol, N-(6-(tert-butylsulfonyl)-7-propoxyquinazolin-4- yl)benzo[(i]thiazo 1-5 -amine,
- N-(6-(tert-butylsulfonyl)-7-(2-(methylthio)ethoxy)quinazolin-4- yl)benzo[(i]thiazo 1-5 -amine,
- N-(7-(2-bromoethoxy)-6-(tert-butylsulfonyl)quinazolin-4-yl)benzo[ ]thiazol-5- amine,
4- (benzo [<i]thiazol-5 -ylamino)-6-(tert-butylthio)quinazo lin-7-o l,N-(6-(tert- butylthio)-7-isopropoxyquinazo lin-4-yl)benzo [(ijthiazo 1-5 -amine,
- N-(6-(terAbutylsulfonyl)-7-isopropoxyquinazolin-4-yl)benzo[(i]thiazol-5- amine,
ethyl 2-((4-(benzo[<i]thiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7- yl)oxy)-2- methylpropanoate,2-((4-(benzo[<i]thiazol-5-ylamino)-6-(tert- butylsulfonyl)quinazolin-7-yl)oxy)-2- methylpropan- 1 -ol,
- N- 1 ,3-benzothiazol-5-yl-6-[(l , 1 -dimethylethyl)sulfonyl]-7-ethenyl-4- quinazo linamine,
- N- 1 ,3-benzothiazol-5-yl-6-[(l , 1 -dimethylethyl)sulfonyl]-7-ethyl-4- quinazo linamine,
- N-(6-(tert-butylsulfonyl)-7-chloroquinazolin-4-yl)benzo[(i]thiazol-5-amine, 6-(tert-butylsulfonyl)-7-chloro-N-(4, 5-dimethyl- lH-pyrazol-3-yl)quinazolin-4- amine, - 6- [( 1 , 1 -dimethylethyl)sulfonyl] -N-(5 -fluoro- IH-pyrazolo [3 ,4-£]pyridin-3 -yl)-7-(methyloxy)- 4-quinazo linamine,
6-(fert-butylsulfonyl)-N-(4-chloro-3 -methoxyphenyl)-7-methoxyquinazolin-4- amine,
5 - ((6-(tert-butylsulfonyl)-7-methoxyquinazo lin-4-yl)amino)-2-chlorophenol,
6- (tert-butylsulfonyl)-7-methoxy-N-(3 -methyl- lH-indazo l-6-yl)quinazo lin-4- amine,
6-(tert-butylsulfonyl)-N-(4-chloro-2-fluorophenyl)-7-methoxyquinazolin-4- amine,
6-(tert-butylsulfonyl)-N-(lH-indazol-6-yl)-7-methoxyquinazolin-4-amine, 6-(tert-butylsulfonyl)-N-(4, 5-dimethyl- lH-pyrazol-3-yl)-7-methoxyquinazolin- 4-amine,
(E)-3 -(4-(benzo [ Jthiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7- yl)prop-2-en-l-o 1, 2-((4-(benzo [ Jthiazo 1-5 -ylamino)-7-methoxyquinazo lin-6- yl)sulfonyl)ethano 1,
(R)-methyl 2-((4-(benzo[<i]thiazol-5-ylamino)-6-(tert- butylsulfonyl)quinazolin-7- yl)oxy)propanoate,
(S)-methyl 2-((4-(benzo[<i]thiazol-5-ylamino)-6-(tert- butylsulfonyl)quinazolin-7- yl)oxy)propanoate,
- methyl 2-((4-(benzo [djthiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7 - yl)oxy)propanoate,
(R)-2-((4-(benzo [<i]thiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7- y l)oxy)prop an- l-o 1,
(S)-2-((4-(benzo[<i]thiazol-5-ylamino)-6-(tert-butylsulfonyl)quinazo lin-7- y l)oxy)prop an- l-o 1,
2-((4-(benzo[<i]thiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7- y l)oxy)prop an- l-o 1,
6-(tert-butylsulfonyl)-4-((4-chloro-2-fluorophenyl)amino)quinazo lin-7-o 1,
- N-(6-(tert-butylsulfinyl)-7-methoxyquinazo lin-4-yl)benzo [(ijthiazo 1-5 -amine,
2-((4-(benzo [ Jthiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7- yl)oxy)acetamide,
2-((4-(benzo[<i]thiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7- yl)oxy)acetic acid, - N-(6-(terAbutylsulfonyl)-7-(2-(methylsulfonyl)ethoxy)quinazolin-4-yl)benzo[ ]thiazol-5- amine,
- N-(6-(terAbutylsulfonyl)-7-(2-(isopropylsulfonyl)ethoxy)quinazolin-4- yl)benzo[(i]thiazol-5- amine,
(E)-methyl 3-((4-(benzo[<i]thiazol-5-ylamino)-6-(tert- butylsulfonyl)quinazolin-7- yl)oxy)acrylate,
(E)-3 -((4-(benzo [ Jthiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7- yl)oxy)acrylamide,
(E)-3 -((4-(benzo [ Jthiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7- yl)oxy)acrylic acid,
- N-(6-(tert-butylsulfonyl)-7-(vinyloxy)quinazolin-4-yl)benzo[(i]thiazol-5- amine,
4-(benzo [ Jthiazo 1-5 -ylamino)-7-methoxy-N,N-dimethylquinazo line-6- sulfonamide,
4-(benzo [ Jthiazo 1-5 -ylamino)-N-isopropyl-7-methoxyquinazo line-6- sulfonamide,
- N-(7-methoxy-6-(pyrrolidin- 1 -ylsulfonyl)quinazolin-4-yl)benzo[<i]thiazol-5 - amine,
- N-(7 -methoxy-6-(morpholino sulfonyl)quinazo lin-4-yl)benzo [(i]thiazol-5 - amine,
4-(benzo [ Jthiazo 1-5 -ylamino)-N-(2-hydroxyethyl)-7-methoxyquinazo line-6- sulfonamide,
4-(benzo[<i]thiazol-5-ylamino)-7-methoxy-N-(tetrahydro-2H-pyran-4- yl)quinazo line-6- sulfonamide,
4-(benzo[<i]thiazol-5-ylamino)-N-(2-hydroxy-2-methylpropyl)-7- methoxyquinazo line-6- sulfonamide,
l-((4-(benzo[ Jthiazo 1-5 -ylamino)-7-methoxy quinazolin-6- yl)sulfonyl)pyrrolidin-3-ol,
4-(benzo[<i]thiazo 1-5 -ylamino)-N-(2-hydroxypropyl)-7-methoxyquinazo line-6- sulfonamide,
4-(benzo [ Jthiazo 1-5 -ylamino)-7-methoxy-N-(2-methoxyethyl)quinazo line-6- sulfonamide,
4-(benzo[<i]thiazo 1-5 -ylamino)-7-methoxy-N-(oxetan-3 -yl)quinazo line-6- sulfonamide, 4-(benzo[<Athiazol-5-ylamino)-N-(2-(dimethylamino)ethyl)-7- methoxyquinazo line-6- sulfonamide,
l-((4-(benzo[<i]thiazol-5-ylamino)-7-methoxyquinazolin-6- yl)sulfonyl)pyrrolidine-2- carboxylic acid,
1 -(4-((4-(benzo[(i]thiazol-5-ylamino)-7-methoxyquinazolin-6- yl)sulfonyl)piperazin- 1 - yl)ethanone,
- N-(2-(lH-tetrazol-5-yl)ethyl)-4-(benzo[ ]thiazol-5-ylamino)-7- methoxyquinazo line-6- sulfonamide,
4-(benzo[<i]thiazol-5-ylamino)-7-methoxy-N-((tetrahydro-2H-pyran-4- yl)methyl)quinazoline-6-sulfonamide,
and pharmaceutically acceptable salts thereof
In some embodiments, the RIPK2 inhibitor is selected from the group consisting of aminoquinolines as described in WO2014043437. In particular, the RIPK2 inhibitor is selected from the group consisting of:
N-(4, 5-dimethyl- 1 H-pyrazol-3-yl)-6-[( 1 -methylethyl) sulfonyl] -7-(methyloxy)- 4- quinolinamine,
6-(isopropyl-sulfonyl)-7-methoxy-N-(4-methyl-5-(trifluoromethyl)-lH-pyrazol- 3- yl)quinolin-4-amine,
6-(tert-butylsulfonyl)-7-methoxy-N-(4-methyl-5-(trifluoromethyl)-lH-pyrazol- 3- yl)quinolin-4-amine,
6-(tert-butylsulfonyl)-N-(4, 5-dimethyl- lH-pyrazo l-3-yl)-7-ethoxyquinolin-4- amine,
7-methoxy-N-(4-methyl-5-(trifluoromethyl)-lH-pyrazol-3-yl)-6-((tetrahydro- 2H-pyran-4-yl)sulfonyl) quinolin-4-amine,
2-((6-(tert-butylsulfonyl)-4-((4, 5-dimethyl- lH-pyrazo l-3-yl)amino)quino lin-7- yl)oxy)ethanol,
6-(tert-butylsulfonyl)-7-(difluoromethoxy)-N-(4, 5-dimethyl- lH-pyrazo 1-3- yl)quinolin-4-amine,
7-(difluoromethoxy)-N-(4, 5-dimethyl- lH-pyrazol-3-yl)-6-((tetrahydro-2H- pyran-4-yl)sulfonyl)quinolin-4-amine, pharmaceutically acceptable salts thereof
In some embodiments, the RIPK2 inhibitor is selected from the group consisting of amino quinazo lines as described in WO2013025958. In particular, the RIPK2 inhibitor is 2- ((4-(benzo [djthiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7-yl)oxy)ethano 1 and is having the following structure:
Figure imgf000045_0001
In some embodiments, the RIPK2 inhibitor is an inhibitor of RIPK2 expression. An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In a preferred embodiment of the invention, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti- sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of RIPK2 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of RIPK2, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding
RIPK2 can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. RIPK2 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that RIPK2 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing RIPK2. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and R A virus such as a retrovirus. One can readily employ other vectors not named but known to the art.
According to the invention, the RIPK2 inhibitor is administered to the subject in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
Typically the active ingredient of the present invention (e.g. RIPK2 inhibitor) is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
A further aspect of the invention relates to a method for screening a plurality of test substances useful for the treatment of hereditary periodic fevers in a subject in need thereof comprising the steps consisting of (a) testing each of the test substances for its ability to inhibit NOD2 mediated pathway and (b) and positively selecting the test substances capable of inhibiting the NOD2 mediated pathway.
In some embodiments, the screening method of the present invention comprises the step of (i) providing a RIPK2 protein; (ii) contacting the RIPK2 protein with a test substance wherein the substance is expected to inhibit the phosphorylation or kinase activity of the RIPK2 protein; and (iii) selecting a test substance as a candidate that decreases the phosphorylation level or the kinase activity of RIPK2 in comparison to a negative control that is not contacted with a test substance.
In some embodiments, the screening method of the present invention comprises the steps of i) bringing into contact the test substance to be tested with a mixture of a first RIPK2 protein (2) a second NOD2 protein, ii) determining the ability of said test substance to inhibit the binding between the RIPK2 protein and the NOD2 protein and iii) positively selected the test substance that is capable to inhibit the binding between the RIPK2 protein and the NOD2 protein.
Typically, RIPK2 proteins come from various sources and sequences in the art may be used for the present disclosure as long as it contains a kinases activity. The sequence of RIPK2 is known in the art, for example as NCBI reference NO. NP— 003812.1. In one embodiment, a full or partial length of RIPK2 can be used.
In some embodiments, RIPK2 proteins are provided as a cell that endogenously or exogenously expressing the protein. For example, mammalian cells are prepared to express the protein of interest such as RIPK2 through a transient or stable transfection or cells that endogenously express the protein of interest may be used. Cells endogenously expressing RIPK2 may include but is not limited to, macrophages, dendritic cells, neutrophils and epithelial cells, which may be obtained from various organs for example such as peritoneal cavity of a mouse. The cells obtained may be cultured in a cell culture dish and treated with a test substance for a certain period time in a suitable medium, from which the whole proteins are extracted and tested/detected for kinase activity of RIPK2 protein. Alternatively established cell lines may be used, in which case the cells are transfected with a plasmid expressing RIPK2. The example of such cells include but is not limited to 293, 293T or 293 A (Graham F L, Smiley J, Russell W C, Naim R (July 1977).“Characteristics of a human cell line transformed by DNA from human adenovirus type 5”. J. Gen. Virol. 36 (1): 59-74; and Louis N, Evelegh C, Graham F L (July 1997).“Cloning and sequencing of the cellular- viral junctions from the human adenovirus type 5 transformed 293 cell line”. Virology 233 (2): 423-9).
The term“test substance” refers generally to a material that is expected to decrease, reduce, suppress or inhibit the kinase activity of RIPK2 or its phosphorylation or to interfere the interaction between RIPK2 and NOD2, which include small molecules, high molecular weight molecules, mixture of compounds such as natural extracts or cell or tissue culture products, biological material such as proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamer, RNAzymes and DNAzymes, or glucose and lipids, but is not limited thereto. The test substances may be polypeptides having amino acid residues of below 20, particularly 6, 10, 12, 20 aa or above 20 such as 50aa. These materials are obtained from synthetic or natural compound libraries and the methods to obtain or construct libraries are known in the art. For example, synthetic chemical library may be obtained from Maybridge Chemical Co. (UK), Comgenex(USA), Brandon Asociates(USA), Microsource(USA) and Sigma-Aldrich(USA). The chemical library of natural origin may be obtained from Pan Laboratories (USA) and MycoSearch(USA). Further test substances may be obtained by various combinatorial library construction methods known in the art including for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries. Test substance of a library may be composed of peptides, peptoides, circular or liner oligomeric compounds, template based compounds such as benzodiazepine, hydantoin, biaryls, carbocyclic and polycyclic compounds such as naphthalene, phenothiazine, acridine, steroids and the like, carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocyclic compounds such as triazine, indole, thiazolidine and the like, but does not limited thereto.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURE:
Figure 1: Oral (but not systemic) route of administration of 2-((4- (benzo[d]thiazol-5-ylamino)-6-(tert-butylsulfonyl)quinazolin-7-yl)oxy)ethanol
(compound 21) protects mice against lethal endotoxin shock. Female mice (n=8) were injected intrap eritoneally with Compound 21 at 2 mg/kg and or with DMSO alone as vehicle. All mice were then injected with murabutide at 10 mg/kg (Invivogen) 24 hours before a secondary challenge with a non-lethal dose of highly purified LPS (10 mg/kg of highly purified E. coli 0111 :B4 purchased from Invivogen). Mice were monitored twice daily over a 6-day period. Statistical significance was assessed by non-parametric Mann- Whitney test. P < 0.05 (*) was considered statistically significant
