WO2010049173A1

WO2010049173A1 - Use of inhibitors of host kinases for the treatment of infectious diseases

Info

Publication number: WO2010049173A1
Application number: PCT/EP2009/007793
Authority: WO
Inventors: Michael Hannus; Cecilie Martin; Maria M. Mota; Miguel Prudencio; Christina Dias Rodrigues
Original assignee: Cenix Bioscience Gmbh; Instituto De Medicina Molecular, Faculdade De Medicina Da Universidade De Lisboa
Priority date: 2008-10-31
Filing date: 2009-10-30
Publication date: 2010-05-06

Abstract

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.

Description

USE OF INHIBITORS OF HOST KINASES FOR THE TREATMENT OF INFECTIOUS DISEASES

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION AND STATE OF THE ART

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.

Death by malaria is almost exclusively caused by P. falciparum, transmitted by the vector Anopheles gambiae, which preferentially feeds on humans. As the mosquito bites, sporozoites are injected into the skin. After finding of a blood vessel, they travel directly to the liver. Once there, they migrate through several hepatocytes before they infect a final one, surrounded by a parasitophorous vacuole where the intrahepatic form of the parasite grows and multiplies. This asymptomatic phase is known as the liver or hepatic stage of disease. During this period there is an amazing parasite multiplication (each parasite gives rise to 10-30 thousand new parasites in 2- 7 days depending on the parasite species). Eventually, the infected hepatocytes burst, releasing the parasites into the bloodstream, where they will target and invade the red blood cells (RBCs). 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.

The hepatic stage of a Plasmodium infection constitutes an appealing target for the development of anti-malarial drugs since these would act before the onset of pathology. Despite the importance of such knowledge, little is known about the parasite's requirements and the strategies it developed in order to successfully invade and develop inside the liver cells.

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.

Both the strong tropism and obligate nature of the events that take place during liver infection suggest an essential requirement for hepatocyte-specific factors in enabling this complex lead-up to the blood stage. It is therefore of primary interest to identify and characterize the role of such host factors, as these may contribute to the design of rational interventional strategies for the development of novel prophylactic agents.

To this end, 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. Using 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.

Using this assay system the inventors surprisingly identified several host kinases which play a role in the infection of the host cells. Thus, the present invention provides novel targets for the prophylaxis and treatment of infectious diseases, in particular malaria.

SUMMARY OF THE INVENTION

According to a first aspect 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 (PKCζ); (b) Serine/threonine-protein kinase WNKl (PRKWNKl); (c) Serine/threonine-protein kinase Sgk2 (SGK2); and (d) Serine/threonine-protein kinase 35 (STK35). According to a second aspect 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%.

According to a third aspect 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.

According to a fourth aspect 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.

According to a fifth aspect the present invention relates to 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.

According to a sixth aspect 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.

BRIEF DESCRIPTION OF THE DRAWINGS 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.

Fig. 2. A kinome-wide 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. (D) Plot comparison of the 2 runs of pass 2 of the RNAi screen. Colour attributions are the same as in panels (B, C). The comparison reveals a high correlation (R = 0.88) between the duplicate runs of pass 2 of the screen (diagonal line). The horizontal and vertical lines represent 100% ± 2.0 s.d. of the average of all the negative controls in the assay.

(E) Plot of pass 3 of the RNAi screen representing the effect of 37 siRNAs targeting 16 human genes on Huh7 cell infection by P. berghei sporozoites and cell nuclei count. Remaining mRNA levels following RNAi were determined for each of these genes by qRT-PCR (see text and Figure 2F). Colour attributions and shading are the same as in (B, C, D). Dark filled circles highlight siRNAs targeting the genes for which at least two independent siRNAs led to an infection increase or decrease above or below ± 3.0 s.d. of the average of all the negative controls in the assay, respectively. The horizontal lines represent 100% ± 3.0 s.d. of the average of all the negative controls in the assay.

(F) Effect of siRNA on infection rates versus remaining mRNA levels for the 7 genes targeted by the siRNAs labelled in dark circles in (E). Each circle represents one siRNA (mean of triplicate values). For all genes except GUKl and HCK, represented in light grey, a positive correlation between infection rate and remaining gene-specific mRNA levels is observed. Shading attributions are the same as in (B, C, E). The horizontal lines represent the same as in E (100% ± 3.0 s.d. of the average of all the negative controls in the assay). The axes on the bottom left of the panel denote the scale of each of the plots in the panel. Fig. 3. PKCζ inhibition by a pseudosubstrate decreases hepatocyte infection without affecting host cell viability.

(A) Representative pictures of cells treated with the PKCζ pseudosubstrate inhibitor and a control peptide. The pictures depict nuclei stained with Hoechst and actin stained with phalloidin, and show that cells are not affected by the inhibitor peptide.

(B, C) Quantification of cell confluency (B) and number of nuclei (C) in 40 microscope fields of cells treated with the PKCζ pseudosubstrate inhibitor and a control peptide. (D, E) Effect of PKCζlnh (20 μM) 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%).

Figure 4. Inhibition of PKCζ impairs invasion of host cells by Plasmodium sporozoites.

(A) PKCζ inhibition by PKCζlnh decreases P. berghei sporozoite infection of Huh7 cells in a dose-dependent manner. PKCζlnh 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.

(B) PKCζ inhibition by PKCζlnh does not affect development of Exo-Erythrocytic Forms (EEF). PKCζlnh was added to Huh7 cells 1 h before addition of GFP-expressing P. berghei sporozoites and GFP intensity (proportional to EEF development) was measured 24 h later by FACS.

(C) PKCζ inhibition by PKCζlnh decreases P. berghei sporozoite invasion of Huh7 cells in a dose-dependent manner. PKCζlnh 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.

(D) PKCζ inhibition does not affect infection after invasion has occurred. PKCζlnh 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.

(E) PKCζlnh does not affect infection by acting on sporozoites directly. Sporozoites were pre- treated with PKCζlnh for 1 hour before addition to the cells and infection rate was measured 24 h later by FACS. All results are expressed as the mean ± s.d. Of GFP⁺ cells (%) in 3 independent infections.

(F) PKCζ inhibition during the period of cell invasion by sporozoites, but not during their intracellular development period, leads to a decrease in infection. The infection rate was determined by qRT-PCR in Huh7 cells incubated with PKCζlnh throughout different periods of the infection process, namely -1 to 2h and 2h to 24h relative to sporozoite addition.

Fig. 5. In vivo PKCζ 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 PKCζ on mouse liver infection by P. berghei (solid bars) and on PKCζ 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 PKCζ were measured by qRT-PCR in the same liver samples. Results are expressed as the mean ± s.d. of all mice in each group. The black bars at the bottom represent the negative control (5 mice treated with luciferase-targeting siRNA). The dark grey bars at the top represent mice treated with the 3 independent siRNAs targeting the PKCζ gene (6 mice per siRNA). (B) Knock-down of PKCζ expression by RNAi delays the onset of blood stage infection, as measured by parasitemia (percentage of infected red blood cells, iRBC) quantification using flow cytometry. Each symbol represents one mouse. Black circles in the left column of each diagram represent the negative controls (5 mice treated with luciferase-targeting siRNA). Dark grey symbols (circle, triangle, square) in the 3 columns to the right of each diagram represent the 6 mice treated with the 3 independent siRNAs targeting the PKCζ gene (6 mice per siRNA).

DETAILED DESCRIPTION

Definitions

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H. G. W, Nagel, B. and Kδlbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

In the following definitions of the terms: 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.

