WO2010049173A1 - Utilisation d’inhibiteurs de kinases hôtes pour traiter les maladies infectieuses - Google Patents

Utilisation d’inhibiteurs de kinases hôtes pour traiter les maladies infectieuses Download PDF

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WO2010049173A1
WO2010049173A1 PCT/EP2009/007793 EP2009007793W WO2010049173A1 WO 2010049173 A1 WO2010049173 A1 WO 2010049173A1 EP 2009007793 W EP2009007793 W EP 2009007793W WO 2010049173 A1 WO2010049173 A1 WO 2010049173A1
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pyrrolo
pyridin
phenyl
benzoic acid
acid
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PCT/EP2009/007793
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English (en)
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Michael Hannus
Cecilie Martin
Maria M. Mota
Miguel Prudencio
Christina Dias Rodrigues
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Cenix Bioscience Gmbh
Instituto De Medicina Molecular, Faculdade De Medicina Da Universidade De Lisboa
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Publication of WO2010049173A1 publication Critical patent/WO2010049173A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11013—Protein kinase C (2.7.11.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of inhibitors of host kinases for the production of a medicament for therapy of and/or prophylaxis against infections, involving liver cells and/or hematopoietic cells, in particular malaria.
  • Malaria is a major health problem, mainly in Sub-Saharan Africa and in some parts of Asia and South America. Each year there are about 600 million new clinical cases and at least one million individuals, mostly children, die from malaria. This reality is even more depressing realising that a death from malaria occurs every 30 seconds. Over 90% of the deaths occur in Africa. Within the last 10 to 15 years the burden of malaria has been increasing mainly because of the emergence of Plasmodium falciparum and P. vivax variants that are resistant to cheap drugs such as chloroquine, mefloquine, and pyrimethamine. In the light of the failure of the development of a malaria vaccine, despite intensive efforts, the development of novel antimalarial drugs is crucial.
  • the infected hepatocytes burst, releasing the parasites into the bloodstream, where they will target and invade the red blood cells (RBCs).
  • RBCs red blood cells
  • the blood or erythrocytic stage of Plasmodium's life cycle corresponds to the symptomatic phase of a malaria infection.
  • the parasites invade and multiply within the RBCs and, upon rupturing the erythrocytic membrane, are released into the blood where they target new erythrocytes.
  • Plasmodium sporozoites only develop in a very restricted type of cell, such as hepatocytes or hepatoma cell lines, strongly suggesting a crucial role of the host cell in sustaining the growth and development of this parasite.
  • the inventors have designed a cultured cell-based assay to study the process of liver infection by Plasmodium parasites at the cellular and molecular level.
  • a cultured cell-based assay to study the process of liver infection by Plasmodium parasites at the cellular and molecular level.
  • Human Huh7 hepatoma cells and sporozoites of the rodent parasite P. berghei freshly isolated from infected Anopheles mosquitoes the inventors have established a high throughput assay system ( Figure IA) that, combined with high content readouts using automated microscopy, and quantitative RT-PCR (qRT-PCR), can be used for RNA interference (RNAi) and/or drug screening experiments.
  • RNAi RNA interference
  • the present invention provides novel targets for the prophylaxis and treatment of infectious diseases, in particular malaria.
  • the present invention relates to the use of a compound for the production of a medicament for the therapy and/or prophylaxis of a protozoal infection, wherein the compound is an inhibitor of a protein kinase, wherein the protein kinase is selected from the group consisting of: (a) protein kinase C zeta (PKC ⁇ ); (b) Serine/threonine-protein kinase WNKl (PRKWNKl); (c) Serine/threonine-protein kinase Sgk2 (SGK2); and (d) Serine/threonine-protein kinase 35 (STK35).
  • PLC ⁇ protein kinase C zeta
  • PRKWNKl Serine/threonine-protein kinase WNKl
  • SGK2 Serine/threonine-protein kinase Sgk2
  • STK35 Serine/threonine-protein kinase 35
  • the present invention relates to a method of identifying compounds for treatment and/or prophylaxis of infectious diseases involving liver or hematopoietic cells comprising the steps of: (i) contacting a protein kinase, a functional variant, or soluble part thereof with a test compound, (ii) selecting a test compound, which specifically binds to said protein kinase, functional variant, or soluble part thereof, (iii) contacting liver or hematopoietic cells with the selected test compound prior, during or after infection of said cell with an infectious agent, and (iv) selecting a test compound inhibiting cell entry and/or development of the infectious agent by at least 10%.
  • the present invention relates to a use of a test compound selected in step (iv) of the method of the second aspect for the production of a medicament for the therapy and/or prophylaxis of infectious diseases, which involve infection of liver and/or hematopoietic cells.
  • the present invention relates to a test compound selected in step (iv) of the method of the second aspect for use in therapy and/or prophylaxis of infectious diseases, which involve infection of liver and/or hematopoietic cells.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising, essentially comprising or consisting of a compound usable according to the first aspect and one or more of a compound selected from the group consisting of a chinine alkaloid, chloroquine-phosphate, hydroxychloroquinesulfate, mefloquine, proguanil, di- aminopyrimidines: pyrimethamine, atovaquone, doxycycline, artemether, and lumefantrine and pharmaceutically acceptable carriers, additives and/or auxiliary substances.
  • the present invention relates to a method for the identification of molecules of pathogens, which are involved in the infection of liver and/or hematopoietic cells, comprising the following steps: (i) contacting one or more protein kinases, functional variants, or soluble parts thereof with one or more molecules present in pathogens, which are involved in the infection of liver and/or hematopoietic cells; and (ii) selecting a molecule, which specifically binds to the protein kinase.
  • Fig. 1 depicts the RNA interference screen strategy for identification of host factors affecting pathogen-caused infection, in particular Plasmodium infection.
  • A Experimental design of a high-throughput RNAi screen to identify host genes that influence Plasmodium sporozoite infection of host cells.
  • B Validation of siRNA-mediated knock-down in Huh7 cells. Knock-down efficiency of 53 genes was evaluated by qRT-PCR following Huh7 cell transfection with 3 independent siRNAs per targeted gene.
  • RNAi screen identifies host genes that influence P. berghei sporozoite infection of Huh7 cells.
  • A Schematic illustration of the three screening passes with increasing stringency criteria.
  • B Plot of pass 1 of the RNAi screen representing the effect of 2181 siRNAs targeting 727 human genes on Huh7 cell infection by P. berghei sporozoites and cell nuclei count. Infection rates for each experimental condition were normalized against cell confluency.
  • the horizontal lines represent 100% ⁇ 2.0 s.d. of the average of all infection data in the assay.
  • Each circle represents one siRNA (mean of triplicate values).
  • Negative controls appear as light and medium grey filled circles, corresponding to untreated cells and cells transfected with a non-specific control siRNA, respectively. Dark filled circles highlight the siRNAs targeting the 73 candidate genes selected to undergo a second screening pass. The shaded areas correspond to cell numbers outside the ⁇ 40% interval centered on the average number of nuclei for the whole dataset.
  • C Plot of 2 independent runs of pass 2 of the RNAi screen representing the effect of 227 siRNAs targeting 73 human genes on Huh7 cell infection by P. berghei sporozoites and cell nuclei count. Shading and colour attributions are the same as in panel (B), with dark filled circles representing the siRNAs targeting the 16 genes selected to undergo a third screening pass.
  • the horizontal lines represent 100% ⁇ 2.0 s.d. of the average of all the negative controls in the assay.
  • the horizontal and vertical lines represent 100% ⁇ 2.0 s.d. of the average of all the negative controls in the assay.
  • B, C Quantification of cell confluency (B) and number of nuclei (C) in 40 microscope fields of cells treated with the PKC ⁇ pseudosubstrate inhibitor and a control peptide.
  • D, E Effect of PKC ⁇ lnh (20 ⁇ M) on P. berghei load in Huh7 cells (D) and mouse primary hepatocytes (E). Parasite loads were measured by qRT-PCR 24 h or 48 h after sporozoite addition, respectively. Results are expressed as the mean ⁇ s.d. of triplicate samples. Cells treated with a myristoylated scrambled peptide were used as controls in each experiment. Infection loads are normalized to the corresponding control infection levels (100%).
  • PKC ⁇ inhibition by PKC ⁇ lnh decreases P. berghei sporozoite infection of Huh7 cells in a dose-dependent manner.
  • PKC ⁇ lnh was added to Huh7 cells 1 h before addition of GFP- expressing P. berghei sporozoites and infection rate was measured 24 h later by FACS.
  • PKC ⁇ inhibition by PKC ⁇ lnh does not affect development of Exo-Erythrocytic Forms (EEF).