Figure 2: The compound 21 inhibits cytokine secretion in response to either NOD2 (eg. bacterial muramyl dipeptide) or NODI (eg. FK565) agonist. (A) Interleukin- 1 beta secretion by peripheral blood monocytes in response to active (eg. MDP) and inactive (eg. MDP-DD) bacterial muramyl dipeptide and lipopolysaccharide (LPS). (B) Interleukin-6 and (C) Interleukin-8 secretion by Beas2B epithelial cells in response to FK565. Cytokine levels were determined by specific ELISA
EXAMPLE 1:
Methods:
Mice. Mr/U2-deficient (. Nlrpl2 ' ) mice were generated through homologous recombination by using the Lex-l ES cells that are derived from the l29SvEvBrd strain (not shown). A gene-targeting vector with a neomycin-resistance cassette was constructed to replace the first two exons of Nlrpl2. The latter is required to encode the Pyrin domain of NLRP12, which is essential for recruiting ASC and subsequently for activating Caspase-l . Genotyping of positive ES clones was accomplished by Southern blotting analysis. Nlrpl2 ' mice were produced at the expected Mendelian ratio by crossing heterozygous animals and were crossed with AGi/2-dcficicnt mice12 for generating animals that are deficient for both Nod2 and Nlrpl2. Genotyping of mouse tail DNA was performed to confirm the presence of the wild-type and/or targeted alleles (data not shown). The absence of Nlrpl2 mRNA in Nlrpl2 ' animals was confirmed by quantitative reverse-transcriptase (RT)-PCR (data not shown). r/?72-deficient (. Nlrpl2 ' ) mice were backcrossed onto a C57BL6/J background. ///72-dcficicnt ( Ifit2 ' ) were generated as described elsewhere and backcrossed onto a C57BL/NCrl background (N10)29. All animal studies were approved by the local investigational review board. Animal experiments were performed in an accredited establishment (N° B59-108) according to governmental guidelines N°86/609/CEE. Age- and gender-matched animals were housed five per cages and had free access to a standard laboratory chow.
Bacterial infection. Age and sex-matched mice were orally inoculated with ~ l x 109 CFU of either C. rodentium strain DBS 100 or kanamycin-resistant C. rodentium strain DBS 120 for CFU counting in feces (kindly provided by D. Schauer, Massachusetts Institute of Technology). Histological scoring of inflammatory cells infiltration and of crypt length damage was blindly performed on hematoxylin and eosin (H&E) stained sections by two investigators.
Bone marrow transplantation experiments. Recipient mice underwent a lethal total- body irradiation. Twenty- four hours post-irradiation, mice received intravenously 5 x 105 fresh bone marrow cells. Blood was collected in heparin-containing tubes 7-8 weeks after bone- marrow transplantation and reconstitution efficiency was checked by flow cytometry using a FACS Canto II (BD biosciences) after cell staining by PE- conjugated anti-CD45. l (A20) and FITC- conjugated anti-CD45.2 (104) from BD biosciences. Two months after bone-marrow transplantation, the colonization resistance towards C. rodentium and the waterfall-shaped flow cytometric distribution of monocyte descendants was analyzed within the colon of chimeric mice.
Septic shock. Male mice (8-10 weeks old) were injected intraperitoneally with a non- lethal dose of highly purified LPS (10 mg/kg of highly purified E. coli 0l l l :B4 purchased from Invivogen) 24 hours before a secondary challenge with murabutide at 10 mg/kg (Invivogen). Alternatively, mice were challenged with a lethal dose of highly purified LPS as a model of acute endotoxin septic shock (54 mg/kg of highly purified E. coli 0l l l :B4 purchased from Invivogen). Mice were monitored twice daily over a 6-day period. The morphology of recruited cells within the peritoneum of mice was determined by cytological examination after centrifugation on glass microscope slides (Cytospin; Shandon), fixation and staining according to the manufacturer's recommendations (Diflf; Dade Behring Inc.). For cytokine measurements, serum was taken at 90’, 180’, 360’ and 540’ after secondary MDP challenge.
Immunoprecipitations. 2.5xl06 HEK293T cells were transfected with the indicated constructs using Lipofectamine2000 (Invitrogen), as indicated in the manufacturers’ protocol. Cells were harvested in RIPA buffer 24h post transfection (150 mM NaCl, 50 mM Tris pH 7.4, 1% Triton X-100, 0.1% SDS, 0.5% Na-deoxycholate) containing phosphatase inhibitors (20 M b-glycerophosphate, 5 mM NaF, 100 mM Na3V04) and protease inhibitors (Complete protease inhibitor cocktail with EDTA; Roche). The lysate was cleared 20 min at 14,000 g for 20 mins and FLAG-tagged NOD2 was subsequently precipitated for 3h at 4°C using anti- FLAG M2 agarose (Sigma-Aldrich). Proteins were separated by Laemmli SDS-PAGE and visualized using either anti- FLAG M2 antibody (Stratagene, 1 :1,000) or anti-MYC antibody (Roche, 1 :1,000).
Generation of NOD2 and NLRP12 knockout THP-1 cells lines using CRISPR/Cas9 gene editing. Analysis of the NOD2 (NP_001280486) and NLRP12 (NM 001277126) locus were deeply examined; a common translational start for all the reported iso forms were selected and used to potential KO CRISPR guide RNA pairs. The gRNAs were subsequently identified using the sanger centre CRISPR webtool (http://www.sanger.ac.uk/htgt/wge/find_crisprs). The chosen guide RNA were designed to cut as far upstream as possible to generate indels in the region containing the ATG start codon; an additional G were added to the 5' end of the guides to maximize expression from the U6 promoter. Complementary oligos were designed and annealed to yield dsDNA inserts with compatible overhangs to BsmBI-digested vectors (Shalem,Sanjana,et ah, Science, 2014), the sense guides were inserted into the puromycin selectable plasmid LentiCRISPR/Cas9v2 (Addgene #52961). HEK-293FT cells were co -transfected with the appropriate Lentivirus plasmids. Following 24 h of recovery and a further 48 hours of puromycin selection (2pg/ml), cells were subjected to a further round of puromycin selection to enrich for transfectants. THP-l cells were infected with the produced lentivirus harboring the gRNA and the Cas9 protein. The cell pools were subsequently single cell sorted by FACS and clones analyzed for NLRP12 or NOD2 depletion by immunob lotting, when applicable, and sequencing. Briefly, genomic DNA was isolated from cell candidates and the region surrounding the ATG start codon of both NOD2 and NLRP12 were amplified by PCR using a forward and a reverse primer. The resulting PCR products were subcloned into the holding vector pUCl9 and around 10 colonies were picked for each clonal line. Plasmid DNAs were isolated and sent for sequencing with primers M13F and M13R to finally select successful cell line. The absence of the NOD2 or NLRP12 protein was then confirmed, when applicable, by western-blotting.
Generation of THP1 cells stably expressing the fusion protein Myc-BirA*-NOD2. The stable THP-l cell line was generated using a retroviral system. The Myc-BirA*-NOD2 was constructed using the pLXN-retro viral vector (ClonTech,USA). The retrovirus vector was transfected into HEK293-FT cell line for the production of viruses. The viruses were harvested after 72h and the THP-l cells were infected and subsequently selected using 250pg/mL of Geneticin® (LifeTechnologies, USA). After 14 days, the surviving cells were cell sorted and individual clones were grown for 2 months. Usually, the individual clones were tested for the proper expression of Myc-BirA*-NOD2 using the <z-NOD2 (2D9) monoclonal anti-body (SantaCruz, USA).
Luciferase Reporter Assays. The NLRP12 coding sequence was inserted into the pcDNA3.l vector and site-directed mutagenesis was performed to generate the plasmids expressing the Arg284X and Arg352Cys mutations. 3xl04 HEK293T cells were seeded in a 96-well format directly prior transfection with the indicated amounts of plasmid using XtremeGene9 (Roche) as indicated in the manufacturers’ protocol. Cells were harvested in luciferase lysis buffer 24h post transfection (25mM Tris pH 8, 8mM MgCk, 1% Triton, 15% glycerol, lmM DTT) and luciferase activity was measured using a standard plate luminometer (Berthold Instruments). Luciferase activity was normalized as a ratio to b-galactosidase activity and standard deviation (SD) was calculated from triplets.
Analysis of human peripheral mononuclear cells. Patients with hereditary recurrent fever carrying the non-sense c.850 C>T (p.R284X) heterozygous mutation in NLRP12 were previously described1. The blood from patients and controls was collected in citrate collection tubes. PBMC were isolated by centrifugation using Ficoll-Paque (GE Healthcare Life Sciences). Fresh cells were stimulated with Ultrapure LPS (lOng/ml; Sigma- Aldrich), MDP (lOpg/ml; Invivogen), MDP-DD (lOpg/ml; Invivogen), or left unstimulated for 24 hours. Supernatants were collected for ELISA analysis. The treatment with 30 pg/ml of cycloheximide (Sigma-Aldrich) for 2 hours was performed on 5 million PBMCs that were isolated from fresh blood of one of the affected patient using Pancoll gradient centrifugation (BioTech) and cultured in RPMI 1640 medium. Flow cytometry analysis. Cells were stained and analyzed using a FACS LSRFortessa system (BD Biosciences). Dead cells were excluded with the LIVE/DEAD Fixable Violet Dead Cell staining kit (Life technologies). Lineage positive cells were excluded using the PerCP5.5 -conjugated anti-CD3 (17A2), anti-NKl.l(PKl36), anti- CDl9(6D5), anti-Ly6G (1A8) (Biolegend). PerCP-conjugated anti-CCR3 (83103) added to the lineage staining to exclude eosinophils was from R&D. Allophycocyanin-Cy7-conjugated anti-CDl lb (Ml/70), PerCP5.5-conjugated anti-Ly6G (1A8), Brilliant violet 5l0-conjugated anti-MHC Class II (I-A/I-E) (M5/114.15.2) and FITC-conjugated and Alexa Fluor 700- conjugated anti-Ly6C (AL21) were from BD Pharmingen. Allophycocyanin-conjugated anti- CDl lc (HL3), PE-conjugated and APC-conjugated anti-CD64 (X54-5/7.1), Alexa-Fluor 700- conjugated anti-MHC Class II (I-A/I-E) (M5/114.15.2), PECF594-conjugated anti-CDl lc (HL3), Brilliant Violet 570-conjugated anti-Ly6G (1A8), Allophycocyanin - conjugated anti- CD64 (X54-5/7.1), PE Cy7-conjugated anti-CD24 (Ml/69), Brilliant violet 650-conjugated anti-CD45.2 (104) and Brilliant violet 71 l-conjugated anti-CD45.l (A20) were all from Biolegend. PE-conjugated anti-CCR2 (475301) was from R&D systems. The data were analyzed using the FlowJo software (Tree Star).
Cytokine measurement. Cytokine levels were determined by ELISA kits, according to protocols provided by R&D Systems.
Microarray and gene-ontology analysis. Caecum specimens from non-infected and infected wild-type and Nlrpl2 ' animals were dissected out and stored at -80°C in RNA/a/er® (Ambion, Applied Biosystems, Foster City, CA), until extraction of total RNA accordingly to manufacturer’s instructions (Qiagen). The quality of the extracted RNA was confirmed by Agilent 2100 Bioanalyzer using RNA Nano 6000 (Agilent Technologies). The 4x44K Whole Mouse Genome Oligo Microarrays (Agilent Technologies) was used to determine the gene expression profile of two biological replicates. For each labelling, 2 pg of total RNA per sample were engaged in the synthesis of a fluorescent probe labelled with Cy5 or Cy3 fluorophores. A 2x2 factorial experimental design and a dye-swap strategy were used (GEO accession number GSE59940). After a within array loess normalization, raw data were analyzed with the LIMMA package and sets of differentially expressed genes were filtered for a p-value < 0.01 and a limit log fold change > 1 by using moderated t-statistic with empirical Bayes shrinkage of the standard errors. Statistics were corrected for multiple testing using False Discovery Rate approach. A gene-ontology analysis using Panther was next performed on up- and down-regulated genes that are referred in Unigene. Gene expression analysis. Isolated RNA was reverse-transcribed with the High- Capacity cDNA Archive kit (Applied Biosystems), according to the manufacturer’s instructions. The resulting cDNA (equivalent to 5ng of total RNA) was amplified using the SYBR Green real-time PCR kit and detected on a Stratagene Mx3005P (Agilent Technologies). RT-PCR was performed with the forward and reverse primers (sequences available upon request) that were designed using Primer express software, version 1.0 (Applied Biosystems, Foster City, CA). On completion of the PCR amplification, a DNA melting curve analysis was carried out in order to confirm the presence of a single and specific amplicon. Actb was used as an internal reference gene in order to normalize the transcript levels. Relative mRNA levels (2 DDa) were determined by comparing (a) the PCR cycle thresholds (Ct) for the gene of interest and Actb (ACt) and (b) ACt values for treated and control groups (AACt). RNA extraction from whole peripheral blood from controls and FCAS2 patients that were collected in PAXgene tubes was performed using PAXgene Blood RNA Kit (QIAGEN) following the manufacturer’s instructions. RNA extraction from PBMCs was performed using the RNeasy Mini Kit (Qiagen) including DNase treatment according to the manufacturer’s instructions. One pg of RNA was reversed transcribed in the presence of 2.5 mM of oligo-dT using the Reverse Transcriptor kit (Roche) following the manufacturer’s instructions. 75 ng of cDNAs were amplified using Q5 High-Fidelity 2X Master Mix (New England BioLabs). Forward and reverse primers used in PCR amplification are located in the 5’UTR and 3’UTR of NLRP12 cDNA respectively (Sequences available upon request), which can amplify the two alleles of NLRP12 in the patients and the healthy donor. Exon 3 of NLRP12 cDNA was sequenced with the Big Dye Terminator sequencing kit (Applied Biosystems) using two different primers (Table 1), and run on an ABI 3730c1 automated sequencer. Sequences were analyzed with SeqScape software (Applied Biosystems). Real time qPCRs were performed using the Mesa Blue qPCR MasterMix Plus for SYBR Assay (Eurogentec) in the Light Cycler LC480 (Roche/Boehringer, Mannheim, USA) and using specific primers for NLRP12 (Sequences available upon request). As template, we used 5 ng of cDNA. RPL13A gene was used as an internal control for normalization (Sequences available upon request).