The term "alkyl" refers to a saturated straight or branched carbon chain. Preferably, 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.

The term "heteroalkyl" refers to a saturated straight or branched carbon chain. Preferably, 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 /sø-propyl), butyl (tt-butyl, wo-butyl, sec-butyl, or tør/-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. Preferably the heteroatoms are selected from O, S, and N, e.g. -O- CH₃, -S-CH₃, -NH-CH₃, -CH₂-O-CH₃, -CH₂-O-C₂H₅, -CH₂-S-CH₃, -CH₂-S-C₂H₅, -CH₂-NH- CH₃, -CH₂-NH-C₂H₅, -C₂H₄-O-CH₃, -C₂H₄-O-C₂H₅, -C₂H₄-S-CH₃, -C₂H₄-S-C₂H₅, -C₂H₄-NH- CH₃, -C₂H₄-NH-C₂H₅, etc. Heteroalkyl groups are optionally substituted.

The terms "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. The terms "cycloalkyl" and "heterocycloalkyl" are also meant to include bicyclic, tricyclic and polycyclic versions thereof. If more than one cyclic ring is present such as in bicyclic, tricyclic and polycyclic versions, then these rings may also comprise one or more aryl- or heteroaryl ring. The term "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 C₃-Ci₀-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, spiro[5,4]decyl, bicyclo[4.1.0]heptyl, bicyclo[3.2.0]heptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[5.1.0]octyl, bicyclo[4.2.0]octyl, octahydro-pentalenyl, octahydro- indenyl, decahydro-azulenyl, adamantyl, or decahydro-naphthalenyl. Preferred examples of heterocycloalkyl include C₃-Ci₀-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, 3,8 diaza-spiro[5,4] decyl, 3,7 diaza-spiro[5,4] decyl, l-aza-7,l l-dioxo-spiro[5,5] undecyl, l,4-diazabicyclo[2.2.2]oct-2-yl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1- piperazinyl, or 2-piperazinyl.

The term "alicyclic system" refers to mono, bicyclic, tricyclic or polycyclic versions of a cycloalkyl or heterocycloalkyl comprising at least one double and/or triple bond. However, an alicyclic system is not aromatic or heteroaromatic, i.e. does not have a system of conjugated double bonds/free electron pairs. Thus, 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. Accordingly, the "cycloalkenyl" as defined below is a preferred embodiment of an alicyclic ring system. Alicyclic systems are optionally substituted.

The term "alkoxy" refers to an -O-alkyl group, i.e. to an oxygen atom substituted by a saturated straight or branched carbon chain. Preferably, the chain comprises from 1 to 10 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Thus, preferred alkoxy groups are methoxy, ethoxy, propoxy (tf-propoxy or /so-propoxy), butoxy (n-butoxy, sec-butoxy, zsø-butoxy, or tert-butoxy), pentoxy, hexoxy, heptoxy, octoxy, nonoxy, or decoxy. Alkoxy groups are optionally substituted. The term "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.

The term "aralkyl" 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. Preferably, in this context 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-propyϊ), butyl (n-butyl, wo-butyl, sec-butyl, or tert-butyl), pentyl, hexyl, heptyl, octyl. Preferably, in this context 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.

The term "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- benzisothiazolyl, 2,1-benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, 1,2,3-benzotriazinyl, or 1,2,4-benzotriazinyl. As used herein, the term "heteroaryl" also encompasses aromatic rings and ring systems as described above fused to non-aromatic rings or ring systems. The term "heteroaralkyl" 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. Preferably, in this context 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 (^-propyl or zso-propyl), butyl (rø-butyl, wo-butyl, sec-butyl, or ter/-butyl), pentyl, hexyl, heptyl, octyl. The heteroaralkyl group is optionally substituted at the alkyl and/or heteroaryl part of the group.

The terms "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. Preferably, the alkenyl chain comprises from 2 to 8 carbon atoms, i.e. 2, 3, 4, 5, 6, 7, or 8, e.g. ethenyl, 1 -propenyl, 2-propenyl, iso- propenyl, 1-butenyl, 2-butenyl, 3-butenyl, wo-butenyl, sec-butenyl, tert-butenyl, 1-pentenyl, 2- pentenyl, 3-pentenyl, 4-pentenyl, hexenyl, heptenyl, octenyl. The term "alkenyl" also comprises

=CH₂, i.e. methenyl, or other alkylidene groups, if the substituent is directly bonded via the double bond. Preferably the cycloalkenyl ring comprises from 3 to 14 carbon atoms, i.e. 3, 4, 5,

6, 7, 8, 9, 10, 1 1, 12, 13 or 14, e.g. cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl, cyclodecenyl, spiro[3,3]heptenyl, spiro[3,4]octenyl, spiro[4,3]octenyl, spiro[3,5]nonenyl, spiro[5,3]nonenyl, spiro[3,6]decenyl, spiro[6,3]decenyl, spiro[4,5]decenyl, spiro[5,4]decenyl, bicyclo [4.1.0] heptenyl, bicyclo[3.2.0]heptenyl, bicyclo[2.2.1]heptenyl, bicyclo[2.2.2]octenyl, bicyclo[5.1.0]octenyl, bicyclo[4.2.0]octenyl, hexahydro-pentalenyl, hexahydro-indenyl, octahydro-azulenyl, or octahydro-naphthalenyl.

The term "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. Preferably, 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,

2-pentynyl, 3-pentynyl, 4-pentynyl, hexynyl, heptynyl, octynyl.

The term "optionally substituted" in each instance if not further specified refers to a substitution by one of the following groups: halogen (in particular F, Cl, Br, or I), -NO₂, -CN, -OR"¹, -NR¹R", -COOR¹", -CONR¹R", -NR¹COR", -NR¹¹COR"¹, -NR¹CONR¹R", -NR¹SO₂A, -COR¹"; -SO₂NR¹R", -OOCR"¹, -CR¹¹¹R¹¹¹¹OH, -R¹¹¹OH, and -E;

R¹ 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-,

6-, or 7-membered heterocycloalkyl; R'" and R"" is each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, aralkyl, heteroaryl, and -NR¹R"; 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. Thus, 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.

Compounds 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". The terms "polypeptide" and "protein" are used interchangeably herein. Peptides 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. Accordingly, in a preferred embodiment 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.

By "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). As will be appreciated by those skilled in the art, all of these nucleic acid analogs may find use in the present invention. In addition, mixtures of naturally occurring nucleic acids, such as DNA and RNA, and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs can be made.

The term "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. Nevertheless, in preferred embodiments 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 μM or less, preferably 50 μM or less, preferably 30 μM or less, preferably 20 μM or less, preferably 10 μM or less, preferably 5 μM or less, more preferably 1 μM 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.

The term "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.

Various assays to measure phosphorylation activity and its inhibition are known from the prior art (Ma et al. 2008). A compound is considered an inhibitor of the phosphorylation activity of a protein kinase, if the compound has an IC₅₀ of < 100 μM in a phosphorylation assay. Preferably the IC₅₀ is < 90μM, < 80μM, < 70μM, < 60μM, < 50μM, < 40μM, < 30μM, < 20μM, < lOμM, < 9μM, < 8μM, < 7μM, < 6μM, < 5μM, < 4μM, < 3μM, < 2μM, < lμM, < 0.9μM, < 0.8μM, < 0.7μM, < 0.6μM, < 0.5μM, < 0.4μM, < 0.3μM, < 0.2μM, < 0.1 μM, < 9OnM, < 8OnM, < 7OnM, < 6OnM, < 5OnM, < 4OnM, < 3OnM, < 20 nM or < 10 nM.