  • EEF Exo-Erythrocytic Forms
  • PKC ⁇ inhibition by PKC ⁇ lnh decreases P. berghei sporozoite invasion of Huh7 cells in a dose-dependent manner.
  • PKC ⁇ lnh was added to Huh7 cells 1 h before addition of GFP- expressing P. berghei sporozoites and infection rate was measured 2 h later, by FACS.
  • PKC ⁇ inhibition does not affect infection after invasion has occurred.
  • PKC ⁇ lnh was added to Huh7 cells 2 h after addition of GFP-expressing P. berghei sporozoites and infection rate was measured 24 h later, by FACS.
  • Fig. 5 In vivo PKC ⁇ down-modulation reduces liver infection by Plasmodium sporozoites confirming the physiological relevance of RNAi screen results.
  • A Effect of siRNA-mediated in vivo silencing of PKC ⁇ on mouse liver infection by P. berghei (solid bars) and on PKC ⁇ mRNA levels (dashed bars), measured by qRT-PCR analysis of liver extracts taken 40 h after sporozoite i.v. injection. Mice were infected 36 h after RNAi treatment. Results are plotted as the percentage of the mean of negative control samples, "C". The remaining mRNA levels for PKC ⁇ were measured by qRT-PCR in the same liver samples. Results are expressed as the mean ⁇ s.d.
  • alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alicyclic system, aryl, aralkyl, heteroaryl, heteroaralkyl, alkenyl and alkynyl are provided. In each instance of their use in the remainder of the specification, these terms will have the respectively defined meaning and preferred meanings.
  • alkyl refers to a saturated straight or branched carbon chain.
  • the chain comprises from 1 to 10 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g. methyl, ethyl, propyl (/7-propyl or /so-propyl), butyl (ft-butyl, sec-butyl, or tert-butyl), pentyl, hexyl, heptyl, octyl, nonyl, or decyl.
  • Alkyl groups are optionally substituted.
  • heteroalkyl refers to a saturated straight or branched carbon chain.
  • the chain comprises from 1 to 9 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, or 9, e.g. methyl, ethyl, propyl (n-propyl or /s ⁇ -propyl), butyl (tt-butyl, wo-butyl, sec-butyl, or t ⁇ r/-butyl), pentyl, hexyl, heptyl, octyl, nonyl, which is interrupted one or more times, e.g. 1, 2, 3, 4, or 5 times, with the same or different heteroatoms.
  • the heteroatoms are selected from O, S, and N, e.g. -O- CH 3 , -S-CH 3 , -NH-CH 3 , -CH 2 -O-CH 3 , -CH 2 -O-C 2 H 5 , -CH 2 -S-CH 3 , -CH 2 -S-C 2 H 5 , -CH 2 -NH- CH 3 , -CH 2 -NH-C 2 H 5 , -C 2 H 4 -O-CH 3 , -C 2 H 4 -O-C 2 H 5 , -C 2 H 4 -S-CH 3 , -C 2 H 4 -S-C 2 H 5 , -C 2 H 4 -NH- CH 3 , -C 2 H 4 -NH-C 2 H 5 , etc.
  • Heteroalkyl groups are optionally substituted.
  • cycloalkyl and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively, with preferably 3, 4, 5, 6, 7, 8, 9 or 10 atoms forming a ring, e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl.
  • cycloalkyl and “heterocycloalkyl” are also meant to include bicyclic, tricyclic and polycyclic versions thereof.
  • heterocycloalkyl preferably refers to a saturated ring having five members of which at least one member is a N, O, or S atom and which optionally contains one additional O or one additional N; a saturated ring having six members of which at least one member is a N, O or S atom and which optionally contains one additional O or one additional N or two additional N atoms; or a saturated bicyclic ring having nine or ten members of which at least one member is a N, O or S atom and which optionally contains one or two additional O or one, two, or three additional N atoms.
  • Cycloalkyl and “heterocycloalkyl” groups are optionally substituted. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
  • Preferred examples of cycloalkyl include C 3 -Ci 0 -cycloalkyl, in particular cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, spiro[3,5]nonyl, spiro[5,3]nonyl, spiro[3,6]decyl, spiro[6,3]decyl, spiro[4,5]decyl,
  • heterocycloalkyl examples include C 3 -Ci 0 -heterocycloalkyl, in particular l-(l,2,5,6-tetrahydropyridyl), 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, 1,8 diaza-spiro-[4,5] decyl, 1,7 diaza-spiro-[4,5] decyl, 1,6 diaza-spiro-[4,5] decyl, 2,8 diaza-spiro[4,5] decyl, 2,7 diaza-spiro[4,5] decyl, 2,6 diaza-spiro[4,5] decyl, 1,8 diaza-spiro-[5,4] decyl, 1,7 diaza-spiro- [5,4] decyl, 2,8 diaza-spiro-[5,4] decyl, 2,7 diaza-spiro[5,4] decyl, 2,
  • alicyclic system refers to mono, bicyclic, tricyclic or polycyclic versions of a cycloalkyl or heterocycloalkyl comprising at least one double and/or triple bond.
  • an alicyclic system is not aromatic or heteroaromatic, i.e. does not have a system of conjugated double bonds/free electron pairs.
  • the number of double and/or triple bonds maximally allowed in an alicyclic system is determined by the number of ring atoms, e.g. in a ring system with up to 5 ring atoms an alicyclic system comprises up to one double bond, in a ring system with 6 ring atoms the alicyclic system comprises up to two double bonds.
  • the "cycloalkenyl" as defined below is a preferred embodiment of an alicyclic ring system. Alicyclic systems are optionally substituted.
  • alkoxy refers to an -O-alkyl group, i.e. to an oxygen atom substituted by a saturated straight or branched carbon chain.
  • the chain comprises from 1 to 10 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • preferred alkoxy groups are methoxy, ethoxy, propoxy (tf-propoxy or /so-propoxy), butoxy (n-butoxy, sec-butoxy, zs ⁇ -butoxy, or tert-butoxy), pentoxy, hexoxy, heptoxy, octoxy, nonoxy, or decoxy.
  • Alkoxy groups are optionally substituted.
  • aryl preferably refers to an aromatic monocyclic ring containing 6 carbon atoms, an aromatic bicyclic ring system containing 10 carbon atoms or an aromatic tricyclic ring system containing 14 carbon atoms. Examples are phenyl, naphthyl, anthracenyl, or phenanthrenyl. The aryl group is optionally substituted. As used herein, the term “aryl” also encompasses aromatic rings or ring systems as described above fused to non-aromatic rings or ring systems.
  • alkyl refers to an alkyl moiety, which is substituted by one or more (e.g. 1, 2, 3) aryl, wherein alkyl and aryl have the meaning as outlined above.
  • An example is the benzyl radical.
  • the alkyl chain comprises from 1 to 8 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, or 8, e.g. methyl, ethyl, propyl (w-propyl or iso-propy ⁇ ), butyl (n-butyl, wo-butyl, sec-butyl, or tert-butyl), pentyl, hexyl, heptyl, octyl.
  • the alkyl chain is substituted by one or more (e.g. 1, 2, 3) phenyl groups, by one or more (e.g. 1, 2, 3) naphthyl groups, by one or more (e.g. 1, 2, 3) anthracenyl groups, or by one or more (e.g. 1, 2, 3) phenanthrenyl groups.
  • the aralkyl group is optionally substituted at the alkyl and/or aryl part of the group.
  • heteroaryl preferably refers to a four-, five-, six-, or seven-membered aromatic monocyclic ring wherein at least one of the carbon atoms are replaced by 1, 2, 3, or 4 (for the five membered ring) or 1, 2, 3, 4, or 5 (for the six-membered ring) of the same or different heteroatoms, preferably selected from O, N and S; an aromatic bicyclic ring system wherein 1, 2, 3, 4, 5, or 6 carbon atoms of the 8, 9, 10, 11 or 12 carbon atoms have been replaced with the same or different heteroatoms, preferably selected from O, N and S; or an aromatic tricyclic ring system wherein 1, 2, 3, 4, 5, or 6 carbon atoms of the 13, 14, 15, or 16 carbon atoms have been replaced with the same or different heteroatoms, preferably selected from O, N and S.
  • Examples are oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,3-oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, thiazolyl, isothiazolyl, 1,2,3,-thiadiazolyl, 1,2,5- thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1- benzofuranyl, 2-benzofuranyl, indolyl, isoindolyl, 1 -benzothiophenyl, 2-benzothiophenyl, IH- indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, 2,1-benzisoxazoyl, benzothiazolyl, 1,2- benzisothi
  • heteroaryl also encompasses aromatic rings and ring systems as described above fused to non-aromatic rings or ring systems.
  • heteroarylkyl refers to an alkyl moiety, which is substituted by one or more (e.g. 1, 2, 3) heteroaryl, wherein alkyl and heteroaryl have the meaning as outlined above.