Assembling of K48 poly-ubiquitin chains on NOD2. Assembling of K48 poly- ubiquitin chains on NOD2 in response to MDP was monitored over time. THP-l Myc-BirA*- NOD2 cells were pre-treated for one hour with DMSO (vehicle) or MG132 (at 12.5mM) before lOpg/niL of MDP (InvivoGen, France) was added. At given time points, samples were taken, spun down and the reaction was stopped using RIPA lysis buffer plus protease and phosphate inhibitors (ROCHE, Germany). Additionally, 50mM of N-ethylmaleimide (Thermofisher scientific, USA) was added to the RIPA buffer to lyse the MGl32-treated samples. A SDS gel was run and membranes were blotted against the NOD2-monoclonal antibody (2D9) (SantaCruz, USA) and RIPK2 (Cell Signalling Technology, USA) and Actin or Tubulin as a controls. NOD2 ubiquitination was assessed using the <z-poly-K48 antibody (Millipore, USA).
Ubiquitination analysis. The THP-l Myc-BirA*-NOD2 stable cell line was grown and a total of lxlO7 of cells were used for these experiments. Cells were incubated for 2 hours with MDP ( 1 Oiig/mL). After the 2 hours incubation with and without proteasome inhibitor MG132 (12.5mM), the cells were placed on ice, washed twice with PBS, spun down and incubated in lmL of lysis buffer (50mM HEPES pH7.5; l50mM NaCl; lx complete-EDTA free protease inhibitor; IX phosphatase inhibitor; 1% iGPAL and lmM PMSF). Samples were incubated for 10 minutes and then spun down at l4,000g for 20 minutes at 4°C. Supernatant was separated and incubated with anti-Myc magnetic beads (Thermofisher, UK) for 3 hours. Beads were washed 5x using the lysis buffer before they were eluted with a 0.1M Glycine solution pH 2.0. Finally, 15 pL were loaded onto the gel and immunoblotted using the monoclonal <z-poly-K48 antibody (Millipore, USA) or the monoclonal NOD2 (2D9) antibody (Santa Cruz, USA).
Statistics. Data were analyzed using Prism4.0 (GraphPad Software, San Diego, CA). The non-parametric Kruskal- Wallis test with Dunn's multiple comparison test or the parametric one-way ANOVA test with Bonferroni's multiple comparison test were used. Values represent the mean of normalized data +/- SEM. Asterisk, significant difference P<0.05.
Results:
NLRP12 interacts with NOD2 through a linker-region proximal to the nucleotide-binding domain that is required for ATP binding.
Since NLRP12 may potentially interact with both Caspase-activating recruitment domains (CARDs) of NOD2 but not that of NOD117, we hypothesized that NLRP12 may promote MDP tolerance by dissociating the NOD2-HSP90 complex, which is required for NF-KB activation in response to bacteria16. To this end, we generated THP-l cells stably expressing the fusion protein Myc-BirA*-NOD2. In accordance with yeast two-hybrid screen data17, we confirmed the NOD2-NLRP12 interaction in monocytic cell line when specifically inhibiting the proteasome degradation of NOD2 that is induced in response to MDP (data not shown). In contrast, the formation the NOD2-NLRP12 complex is not observed in response to bacterial lipopolysaccharide (LPS) that is not sensed by either NOD2 nor NLRP12 (data not shown). HEK-293T cells were next transfected with plasmids transiently expressing FLAG-tagged NOD2 and Myc-tagged NLRP12 (data not shown). Overexpression of full- length FLAG-tagged NOD2 together with Myc-tagged NFRP12 resulted in an interaction with RIPK2, which was underrepresented in the complex at high NFRP12 concentration as shown by immunoprecipitation using an anti-FFAG antibody (data not shown). Mapping of the interaction domain from human NFRP12 further showed that the assembly of a protein complex with NOD2 involved a linker region of NFRP12 (residues 200-224) in which ATP binding is required for the inhibition to occur2 (data not shown). In contrast, the N-terminal Pyrin domain of NFRP12 (residues 1-98) that is required for apoptotic signaling18 was found dispensable for specifically interacting with NOD2 (data not shown). Together, these results suggested that NFRP12 might interfere with NOD2 signaling in monocyte-derived cells where NFRP12 is primarily expressed (data not shown).
NLRP12 dominantly suppress MDP-induced NF-kB activation by promoting degradation of the NOD2/RIPK2 complex.
To corroborate the role of NFRP12 as a potential checkpoint blocker of NOD2 signaling in monocytes, we examined the influence of NFPR12 on the stability and the activity of the NOD2/RIPK2 complex. Co-immunoprecipitation experiments revealed that NFRP12 expression promotes poly-ubiquitination of the NOD2/RIPK2 complex in HEK- 293T cells (data not shown). In contrast, the ubiquitination status of NOD1 was not influenced by full-length NFRP12 (data not shown). Consistent with a regulation of NOD2 activity at the protein level though its interaction with NFRP12, blocking of protein neosynthesis using cycloheximide led us to reveal that NFRP12 expression reduced the half- live of the NOD2 protein (data not shown). This inhibitory effect of NFRP12 on the stability ofNOD2 was compromised upon inhibition of the ubiquitin-proteasome pathway by MG132 (data not shown). Accordingly, transient expression of full-length human NFRP12 greatly reduced the NOD2-mediated p50/p65 reporter activation in response to MDP in HEK-293T cells (data not shown). While increasing amounts of transfected NFRP12 further reduced MDP-induced NF-kB activation by about 70% (data not shown), this correlated with the lowered protein levels of both NOD2 and RIPK2 when NFRP12 is overexpressed (data not shown). The formation of such protein complex between NOD2 and NFRP12 coincided with a greater assembling of K48 poly-ubiquitin chains on NOD2 when pretreating THP-l cells stably expressing the fusion protein Myc-BirA*-NOD2 with the proteasome inhibitor MG 132 (data not shown). As reported previously19, co-immunoprecipitation experiments confirmed that an interaction between NOD2 and the chaperone protein HSP90 in response to MDP that is critical for the inhibition to occur (data not shown). Indeed, pretreating THP1 -cells with MG132 strongly inhibited the formation of the NOD2/HSP90 complex in response to MDP, while LPS stimulation expectedly failed to do so (data not shown). Consequently, NOD2 was migrating at higher molecular weights (visible as a smear) when treating THP-l cells with MDP (data not shown), in which a greater amount of NLRP12 was detected by western blotting (data not shown). Collectively, we identified NLRP12 as a NOD2-interacting protein that may promote NOD2 degradation and subsequently MDP tolerance of monocytes and potentially in their daughter cells arising from their differentiation.
NLRP12 deficiency impairs MDP tolerance in mice.
A lowered NLRP12 gene expression is observed in patients with septic shock20, suggesting a potential feed-back regulatory loop on NLRP12 function during sepsis. Indeed, endotoxin treatment negatively regulates NLRP12 promoter activity in human monocytes through the PR domain-containing 1, with ZNF domain21. Given that knocking-down NLRP12 expression may enhance TLR4 signaling in vitro3, we next aimed to confirm whether Nlrpl2 expression may indirectly influence host responsiveness to bacterial LPS in vivo. To this end, we made use of Mr/U2-deficient mice that were generated by replacing the second exon encoding the Pyrin domain of NLRP12 with a neomycin selection cassette through homologous recombination (data not shown). Wild-type and mutant mice were challenged with a lethal dose of highly purified LPS from Escherichia coli 011 LB4 as a model of acute endotoxin septic shock. Unlike Caspasell- and 77r4-dcficicnt mice22, no difference in survival of wild-type and Mr/U2-deficient mice was observed (data not shown). We next tested the potential role of NLRP12 in the susceptibility of LPS-sensitized mice to a secondary challenge with MDP23. Wild-type and Nlrpl2- deficient mice were primed with a non-lethal dose of highly purified LPS from E coli 0111 :B4. As a consequence of a failure to negatively regulate NOD2 signaling, LPS-primed Nlrp 12 -deficient mice were significantly more susceptible to secondary MDP challenge when compared to similarly treated control animals (data not shown). We next generated mice that are deficient for both Nod2 and Nlrpl2 and compared the survival rate of this compound mutant mice to the one of Nlrpl2- deficient mice. Expectedly, a greater survival rate was observed in the absence of both NOD2 and NLRP12 (data not shown). Collectively, our results indicate that NLRP12 is dispensable for protecting mice against endotoxemia, but rather function as a negative regulator of NOD2 signaling in mice. MDP Tolerance is lost in monocytes that are either deficient for NLRP12 or expressing the NLRP12 mutation that is causing FCAS2.
Given that unrestrained human NOD2 signaling results in auto -inflammatory syndromes24, we next assessed whether the FCAS2-causing mutation in the NLRP12 gene may fail to dominantly repress activation of NF-kB in response to MDP. To this end, plasmids encoding the most frequent mutations in NLRP12 were generated. Each mutant was transiently transfected in HEK-293T cells together with full-length NOD2. All mutants with single amino-acid replacements were found to efficiently repress MDP-induced activation of NF-KB (data not shown) and to interact with HSP90 (data not shown), as what observed in cells expressing wild-type NLRP12. In contrast, the R284X nonsense mutation failed to inhibit the activation of NF-kB in response to MDP (data not shown) and of the JAK/STAT pathway by the S/T kinase TANK-binding kinase 1 that is commonly referred as TBK-l (data not shown). It coincided with a barely detectable recruitment of HSP90 by such mutation (data not shown) even if this was not related to a failure of the R284X nonsense mutation to interact with NOD2 (data not shown). As a consequence, loss of NLRP12 expression by CRISPR/Cas9 system in human monocytic THP-l cells enhanced secretion of TNF-a in response to MDP when compared to parental cells (data not shown). Likewise, MDP induced a greater secretion of either tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL-6) by PBMCs from patients bearing the R284X nonsense mutation when compared to control cells (data not shown). Such lack of MDP tolerance was the consequence of the activation of a surveillance pathway referred to as nonsense-mediated mRNA decay, which can be blocked by cycloheximide (data not shown). This provided a potential explanation for the loss of tolerance to MDP that account for the failure to detect the truncated protein in such mutant cells by western blotting (data not shown). Expectedly, the possibility of a dominant negative effect was further ruled out by confirming that the protein level of the full-length isoform of NLRP12 was lowered by about 30% in PBMCs from twin patients bearing the R284X nonsense mutation when compared to control lysates (data not shown). Meanwhile, a greater activation of the NF-kB transcriptional complex by MDP was observed in monocytes from the bone marrow of Nlrp 12 -deficient mice but not in those from Nlrpl2 / :Nod2 / mice (data not shown). Consequently, a greater secretion of both TNF-a and IL-6 was also detectable in the supernatant of LPS-primed monocytes that were subsequently treated by MDP (data not shown). Likewise, similar findings were observed in macrophages that were derived from the bone marrow of Nlrp 12 -deficient mice (data not shown), even if the difference was less pronounced when compared to what observed in monocytes (data not shown). A potential explanation for this difference may result from the down-regulation of NLRP12 during differentiation of monocytes into macrophages (data not shown and 21). Collectively, these results suggested that the serosal inflammation of FCAS2 patients might result from an impaired MDP tolerance of monocytes.