The term "infectious agent" as it is used in the present application refers to an organism capable of causing an infectious disease in a subject. In particular, said "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. 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.

Embodiments of the Invention

The present invention will now be further described. In the following passages different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

In a first aspect, 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 (PKCζ); (b) Serine/threonine-protein kinase WNKl (PRKWNKl); (c) Serine/threonine-protein kinase Sgk2 (SGK2); and (d) Serine/threonine-protein kinase 35 (STK35).

PKCζ (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 PKCζ (Ono et al, 1989) and PKCλ/i (PKClambda/iota) (Akimoto et al, 1994) isoforms. The aPKCs have been implicated in numerous processes, including cell growth and survival, regulation of NF-κB 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). Down-modulation of both of these osmotic and oxidative stress-responsive proteins led to a reduced infection in the screen shown in the Example section of the present application. Although their role in Plasmodium infection remains unclear, the present data may be highlighting the importance of maintaining an optimal osmotic balance in the host cell to permit successful infection.

STK35 (UniProtKB/Swiss-Prot Q8TDR2 (STK35_HUMAN); EC=2.7.11.1 ; alternative names: "CLP-36-interacting kinase", "Clikl", "PDLIMl -interacting kinase 1") is known to interact with CLP-36, a PDZ-LIM protein and re-localize from the nucleus to actin stress fibres (Vallenius and Makela, 2002). This has led to the suggestion that STK35 may act as a regulator of the actin-myosin cytoskeleton in non-muscle cells (Vallenius and Makela, 2002). Thus, without wishing to be bound by a particular theory the inventors hypothesize that recruitment of STK35 may influence Plasmodium infection by playing a role in this process.

As set out below, siRNAs were also used to identify protein kinases as a potential target to interfere with the cell entry and/or development of pathogens, in particular malaria. Accordingly, in a preferred embodiment of the present invention 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. It is further preferred that 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. In preferred embodiments, both stands of the siRNA duplex have a 3' overhang of 2 nucleotides each. In further preferred embodiments, the 3' overhang of the sense strand, of the antisense strand or of both strands comprises at least one dT nucleotide. Preferably, 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. Thus, in particularly preferred embodiments, 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. In the duplex region 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. Preferably, one or more C ribonucleotides are replaced by nucleotide analogs, e.g. by the corresponding 2'0Me nucleotide(s). Preferably, 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.

In a preferred embodiment of the first aspect the protein kinase is (a) PKCζ 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;

(c) SGK2 and the 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

(d) STK35 and 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. As noted before, it is also preferred for the siRNA duplexes described in paragraphs (a) to

(d) above that the antisense strand has a 3' overhang from 1 to 3 nucleotides, most preferably of 2 nucleotides. Preferably 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.

In a further preferred embodiment of the present invention, 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 IC₅₀ as defined above in the general definition of inhibitors of the phosphorylation activity of a protein kinase.

In a further preferred embodiment of the present invention, 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). Without wishing to be bound by a specific theory, the inventors believe that such autoinhibitory pseudosubstrate domain sequences exist also for other protein kinases so that all protein kinases usable in the methods and uses of the present application can be inhibited by peptides. Furthermore, 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. In a particularly preferred embodiment the protein kinase is PKCζ 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. Thus, in one embodiment the protein kinase is PKCζ 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). The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, preferably with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson J.D. et al., 1994) available e.g. on http://www.ebi.ac.uk/clustalw/ or on http://npsa-pbil.ibcp.fr/cgi- bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html. Preferred parameters used are the default parameters as they are set on http://www.ebi.ac.uk/clustalw/index.htmW. The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). Preferably, sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., 2003) or Markov random fields. When percentages of sequence identity are calculated in the context of the present invention, these percentages are to be calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.

In a further preferred embodiment of the present invention, 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.

In a preferred embodiment of the present invention, the protein kinase is SGK2 and the small molecule is a compound of formula (I) or a pharmaceutically acceptable salt thereof

(I) wherein R₃ is

wherein

X and Y are each independently CR , 1ⁱa^aor N;

Z is NR, O or S;

A, B, D, and R^la are each independently hydrogen; OR; CN; halogen; CO₂R; CONR,R₂; NRiR₂;

NR₃R₄; aryl; heteroaryl; (Ci_-3)alkyl-NR,R₂; (C,_-6)alkyl, or (Ci_-6)haloalkyl; wherein R_b is

(k) G) (1)

(n) (o)

(P) (q) (r)

(S)

(t) wherein

M is independently hydrogen; (Ci.₃)alkyl-NRiR₂; (C,.₃)alkyl-OR; halogen; CO₂R; OR; NR₁R₂;

CONR₁R₂; (C,_-6)alkyl-CONRiR₂; CHO; (C_I-6)alkylCO₂R; (C,_-6)alkyl; or

NR₃R₄;

M' is independently hydrogen; (C,_-3)alkyl-NRiR₂; (Ci_-3)alkyl-OR; halogen; CO₂R; OR; NRiR₂;

(C,.₃)alkyl-NR₃R₄; CONRiR₂; (Ci_-6)alkyl-CONR,R₂; CHO; (Ci_-6)alkylCO₂R; NR₃R₄; (C-

₆)alkyl; or phenyl;

P, Q, T, U, V and W are each independently hydrogen; halogen; (Ci_-6)alkyl; (Ci_-3)alkylOR; (Ci-

₆)haloalkyl; CO₂R; CHO; (C,_-6)alkyl-CO₂R; (Ci_-3)alkyl-NR,R₂; OR; NRiR₂; NR₃R₄; CONRiR₂;

(Ci_-6)alkyl-CONRiR₂; aryl; or heteroaryl;

M" and M'" are independently at each occurrence hydrogen; (Ci_-3)alkylaryl; (Ci-

₃)alkylheteroaryl; (Ci_-6)alkyl; or (Ci_-6)haloalkyl;

Ri and R₂ are independently at each occurrence hydrogen; (Ci_-6)alkyl; (Cj.₃)alkylNRR'; (Ci.

₃)alkyl0R; (Ci_-6)cyanoalkyl; NRR'; (Ci_-3)alkylaryl; (Ci.₃)alkylheteroaryl; (Ci_-6)haloalkyl; or together with the nitrogen that they are attached to form a 4, 5, 6, or 7 member non-aromatic ring, said ring optionally containing up to 2 additional heteroatoms selected from the group consisting of NR; O; or S(O)_n; and said ring is unsubstituted or substituted with from 1-3 substituents selected from the group consisting of halogen; (Ci_-6)alkyl; OR; NRR'; CN; halogen; (Ci_-6)haloalkyl; phenyl; heteroaryl and heterocyclyl; n is independently at each occurrence 0, 1 or 2;

R₃ is independently at each occurrence hydrogen; (Ci_-6)alkyl; or (Ci_-6)haloalkyl; R₄ is C(=O)(Ci. ₆)alkyl; C(=O)(Ci_-3)alkyl-NRR'; or C(=O) — (Ci_-3)alkylaryl wherein said aryl is unsubstituted or substituted with 1-3 substituents selected from the group consisting of halogen, (Ci_-3)alkyl and (Ci_-3)alkoxy); C(=O) — (Ci_-3)alkylheteroaryl; C(=O)phenyl wherein the phenyl group is unsubstituted or substituted with 1 -3 substituents selected from the group consisting of halogen, (C,_-6)alkyl and OR.