  • An example is the 2-alkylpyridinyl, 3-alkylpyridinyl, or 2-methylpyridinyl.
  • the alkyl chain comprises from 1 to 8 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, or 8, e.g.
  • heteroaralkyl group is optionally substituted at the alkyl and/or heteroaryl part of the group.
  • alkenyl and cycloalkenyl refer to branched or straight carbon chains containing olefinic unsaturated carbon atoms and to rings with one or more double bonds, respectively. Examples are propenyl and cyclohexenyl.
  • the alkenyl chain comprises from 2 to 8 carbon atoms, i.e. 2, 3, 4, 5, 6, 7, or 8, e.g.
  • alkenyl also comprises
  • the cycloalkenyl ring comprises from 3 to 14 carbon atoms, i.e. 3, 4, 5,
  • alkynyl and cycloalkynyl refers to branched or straight carbon chains or rings containing unsaturated carbon atoms with one or more triple bonds.
  • An example is the propargyl radical.
  • the alkynyl chain comprises from 2 to 8 carbon atoms, i.e. 2, 3, 4, 5, 6, 7, or 8, e.g. ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl,
  • R 1 and R" is each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, and heteroaryl or together with the nitrogen that they are attached to form a 4-, 5-, 6-, or 7-membered heteroaryl or a 4-, 5-,
  • R'" and R"" is each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, aralkyl, heteroaryl, and -NR 1 R";
  • E is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkoxyalkyl, heterocycloalkyl, an alicyclic system, aryl and heteroaryl; optionally substituted.
  • a “compound” or “test compound” within the context of the present application is not particularly limited regarding its structural requirements.
  • a compound or test compound may refer to a peptide, a protein, a nucleic acid or any other chemical substance as further defined below.
  • a “compound” of the present invention is characterized in functional terms as being an inhibitor of a protein kinase.
  • a “test compound”, as used in the present application, is suspected of being an inhibitor of a protein kinase.
  • Proteins and test compounds that can be used in the context of the present invention are not particularly limited and comprise without limitation peptides, proteins, peptidomimetics, small molecules, and/or nucleic acids.
  • “Peptides” in this sense are chains of naturally and/or non-naturally occurring amino acids with 2 to 50 amino acids connected by peptide bonds, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. Chains with 50 or more naturally and/or non-naturally occurring amino acids are referred to as "proteins".
  • polypeptide and proteins usable in the present invention may contain post-translational modifications.
  • Preferred peptides used in the methods of the present invention are peptides interfering with the interaction of the protein kinase with the structure on the respective pathogen, e.g. plasmodiidae, preferably Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium semiovale and Plasmodium knowlesi, required for binding to the protein kinase.
  • peptides are fragments of protein kinase.
  • Other preferred peptides usable in the present invention may not interfere with the above interaction but are peptides which inhibit the enzyme activity of a protein kinase usable in the present invention.
  • peptidomimetics are well known in the art and refer to compounds, which are designed based on the primary structure of a given peptide to be modelled, e.g. like one of the peptides mentioned above, and which take on a similar secondary structure. Thus, peptidomimetics can be designed to be, e.g. more protease resistant, have a different half life, improved pharmacokinetics or pharmacodynamics etc.
  • "Small molecules” within the meaning of the present invention are preferably non-peptidyl (no peptide bonds and/or not formed from amino acids), non nucleic acid compounds, of a molar mass lower than 1000 g/mol, preferably lower than 500 g/mol. In most cases the small molecules used in the methods of the present invention are hydrocarbons or mixtures thereof, e.g. plant extracts.
  • nucleic acid or “oligonucleotide” or grammatical equivalents thereof is meant at least two nucleotides covalently linked together.
  • a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide, phosphorothioate, phosphorodithioate, O-methylphosphoroamidite linkages, and peptide nucleic acid backbones and linkages.
  • Other analog nucleic acids include those with positive backbones, non-ionic backbones and non-ribose backbones.
  • Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of labels, or to increase the stability and half-life of such molecules in physiological environments.
  • Nucleic acids usable in the context of the present invention can consist of DNA, RNA, peptide nucleic acid (PNA), phosphorothioate DNA (PS-DNA), 2'-O-methyl RNA (OMe-RNA), 2'-O-methoxy- ethyl RNA (MOE-RNA), N3'-P5' phosphoroamidate (NP), 2'-fluoro-arabino nucleic acid (FANA), locked nucleic acid (LNA), morpholino phosphoroamidate (MF), cyclohexene nucleic acid (CeNA), or tricycle-DNA (tcDNA) or of mixtures of any of these naturally occurring nucleic acids and nucleic acid analogs (for a review see Kurreck J., 2003).
  • PNA peptide nucleic acid
  • PS-DNA phosphorothioate DNA
  • OMe-RNA 2'-O-methyl RNA
  • MOE-RNA 2'-O-methoxy- e
  • nucleic acid analogs may find use in the present invention.
  • mixtures of naturally occurring nucleic acids such as DNA and RNA, and analogs can be made.
  • mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs can be made.
  • antibody as used herein comprises monoclonal and polyclonal antibodies and binding fragments thereof, in particular Fc-fragments as well as so called “single-chain- antibodies” (Bird R.E. et al., 1988), chimeric, humanized, in particular CDR-grafted antibodies, and diabodies or tetrabodies (Holliger P. et al., 1993). Also comprised are immunoglobulin-like proteins that are selected through techniques including, for example, phage display to specifically bind to their target molecules. Such target molecules in the context of the present invention may be host protein kinases which play a role in the infection of a host cell by a pathogen.
  • a "functional variant" of a protein kinase is a protein, which has been modified by N- terminal, C-terminal and/or internal deletions and/or amino acid additions and or mutations, preferably conservative mutations and which has at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the phosphorylation activity, if compared to the respective wild-type protein kinase on which the variant is based.
  • a “functional variant” can also be defined in structural terms in that it exhibits an amino acid sequence identity of preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 98 % and even more preferably at least 99% to the amino acid sequence of the wild-type protein kinase on which said variant is based.
  • soluble parts of protein kinases are fragments of protein kinases, which do not comprise hydrophobic membrane spanning regions of the protein kinase, if such are present in the wild-type protein kinase on which the soluble parts are based, and which are soluble in an aqueous solution without the addition of detergents. For some applications, e.g. the generation of antibodies, it is not necessary that a soluble part exhibits phosphorylation activity.
  • a soluble part of a protein kinase exhibits phosphorylation activity of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the phosphorylation activity, if compared to the respective wild-type protein kinase on which the soluble part is based.
  • a test compound is considered to "specifically bind" to a protein kinase, if it has a binding constant to the respective protein kinase of 100 ⁇ M or less, preferably 50 ⁇ M or less, preferably 30 ⁇ M or less, preferably 20 ⁇ M or less, preferably 10 ⁇ M or less, preferably 5 ⁇ M or less, more preferably 1 ⁇ M or less, more preferably 900 nM or less, more preferably 800 nM or less, more preferably 700 nM or less, more preferably 600 nM or less, more preferably 500 nM or less, more preferably 400 nM or less, more preferably 300 nM or less, more preferably 200 nM or less, and even more preferably 100 nM or less.
  • inhibitor of a protein kinase within the present invention refers to compounds which can inhibit the phosphorylation activity of a protein kinase in vitro or in vivo or which can inhibit production of the protein kinase. Said inhibition can be accomplished by inhibition of the protein kinase enzyme or by inhibiting the translation of an mRNA coding for a protein kinase or by inhibiting transcription of a protein kinase gene to the corresponding mRNA.
  • a compound is considered an inhibitor of the phosphorylation activity of a protein kinase, if the compound has an IC 50 of ⁇ 100 ⁇ M in a phosphorylation assay.
  • the IC 50 is ⁇ 90 ⁇ M, ⁇ 80 ⁇ M, ⁇ 70 ⁇ M, ⁇ 60 ⁇ M, ⁇ 50 ⁇ M, ⁇ 40 ⁇ M, ⁇ 30 ⁇ M, ⁇ 20 ⁇ M, ⁇ lO ⁇ M, ⁇ 9 ⁇ M, ⁇ 8 ⁇ M, ⁇ 7 ⁇ M, ⁇ 6 ⁇ M, ⁇ 5 ⁇ M, ⁇ 4 ⁇ M, ⁇ 3 ⁇ M, ⁇ 2 ⁇ M, ⁇ l ⁇ M, ⁇ 0.9 ⁇ M, ⁇ 0.8 ⁇ M, ⁇ 0.7 ⁇ M, ⁇ 0.6 ⁇ M, ⁇ 0.5 ⁇ M, ⁇ 0.4 ⁇ M, ⁇ 0.3 ⁇ M, ⁇ 0.2 ⁇ M, ⁇ 0.1 ⁇ M, ⁇ 9OnM, ⁇ 8OnM, ⁇ 7OnM, ⁇ 6OnM, ⁇ 5OnM, ⁇ 4OnM, ⁇ 3OnM, ⁇ 20 nM or ⁇ 10 nM.