Loss of tolerance in the intestine of 7Wfy /2-deficient mice is caused by type I and/or III interferon downstream of NOD2 signaling.
We next examined at high-resolution the colon and caecum of Nlrp 12 -deficient mice for any spontaneous signs of inflammation as a consequence of an impaired tolerance to MDP derived from the gut microbiota. To this end, whole-genome expression analysis was performed on RNA extracted from the caecum of wild-type and mutant mice. A set of 106 genes were differentially regulated as a result of Nlrpl2 deficiency, among which 35 genes were significantly up-regulated by at least one log2-fold change in A7/y /2-dcficicnt mice compared to controls (data not shown). Of these, gene-ontology analysis identified a significant overrepresentation of ISG (data not shown), including those encoding interferon- induced protein 44 (IFI44), interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), Apo lipoprotein L9 (APOL9a/b) and 2’-5’-Oligoadenylate synthetase 2 (OAS2). Transcriptional activation of the promoters of those ISG is allowed through the binding to Interferon Stimulated Response Elements (ISRE) of the heteromeric IFN-stimulated gene factor 3 complex (ISGF3) that is composed of the signal transducer and activator of transcription 1 (STAT1). In agreement with this, a tonic activation of the transcription factor STAT1 was observed in the caecum of A7/y /2-dcficicnt mice when compared to that in controls (data not shown), while it was lowered in the absence of NOD2 signaling at steady state (data not shown). Consistently, qRT-PCR analysis confirmed a greater expression of the aforementioned differentially expressed genes (namely lfi44, Ifit2, Oas2 and Apol9a/b ) in the caecum from A7/y /2-dcficicnt mice (data not shown), but not in the intestine of either Nlrpl2 / :Nod2 / or Nod2 / mice (data not shown). Further, the use of the ISRE-luciferase reporter gene assay led us to reveal that overexpression of NFRP12 significantly lowered the activation of the JAK/STAT signaling pathway by TBK-l (data not shown). To further understand the sequence of events leading to this robust antiviral response at baseline, RNA was next extracted from isolated intestinal epithelial cells (IECs) and from lamina propria mononuclear cells (FPMCs) of wild-type and mutant intestine. In line with previous findings, the transcript level of the above-mentioned differentially expressed genes (including lfi44, Ifit2, Oas2 and Apol9a/b ) was primarily enriched in primary IECs isolated from both the colon and the caecum of Nlrp 12 -deficient mice (data not shown). Consistently, immunohistochemical analysis revealed an enhanced production of either IFIT2 or OAS2 that is essentially restricted to the epithelium of the caecum and proximal colon from Nlrpl2- deficient mice (data not shown). This is in line with the idea that NOD2-mediated inflammasome activation is enhanced by IL-3225, which subsequently triggers type I/III IFNs activation2627. In contrast, the transcript levels of numerous genes with ascribed function on mucosal adaptive immunity (including Ighm, Ptprc, Anp32a and Mcptl ) were not significantly changed (data not shown). To specifically elucidate the cell type responsible for the negative effect of NLRP12 on epithelial expression of IFIT228, we generated bone- marrow chimeras. While the expression of IFIT2 was barely detectable in the proximal colon of wild-type chimeric mice (WT WT) (data not shown), the intestinal epithelium of Nlrp 12 -deficient recipients that were reconstituted with hematopoietic cells from mutant mice ( KO KO ) was characterized by higher protein level of IFIT2 (data not shown) as what observed in non-chimeric mutant mice (data not shown). Surprisingly, the epithelial expression of IFIT2 was observed in wild-type recipients that were adoptively transplanted with leukocytes from Nlrp 12 -deficient mice ( KO WT) (data not shown). This coincided with a lowered abundance of macrophages in the intestine 8 weeks after reconstitution of wild-type and mutant recipients with Nlrp 12 -deficient leukocytes, compared to animals that received wild-type bone marrow cells (data not shown). By contrast, transfer of wild-type bone marrow into mutant mice (WT /f<9) failed to induce epithelial expression of IFIT2 (data not shown). Similar results were observed in the caecum of chimeric mice (data not shown). In accordance with our data in the model of acute endotoxin septic shock (data not shown), IFIT2 primarily functions as a downstream effector of the IFN-l receptor28 that may subsequently influence the outcome of Nlrp 12 -deficient mice in response to endotoxin shock29. While IFIT2 is thought to regulate cell death and inflammation29, epithelial proliferation is also orchestrated by several additional ISG that were upregulated within the epithelium of Nlrp 12 -deficient mice30, such as Apol9a/b31. In this context, it is worth noting that the effect of IFN alpha/beta on IFIT2 induction is far less compartmentalized than the one of the IFN lambda receptor that is primarily expressed to epithelial cells32. In line with this finding, intrap eritoneal injection of recombinant type III IFN specifically triggered epithelial expression of IFIT2 in the intestine (data not shown). Altogether, the bone marrow experiments demonstrated that NLRP 12 deficiency in leukocytes is responsible for a greater inflammatory response driven by type I/III interferons downstream of NOD2 signaling in monocytes. This suggested us such epithelial ISG induction is likely the consequence of greater NOD2-dependent secretion of type I/III interferons by monocytes that migrate to the tissue from the bone marrow, but it is still not clear whether it functions independently.
NLRP12 deficiency contributes to a greater colonization resistance against attaching-and-effacing bacterial pathogen through activation of NOD2 signaling in monocytes.
Previous work showed that NOD2 signaling is required for optimal eradication of C. rodentium33 34, which is a mouse-restricted model for attaching and effacing (A/E) enteric bacterial-induced diarrhea such as those caused by EPEC and EHEC. C. rodentium colonizes the caecum and the colon of mice through attachment to the epithelium, effacement of microvilli-covered surface and the formation of pedestal-like structure9. Given that NLRP12 negatively MDP tolerance by regulating the stability of NOD2/RIPK2 complex, we next determined whether loss of NLRP12 may protect mice from C. rodentium as a potential consequence of a greater epithelial expression of IFIT2 n’ 34. To this end, we orogastrically inoculated wild-type, ///72-dcficicnt and Nlrp 12 -deficient mice with lxlO9 colony- forming units of C. rodentium. A kanamycin-resistant strain of C. rodentium was used for non- invasive monitoring of bacterial growth in the feces. As expected35, a typical bacterial shedding curve was observed in wild-type mice (data not shown), however the intestine of Mr/?72-deficient mice was less rapidly colonized by C. rodentium (data not shown). In contrast, the peak of bacterial colonization occurred earlier in Ifit2- deficient mice (data not shown) as what observed in the absence of STAT1 that is required for Caspasel 1 -dependent inflammasome activation in response to C. rodentium36. The latter results suggested to us that enhanced IFIT2 secretion at steady state may protect the intestine of A7/y /2-dcficicnt mice against C. rodentium, which is most likely caused by a loss of tolerance to MDP. We next compared the colonization dynamic of C. rodentium in mice that are deficient for both NLRP12 and NOD2. As what observed in Abi/2-dcficicnt mice (data not shown), bacterial burden among compound mutant mice was persisting even two weeks post-infection (data not shown). Consistent with a tolerogenic property of NLRP12 on NOD2 signaling in monocytes, intraluminal elimination of C. rodentium was improved in lethally irradiated mice that were specifically engrafted with the bone marrow of A7/y /2-dcficicnt mice although this difference failed to reach statistical significance (data not shown). Conversely, the colonization resistance was found similar in both control and mutant mice that were reconstituted with wild-type bone marrow cells (data not shown). To obtain additional evidence that such colonization resistance result from a loss of MDP tolerance in monocytes, controls and Ly s M -C rc ; Nod2fi n mice, in which NOD2 is specifically deleted in LysM- expressing cells, were orally infected with C. rodentium for analyzing the intraluminal bacterial load. A similar number of the pathogen to what seen in AΆ/2-dcficicnt mice was recovered from feces of Ly s M -C rc ; No 211 11 mice compared to Nod2fm animals and to those that express the Cre recombinase in IECs (data not shown). Such delayed clearance of C. rodentium from the gut lumen resulted in a greater bacterial dissemination in the spleen (data not shown). This was associated with spenomegaly (data not shown) and tissue pathology as evidenced respectively by about 47 percent increase in spleen weight (data not shown) and by the enhanced histological score (data not shown) and a 30 percent increase in the crypt length (data not shown). To further examine the basis of how NOD2-mediated host defense may protect Nlrp 12 -deficient mice against C. rodentium, R A was extracted from the caecum of infected mice at day 0 and day 7 post-infection and a genome-wide analysis of the acute transcriptional response to the pathogen was performed (data not shown). As expected, C. rodentium infection was found to robustly induce expression of many genes involved in Thl- and Thl7-mediated host defense37. We identified a set of 551 transcripts whose expression was modulated at least one log2-fold change in response to the infection independently of NLRP12 expression (overlapping zone in Venn diagram; data not shown). By contrast, the expression of 427 genes showed a greater degree of change as a consequence of NLRP12 deficiency. Gene ontology analysis of these 427 differentially expressed genes revealed a significant enrichment of up-regulated molecules that were primarily assigned to the functional categories of T helper cell differentiation and granulocyte adhesion and diapedesis (data not shown). In agreement with these results, the colon lengths of Nlrpl2- deficient mice was significantly shortened at day 7 post-infection when compared to controls (data not shown). An enhanced luminal level of lipocalin-2 that is primarily secreted by neutrophils was subsequently found in the absence of NLRP12 as early as day 8 after bacterial infection (data not shown). This was correlated with a greater thickening of the colonic mucosa (data not shown) and with an increased length of the colonic crypts of Nlrpl2- deficient mice (data not shown) that is most likely caused by greater recruitment of leukocytes in response to the pathogen. To further explore this possibility, the mononuclear phagocyte system from the colon of mice was examined in response to the pathogen (data not shown). Cytofluorometry analysis confirmed enhanced accumulation of Ly6ChlMHCIIhlCCR2+ cells within cell suspensions of the colonic lamina propria of infected AU /2-dcficicnt mice when compared to that in controls on day 4 (data not shown). Of note, the frequency of residing macrophages (Ly6C MHCIIlo to hlCCR2 ) was similar in mutant and control mice (data not shown). When compared to that in Nlrpl2 / :Nod2 / mice, the colon of AY/y /2-dcficicnt mice showed an increased accumulation of macrophages (265±62 vs 54±49 of Ly6C CD64+CDl lb+ cells among a pool of 10,000 Lin MHCII+ mononuclear cells) (data not shown) in which NLRP12 has been shown to negatively regulate extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling38. This was followed by an increased phosphorylation of ERK1/2 as what observed in response to Salmonella enterica serovar Typhimurium6 (data not shown). As a consequence, such imbalance in the tolerogenic milieu suggested to us that C. rodentium may exploit NLRP12 signaling for limiting the accumulation of monocytes when being recruited at the site of the infection. By contrast, no change in autophagy induction was observed at either day 7 or 14 post-infection (data not shown) and the monocytes isolated from intestine of ///72-dcficicnt mice showed a similar MHCII expression with a progressive loss of Ly6C marker when compared to that in control mice (data not shown). Altogether, these results demonstrated that NOD2 signaling promotes accumulation of phagocytes to the site of infection in A7/y /2-dcficicnt mice, which may subsequently contribute to the improved clearance of C. rodentium 9.