In a preferred embodiment of the present invention, 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}propanoic acid; f) {3-[5-(2-naphthyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}methanol; g) 4-{5-[3-(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; h) 3 -(4- { 5- [3 -(methyloxy)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } phenyl )propanoic acid; i) 3 - { 3 - [4-(aminomethyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-5 -yl } benzonitrile; j) 4-{5-[3-(aminocarbonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; k) 4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; 1) 4-{5-[6-(methyloxy)-3-pyridinyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; m) 3-{3-[3-(aminomethyl)phenyl]-l-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; n) 4-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; o) 2-fluoro-4-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; p) 3-amino-5-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; q) 3-{4-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; r) 3 -(4- { 5 - [3 -(aminocarbonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -y 1 } phenyl)propanoic acid; s) 3-{4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; t) 3-(4-{5-[6-(methyloxy)-3-pyridinyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}phenyl)propanoic acid; u) {4-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; v) (4-{5-[3-(aminocarbonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; w) {4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; χ) (4- { 5-[6-(methyloxy)-3 -pyridinyl] - 1 H-pyrrolo[2,3 -b]pyridin-3 -yl }phenyl)acetic acid; y) 3-{4-[5-(3 -Methanesulfonylamino-phenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } -phenyl] - propionic acid; z) {4-[5-(3-Methanesulfonylamino-phenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-acetic acid; aa) 3 - { 4- [5 -(3 -Methanesulfonyl-phenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl] -phenyl } -propionic acid; ab) {4-[5-(3-Methanesulfonyl-phenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-acetic acid; ac) 3-[3-fluoro-4-(methyloxy)phenyl]-5-phenyl-lH-pyrrolo[2,3-b]pyridine; ad) 5-phenyl-3-pyridin-4-yl-lH-pyrrolo[2,3-b]pyridine; ae) 3-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; af) N-[3-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; ag) 4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenylamine; ah) 4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenol; ai) 4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzylamine; aj) 3-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenol; ak) 5-(3,4-dimethoxyphenyl)-3-pyridin-4-yl-lH-pyrrolo[2,3-b]pyridine; al) 4- [5 -(3 ,4-dimethoxyphenyl)- 1 H-pyrrolo [2,3 -b]pyridin-yl] -phenol ; am) 4-[5-(3,4-dimethoxyphenyl)-lH-pyrrolo[2,3-b]pyridin-yl]-phenylamine; an) 4-[5-(3,4-dimethoxyphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; ao) 4-[5-(4-chlorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; ap) N-[4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; aq) 3,5-bis-(4-hydroxyphenyl)-lH-pyrrolo[2,3-b]pyridine; ar) 3,5-bis-(4-carboxyphenyl)-lH-pyrrolo[2,3-b]pyridine; as) 4-[5-(4-aminophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzylamine; at) 4-[5(4-aminophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; au) 4-[5-(2-fluoro-biphen-4-yl)-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; av) N-P-CS-thiophen-S-yl-lH-pyiroloP^-bJpyridin-S-yO-phenylJ-acetamide; aw) 4-(5-thiophen-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; ax) 4-(5 -thiophen-3 -yl- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl)-phenol ; ay) 4-(5-thiophen-3-yl-lH-pyiτolo[2,3-b]pyridin-3-yl)-benzamide; az) N-[3-(5-pyridin-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; ba) 4-(5-pyridin-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; bb) 4-(5-pyridin-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenol; be) 4-(5-thiophen-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzylamine; bd) 3-(lH-indol-5-yl)-5-thiophen-3-yl-lH-pyrrolo[2,3-b]pyridine; be) N- [4-(5 -thiophen-3 -yl- 1 H-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; bf) 5-(3-pyridinyl)-3-(4-pyridinyl)-lH-indole; bg) 4-(5-pyridin-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzamide; bh) 4-[3-(2-fluorobiphenyl-4-yl-lH-pyrrolo[2,3-b]pyridin-5-yl]-benzylamine; bi) 4-(5-pyridin-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenylamine; bj ) {3 - [5 -(4-methanesulfonylphenyl- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl] -phenyl } -acetic acid; bk) N-[3-(3-thiophen-3-yl-l//-pyrrolo[2,3-b]pyridin-5-yl)-phenyl]-acetamide; bl) N-{3-[3-(3-pyridinyl)-lH-pyrrolo[2,3-b]pyridin-5-yl]phenyl}acetamide; bm) 4- [5 -(3 -acetylaminophenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -y 1] -benzoic acid; bn) N-{3-[3-(2,3-difluorophenyl)-lH-pyrrolo[2,3-b]pyridin-5-yl]-phenyl}-acetamide; bo) N-{3-[3-(4-hydroxyphenyl)-lH-pyrrolo[2,3-b]pyridin-5-yl]-phenyl}-acetamide; bp) N- { 3 - [3 -(4-aminomethylpheny I)- 1 H-pyrrolo [2,3 -b] pyridin-5 -yl] -phenyl } -acetamide; bq) N- { 3 -[3 -(4-aminophenyl)- 1 H-pyrrolo [2,3 -b]pyridin-5-yl] -phenyl } -acetamide; br) N- {3-[3-(lH-indol-5-yl)-lH-pyrrolo[2,3-b]pyridin-5-yl]-phenyl} -acetamide; bs) 4-[3-(2-fluorobiphenyl-4-yl)-lH-pyrrolo[2,3-b]pyridin-5-yl]-benzoic acid; bt) N-{3-[3-(4-pyridinyl)-lH-pyrrolo[2,3-b]pyridin-5-yl]phenyl}acetamide; bu) N- { 3 - [5 -(3 -fluorophenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl] -phenyl } -acetamide; by) 4- [5 -(3 -acetylaminophenyl)- 1 H-pyrrolo[2,3-b]pyridin-3-yl]-benzamide; bw) 4-[S(3-fluorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; bx) 4-[S(3-fluorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-phenylamine; by) N- {4- [5 -(3 -fluorophenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl] -phenyl } -acetamide; bz) 4-[5-(3-fluorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-benzamide; ca) 2-chloro-N-[4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-benzamide; cb) 2-phenyl-N-[4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; cc) 2-chloro-N-[3-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzyl] l-benzamide; cd) 2-phenyl-N-[3-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzyl]-acetamide; ce) (4- { 5 - [3 -(methyloxy)pheny 1] - 1 H-pyrrolo [2,3 -b]pyridin-3 -y 1 } phenyl)acetic acid ; cf) 3-[4-(5-{4-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3- yl)phenyl]propanoic acid; cg) [4-(5-{4-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3-yl)phenyl]acetic acid; ch) (4-{5-[3,4,5-tris(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid;

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-[5-(5-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; co) 3-(4-{5-[6-(methyloxy)-2-naphthalenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl}phenyl)propanoic acid; cp) 3-{4-[5-(3,4-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; cq) {4-[5-(3,4-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; cr) {4-[5-(2,3-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; cs) 3 - { 4- [5 -(2,3 -dimethylphenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yljphenyl } propanoic acid;

Ct) {4-[5-(2,3-dichlorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; cu) 3-{4-[5-(2,3-dichlorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; cv) {4-[5-(l-benzothien-3-yl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; cw) [(3 - { 5 - [3 -(methy loxy)pheny 1] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } phenyl )methy 1] amine ; ex) 7-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-l,2,3,4-tetrahydroisoquinoline; cy) 2-fluoro-4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid;