  • infectious agent refers to an organism capable of causing an infectious disease in a subject.
  • infectious agent is a protozoal organism as defined throughout this specification.
  • “Infectious diseases involving liver cells and/or hematopoietic cells” are diseases wherein the pathogen in one or more stages of its life cycle in the respective host attacks and/or enters liver cells and/or hematopoietic cells in order to, e.g. proliferate, develop or evade the immune system in those cells, in particular protozoal infections.
  • “Pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • a “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous acid and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous acid and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • the present invention is directed to a use of a compound for the production of a medicament for the therapy and/or prophylaxis of a protozoal infection, wherein the compound is an inhibitor of a protein kinase, wherein the protein kinase is selected from the group consisting of: (a) protein kinase C zeta (PKC ⁇ ); (b) Serine/threonine-protein kinase WNKl (PRKWNKl); (c) Serine/threonine-protein kinase Sgk2 (SGK2); and (d) Serine/threonine-protein kinase 35 (STK35).
  • PLC ⁇ protein kinase C zeta
  • PRKWNKl Serine/threonine-protein kinase WNKl
  • SGK2 Serine/threonine-protein kinase Sgk2
  • STK35 Serine/threonine-protein kinase 35
  • PKC ⁇ (UniProtKB/Swiss-Prot Q05513 (KPCZ_HUMAN); EC 2.7.1 1.13; alternative name: "nPKC-zeta”) is part of the large family of PKCs that has been implicated in numerous cellular processes.
  • PKC isotypes include 10-15 members, divided into 4 groups (Newton, 2003; Mellor and Parker, 1998). One of these groups, known as the atypical PKCs (aPKCs) (Moscat and Diaz-Meco, 2000), comprises the PKC ⁇ (Ono et al, 1989) and PKC ⁇ /i (PKClambda/iota) (Akimoto et al, 1994) isoforms.
  • aPKCs have been implicated in numerous processes, including cell growth and survival, regulation of NF- ⁇ B activation and polarity (reviewed in (Moscat et al, 2006; Suzuki and Ohno, 2006; Moscat and Diaz-Meco, 2000)).
  • PRKWNKl UniProtKB/Swiss-Prot Q9H4A3 (WNK1_HUMAN); EC 2.7.11.1 ; alternative names: "protein kinase, lysine-deficient 1", “erythrocyte 65 kDa protein”; “p65”) and SGK2 (UniProtKB/Swiss-Prot Q9HBY8 (SGK2_HUMAN); EC 2.7.1 1.1 ; alternative name: "Serum/glucocorticoid-regulated kinase 2" are serine/threonine kinases that have been implicated in osmotic control through the regulation of Na + and K + transport channels (Anselmo et al, 2006; Moriguchi et al, 2005; Friedrich et al, 2003; Gamper et al, 2002).
  • the inhibitor of the protein kinase is a small interfering RNA (siRNA) capable of inhibiting expression of a protein kinase. It is preferred that each RNA strand of the siRNA has a length from 19 to 30, particularly from 19 to 23 nucleotides, wherein said RNA molecule is capable of mediating target-specific nucleic acid modifications, particularly RNA interference and/or DNA methylation.
  • siRNA small interfering RNA
  • At least one strand has a 3' overhang from 1 to 5 nucleotides, more preferably from 1 to 3 nucleotides and most preferably of 2 nucleotides.
  • the other strand may be blunt-ended or may have up to 6 nucleotides 3' overhang.
  • both stands of the siRNA duplex have a 3' overhang of 2 nucleotides each.
  • the 3' overhang of the sense strand, of the antisense strand or of both strands comprises at least one dT nucleotide.
  • the 3' overhang of the sense strand, of the antisense strand or of both strands comprises or consists of two dT nucleotides, more preferably two adjacent dT nucleotides, which may be optionally linked by a phosphorothioate linkage.
  • the 3' overhang of the sense strand, of the antisense strand or of both strands comprises or consists of 2 dT nucleotides, which may be optionally linked by a phosphorothioate linkage.
  • one or more ribonucleotides may be replaced by one or more nucleotide analogs, e.g.
  • 2' O-methyl-ribonucleotides (2'0Me).
  • This replacement is particularly preferred, when the siRNA duplex is to be used in vivo.
  • one or more C ribonucleotides are replaced by nucleotide analogs, e.g. by the corresponding 2'0Me nucleotide(s).
  • one or more U ribonucleotides are replaced by nucleotide analogs, e.g. by the corresponding 2'0Me nucleotide(s). More preferably, all C ribonucleotides and/or all U ribonucleotides are replaced by the corresponding 2'0Me nucleotides.
  • the protein kinase is (a) PKC ⁇ and the siRNA is a duplex comprising, essentially comprising or consisting of a sense strand selected from the group consisting of (al) the nucleotide sequence according to SEQ ID NO: 38; (a2) the nucleotide sequence according to SEQ ID NO: 39; (a3) the nucleotide sequence according to SEQ ID NO: 40; (a4) the nucleotide sequence according to SEQ ID NO: 18; (a5) the nucleotide sequence according to SEQ ID NO: 20; and an antisense strand which is complementary to nucleotides 1 to 19 of its corresponding sense strand, the antisense strand optionally having a 3' overhang of between 1 and 5 nucleotides;
  • (b) PRKWNKl and the siRNA is a duplex comprising, essentially comprising or consisting of a sense strand selected from the group consisting of (bl) the nucleotide sequence according to SEQ ID NO: 22; (b2) the nucleotide sequence according to SEQ ID NO: 24; and an antisense strand which is complementary to nucleotides 1 to 19 of its corresponding sense strand, the antisense strand optionally having a 3' overhang of between 1 and 5 nucleotides;
  • siRNA is a duplex comprising, essentially comprising or consisting of a sense strand selected from the group consisting of (cl) the nucleotide sequence according to SEQ ID NO: 26; (c2) the nucleotide sequence according to SEQ ID NO: 28; (c3) the nucleotide sequence according to SEQ ID NO: 30; and an antisense strand which is complementary to nucleotides 1 to 19 of its corresponding sense strand, the antisense strand optionally having a 3' overhang of between 1 and 5 nucleotides; or
  • the siRNA is a duplex comprising, essentially comprising or consisting of a sense strand selected from the group consisting of (dl) the nucleotide sequence according to SEQ ID NO: 32; (d2) the nucleotide sequence according to SEQ ID NO: 34; (d3) the nucleotide sequence according to SEQ ID NO: 36; and an antisense strand which is complementary to nucleotides 1 to 19 of its corresponding sense strand, the antisense strand optionally having a 3' overhang of between 1 and 5 nucleotides.
  • siRNA duplexes described in paragraphs (a) it is also preferred for the siRNA duplexes described in paragraphs (a) to
  • the antisense strand has a 3' overhang from 1 to 3 nucleotides, most preferably of 2 nucleotides.
  • the nucleotides in the 3' overhang of the sense strand and/or the antisense strand are linked by other bonds than the naturally occurring phosphodiester bonds.
  • the nucleotides in the 3' overhang may be linked by a phosphorothioate linkage. It is also preferred for the siRNA duplexes described in paragraphs (a) to (d) above that one or more ribonucleotides in the sense strand or in the antisense strand is replaced by nucleotide analogs, e.g. by 2' O- methyl-ribonucleotides.
  • the inhibitor of the protein kinase is an antibody specifically binding to said protein kinase, whereby the phosphorylation activity of the protein kinase is reduced.
  • Said antibody having an inhibitory activity has an IC 50 as defined above in the general definition of inhibitors of the phosphorylation activity of a protein kinase.
  • the inhibitor of the protein kinase is a peptide.
  • Peptides in the context of the present invention are chains of naturally and/or non-naturally occurring amino acids with 2 to 50 amino acids connected by peptide bonds. At least for the PKC isoenzymes it has been shown that they have an autoinhibitory pseudosubstrate domain sequence that can bind to the substrate-binding cavity and prevent catalysis (Newton, 2003). This inhibitory effect can be mimicked in vitro by addition of a corresponding synthetic peptide (House and Kemp, 1987).
  • inhibitory peptides are also encompassed within the present invention when they exert their inhibitory activity by other mechanisms than binding to the substrate-binding cavity of the protein kinase.