EXAMPLE2:
We also test prophylactic effect of orally administered compound of administration of 2-((4-(benzo[d]thiazo 1-5 -ylamino)-6-(tert-butylsulfonyl)quinazo lin-7-yl)oxy)ethano 1
(compound 21) in a mouse model of sepsis. We show that oral route protects mice against lethal endotoxin shock (Figure 1). We also show that the compound 21 inhibits cytokine secretion in response to either NOD2 (eg. bacterial muramyl dipeptide) or NOD1 (eg. FK565) agonist (Figures 2A, 2B and 2C).
Discussion:
Herein, we unveil a novel function of NLRP12 as a checkpoint blocker of NOD2 signaling in monocytes that provides a potential explanation for the recurrent episodes of serosal inflammation (including peritonitis and abdominal pains) in patients bearing non sense mutations in the NLRP12-encoding gene1. Such paradigm indicates that disease manifestation in patients with NLRP12 mutations is likely initiated by the influence of some specific interactions with the gut microbiota that are regulated by the Crohn’s disease predisposing NOD2 gene41. In mice, this may subsequently account for the colitis-prone changes in the composition of the gut microbiota that are caused by NLRP12 deficiency40. In this context, the exact role of NLRP12 signaling on NOD2-mediated resilience of the gut microbiota now deserves further experimental studies with littermate controls and co-housed mice.
Equally important is that protective immunity against enteric bacterial pathogens in Mr/?72-deficient mice relies on the accumulation of inflammatory monocytes through NOD2 signaling in mice, although it is unclear whether this reflects an improved NOD2-dependent survival of newly extravasated blood monocytes. Such non-resolving inflammatory conditions may also reflect the impact of some NOD2-mediated changes in the composition of the gut microbiota that were observed in mice that are deficient for NLRP1240. We are currently investigating the cause of such influx of inflammatory monocytes that correlated with ISG induction within the epithelium of Nlrp 12 -deficient mice. Further, we provide evidence that NLRP12 signaling within the hematopoietic compartment is exploited by C. rodentium in mice as what observed with Salmonella enterica serovar Typhimurium34. This agrees with a recent report showing enhanced NOD2-mediated protective immune response against C. rodentium in animals that are hypomorphic for Atgl6Ll protein expression 15. One may also consider that loss of NLRP12 may influence the persistence of some viruses as a consequence of the greater epithelial expression of several ISG, including IFIT2 that is involved in antiviral immunity and OAS2 is a RNA-binding protein that interacts with NOD242.
Mutational analysis led us to identify the essential domain (residues 200-224) of NFRP12 that is interacting with the Crohn’s disease predisposing NOD2 protein. Our finding indicated that the nucleotide-bound state of NFRP12 may repress the stability of NOD2 by sequestrating HSP90 that is required for stabilizing the NOD2/RIPK2 complex in response to MDP16. This highly conserved region of NFRP12 may contribute to some conformational changes of HSP90. It shares similarities with the fish- specific NACHT associated domain (referred as PF 14484) and is responsible for degradation of NIK2, which is known to regulate the NOD2-mediated signaling pathway in monocytes43. Noteworthy, it includes the ATP/GTP-specific phosphate binding loop called Walker A and a Mg2+ coordination site called Walker B. This agrees with previous reports indicating a suppressive function of ATP- binding domain on the activity of the plant R protein 1-2 that shows similarities with NFRP1244. As a consequence of its interaction with NFRP12, NOD2 was ubiquitinated and the NOD2/RIPK2 complex showed lowered stability in vitro.
Collectively, we provide the rational for targeting therapeutically NOD2 signaling in familial cold auto -inflammatory syndrome by unveiling an unappreciated molecular link with the pathogenesis of Crohn’s disease. As proposed for some patients with Familial Mediterranean Fever45, our results suggest a potential selective advantage of disease-causing NLRP12 mutations against some enteric bacterial infections as a consequence of a loss of MDP tolerance.
REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
1. Jeru, I. et al. Mutations in NALP12 cause hereditary periodic fever syndromes. Proceedings of the National Academy of Sciences of the United States of America 105, 1614- 1619 (2008).
2. Ye, Z. et al. ATP binding by monarch- l/NLRP 12 is critical for its inhibitory function. Molecular and cellular biology 28, 1841-1850 (2008).
3. Williams, K.L. et al. The CATERPILLER protein monarch- 1 is an antagonist of toll-like receptor-, tumor necrosis factor alpha-, and Mycobacterium tuberculosis-induced pro-inflammatory signals. The Journal of biological chemistry 280, 39914-39924 (2005).
4. Allen, I.C. et al. NLRP12 suppresses colon inflammation and tumorigenesis through the negative regulation of noncanonical NF-kappaB signaling. Immunity 36, 742-754 (2012).
5. Zaki, M.H. et al. The NOD-like receptor NLRP12 attenuates colon inflammation and tumorigenesis. Cancer cell 20, 649-660 (2011).
6. Zaki, M.H., Man, S.M., Vogel, P., Lamkanfi, M. & Kanneganti, T.D. Salmonella exploits NLRP12-dependent innate immune signaling to suppress host defenses during infection. Proceedings of the National Academy of Sciences of the United States of America (2013).
7. Vladimer, G.I. et al. The NLRP12 inflammasome recognizes Yersinia pestis. Immunity 37, 96-107 (2012).
8. Kaper, J.B., Nataro, J.P. & Mobley, H.L. Pathogenic Escherichia coli. Nature reviews. Microbiology 2, 123-140 (2004).
9. Mundy, R., MacDonald, T.T., Dougan, G., Frankel, G. & Wiles, S. Citrobacter rodentium of mice and man. Cellular microbiology 7, 1697-1706 (2005).
10. Poulin, L.F. & Chamaillard, M. The battlefield in the war against attaching- and-effacing bacterial pathogens: Monocytes, macrophages and dendritic cells in action. Vet Microbiol 202, 47-51 (2017). 11. Kim, Y.G. et al. The cytosolic sensors Nodl and Nod2 are critical for bacterial recognition and host defense after exposure to Toll-like receptor ligands. Immunity 28, 246- 257 (2008).
12. Kobayashi, K.S. et al. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science 307, 731-734 (2005).
13. Homer, C.R., Richmond, A.L., Rebert, N.A., Achkar, J.P. & McDonald, C. ATG16L1 and NOD2 interact in an autophagy-dependent antibacterial pathway implicated in Crohn's disease pathogenesis. Gastroenterology 139, 1630-1641, 1641 el631-1632 (2010).
14. Sorbara, M.T. et al. The protein ATG16L1 suppresses inflammatory cytokines induced by the intracellular sensors Nodl and Nod2 in an autophagy-independent manner. Immunity 39, 858-873 (2013).
15. Marchiando, A.M. et al. A deficiency in the autophagy gene Atgl6Ll enhances resistance to enteric bacterial infection. Cell host & microbe 14, 216-224 (2013).
16. Lee, K.H., Biswas, A., Liu, Y.J. & Kobayashi, K.S. Proteasomal degradation of Nod2 protein mediates tolerance to bacterial cell wall components. The Journal of biological chemistry 287 , 39800-39811 (2012).
17. Wagner, R.N., Proell, M., Kufer, T.A. & Schwarzenbacher, R. Evaluation of Nod-like receptor (NLR) effector domain interactions. PloS one 4, e493l (2009).
18. Ting, J.P., Willingham, S.B. & Bergstralh, D.T. NLRs at the intersection of cell death and immunity. Nature reviews. Immunology 8, 372-379 (2008).
19. Arthur, J.C., Lich, J.D., Aziz, R.K., Kotb, M. & Ting, J.P. Heat shock protein 90 associates with monarch- 1 and regulates its ability to promote degradation of NF-kappaB- inducing kinase. Journal of immunology 179, 6291-6296 (2007).
20. Fahy, R.J. et al. Inflammasome mRNA expression in human monocytes during early septic shock. American journal of respiratory and critical care medicine 177, 983-988 (2008).
21. Ford, C.A. et al. Blimp- 1/PRDM1 mediates transcriptional suppression of the NFR gene NFRP12/Monarch- 1. Journal of immunology 182, 2948-2958 (2009).
22. Kayagaki, N. et al. Noncanonical inflammasome activation by intracellular EPS independent of TFR4. Science 341, 1246-1249 (2013).
23. Kobayashi, S., Nantz, R., Kitamura, T., Higashikubo, R. & Horikoshi, N. Combined inhibition of extracellular signal-regulated kinases and HSP90 sensitizes human colon carcinoma cells to ionizing radiation. Oncogene 24, 3011-3019 (2005). 24. Miceli-Richard, C. et al. CARD15 mutations in Blau syndrome. Nature genetics 29, 19-20 (2001).
25. Netea, M.G. et al. IL-32 synergizes with nucleotide oligomerization domain (NOD) 1 and NOD2 ligands for IL-lbeta and IL-6 production through a caspase 1 -dependent mechanism. Proceedings of the National Academy of Sciences of the United States of America 102, 16309-16314 (2005).
26. Li, Y. et al. Inducible interleukin 32 (IL-32) exerts extensive antiviral function via selective stimulation of interferon lambdal (IFN-lambdal). The Journal of biological chemistry 288, 20927-20941 (2013).
27. Park, G.B. et al. TLR3/TRIF signalling pathway regulates IL-32 and IFN-beta secretion through activation of RIP- 1 and TRAF in the human cornea. J Cell Mol Med 19, 1042-1054 (2015).
28. Pott, J. et al. IFN-lambda determines the intestinal epithelial antiviral host defense. Proceedings of the National Academy of Sciences of the United States of America 108, 7944-7949 (2011).
29. Siegfried, A. et al. IFIT2 is an effector protein of type I IFN-mediated amplification of lipopolysaccharide (LPS)-induced TNF-alpha secretion and LPS-induced endotoxin shock. Journal of immunology 191, 3913-3921 (2013).
30. Katlinskaya, Y.V. et al. Type I Interferons Control Proliferation and Function of the Intestinal Epithelium. Molecular and cellular biology 36, 1124-1135 (2016).
31. Sun, L. et al. Type I interferons link viral infection to enhanced epithelial turnover and repair. Cell host & microbe 17, 85-97 (2015).
32. Sommereyns, C., Paul, S., Staeheli, P. & Michiels, T. IFN-lambda (IFN- lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo. PLoS pathogens 4, el0000l7 (2008).
33. Kim, Y.G. et al. The Nod2 sensor promotes intestinal pathogen eradication via the chemokine CCL2-dependent recruitment of inflammatory monocytes. Immunity 34, 769- 780 (2011).
34. Geddes, K. et al. Identification of an innate T helper type 17 response to intestinal bacterial pathogens. Nature medicine 17, 837-844 (2011).
35. Collins, J.W. et al. Citrobacter rodentium: infection, inflammation and the microbiota. Nature reviews. Microbiology 12, 612-623 (2014).
36. Rathinam, V. A. et al. TRIF licenses caspase- 11 -dependent NLRP3 inflammasome activation by gram-negative bacteria. Cell 150, 606-619 (2012). 37. Spehlmann, M.E. et al. CXCR2-dependent mucosal neutrophil influx protects against colitis-associated diarrhea caused by an attaching/effacing lesion-forming bacterial pathogen. Journal of immunology 183, 3332-3343 (2009).
38. Lich, J.D. et al. Monarch-l suppresses non-canonical NF-kappaB activation and p52-dependent chemokine expression in monocytes. Journal of immunology 178, 1256-
1260 (2007).
39. Satpathy, A.T. et al. Notch2-dependent classical dendritic cells orchestrate intestinal immunity to attaching-and-effacing bacterial pathogens. Nature immunology 14, 937-948 (2013).
40. Chen, L. et al. NLRP12 attenuates colon inflammation by maintaining colonic microbial diversity and promoting protective commensal bacterial growth. Nature immunology 18, 541-551 (2017).
41. Couturier-Maillard, A. et al. NOD2-mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer. J Clin Invest 123, 700-711 (2013).
42. Dugan, J.W. et al. Nucleotide oligomerization domain-2 interacts with 2'-5'- oligoadenylate synthetase type 2 and enhances RNase-L function in THP-l cells. Molecular immunology 47, 560-566 (2009).
43. Pan, Q. et al. NF-kappa B-inducing kinase regulates selected gene expression in the Nod2 signaling pathway. Infection and immunity 74, 2121-2127 (2006).
44. Tameling, W.I. et al. Mutations in the NB-ARC domain of 1-2 that impair ATP hydrolysis cause autoactivation. Plant physiology 140, 1233-1245 (2006).
45. Cattan, D. MEFV mutation carriers and diseases other than familial Mediterranean fever: proved and non-proved associations; putative biological advantage. Current drug targets. Inflammation and allergy 4, 105-112 (2005).