CZ) 2-fluoro-4- { 5 - [3 -(methyloxy)phenyl] - 1 H-pyrrolo [2 ,3 -b]pyridin-3 -yl } benzoic acid; da) 2-methyl-4-{5-[3-(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; db) 5 - [5-(2-naphthalenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl] -2-thiophenecarbaldehyde ; dc) 5-{5-[3-(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}-2-thiophenecarbaldehyde; dd) 2-methyl-4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; de) (4-{5-[6-(methyloxy)-2-naphthalenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; df) 2-fluoro-4-{5-[6-(methyloxy)-2-naphthalenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; dg) 2-fluoro-4-[5-(5-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; dh) 4-[5-(5-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; di) 2-methyl-4-[5-(5-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; dj) 4-[5-(3,4-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; dk) 4-[5-(3,4-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; dl) 4- [5 -(3 ,4-dimethy lphenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl] -2-methylbenzoic acid; dm) 4-[5-(2,3-dichlorophenyl)-lH-pyiτolo[2,3-b]pyridin-3-yl]benzoic acid; dn) 4-[5-(2,3-dichlorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; do) 4-[5-(l-benzothien-3-yl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; dp) 4-[5-(l-benzothien-3-yl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; dq) 4-[5-(l-benzothien-3-yl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; dr) 6-{3-[4-(ethylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5-yl}quinoline; ds) 4-(5-(3-[(methylsulfonyl)amino]phenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; dt) 3-[4-(butyloxy)phenyl]-5-[3-(methylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridine; du) N-(3 - { 3 - [4-(aminomethy l)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-5 - yl}phenyl)methanesulfonamide; dv) N-(3-{3-[3-(aminomethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5- yl}phenyl)methanesulfonamide; dw) 3 -amino-5 -(5 - { 3 - [(methylsulfonyl)amino]phenyl } - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl)benzoic acid; dx) 2-fluoro-4-(5 - { 3 - [(methylsulfonyl)amino]phenyl } - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl)benzoic acid; dy) S-amino-S-IS-tS-CmethylsulfonyOpheny^-lH-pyrroloPjS-^pyridin-S-ylJbenzoic acid; dz) 2-fluoro-4-{5-[3-(methylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; ^ea) [(4- { 5 - [3 -(methy lsulfonyl)phenyl] - 1 H-pyrrolo [2 ,3 -b]pyridin-3 -yl } phenyl)methyl] amine; eb) [(3-{5-[3-(methylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}phenyl)methyl]amine; ec) 5 - { 5 - [3 -(methylsulfonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -y 1 } -2- thiophenecarbaldehyde; ed) 4- { 5 - [3 -(methylsulfonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } benzoic acid; ee) N-(4- { 3 - [4-(buty loxy)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-5 - yl}phenyl)methanesulfonamide; ef) 1,1-dimethylethyl p-CS-IS-tS-CmethylsulfonyOphenylj-lH-pyrroloP^-bJpyridin-S- y 1 } phenyl)ethyl] carbamate; eg) 3-amino-5-(5-{4-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; eh) 2-fluoro-4-(5-{4-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; ei) 4-(5-{4-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; ej) N-(4-{3-[4-(aminomethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5- yl}phenyl)methanesulfonamide; ek) N-(4- { 3 - [3 -(aminomethy l)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-5- yl }phenyl)methanesulfonamide; el) N-{4-[3-(5-formyl-2-thienyl)-lH-pyrrolo[2,3-b]pyridin-5- yl]phenyl}methanesulfonamide; em) 7-{5-[3-(methylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}-l, 2,3,4- tetrahydroisoquinoline; en) N- { 3 -[3 -( 1 ,2,3 ,4-tetrahydro-7-isoquinolinyl)- 1 H-pyrrolo [2,3 -b]pyridin-5- y 1] phenyl } methanesulfonamide ; eo) N-{4-[3-(l,2,3,4-tetrahydro-7-isoquinolinyl)-lH-pyrrolo[2,3-b]pyridin-5- yl]phenyl}methanesulfonamide; ep) 2-methyl-4-{5-[3-(methylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; eq) 5-{5-[3-(methylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}-2-thiophenecarboxylic acid; er) 3-{3-[3-(aminomethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; es) 2-fluoro-4-{5-[6-(methyloxy)-3-pyridinyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; et) 3-[3-(l,2,3,4-tetrahydro-7-isoquinolinyl)-lH-pyrrolo[2,3-b]pyridin-5-yl]benzonitrile; eu) 7-{5-[3,4,5-tris(methyloxy)phenyl]-lH-pyπOlo[2,3-b]pyridin-3-yl}-l,2,3,4- tetrahydroisoquinoline; ev) 4-{5-[3,4,5-tris(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; ew) 2-fluoro-4-{5-[3,4,5-tris(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; ex) 2-amino-4-{5-[3,4,5-tris(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; ey) 4-[5-(6-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; ez) 4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; fa) 4-[5-(2,3-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; fb) 4-[5-(2,3-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; fc) 2-methyl-4-[5-(6-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; fd) 2-methyl-4-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; fe) 4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; ff) 2-methyl-4- { 5 - [6-(methyloxy)-3 -pyridinyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } benzoic acid; fg) 2-methyl-4-{5-[3,4,5-tris(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; fh) 2-(l-methylethyl)-4-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; fi) 4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-(l-methylethyl)benzoic acid; fj) 2-(l-methylethyl)-4-[5-(6-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; fk) 4-[5-(2,3-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; fl) 2-chloro-4- { 5 - [3 -(methylsulfonyl)phenyl] - 1 -H-pyrrolo- [2,3 -b]pyridin-3 -yl } benzoic acid; fm) 4- { 5 - [3 -(methylsulfonyl)phenyl] - 1 -H-pyrrolo- [2,3 -b]pyridin-3 -yl } -2,6- bis(trifluoromethyl)benzoic acid; fn) methyl 2-(azidomethyl)-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoate; fo) 2-ethyl-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fp) 2-(methylamino)-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fq) 2-(dimethylamino)-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fr) 2-cyclopentyl-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fs) 4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-2-propylbenzoic acid; ft) 2,6-difluoro-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fu) 2,6-dimethyl-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fv) 2-(2-propyl)-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fw) 6-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-lH-indazole; fx) 2-(2-methylpropyl)-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fy) 2-methyl-4-(5 -phenyl- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl)benzoic acid; fz) 4-[5-(3-hydroxyphenyl)-l-H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; ga) 3 -amino-5 - [5 -(3 -hydroxyphenyl) 1 H-pyrrolo [2,3 -b] -pyridin-3 -yl] -benzoic acid; gb) {4-[5-hydroxyphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}acetic acid; gc) 4-[5-(3-aminophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; gd) 3-{4-[5-(3-hydroxyphenyl)-l-H-pyrrolo[2,3-b]pyridine-3-yl]-phenyl}-propionic acid; gd) 3-{4-[5-(3-aminophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; gf) {4-[5-(3-aminophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; gg) 4- { 5 - [3 -(aminomethyl)pheny 1] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } benzoic acid; gh) (4-{5-[3-(aminomethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; gi) 4-[5-(3-hydroxyphenyl)-l-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; gj) 2-fluoro-4-[5-(3-hydroxyphenyl)-l-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; gk) 5 -[5 -(3 -hydroxyphenyl)- 1 -H-pyrrolo-[2,3-b]pyridin-3-yl]thiophene-2-carbaldehyde; gl) 3-{3-[3-(2-aminoethyl)phenyl]-l-H-pyrrolo-[2,3-b]pyridin-5-yl}phenol; gm) 3-[3-(l,2,3,4-tetrahydroisoquinolin-7-yl)-l-H-pyrrolo-[2,3-b]pyridin-5-yl]phenol; gn) 3-amino-5-[5-(3-aminophenyl)-l-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; go) 4-[5-(3-aminophenyl)-l-H-pyrτolo-[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; gp) 2-fluoro-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)phenol; gq) 2,6-difluoro-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)phenol; gr) 5-phenyl-3-[4-(lH-tetrazol-5-yl)phenyl]-lH-pyrrolo[2,3-b]pyridine; gs) 5-(2-naphthalenyl)-3-[4-(lH-tetrazol-5-yl)phenyl]-lH-pyrrolo[2,3-b]pyridine; gt) 3-[3-fluoro-4-(lH-tetrazol-5-yl)phenyl]-5-phenyl-lH-pyrrolo[2,3-b]pyridine; gu) 2-methyl-2-(4- { 5 - [3 -(methylsulfonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 - yl}phenyl)propanoic acid; gv) 2-{4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; gw) 2-(4- { 5 - [3 -(aminocarbonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } phenyl)-2- methylpropanoic acid; gx) 2-{4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}-2-methylpropanoic acid; gy) 2-methyl-2-{4-[5-(6-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; gz) 2-methyl-2-{4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; ha) 2-methyl-2- [4-(5 - { 3 - [(methylsulfonyl)amino]phenyl } - 1 H-pyrrolo [2,3 -b]pyridin-3 - yl)phenyl]propanoic acid; hb) 2-methyl-2-[4-(5-{4-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3- yl)phenyl]propanoic acid; he) 2-methyl-2-(4-{5-[6-(methyloxy)-2-naphthalenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl}phenyl)propanoic acid; hd) 2-methyl-2-{4-[5-(5-quinolinyl)-lH-pyπOlo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; he) 2-methyl-2-{4-[5-(3-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; hf) 2-{4-[5-(6-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; hg) 2-{4-[5-(5-quinolinyl)-lH-pyπOlo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; hh) 2-{4-[5-(3-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; hi) 2-(4- { 5 - [3 -(methy lsulfonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } pheny l)propanoic acid; hj) 2-(4-{5-[6-(methyloxy)-2-naphthalenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl}phenyl)propanoic acid; hk) 2-methyl-2-[4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)phenyl]propanoic acid; hi) 2-methyl-2-{4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; hm) 4-[5-(6-amino-3-pyridinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; hn) 4-{5-[6-(β-alanylamino)-3-pyridinyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; ho) 4-(5-(6-indolyl)-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; hp) [2-(3 - { 5 - [3 -(methylsulfonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } pheny l)ethyl] amine; hq) N-(3-{3-[3-(2-aminoethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5- yl}phenyl)methanesulfonamide; hr) N-(4- { 3 - [3 -(2-aminoethy l)pheny 1] - 1 H-pyrrolo [2,3 -b]pyridin-5 - yl}phenyl)methanesulfonamide; hs) 4- { 5- [3 -(2-aminoethyl)phenyl]- 1 -H-pyrrolo- [2,3 -b]pyridin-3-yl } benzoic acid; ht) 4-{5-[3-(2-aminoethyl)phenyl]-l-H-pyrrolo-[2,3-b]pyridin-3-yl}-2-methylbenzoic acid; hu) 4-{5-[3-({ [2-(dimethylamino)ethyl]amino)carbonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 - yl} benzoic acid; hv) 4-(5-(3-[4-(l,l-dimethylethyloxycarbonyl)aminobutanoyl]amino)phenyl-lH-pyrrolo[2,3- b]pyridin-3-yl)benzoic acid; hw) 4-(5 -(3 - [3 -( 1 , 1 -dimethyl ethy loxycarbony l)aminopropanoy 1] amino)phenyl- 1 H- pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; hx) 2-(2-propyl)-4-[5-(3-[3-(l,l-dimethylethyloxycarbonyl)aminopropanoyl]amino)phenyl- lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; hy) 4-{5-[3-(β-alanylamino)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; hz) 4-(5-{3-[(4-aminobutanoyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; ia) 4-{5-[3-(beta-alanylamino)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}-2-methylbenzoic acid; ib) 4- { 5 - [3 -(beta-alanylamino)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } -2-( 1 - methylethyl)benzoic acid; ic) 4- [5 -(3 - { [(2-aminoethyl)amino] carbonyl } phenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yljbenzoic acid; id) 4- [5 -(3 - { [(3 -aminopropy l)amino] carbonyl } phenyl)- 1 H-pyrrolo [2,3 -b] pyridin-3 - yl]benzoic acid; ie) 3-amino-5-[5-(3-aminophenyl)-l-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; if) 3-amino-5-{5-[3-(aminomethyl)phenyl]-l-H-pyrrolo-[2,3-b]pyridin-3-yl}benzoic acid; ig) 4- { 5 - [3 -(aminomethyl)phenyl] - 1 -H-pyrrolo- [2,3 -b]pyridin-3 -yl } -2-fluorobenzoic acid; ih) 2-(aminomethyl)-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; ii) 3-{3-[3-(2-aminoethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; and