  • the protein kinase is PKC ⁇ and the peptide is selected from (a) SIYRRGARR WRXL YRAN (SEQ ID NO: 1); or (b) a peptide comprising, essentially comprising or consisting of a peptide having at least 90% sequence identity to the amino acid sequence according to (a), wherein the sequence identity is calculated over the entire length of the amino acid sequence according to (a). It is further preferred that said peptide is acylated with a saturated or non-saturated fatty acid comprising or essentially comprising between 8 and 20 carbon atoms. Preferably said peptide is acylated at its N-terminal amino acid.
  • Preferred fatty acids include lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (Cl 8:0).
  • An especially preferred fatty acid is myristic acid.
  • the protein kinase is PKC ⁇ and the peptide is selected from (a) myr-SIYRRGARRWRKLYRAN (SEQ ID NO: 2); or (b) a peptide comprising, essentially comprising or consisting of a peptide myristoylated at its N-terminus having at least 90% sequence identity to the amino acid sequence according to (a), wherein the sequence identity is calculated over the entire length of the amino acid sequence according to (a).
  • HMMER package http://hmmer.wustl.edu/
  • CLUSTAL algorithm Thimpson J.D. et al., 1994
  • sequence matching may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX).
  • sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., 2003) or Markov random fields.
  • the inhibitor of the protein kinase is a small molecule.
  • Said small molecule having an inhibitory activity has an IC 5O as defined above in the general definition of inhibitors of the phosphorylation activity of a protein kinase.
  • the protein kinase is SGK2 and the small molecule is a compound of formula (I) or a pharmaceutically acceptable salt thereof
  • X and Y are each independently CR , 1 i a a or N;
  • Z is NR, O or S
  • A, B, D, and R la are each independently hydrogen; OR; CN; halogen; CO 2 R; CONR,R 2 ; NRiR 2 ;
  • M is independently hydrogen; (Ci. 3 )alkyl-NRiR 2 ; (C,. 3 )alkyl-OR; halogen; CO 2 R; OR; NR 1 R 2 ; CONR 1 R 2 ; (C, -6 )alkyl-CONRiR 2 ; CHO; (C I-6 )alkylCO 2 R; (C, -6 )alkyl; or
  • M' is independently hydrogen; (C, -3 )alkyl-NRiR 2 ; (Ci -3 )alkyl-OR; halogen; CO 2 R; OR; NRiR 2 ;
  • P, Q, T, U, V and W are each independently hydrogen; halogen; (Ci -6 )alkyl; (Ci -3 )alkylOR; (Ci-
  • M" and M'" are independently at each occurrence hydrogen; (Ci -3 )alkylaryl; (Ci-
  • Ri and R 2 are independently at each occurrence hydrogen; (Ci -6 )alkyl; (Cj. 3 )alkylNRR'; (Ci.
  • R 3 is independently at each occurrence hydrogen; (Ci -6 )alkyl; or (Ci -6 )haloalkyl;
  • the protein kinase is SGK2 and the small molecule is a compound selected from the group consisting of: a) 4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; b) ⁇ 3-[5-(2-naphthyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzyl ⁇ amine; c) 4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; d) ⁇ 4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl ⁇ acetic acid; e) 3- ⁇ 4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl ⁇
  • Ci 3-(4- ⁇ 5-[3,4,5-tris(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl ⁇ phenyl)propanoic acid; cj) ⁇ 4-[5-(6-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl ⁇ acetic acid; ck) 3- ⁇ 4-[5-(6-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl ⁇ propanoic acid; cl) ⁇ 4-[5-(3-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl ⁇ acetic acid; cm) ⁇ 4-[5-(5-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl ⁇ acetic acid; en) 3- ⁇ 4
  • the 244 compounds a) to ij) listed above were identified as inhibitors of Serum and Glucocorticoid-Regulated Kinase 1 (SGK-I) in WO 2006/063167 Al. All compounds displayed IC 50 values of less than 1.5 ⁇ M. Given the relatedness between SGK-I and SGK-2 it can be assumed that these compounds exhibit an inhibitory activity to SGK-2, too.
  • above compound fr) (2-cyclopentyl-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid) is commercially available from Tocris Bioscience (Bristol, UK; Catalogue No. 3572) and displays IC 50 values for SGK-I and SGK-2 of 62 nM and 103 nM, respectively.
  • the protein kinase is SGK2 and the small molecule is 2-cyclopentyl-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3- yl)benzoic acid.
  • the protein kinase is PKC ⁇ and the small molecule is a compound of formula (II) or a pharmaceutically acceptable salt thereof:
  • R 5 is aryl or heteroaryl, optionally substituted once, twice or three times.
  • R 5 is aryl or heteroaryl, optionally substituted once, twice or three times.
  • optionally substituted has the meaning as defined above in the section "Definitions”.
  • R 5 is a phenyl group that is optionally substituted once, twice or three times.
  • the term “optionally substituted” has the meaning as defined above in the section "Definitions”.
  • R 5 is
  • R 6 is hydrogen, -OH, or -NH 2 ; preferably -OH or -NH 2 ; most preferably-NH 2 ;
  • R 7 is hydrogen, methoxy, or F; preferably hydrogen or F, most preferably hydrogen:
  • R 8 is hydrogen or methoxy; preferably hydrogen.
  • R 6 is -NH 2
  • R 7 is hydrogen
  • R 8 is hydrogen
  • the protein kinase is PKC ⁇ and the small molecule is a compound of formula (III) or a pharmaceutically acceptable salt thereof:
  • Ring(A) is aryl or heteroaryl, optionally substituted once, twice or three times
  • Ring(B) is cycloalkyl, aryl, or heteroaryl, optionally substituted once, twice or three times;
  • R 9 is hydrogen or C 1 -C 5 alkyl (e.g. methyl, ethyl, /7-propyl, iso-propy ⁇ , «-butyl, sec-butyl, tert- butyl, pentyl) or C 3 -C 5 cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl); preferably hydrogen or Ci-C 3 alkyl or C 3 cycloalkyl; and
  • Ri 0 is hydrogen or CpC 5 alkyl (e.g. methyl, ethyl, /7-propyl, /.s ⁇ -propyl, n-butyl, sec-butyl, tert- butyl, pentyl) or C 3 -C 5 cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl); preferably hydrogen or Ci-C 3 alkyl or C 3 cycloalkyl.
  • CpC 5 alkyl e.g. methyl, ethyl, /7-propyl, /.s ⁇ -propyl, n-butyl, sec-butyl, tert- butyl, pentyl
  • C 3 -C 5 cycloalkyl e.g. cyclopropyl, cyclobutyl, cyclopentyl
  • Ci-C 3 alkyl or C 3 cycloalkyl preferably
  • Ring(A) is N-(A)
  • Ring(B) is N-(2-aminoethyl)-2-aminoethyl
  • Ring(A) is a group according to formula (v) and Ring(B) is a group according to formula (x), formula (y), formula (z), or formula (aa).
  • Ring(A) is a group according to formula (w) and Ring(B) is a group according to formula (x), formula (y), formula (z), or formula (aa).
  • one of R 9 and Ri 0 is hydrogen and the other one is selected from the group consisting of hydrogen, Ci-C 5 alkyl (e.g. methyl, ethyl, ⁇ -propyl, wo-propyl, n- butyl, sec-butyl, pentyl), and C 3 -C 5 cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl).
  • R 9 may be hydrogen and Ri 0 is selected from the group consisting of hydrogen, Ci-C 5 alkyl and C 3 -C 5 cycloalkyl.
  • one of R 9 and Ri 0 is hydrogen and the other one is selected from the group consisting of hydrogen, C)-C 3 alkyl (e.g. methyl, ethyl, n- propyl, /so-propyl) and C 3 cycloalkyl (i.e. cyclopropyl).
  • C)-C 3 alkyl e.g. methyl, ethyl, n- propyl, /so-propyl
  • C 3 cycloalkyl i.e. cyclopropyl
  • kinases in particular hepatic and haematopoietic cells, preferably hepatic cells.
  • these diseases are all amenable to the treatment and/or prophylaxis with inhibitors of protein kinases. Accordingly, in a preferred use of the invention the infectious disease is a protozoal infection.
  • the pathogenic protozoa is selected from the group consisting of Entamoeba histolytica, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Toxoplasma gondii, Theileria lawrenci, Theileria parva, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium semiovale and Plasmodium knowlesi.
  • the protozoa is a member of the family of plasmodiidae, preferably Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium semiovale and Plasmodium knowlesi.
  • the infectious disease for which treatment and/or prophylaxis is provided is malaria.
  • the present invention further relates to a compound as defined in the first aspect and in the preferred embodiments for use in medicine.
  • the present invention relates to a compound as defined in the first aspect for use in therapy and/or prophylaxis of a protozoal infection.
  • said protozoal infection is malaria.