Claims

CLAIMS:
1. A method for treating hereditary periodic fevers in a subject in need thereof comprising administering the subject with a therapeutically effective amount of at least one inhibitor of NOD2-mediated signaling pathway.
2. The method of claim 1 wherein the hereditary periodic fever is a NLRPl2-associated hereditary periodic fever
3. The method of claim 1 wherein the hereditary periodic fever is a familial cold- induced auto -inflammatory syndrome
4. The method of claim 1 wherein the inhibitor of NOD2-mediated signaling pathway is a RIP2 inhibitor.
5. A method for screening a plurality of test substances useful for the treatment of hereditary periodic fevers in a subject in need thereof comprising the steps consisting of (a) testing each of the test substances for its ability to inhibit NOD2-mediated signaling pathway and (b) and positively selecting the test substances capable of inhibiting the NOD2-mediated signaling pathway.
6. The method of claim 5 which comprises the step of (i) providing a RIP2 protein; (ii) contacting the RIP2 protein with a test substance wherein the substance is expected to inhibit the phosphorylation or kinase activity of the RIP2 protein; and (iii) selecting a test substance as a candidate that decreases the phosphorylation level or the kinase activity of RIP2 in comparison to a negative control that is not contacted with a test substance.
7. The method of claim 5 which comprises the steps of i) bringing into contact the test substance to be tested with a mixture of a first RIP2 protein (2) a second NOD2 protein, ii) determining the ability of said test substance to inhibit the binding between the RIP2 protein and the NOD2 protein and iii) positively selected the test substance that is capable to inhibit the binding between the RIP2 protein and the NOD2 protein.
PCT/EP2019/080183 2018-11-06 2019-11-05 Nod2 inhibitors for the treatment of hereditary periodic fevers WO2020094613A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP18306453 2018-11-06
EP18306453.4 2018-11-06