U) 4-[5-(3-amino-l-isoquinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; or a pharmaceutically acceptable salt thereof.

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 IC₅₀ values of less than 1.5 μM. Given the relatedness between SGK-I and SGK-2 it can be assumed that these compounds exhibit an inhibitory activity to SGK-2, too. In particular, 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 IC₅₀ values for SGK-I and SGK-2 of 62 nM and 103 nM, respectively.

Thus, in a particularly preferred embodiment of the present invention, 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.

In a preferred embodiment of the present invention, the protein kinase is PKCζ and the small molecule is a compound of formula (II) or a pharmaceutically acceptable salt thereof:

(H) wherein R₅ is aryl or heteroaryl, optionally substituted once, twice or three times. The term "optionally substituted" has the meaning as defined above in the section "Definitions".

Various 2-(6-phenyl-lH-indazol-3-yl)-lH-benzo[d]imidazoles and their inhibitory activity on PKC-ζ were described by Trujillo and co-workers (Trujillo et al. (2009) Bioorg. Med. Chem. Lett. 19(3):908-l l; electronic publication: 2008 Dec 6). The compounds displayed IC₅₀ values between 5.18 nM and 31,100 nM (= 31.1 μM).

In a preferred embodiment, R₅ 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".

In a more preferred embodiments, R₅ is

(U) wherein

R₆ is hydrogen, -OH, or -NH₂; preferably -OH or -NH₂; most preferably-NH₂; R₇ is hydrogen, methoxy, or F; preferably hydrogen or F, most preferably hydrogen: R₈ is hydrogen or methoxy; preferably hydrogen.

In a particularly preferred embodiment, R₆ is -NH₂, R₇ is hydrogen and R₈ is hydrogen.