  • the present invention is directed to a method of identifying compounds for treatment and/or prophylaxis of infectious diseases involving liver or hematopoietic cells comprising the steps of: (i) contacting a protein kinase, a functional variant, or soluble part thereof with a test compound, (ii) selecting a test compound, which specifically binds to said protein kinase, functional variant, or soluble part thereof, (iii) contacting liver or hematopoietic cells with the selected test compound prior, during or after infection of said cell with an infectious agent, and (iv) selecting a test compound inhibiting cell entry and/or development of the infectious agent by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%.
  • the protein kinase is selected from the group consisting of: (a) protein kinase C zeta (PKC ⁇ ); (b) Serine/threonine-protein kinase WNKl (PRKWNKl); (c) Serine/threonine-protein kinase Sgk2 (SGK2); and (d) Serine/threonine-protein kinase 35 (STK35).
  • PLC ⁇ protein kinase C zeta
  • PRKWNKl Serine/threonine-protein kinase WNKl
  • SGK2 Serine/threonine-protein kinase Sgk2
  • STK35 Serine/threonine-protein kinase 35
  • the method further comprises the step of formulating the test compound selected in step (iv) with pharmaceutically acceptable additives and/or auxiliary substances.
  • the present invention is directed to a use of a test compound selected in step (iv) of the method according to the second aspect for the production of a medicament for the therapy and/or prophylaxis of infectious diseases, which involve infection of liver and/or hematopoietic cells.
  • infectious disease is malaria.
  • the present invention is directed to a test compound selected in step (iv) of the method according to the second aspect for use in medicine, in particular for use in therapy and/or prophylaxis of infectious diseases, which involve infection of liver and/or hematopoietic cells.
  • infectious disease is malaria.
  • the present invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising, essentially comprising or consisting of one or more of a compound usable according to the present invention (in particular usable according to the first aspect of the invention) and one or more selected from the group consisting of chinine alkaloids, chloroquine (-phosphate, hydroxychloroquinesulfate), mefloquine (Lariam), bi-guanides: proguanil (Paludrine), di-aminopyrimidines: pyrimethamine, atovaquone, doxycycline, artemether, and lumefantrine and pharmaceutically acceptable carriers, additives and/or auxiliary substances.
  • the present invention also relates to the use of the compounds usable according to the present invention and one or more malaria medicament, preferably chinine alkaloids, chloroquine (-phosphate, hydroxychloroquinesulfate), mefloquine (Lariam), bi-guanides: proguanil (Paludrine), di-aminopyrimidines: pyrimethamine, atovaquone, doxycycline, artemether, and lumefantrine for the manufacture of a pharmaceutical composition for the treatment of diseases involving liver and/or hematopoietic cells, preferably malaria.
  • the two medicaments are administered simultaneously, e.g. combined in one administration form.
  • the two medicaments in said pharmaceutical compositions may be administered subsequently in separate administration forms.
  • the present invention is directed to a method for the identification of molecules of pathogens, which are involved in the infection of liver and/or hematopoietic cells, comprising the following steps: (i) contacting one or more protein kinases, functional variants, or soluble parts thereof with one or more molecules present in pathogens, which are involved in the infection of liver and/or hematopoietic cells; and (ii) selecting a molecule, which specifically binds to the protein kinase.
  • the protein kinase is selected from the group consisting of: (a) protein kinase C zeta (PKC ⁇ ); (b) Serine/threonine-protein kinase WNKl (PRKWNKl); (c) Serine/threonine-protein kinase Sgk2 (SGK2); and (d) Serine/threonine-protein kinase 35 (STK35).
  • PLC ⁇ protein kinase C zeta
  • PRKWNKl Serine/threonine-protein kinase WNKl
  • SGK2 Serine/threonine-protein kinase Sgk2
  • STK35 Serine/threonine-protein kinase 35
  • the pathogen is selected from the group consisting of Entamoeba histolytica, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Toxoplasma gondii, Theileria lawrenci, Theileria parva, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium semiovale and Plasmodium knowlesi.
  • RNAi screen implicates at least 5 host kinases in Plasmodium infection of human hepatoma cells
  • Huh7 cells a human hepatoma cell line, were cultured in RPMI medium supplemented with 10% fetal calf serum (FCS, Gibco/Invitrogen), 1% non-essential amino acid (Gibco/Invitrogen), 1% penicillin/streptomycin (pen/strep, Gibco/Invitrogen), 1% glutamine (Gibco/Invitrogen) and 1% HEPES, pH 7 (Gibco/Invitrogen) and maintained at 37°C with 5%
  • liver perfusion medium Gibco/Invitrogen
  • mice were isolated by perfusion of mouse liver lobule with liver perfusion medium (Gibco/Invitrogen) and purified using a 1.12 g/ml; 1.08 g/ml and 1.06 g/ml Percoll gradient.
  • Cells were cultured in William's E medium containing 4% FCS, 1% pen/strep, 50 mg/ml epidermal growth factor (EGF), 10 ⁇ g/ml transferrin, 1 ⁇ g/ml insulin and 3.5 ⁇ M hydrocortisone in 24 well plates coated with 0.2% gelatine in PBS. Cells were maintained in culture at 37°C and 5% CO 2 .
  • C57BL/6 mice were bred in the pathogen-free facilities of the Instituto de Gulbenkian de
  • Ciencia IRC and housed in the pathogen-free facilities of the Instituto de Medicina Molecular
  • Green fluorescent protein expressing P. berghei (parasite line 259cl2) sporozoites
  • Negative control samples included untransfected cells and cells transfected with a negative control siRNA not targeting any annotated genes in the human genome.
  • Huh7 cells (4500 per well) were seeded in 100 ⁇ l complete RPMI medium in optical 96- well plates (Costar) and incubated at 37°C in 5% CO 2 . 24 h after seeding, cells were transfected with individual siRNAs in a final concentration of 10OnM per lipofection. Each siRNA was transfected in triplicate. Briefly, for each well, cell supernatant was replaced by 80 ⁇ l of serum- free culture medium without antibiotics.
  • cells were fixed with 4% paraformaldehyde (PFA) in PBS and permeabilized with 0.2% saponin in PBS.
  • PFA paraformaldehyde
  • Cell nuclei were stained with Hoechst-33342 (Molecular Probes/Invitrogen), filamentous actin was stained with Phalloidin AlexaFluor488 (Molecular Probes/Invitrogen), EEFs were detected using the mouse monoclonal antibody 2E6 and an AlexaFluor555 labeled goat anti-mouse secondary antibody (Molecular Probes/Invitrogen).
  • qRT- PCR used the SybrGreen method with Quantace qPCR mastermix at 1 1 ⁇ l total reaction volume, containing 500 nM of the target- specific primers, and primers that were designed to specifically amplify a fragment of the selected genes.
  • Real-time PCR reactions were performed on an ABI Prism 7900HT system.
  • Relative amounts of remaining mRNA levels of RNAi targets were calculated against the level of RPLl 3 A or 18S rRNA, as housekeeping genes. Remaining mRNA levels of RNAi-treated samples were compared with those of samples transfected with negative unspecific siRNA.
  • RPL13A-specific primer sequences were: 5'-CCT GGA GGA GAA GAG GAA AGA GA-3' (SEQ ID NO: 4) and 5'-TTG AGG ACC TCT GTG TAT TTG TCA A-3' (SEQ ID NO: 5).
  • 18S rRNA-specific primer sequences were: 5'-CGG CTT AAT TTG ACT CAA CAC G-3' (SEQ ID NO: 6) and 5'-TTA GCA TGC CAG AGT CTC GTT C-3' (SEQ ID NO: 7).
  • total RNA was isolated from livers or primary hepatocytes using Qiagen's RNeasy Mini or Micro kits, respectively, following the manufacturer's instructions. The determination of liver parasite load in vivo, was performed according to the method developed for P. yoelii infections (Bruna-Romero et ah, 2001).
  • the qRT-PCR reactions used Applied Biosystems' Power SYBR Green PCR Master Mix and were performed according to the manufacturer's instructions on an ABI Prism 7000 system (Applied Biosystems).
  • Amplification reactions were carried out in a total reaction volume of 25 ⁇ l, containing 0.8 pmoles/ ⁇ l or 0.16 pmoles/ ⁇ l of the PbA 18 S- or housekeeping gene- specific primers, respectively. Relative amounts of PbA mRNA were calculated against the Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) housekeeping gene.