Publications (1)

Publication Number Publication Date
WO2020094613A1 true WO2020094613A1 (en) 2020-05-14

Family

ID=64270785

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2019/080183 WO2020094613A1 (en) 2018-11-06 2019-11-05 Nod2 inhibitors for the treatment of hereditary periodic fevers

Country Status (1)

Country Link
WO (1) WO2020094613A1 (en)

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981732A (en) 1998-12-04 1999-11-09 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-13 expression
US6046321A (en) 1999-04-09 2000-04-04 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-i1 expression
US6107091A (en) 1998-12-03 2000-08-22 Isis Pharmaceuticals Inc. Antisense inhibition of G-alpha-16 expression
US6365354B1 (en) 2000-07-31 2002-04-02 Isis Pharmaceuticals, Inc. Antisense modulation of lysophospholipase I expression
US6410323B1 (en) 1999-08-31 2002-06-25 Isis Pharmaceuticals, Inc. Antisense modulation of human Rho family gene expression
US6566135B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of caspase 6 expression
US6566131B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of Smad6 expression
WO2011120025A1 (en) 2010-03-26 2011-09-29 Glaxo Group Limited Indazolyl-pyrimidines as kinase inhibitors
WO2011120026A1 (en) 2010-03-26 2011-09-29 Glaxo Group Limited Pyrazolyl-pyrimidines as kinase inhibitors
WO2011123609A1 (en) 2010-03-31 2011-10-06 Glaxo Group Limited Imidazolyl-imidazoles as kinase inhibitors
WO2011140442A1 (en) 2010-05-07 2011-11-10 Glaxo Group Limited Amino-quinolines as kinase inhibitors
WO2012021580A1 (en) 2010-08-10 2012-02-16 Glaxo Group Limited Quinolyl amines as kinase inhibitors
WO2012122011A2 (en) 2011-03-04 2012-09-13 Glaxosmithkline Llc Amino-quinolines as kinase inhibitors
WO2013025958A1 (en) 2011-08-18 2013-02-21 Glaxo Group Limited Amino quinazolines as kinase inhibitors
WO2014043437A1 (en) 2012-09-13 2014-03-20 Glaxosmithkline Llc Amino-quinolines as kinase inhibitors
WO2014043446A1 (en) 2012-09-13 2014-03-20 Glaxosmithkline Llc Prodrugs of amino quinazoline kinase inhibitor

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6107091A (en) 1998-12-03 2000-08-22 Isis Pharmaceuticals Inc. Antisense inhibition of G-alpha-16 expression
US5981732A (en) 1998-12-04 1999-11-09 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-13 expression
US6046321A (en) 1999-04-09 2000-04-04 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-i1 expression
US6410323B1 (en) 1999-08-31 2002-06-25 Isis Pharmaceuticals, Inc. Antisense modulation of human Rho family gene expression
US6365354B1 (en) 2000-07-31 2002-04-02 Isis Pharmaceuticals, Inc. Antisense modulation of lysophospholipase I expression
US6566135B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of caspase 6 expression
US6566131B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of Smad6 expression
WO2011120026A1 (en) 2010-03-26 2011-09-29 Glaxo Group Limited Pyrazolyl-pyrimidines as kinase inhibitors
WO2011120025A1 (en) 2010-03-26 2011-09-29 Glaxo Group Limited Indazolyl-pyrimidines as kinase inhibitors
WO2011123609A1 (en) 2010-03-31 2011-10-06 Glaxo Group Limited Imidazolyl-imidazoles as kinase inhibitors
WO2011140442A1 (en) 2010-05-07 2011-11-10 Glaxo Group Limited Amino-quinolines as kinase inhibitors
WO2012021580A1 (en) 2010-08-10 2012-02-16 Glaxo Group Limited Quinolyl amines as kinase inhibitors
WO2012122011A2 (en) 2011-03-04 2012-09-13 Glaxosmithkline Llc Amino-quinolines as kinase inhibitors
WO2013025958A1 (en) 2011-08-18 2013-02-21 Glaxo Group Limited Amino quinazolines as kinase inhibitors
WO2014043437A1 (en) 2012-09-13 2014-03-20 Glaxosmithkline Llc Amino-quinolines as kinase inhibitors
WO2014043446A1 (en) 2012-09-13 2014-03-20 Glaxosmithkline Llc Prodrugs of amino quinazoline kinase inhibitor

Non-Patent Citations (57)