In a preferred embodiment of the present invention, the protein kinase is PKCζ and the small molecule is a compound of formula (III) or a pharmaceutically acceptable salt thereof:

(III) wherein

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;

R₉ is hydrogen or C₁-C₅ alkyl (e.g. methyl, ethyl, /7-propyl, iso-propy\, «-butyl, sec-butyl, tert- butyl, pentyl) or C₃-C₅ cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl); preferably hydrogen or Ci-C₃ alkyl or C₃ cycloalkyl; and

Ri₀ is hydrogen or CpC₅ alkyl (e.g. methyl, ethyl, /7-propyl, /.sø-propyl, n-butyl, sec-butyl, tert- butyl, pentyl) or C₃-C₅ cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl); preferably hydrogen or Ci-C₃ alkyl or C₃ cycloalkyl.

The term "optionally substituted" used above with regard to Ring(A) and Ring(B) has the meaning as defined above in the section "Definitions".

In US 2007/0191420 Al the synthesis of 44 compounds falling within the general formula (III) shown above was described and the inhibitory activity on different isoforms of protein kinase C (PKC) was examined. Several of these compounds exhibit an IC₅₀ value for the inhibition of PKCζ of 70 μM or less.

Thus, in preferred embodiments, Ring(A) is

(V) (W).

In preferred embodiments, Ring(B) is

(X) (y) (Z)

(aa)

In preferred embodiments, 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).

In preferred embodiments, 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).

In preferred embodiments, one of R₉ and Ri₀ is hydrogen and the other one is selected from the group consisting of hydrogen, Ci-C₅ alkyl (e.g. methyl, ethyl, ^-propyl, wo-propyl, n- butyl, sec-butyl,

pentyl), and C₃-C₅ cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl). For example, R₉ may be hydrogen and Ri₀ is selected from the group consisting of hydrogen, Ci-C₅ alkyl and C₃-C₅ cycloalkyl. Preferably, one of R₉ and Ri₀ is hydrogen and the other one is selected from the group consisting of hydrogen, C)-C₃ alkyl (e.g. methyl, ethyl, n- propyl, /so-propyl) and C₃ cycloalkyl (i.e. cyclopropyl).

In a particularly preferred embodiment of the present invention, the protein kinase is PKCζ and the small molecule is a compound selected from the group consisting of: (1) trans-4-aminomethyl-cyclohexanecarboxylic acid phenylamide (IC₅₀ = 40.3 μM);

(2) (+)-trans-N-(4-pyridyl)-4-(l-aminoethyl)-cyclohexanecarboxamide (IC₅₀ = 37.68 μM);

(3) trans-4-aminomethyl-cyclohexanecarboxylic acid pyridine-4-ylamide (IC₅₀ = 24.33 μM);

(4) 4-aminomethyl-N-pyridin-4-yl-benzamide (IC₅₀ = 51.40 μM);

(5) 5-(l-amino-ethyl)-thiophene-2-carboxylic acid pyridin-4-ylamide (IC₅₀ = 45.9 μM); (6) 4-(l-amino-ethyl)-N-pyridin-4-yl-benzamide; (IC₅₀ = 22.14 μM);

(7) 4-(l-amino-propyl)-N-pyridin-4-yl-benzamide; (IC₅₀ = 25.4 μM); (8) 4-(l-amino-cyclopropyl-methyl)-N-pyridin-4-yl-benzamide; (IC₅₀ = 41.5 μM);

(9) 4-(l-amino-ethyl)-naphthalene-l-carboxylic acid pyridin-4-yl-amide; (IC₅₀ = 6.4 μM); and

(10) 4-(l-amino-cyclopentyl-methyl)-N-pyridin-4-yl-benzamide; (IC₅₀ = 70 μM); or a pharmaceutically acceptable salt of (1) to (10), in particular a hydrochloric acid addition salt.

A large number of protozoal pathogens is known, which require during their life cycle in their host, e.g. a human, to attach to and/or enter receptor cells expressing 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. Preferably 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. In a particular preferred embodiment of the invention the protozoa is a member of the family of plasmodiidae, preferably Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium semiovale and Plasmodium knowlesi. In the most preferred use of the invention, 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. In particular, the present invention relates to a compound as defined in the first aspect for use in therapy and/or prophylaxis of a protozoal infection. Preferably, said protozoal infection is malaria.

In a second aspect, 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%.

In preferred embodiments of the second aspect, the protein kinase is selected from the group consisting of: (a) protein kinase C zeta (PKCζ); (b) Serine/threonine-protein kinase WNKl (PRKWNKl); (c) Serine/threonine-protein kinase Sgk2 (SGK2); and (d) Serine/threonine-protein kinase 35 (STK35).

In a preferred embodiment of the second aspect, the method further comprises the step of formulating the test compound selected in step (iv) with pharmaceutically acceptable additives and/or auxiliary substances. In a third aspect, 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. Preferably said infectious disease is malaria.

In a fourth aspect, 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. Preferably said infectious disease is malaria.

The increasing speed of the development of resistance of protozoal pathogens, in particular malaria, to the various medicaments used to treat these diseases makes it necessary to efficiently eradicate the pathogen without giving it a chance to develop a resistance. To that end it is desirable to interfere with distinct pathways required for the lifecycle of a given pathogen. Since the present inventors could demonstrate that inhibitors of host cell protein kinases can interfere with development/proliferation of malaria, the compounds according to the present invention present a hitherto unknown route of attack on pathogens, in particular pathogens causing malaria, which can beneficially be combined with the known treatments of malaria.

Accordingly, in a fifth aspect the present invention is directed to 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. Accordingly, 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. Preferably, the two medicaments are administered simultaneously, e.g. combined in one administration form. Alternatively, the two medicaments in said pharmaceutical compositions may be administered subsequently in separate administration forms.

In a sixth aspect, 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. Preferably, the protein kinase is selected from the group consisting of: (a) protein kinase C zeta (PKCζ); (b) Serine/threonine-protein kinase WNKl (PRKWNKl); (c) Serine/threonine-protein kinase Sgk2 (SGK2); and (d) Serine/threonine-protein kinase 35 (STK35). It is further preferred that 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.

EXAMPLES

In the following, the invention is explained in more detail by non-limiting examples:

Example 1: Kinome-wide RNAi screen implicates at least 5 host kinases in Plasmodium infection of human hepatoma cells

Materials and Methods Cells, Mice and Parasites

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%

CO₂.

Mouse primary hepatocytes were obtained as previously described (Goncalves et al,

2007). Briefly, they 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 μg/ml transferrin, 1 μg/ml insulin and 3.5 μM hydrocortisone in 24 well plates coated with 0.2% gelatine in PBS. Cells were maintained in culture at 37°C and 5% CO₂. C57BL/6 mice, were bred in the pathogen-free facilities of the Instituto de Gulbenkian de

Ciencia (IGC) and housed in the pathogen-free facilities of the Instituto de Medicina Molecular

(IMM). All protocols were approved by the Animal Care Committees of both Institutes.

Green fluorescent protein (GFP) expressing P. berghei (parasite line 259cl2) sporozoites

(Franke-Fayard et al, 2004) were obtained from dissection of infected female Anopheles stephensi mosquito salivary glands.

siRNA design, siRNA library and screening controls

All siRNAs were purchased from Ambion's Silencer genome wide library

(Ambion/ Applied Biosystems, Austin USA). Each gene was targeted by using distinct siRNAs used individually in all cases. Negative control samples included untransfected cells and cells transfected with a negative control siRNA not targeting any annotated genes in the human genome.