  • HPRT Hypoxanthine Guanine Phosphoribosyltransferase
  • PbA 18 S-, mouse and human HPRT-specific primer sequences were 5'- AAG CAT TAA ATA AAG CGA ATA CAT CCT TAC - 3' (SEQ ID NO: 8) and 5' - GGA GAT TGG TTT TGA CGT TTA TGT G - 3' (SEQ ID NO: 9) and 5' - TGC TCG AGA TGT GAT GAA GG - 3' (SEQ ID NO: 10) and 5' - TCC CCT GTT GAC TGG TCA TT - 3' (SEQ ID NO: 11) and 5' - TGC TCG AGA TGT GAT GAA GG - 3 ' (SEQ ID NO: 12) and 5' - TCC CCT GTT GAC TGG TCA TT - 3' (SEQ ID NO: 13), respectively.
  • PKC ⁇ mRNA level determination by qRT-PCT PKC ⁇ - specific primers were used (RT2 qPCR Primer Assay for Mouse Prk
  • RNAi screening was used to selectively silence the expression of 727 genes encoding proteins with known or putative kinase activity, as well as kinase-interacting proteins, thereby covering the entire annotated kinome.
  • the effect of each gene-specific knock-down on the infection of Huh7 cells by Plasmodium sporozoites was then monitored using the high- throughput, high-content immunofluorescence microscopy-based assay depicted in Fig. IA. Briefly, short interfering RNA duplexes (siRNAs) targeting each of the chosen genes were transfected into Huh7 cells 24 h after seeding in 96-well plates. 48 h later, cells were infected with P. berghei sporozoites.
  • siRNAs short interfering RNA duplexes
  • EEFs intracellular parasites
  • host cell nuclei and actin to estimate cell numbers and confluency, respectively.
  • customized image analysis algorithms were used to automatically quantify infection rates, normalizing the number of EEFs against the cell confluency in each well.
  • a plate- wise normalization was also used to facilitate comparisons between plates in the first pass of the screen, where the low rate of positive hits yields minimal expectation of variability in the mean infection values between different plates.
  • the infection rate in each experimental well was calculated as a percentage of the mean infection rate from all experimental wells on that plate.
  • infection rate data were plotted against the number of nuclei, also expressed as a percentage of the mean number of nuclei for that plate.
  • RNAi strategy employed was validated by targeting 53 randomly chosen genes with 3 siRNAs each and performing quantitative real-time PCR (qRT-PCR) analysis to determine the level of knock-down achieved in each case. For 13 of these genes either expression was too low to be correctly assessed or primer specificity was insufficient. Most importantly, for 85% of the genes whose expression could be determined, at least 1 of the siRNAs led to an expression knock-down greater than 70% (Figure IB).
  • candidate gene hits were selected for follow-up in pass 2 if any single one of the three siRNAs yielded an increase or decrease on infection greater than 2 standard deviations (s.d.) of the average of the infection of the whole data set, within a defined range of nuclei number ( ⁇ 40% of the average number of nuclei in each experimental plate) (Figure 2B).
  • the latter precaution while relatively inclusive, allowed to exclude from further analysis those siRNAs yielding strong effects on cell proliferation or survival.
  • 73 genes were selected to undergo a second pass of confirmation screening, in which up to 2 additional siRNAs were included to maximize the detection sensitivity for those genes that had yielded only a single siRNA hit in pass 1.
  • siRNAs were noted as “positive candidates” if they yielded infection rates more than 2 s.d. above or below the mean of all the negative controls in this pass. Negative controls replaced whole data set mean for s.d. calculation, since the selected subset of genes in this pass 2 was expected to have a significantly higher hit rate than in pass 1.
  • the selection of candidate genes for follow-up beyond pass 2 required that at least two independent siRNAs targeting the same gene be "positive candidates" according to the above selection criteria (Figure 2C).
  • genes for which different siRNAs yielded conflicting phenotypic results were also excluded from further analysis.
  • RNAi screens are generally inconclusive (Echeverri et al, 2006), and certain genes showing phenotypes with lower than 3 s.d. from mean levels in our assays may provide real, though perhaps more subtle, functionalities in this context.
  • Example 2 PKC ⁇ inhibition leads to a decrease in host cell infection by Plasmodium sporozoites
  • aPKCs atypical PKCs
  • PKC ⁇ Ono et al, 1989
  • PKC ⁇ A PKC lambda/iota
  • NF-kappaB NF-kappaB activation and polarity
  • PKC isoenzymes have an autoinhibitory pseudosubstrate domain sequence that can bind to the substrate-binding cavity and prevent catalysis (Newton, 2003). This inhibitory effect can be mimicked in vitro by addition of a corresponding synthetic peptide (House and Kemp, 1987).
  • Huh7 cells were incubated overnight with either scrambled or pseudosubstrate peptides and then harvested in modified RIPA buffer (150 mM NaCl; 50 mM Tris, pH 7.5; 1% Triton X-100; 50 mM NaF; 1 mM Na 3 VO 4 ; complete EDTA-free protease inhibitor cocktail).
  • modified RIPA buffer 150 mM NaCl; 50 mM Tris, pH 7.5; 1% Triton X-100; 50 mM NaF; 1 mM Na 3 VO 4 ; complete EDTA-free protease inhibitor cocktail).
  • proteins were transferred to a nitrocellulose membrane (BIO-RAD), which was probed with anti-phospho-PKC (pan) ( ⁇ ll Ser660) (Cell Signaling Technology) or anti-phospho- aPKC (Thr555/PKC ⁇ ; Thr560/PKC ⁇ ) (Upstate) plus HRP-conjugated anti-rabbit (Amersham).
  • BIO-RAD nitrocellulose membrane
  • the membrane was developed with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).
  • Huh7 cells were transfected (Lipofectamine 2000, Invitrogen) with plasmids encoding
  • GFP-PKC ⁇ or GST-PKCv 48 hours after transfection the cells were incubated with either scrambled or pseudosubstrate peptides for 1 hour and then harvested as before.
  • the relative expression levels of GFP-PKC ⁇ and GST-PKCi were determined by probing the membrane with anti-aPKC ⁇ (C20, Santa Cruz Biotechnology), which recognizes the two isoenzymes.
  • the % of inhibition of PKC ⁇ versus PKCi was calculated from the anti-phospho-aPKC signals. All signals were normalized to those of actin.
  • Example 2 The cell-based assay described above in Example 1 was used to test the effects of a myristoylated PKC ⁇ pseudosubstrate (myr-SIYRRGARRWRKLYRAN, SEQ ID NO: 2), previously characterized as a specific PKC ⁇ inhibitor (PKC ⁇ lnh) (Laudanna et al., 1998; Standaert et al., 1997), on P. berghei infection. A scrambled myristolated peptide was used as control in all PKC ⁇ inhibition experiments (Laudanna et al, 1998).
  • myristoylated PKC ⁇ pseudosubstrate myr-SIYRRGARRWRKLYRAN, SEQ ID NO: 2
  • PKC ⁇ lnh a specific PKC ⁇ inhibitor
  • a scrambled myristolated peptide was used as control in all PKC ⁇ inhibition experiments (Laudanna et al, 1998).
  • Example 3 Inhibition of PKC ⁇ impairs invasion of host cells by Plasmodium sporozoites
  • Green fluorescent protein (GFP) expressing P. berghei parasite line 259cl2
  • sporozoites were obtained from dissection of infected female Anopheles stephensi mosquito salivary glands.
  • FACS analysis at 2 h and 24 h after sporozoite addition was performed to determine the percentage of parasite-containing cells and parasite-GFP intensity within infected cells.
  • For infection level measurement at 2 h 1 mg/ml Dextran tetramethylrhodamine 10,000 MW, lysine fixable (fluoro-ruby) (Molecular Probes/ Invitrogen) was added to the cells immediately prior to sporozoite addition.
  • Cell samples for FACS analysis were processed as previously described (Prudencio et al. , 2007).
  • PKC ⁇ -specific primers were used (RT2 qPCR Primer Assay for Mouse Prkcz, SuperArray Bioscience Corporation).
  • Example 4 PKC ⁇ knock-down in mouse livers confirms the physiological relevance of PKC ⁇ role in malaria infection in vivo
  • siRNA#l - S'-GGGAcAGcAAcAAcuGcuudTsdT-S ' SEQ ID NO: 14
  • siRNA#2 - S ' -GGccucAcAcGucuuAAAAdTsdT-S' SEQ ID NO: 15
  • siRNA#3 - 5 ' -cccuuAAcuAcAGcAuAuGdTsdT-3 SEQ ID NO: 16.
  • a modified siRNA targeting luciferase was used as control (5 ' - cuuAcGcuGAGuAcuucGAdTsdT-3 ', SEQ ID NO: 17).
  • Lower case letters (c and u) represent
  • dT 2'OMe nucleotides
  • s 2'OMe nucleotides
  • mice 36 h after siRNA administration mice were infected by i.v. injection of 2 x 10 4 P. berghei sporozoites. Remaining PKC ⁇ mRNA levels, parasite load in the livers of infected mice were determined by qRT-PCR 40 h after sporozoite injection, 76 h after siRNA administration.