* Cited by examiner, † Cited by third party
Title
"NCBI", Database accession no. NP—003812.1
ALLEN, I.C. ET AL.: "NLRP12 suppresses colon inflammation and tumorigenesis through the negative regulation of noncanonical NF-kappaB signaling", IMMUNITY, vol. 36, 2012, pages 742 - 754
ARTHUR, J.C.LICH, J.D.AZIZ, R.K.KOTB, M.TING, J.P.: "Heat shock protein 90 associates with monarch-1 and regulates its ability to promote degradation of NF-kappaB-inducing kinase", JOURNAL OF IMMUNOLOGY, vol. 179, 2007, pages 6291 - 6296
BIELIG H ET AL: "Anti-inflammatory arene-chromium complexes acting as specific inhibitors of NOD2 signalling", CHEMMEDCHEM 20101203 JOHN WILEY AND SONS LTD GBR, vol. 5, no. 12, 3 December 2010 (2010-12-03), pages 2065 - 2071, XP002737523, ISSN: 1860-7179 *
BORGHINI STASSI SCHIESA SCAROLI FCARTA SCAORSI R ET AL.: "Clinical presentation and pathogenesis of cold-induced autoinflammatory disease in a family with recurrence of an NLRP12 mutation", ARTHRITIS AND RHEUMATISM, vol. 63, no. 3, 2011, pages 830 - 839, XP055177305, DOI: 10.1002/art.30170
CATTAN, D.: "MEFV mutation carriers and diseases other than familial Mediterranean fever: proved and non-proved associations; putative biological advantage. Current drug targets", INFLAMMATION AND ALLERGY, vol. 4, 2005, pages 105 - 112
CHEN, L. ET AL.: "NLRP12 attenuates colon inflammation by maintaining colonic microbial diversity and promoting protective commensal bacterial growth", NATURE IMMUNOLOGY, vol. 18, 2017, pages 541 - 551
COLLINS, J.W. ET AL.: "Citrobacter rodentium: infection, inflammation and the microbiota", NATURE REVIEWS. MICROBIOLOGY, vol. 12, 2014, pages 612 - 623
COUTURIER-MAILLARD, A. ET AL.: "NOD2-mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer", J CLIN INVEST, vol. 123, 2013, pages 700 - 711
CURRENT BIOLOGY, vol. 8, 1998, pages 885 - 889
DUGAN, J.W. ET AL.: "Nucleotide oligomerization domain-2 interacts with 2'-5'-oligoadenylate synthetase type 2 and enhances RNase-L function in THP-1 cells", MOLECULAR IMMUNOLOGY, vol. 47, 2009, pages 560 - 566, XP026886979, DOI: 10.1016/j.molimm.2009.09.025
FAHY, R.J. ET AL.: "Inflammasome mRNA expression in human monocytes during early septic shock", AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, vol. 177, 2008, pages 983 - 988
GEDDES, K. ET AL.: "Identification of an innate T helper type 17 response to intestinal bacterial pathogens", NATURE MEDICINE, vol. 17, 2011, pages 837 - 844
GRAHAM F LSMILEY JRUSSELL W CNAIRN R: "Characteristics of a human cell line transformed by DNA from human adenovirus type 5", J. GEN. VIROL., vol. 36, no. 1, July 1977 (1977-07-01), pages 59 - 74, XP008153803, DOI: 10.1099/0022-1317-36-1-59
HOMER, C.R.RICHMOND, A.L.REBERT, N.A.ACHKAR, J.P.MCDONALD, C.: "ATG16L1 and NOD2 interact in an autophagy-dependent antibacterial pathway implicated in Crohn's disease pathogenesis", GASTROENTEROLOGY, vol. 139, 2010, pages 1630 - 1641,1641 e1631-1632
J. BIOL. CHEM., vol. 273, 1998, pages 16968 - 16975
JERU I ET AL: "Mutations in NALP12 cause hereditary periodic fever syndromes", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 105, no. 5, 5 February 2008 (2008-02-05), pages 1614 - 1619, XP008175506, ISSN: 0027-8424, [retrieved on 20080129], DOI: 10.1073/PNAS.0708616105 *
JERU IDUQUESNOY PFERNANDES-ALNEMRI TCOCHET EYU JWLACKMY-PORT-LIS M ET AL.: "Mutations in NALP12 cause hereditary periodic fever syndromes", PROC NATL ACAD SCI U S A, vol. 105, no. 5, 2008, pages 1614 - 1619, XP008175506, DOI: 10.1073/pnas.0708616105
JERU ILE BORGNE GCOCHET EHAYRAPETYAN HDUQUESNOY PGRATEAU G ET AL.: "Identification and functional consequences of a recurrent NLRP12 missense mutation in periodic fever syndromes", ARTHRITIS AND RHEUMATISM, vol. 63, no. 5, 2011, pages 1459 - 1464, XP055177298, DOI: 10.1002/art.30241
JERU, I. ET AL.: "Mutations in NALP12 cause hereditary periodic fever syndromes", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 105, 2008, pages 1614 - 1619, XP008175506, DOI: 10.1073/pnas.0708616105
KAPER, J.B.NATARO, J.P.MOBLEY, H.L.: "Pathogenic Escherichia coli", NATURE REVIEWS. MICROBIOLOGY, vol. 2, 2004, pages 123 - 140
KATLINSKAYA, Y.V. ET AL.: "Type I Interferons Control Proliferation and Function of the Intestinal Epithelium", MOLECULAR AND CELLULAR BIOLOGY, vol. 36, 2016, pages 1124 - 1135
KAYAGAKI, N. ET AL.: "Noncanonical inflammasome activation by intracellular LPS independent of TLR4", SCIENCE, vol. 341, 2013, pages 1246 - 1249, XP055488934, DOI: 10.1126/science.1240248
KIM, Y.G. ET AL.: "The cytosolic sensors Nodl and Nod2 are critical for bacterial recognition and host defense after exposure to Toll-like receptor ligands", IMMUNITY, vol. 28, 2008, pages 246 - 257, XP009118468, DOI: 10.1016/j.immuni.2007.12.012
KIM, Y.G. ET AL.: "The Nod2 sensor promotes intestinal pathogen eradication via the chemokine CCL2-dependent recruitment of inflammatory monocytes", IMMUNITY, vol. 34, 2011, pages 769 - 780, XP028218518, DOI: 10.1016/j.immuni.2011.04.013
KOBAYASHI, K.S. ET AL.: "Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract", SCIENCE, vol. 307, 2005, pages 731 - 734
KOBAYASHI, S.NANTZ, R.KITAMURA, T.HIGASHIKUBO, R.HORIKOSHI, N.: "Combined inhibition of extracellular signal-regulated kinases and HSP90 sensitizes human colon carcinoma cells to ionizing radiation", ONCOGENE, vol. 24, 2005, pages 3011 - 3019
LEE, K.H.BISWAS, A.LIU, Y.J.KOBAYASHI, K.S.: "Proteasomal degradation of Nod2 protein mediates tolerance to bacterial cell wall components", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 287, 2012, pages 39800 - 39811
LI, Y. ET AL.: "Inducible interleukin 32 (IL-32) exerts extensive antiviral function via selective stimulation of interferon lambdal (IFN-lambdal", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 288, 2013, pages 20927 - 20941
LICH, J.D. ET AL.: "Monarch-1 suppresses non-canonical NF-kappaB activation and p52-dependent chemokine expression in monocytes", JOURNAL OF IMMUNOLOGY, vol. 178, 2007, pages 1256 - 1260
LORD, C.A. ET AL.: "Blimp-1/PRDMI mediates transcriptional suppression of the NLR gene NLRP12/Monarch-l", JOURNAL OF IMMUNOLOGY, vol. 182, 2009, pages 2948 - 2958
LOUIS NEVELEGH CGRAHAM F L: "Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line", VIROLOGY, vol. 233, no. 2, July 1997 (1997-07-01), pages 423 - 9, XP004452203, DOI: 10.1006/viro.1997.8597
MARCHIANDO, A.M. ET AL.: "A deficiency in the autophagy gene Atgl6Ll enhances resistance to enteric bacterial infection", CELL HOST & MICROBE, vol. 14, 2013, pages 216 - 224
MICELI-RICHARD, C. ET AL.: "CARD15 mutations in Blau syndrome", NATURE GENETICS, vol. 29, 2001, pages 19 - 20, XP002234637, DOI: 10.1038/ng720
MUNDY, R.MACDONALD, T.T.DOUGAN, G.FRANKEL, G.WILES, S.: "Citrobacter rodentium of mice and man", CELLULAR MICROBIOLOGY, vol. 7, 2005, pages 1697 - 1706
NETEA, M.G. ET AL.: "IL-32 synergizes with nucleotide oligomerization domain (NOD) 1 and NOD2 ligands for IL-lbeta and IL-6 production through a caspase 1-dependent mechanism", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 102, 2005, pages 16309 - 16314
PAN, Q. ET AL.: "NF-kappa B-inducing kinase regulates selected gene expression in the Nod2 signaling pathway", INFECTION AND IMMUNITY, vol. 74, 2006, pages 2121 - 2127
PARK, G.B. ET AL.: "TLR3/TRIF signalling pathway regulates IL-32 and IFN-beta secretion through activation of RIP-1 and TRAF in the human cornea", J CELL MOL MED, vol. 19, 2015, pages 1042 - 1054
POTT, J. ET AL.: "IFN-lambda determines the intestinal epithelial antiviral host defense", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 108, 2011, pages 7944 - 7949
POULIN, L.F.CHAMAILLARD, M.: "The battlefield in the war against attaching-and-effacing bacterial pathogens: Monocytes, macrophages and dendritic cells in action", VET MICROBIOL, vol. 202, 2017, pages 47 - 51
RATHINAM, V.A. ET AL.: "TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by gram-negative bacteria", CELL, vol. 150, 2012, pages 606 - 619
SATPATHY, A.T. ET AL.: "Notch2-dependent classical dendritic cells orchestrate intestinal immunity to attaching-and-effacing bacterial pathogens", NATURE IMMUNOLOGY, vol. 14, 2013, pages 937 - 948
SHALEM,SANJANA, SCIENCE, 2014
SIEGFRIED, A. ET AL.: "IFIT2 is an effector protein of type I IFN-mediated amplification of lipopolysaccharide (LPS)-induced TNF-alpha secretion and LPS-induced endotoxin shock", JOURNAL OF IMMUNOLOGY, vol. 191, 2013, pages 3913 - 3921
SOMMEREYNS, C.PAUL, S.STAEHELI, P.MICHIELS, T.: "IFN-lambda (IFN-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo", PLOS PATHOGENS, vol. 4, 2008, pages el000017
SORBARA, M.T. ET AL.: "The protein ATG16L1 suppresses inflammatory cytokines induced by the intracellular sensors Nodl and Nod2 in an autophagy-independent manner", IMMUNITY, vol. 39, 2013, pages 858 - 873
SPEHLMANN, M.E. ET AL.: "CXCR2-dependent mucosal neutrophil influx protects against colitis-associated diarrhea caused by an attaching/effacing lesion-forming bacterial pathogen", JOURNAL OF IMMUNOLOGY, vol. 183, 2009, pages 3332 - 3343
SUN, L. ET AL.: "Type I interferons link viral infection to enhanced epithelial turnover and repair", CELL HOST & MICROBE, vol. 17, 2015, pages 85 - 97, XP029132254, DOI: 10.1016/j.chom.2014.11.004
TAMELING, W.I. ET AL.: "Mutations in the NB-ARC domain of 1-2 that impair ATP hydrolysis cause autoactivation", PLANT PHYSIOLOGY, vol. 140, 2006, pages 1233 - 1245
TING, J.P.WILLINGHAM, S.B.BERGSTRALH, D.T.: "NLRs at the intersection of cell death and immunity", NATURE REVIEWS. IMMUNOLOGY, vol. 8, 2008, pages 372 - 379
VLADIMER, G.I. ET AL.: "The NLRP12 inflammasome recognizes Yersinia pestis", IMMUNITY, vol. 37, 2012, pages 96 - 107
WAGNER, R.N.PROELL, M.KUFER, T.A.SCHWARZENBACHER, R.: "Evaluation of Nod-like receptor (NLR) effector domain interactions", PLOS ONE, vol. 4, 2009, pages e4931
WILLIAMS, K.L. ET AL.: "The CATERPILLER protein monarch-1 is an antagonist of toll-like receptor-, tumor necrosis factor alpha-, and Mycobacterium tuberculosis-induced pro-inflammatory signals", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 280, 2005, pages 39914 - 39924
YE, Z. ET AL.: "ATP binding by monarch-1/NLRP12 is critical for its inhibitory function", MOLECULAR AND CELLULAR BIOLOGY, vol. 28, 2008, pages 1841 - 1850
YUN ZHAOJOO HYE SONGALFRED M AJAMIHANS-CHRISTIAN REINECKER: "A New RIPK2 Kinase Inhibitor for the Treatment of Intestinal Inflammation", THE JOURNAL OF IMMUNOLOGY, vol. 188, 2012
ZAKI, M.H. ET AL.: "The NOD-like receptor NLRP12 attenuates colon inflammation and tumorigenesis", CANCER CELL, vol. 20, 2011, pages 649 - 660, XP028112777, DOI: 10.1016/j.ccr.2011.10.022
ZAKI, M.H.MAN, S.M.VOGEL, P.LAMKANFI, M.KANNEGANTI, T.D.: "Salmonella exploits NLRP12-dependent innate immune signaling to suppress host defenses during infection", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2013

Similar Documents

Publication Publication Date Title
Biswas et al. Phosphorylation of IRF4 by ROCK2 regulates IL-17 and IL-21 production and the development of autoimmunity in mice
US20200108141A1 (en) Fgfr/pd-1 combination therapy for the treatment of cancer
Hou et al. A novel selective autophagy receptor, CCDC50, delivers K63 polyubiquitination-activated RIG-I/MDA5 for degradation during viral infection
Normand et al. Proteasomal degradation of NOD2 by NLRP12 in monocytes promotes bacterial tolerance and colonization by enteropathogens
US11865176B2 (en) Compositions and methods of modulating anti-tumor immunity
Tian et al. The spleen contributes importantly to myocardial infarct exacerbation during post-ischemic reperfusion in mice via signaling between cardiac HMGB1 and splenic RAGE
US20220313700A1 (en) Methods for treating map3k8 positive cancers
Brown et al. Forward genetic screening identifies a small molecule that blocks Toxoplasma gondii growth by inhibiting both host-and parasite-encoded kinases
Stafford et al. IAPs regulate distinct innate immune pathways to co-ordinate the response to bacterial peptidoglycans
Liu et al. NLRP3 inflammasome induced liver graft injury through activation of telomere‐independent RAP1/KC axis
Hellvard et al. Inhibition of CDK9 as a therapeutic strategy for inflammatory arthritis
Coux et al. The proteasome system in health and disease
Dixit et al. Targeting TNF-α–producing macrophages activates antitumor immunity in pancreatic cancer via IL-33 signaling
US20170198011A1 (en) Composition for treating sepsis or septic shock comprising the peptide originated from the smad6
Ma et al. Mutual amplification of HNF4α and IL-1R1 composes an inflammatory circuit in Helicobacter pylori associated gastric carcinogenesis
Gao et al. The role of NFAT in the pathogenesis and targeted therapy of hematological malignancies
EP3275444A2 (en) Composition for inhibiting growth or proliferation of chronic myelogenous leukemia cancer stem cells, and screening method therefor
WO2020094613A1 (en) Nod2 inhibitors for the treatment of hereditary periodic fevers
EP3289104A2 (en) Method for treating high-grade gliomas
Devi et al. CARD-only proteins regulate in vivo inflammasome responses and ameliorate gout
WO2013020372A1 (en) Methods and reagents for preventing and curing insulin resistance and diabetes mellitus
US20130251702A1 (en) Method for screening compounds for treating sepsis targeting nod2 signalling pathway and composition for treating sepsis comprising nod2 signalling pathway inhibitors
CN105695562B (en) Use of MST4 gene diagnosis and cell treatment for infectious diseases and related medicine thereof
Wang et al. hnRNPA2B1 represses the disassembly of arsenite-induced stress granules and is essential for male fertility
US20240026461A1 (en) Bcl10 mutations as biomarkers for targeted therapy in b cell lymphomas

Legal Events

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

Ref document number: 19795581

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19795581

Country of ref document: EP

Kind code of ref document: A1