  • mice treated with one PKC ⁇ siRNA were allowed to proceed onto the blood stage and parasitemia (% of infected red blood cells) was measured daily.
  • the PKC ⁇ protein level in the liver of siRNA-treated mice was determined by Western blot.
  • PKC ⁇ protein level in the liver of mice treated with a PKC ⁇ siRNA was quantified by Western blot using the primary antibody (rabbit anti-PKC ⁇ (C20): sc-216, Santa Cruz Biotechnology) and normalised against actin level detected using rabbit anti-actin (A2066, Sigma).
  • Anti-rabbit horseradish peroxidase-conjugated NA934V, GE Healthcare, UK Ltd. was used as secondary antibody.
  • the membrane was developed using the ECL Western Blotting Analysis System, according to the manufacturer's instructions (Amersham Bioscience, Germany). Signal quantification was performed using the ImageJ software package (NIH, USA).
  • mice from the same litter were given an initial intravenous (i.v.) injection of either test or control siRNAs and, infection was initiated 36 h later by i.v. injection of freshly isolated P. berghei sporozoites. Mice were sacrificed 40 h after infection to permit parallel analyses of gene silencing and infection load.
  • three distinct siRNA sequences targeting PKC ⁇ were tested individually, while a siRNA targeting luciferase, a transcript known to be absent in these mice, was used to address sequence- independent off-target effects that may arise from these treatments.
  • the serine/threonine kinases SGK2 and SGK3 are potent stimulators of the epithelial Na+ channel alpha,beta,gamma-ENaC. Pflugers Arch. 445: 693-696.
  • Pflugers Arch. 445 60-66.
  • Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science. 238: 1726-1728.
  • Prudencio M., Rodrigues, CD., Ataide, R. and Mota, M. M. (2007) Dissecting in vitro host cell infection by Plasmodium sporozoites using flow cytometry. Cell Microbiol.
  • Host SR-BI plays a dual role in the establishment of malaria liver infection Cell Host Microbe. In Press. Standaert, M.L., Galloway, L., Karnam, P., Bandyopadhyay, G., Moscat, J. and Farese, R.V. (1997) Protein kinase C-zeta as a downstream effector of phosphatidylinositol 3-kinase during insulin stimulation in rat adipocytes. Potential role in glucose transport. J Biol Chem. 272: 30075-30082. Suzuki, A. and Ohno, S. (2006) The PAR-aPKC system: lessons in polarity. J Cell Sci.
  • SEQ ID NO: 2 Inhibitor of PKC zeta, myristoylated
  • SEQ ID NO: 3 control peptide, scrambled sequence of SEQ ID NO: 1 ; myristoylated
  • SEQ ID NO: 4 RPLl 3 A- specific primer sequence
  • SEQ ID NO: 5 RPL13A-specific primer sequence
  • SEQ ID NO: 6 18 S rRNA-specific primer sequence
  • SEQ ID NO: 7 18 S rRNA-specific primer sequence
  • SEQ ID NO: 8 PbA 18 S-specific primer
  • SEQ ID NO: 14 siRNA#l targeting PKCzeta
  • SEQ ID NO: 15 siRNA#2 targeting PKCzeta
  • SEQ ID NO: 16 siRNA#3 targeting PKCzeta
  • SEQ ID NO: 17 siRNA targeting luciferase
  • SEQ ID NO: 18 siRNA#4 targeting PKCzeta, sense strand
  • SEQ ID NO: 19 siRNA#4 targeting PKCzeta, antisense strand
  • SEQ ID NO: 20 siRNA#5 targeting PKCzeta, sense strand
  • SEQ ID NO: 21 siRNA#5 targeting PKCzeta, antisense strand
  • SEQ ID NO: 23 siRNA#l targeting WNKl, antisense strand
  • SEQ ID NO: 24 siRNA#2 targeting WNKl
  • sense strand SEQ ID NO: 25 siRNA#2 targeting WNKl
  • SEQ ID NO: 26 siRNA#l targeting SGK2, sense strand
  • SEQ ID NO: 27 siRNA#l targeting SGK2, antisense strand
  • SEQ ID NO: 28 siRNA#2 targeting SGK2, sense strand
  • SEQ ID NO: 29 siRNA#2 targeting SGK2, antisense strand SEQ ID NO: 30: siRNA#3 targeting SGK2, sense strand
  • SEQ ID NO: 31 siRNA#3 targeting SGK2, antisense strand
  • SEQ ID NO: 32 siRNA#l targeting STK35, sense strand
  • SEQ ID NO: 33 siRNA#l targetingSTK35, antisense strand
  • SEQ ID NO: 34 siRNA#2 targetingSTK35
  • sense strand SEQ ID NO: 35 siRNA#2 targetingSTK35
  • SEQ ID NO: 36 siRNA#3 targetingSTK35, sense strand
  • SEQ ID NO: 37 siRNA#3 targetingSTK35, antisense strand
  • SEQ ID NO : 38 siRNA# 1 targeting PKCzeta, sense strand
  • SEQ ID NO: 39 siRNA#2 targeting PKCzeta, sense strand
  • SEQ ID NO: 40 siRNA#3 targeting PKCzeta, sense strand

Abstract

L’invention concerne l’utilisation d’inhibiteurs de kinases hôtes pour produire un médicament destiné à traiter et/ou prévenir les infections  impliquant les cellules hépatiques et/ou les cellules hématopoïétiques, en particulier la malaria.
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WO2015006752A1 (fr) * 2013-07-12 2015-01-15 The Regents Of The University Of California Polythérapies contre la malaria
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WO2020103896A1 (fr) * 2018-11-22 2020-05-28 Beigene, Ltd. Pyrrolo[2,3-b]pyridines utilisés en tant qu'inhibiteur de hpk1 et leurs utilisations

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WO2012000095A1 (fr) * 2010-06-30 2012-01-05 Tony Kusalik Méthodes d'analyse de kinome
EP2588428A1 (fr) * 2010-06-30 2013-05-08 University of Saskatchewan Méthodes d'analyse de kinome
EP2588428A4 (fr) * 2010-06-30 2014-03-05 Univ Saskatchewan Méthodes d'analyse de kinome
JP2016505584A (ja) * 2012-12-18 2016-02-25 サントル ナスィオナル ド ラ ルシェルシュ スィアンティフィク（セ．エン．エル．エス．） ダウン症候群及びアルツハイマー病に関連する認知欠損の治療のための、ｄｙｒｋ１ａタンパク質阻害剤としての３，５−ジアリールアザインドール類
US9206188B2 (en) * 2013-04-18 2015-12-08 Arrien Pharmaceuticals Llc Substituted pyrrolo[2,3-b]pyridines as ITK and JAK inhibitors
US20140315909A1 (en) * 2013-04-18 2014-10-23 Arrien Pharmaceuticals Llc 3,5-(Un)substituted-1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine and 5H- pyrrolo[2,3-b]pyrazine dual ITK and JAK3 Kinase Inhibitors
JP2016516823A (ja) * 2013-04-18 2016-06-09 アーリーン ファーマシューティカルズ エルエルシー ３，５−（非）置換−１Ｈ−ピロロ［２，３−ｂ］ピリジン、１Ｈ−ピラゾロ［３，４−ｂ］ピリジン、及び５Ｈ−ピロロ［２，３−ｂ］ピラジン、ＩＴＫ及びＪＡＫ３キナーゼの阻害剤
US9834551B2 (en) 2013-04-18 2017-12-05 Arrien Pharmaceuticals Llc Substituted pyrrolo[2,3-b]pyrazines and substituted pyrazolo[3,4-b]pyridines as ITK and JAK kinase inhibitors
WO2015006752A1 (fr) * 2013-07-12 2015-01-15 The Regents Of The University Of California Polythérapies contre la malaria
US20160151379A1 (en) * 2013-07-12 2016-06-02 The Regents Of The University Of California Combination therapies for malaria
US9918989B2 (en) 2013-07-12 2018-03-20 The Regents Of The University Of California Combination therapies for malaria
WO2015146929A1 (fr) * 2014-03-24 2015-10-01 武田薬品工業株式会社 Composé hétérocyclique
CN106459040A (zh) * 2014-03-24 2017-02-22 武田药品工业株式会社 杂环化合物
WO2020103896A1 (fr) * 2018-11-22 2020-05-28 Beigene, Ltd. Pyrrolo[2,3-b]pyridines utilisés en tant qu'inhibiteur de hpk1 et leurs utilisations
CN113166139A (zh) * 2018-11-22 2021-07-23 百济神州有限公司 作为HPK1抑制剂的吡咯并[2,3-b]吡啶及其用途

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