High-throughput siRNA screening of Plasmodium infection

Huh7 cells (4500 per well) were seeded in 100 μl complete RPMI medium in optical 96- well plates (Costar) and incubated at 37°C in 5% CO₂. 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 80μl of serum- free culture medium without antibiotics. One μl of 10 μM siRNA diluted in 16 μl of Opti-MEM (Invitrogen) was complexed with 0.4 μl Oligofectamine (Invitrogen) diluted with 2.6 μl Opti- MEM and added onto the cells following the manufacturer's protocol. Four hours after addition of the complex, 50 μl of fresh RPMI medium, supplemented with 30% FCS, 3% pen/strep, 3% non-essential amino acid, 3% glutamine and 3% HEPES were added to the cells. Two days after siRNA transfection, cells were infected with 10⁴ P. berghei sporozoites/well. 24 h after infection, cells were fixed with 4% paraformaldehyde (PFA) in PBS and permeabilized with 0.2% saponin in PBS. 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).

Automated image acquisition and analysis

Plates were acquired with a Discovery 1 automated fluorescence microscope (Molecular Devices Corporation, CA, USA) using a 1Ox lens. In each well, cell nuclei, actin and EEFs were imaged in 9 fields covering a total area of 2.7 x 2.0 mm. Image data was analyzed using a custom MetaMorph (Molecular Devices Corporation, CA, USA) based algorithm extracting the following values for each imaged field: Cell proliferation as measured by the number of nuclei per imaged field (Hoechst staining), cell confluency as measured by the percentage of the imaged field covered by actin staining and number of EEFs as number of compact, high contrast objects in a size range from 16 to 150 pixels. Within each field, the number of EEFs was normalized to the cell confluency. Normalized EEF numbers and number of nuclei were averaged between the 9 imaged fields within each well. Mean and standard deviations were calculated for each experimental triplicate.

Gene-specific expression and infection quantification by qRT-PCR For gene-specific expression in vitro, total RNA was isolated from Huh7 cells 48 h post- transfection (Invitek Invisorb 96-well plate kit) and converted into cDNA (ABI's HighCapacity cDNA reagents) with random hexamers, following the manufacturer's recommendations. qRT- PCR used the SybrGreen method with Quantace qPCR mastermix at 1 1 μl 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). For infection determination in vivo or ex vivo, 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). Livers were collected and homogenized in denaturing solution (4 M guanidine thiocyanate; 25 mM sodium citrate pH 7, 0.5 % sarcosyl and 0.7 % β-Mercaptoethanol in DEPC-treated water), 40 h after sporozoite injection. Total RNA was extracted using Qiagen's RNeasy Mini kit, following the manufacturer's instructions. RNA for infection measurements was converted into cDNA using Roche's Transcriptor First Strand cDNA Synthesis kit, according to the manufacturer's protocol. 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 μl, containing 0.8 pmoles/μl or 0.16 pmoles/μl 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. 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. For PKCζ mRNA level determination by qRT-PCT, PKCζ- specific primers were used (RT2 qPCR Primer Assay for Mouse Prkcz, SuperArray Bioscience Corporation).

Statistical analysis

For samples in which n > 5, statistical analyses were performed using unpaired Student t or ANOVA parametric tests. Normal distributions were confirmed using the Kolmogorov- Smirnov test. For samples in which n < 5, statistical analyses were performed using Kruskall- Wallis or Wilcoxon non-parametric tests, p < 0.05 was considered significant, p < 0.001 was considered highly significant. The same statistical tests were also used in Examples 2 to 4.

Results

Systematic 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. Cells were fixed 24 h after infection and immuno-stained to detect intracellular parasites (EEFs), as well as host cell nuclei and actin to estimate cell numbers and confluency, respectively. Following image acquisition, 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. To this end, the infection rate in each experimental well was calculated as a percentage of the mean infection rate from all experimental wells on that plate. In order to assess possible siRNA effects on cell proliferation, infection rate data were plotted against the number of nuclei, also expressed as a percentage of the mean number of nuclei for that plate.

The 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).

Sporozoite infection assays inevitably have considerable levels of variation, a problem that cannot currently be overcome and which exacerbates difficulties generally associated with siRNA screens. In order to reduce the risk of reporting false positives, a multi-step screening system was devised in which candidate genes were subjected to three screening passes with increasingly stringent selection criteria (Figure 2A). In the first pass, the 727 selected genes were screened by targeting each with three distinct siRNAs used individually. In order to minimize the number of false negative results, 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. As a result, 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. In this round of analysis, 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. To minimize the risk of false positives due to siRNA sequence-dependent off-target effects, 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). Furthermore, genes for which different siRNAs yielded conflicting phenotypic results were also excluded from further analysis. In order to further minimize any bias due to experimental variability, all pass 2 siRNAs were assayed in two independent experiments, and were selected for follow-up only if the criteria were met in both experiments (Figure 2D). It is worthwhile noting that while in Pass 1 only 3.6 % of the siRNAs met the selection threshold, 18.4% of the siRNAs tested for the first time in Pass 2 met similar criteria, while the distribution of infection levels in controls is not statistically different between pass 1 and pass 2 experiments, showing that a 5 -fold enrichment has taken place from Pass 1 to Pass 2.

The 16 genes thus selected for further verification in pass 3 were targeted with the siRNAs yielding the strongest phenotypes in the second pass. This third pass was used to further restrict our selection to those genes showing clearest functionality, i.e. those with at least two siRNAs yielding infection rates more than 3 s.d. above or below the mean of all the negative controls in the assay, respectively (Figure 2E). Secondly, target mRNA knock-down levels attained for these genes were also assessed in this pass by qRT-PCR. This allowed the selection of positive hit candidates to be refined further yet by excluding genes for which a correlation between phenotypic severity and decreased mRNA levels could not be confirmed (Figure 2F).

Based on these data, the following 5 genes have emerged from the above screen as the clearest and strongest positive hits, showing RNAi-induced loss-of-function phenotypes with specific, reproducible and marked effects on P. berghei infection rates in the Huh7-based assay: MET (hepatocyte growth factor receptor), PKCζ (PKCzeta), PRKWNKl, SGK2 and STK35. Knock-down of the expression of these genes did not lead to any significant effects in terms of cell proliferation or morphology. It should also be noted that the present data do not rule out the possible involvement of other genes among those tested here, since negative results in 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: PKCζ inhibition leads to a decrease in host cell infection by Plasmodium sporozoites

In order to further characterize the functionalities identified in the cell-based infection model described in Example 1 and to validate their relevance both from a physiological point of view and in terms of human malaria, detailed follow-up studies for all 5 of the top hits from the above RNAi screen were initiated. In the following, results for PKCζ are presented, the first of these to be prioritized due to its role in several liver pathological processes (Duran et al, 2004; McConkey et al, 2004). PKCζ is part of the large family of PKCs, which has been implicated in a wide range of cellular processes. PKC isotypes include 10-15 members, divided into 4 groups (Mellor and Parker, 1998). One of these groups, known as the atypical PKCs (aPKCs) (Moscat and Diaz-Meco, 2000), comprises the PKCζ (Ono et al, 1989) and PKCλA (PKC lambda/iota ) (Akimoto et al, 1994) isoforms. The aPKCs have been implicated in numerous processes, including cell growth and survival, regulation of NF-κB (NF-kappaB) activation and polarity (reviewed in (Moscat et al, 2006; Suzuki and Ohno, 2006; Moscat and Diaz-Meco, 2000)).

All 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).

Materials and Methods Inhibition of PKCζ was carried out by incubation of the cells with a myristoylated PKCζ peptide (myr-SI YRRG ARR WRKL YRAN, SEQ ID NO: 2), whose sequence corresponds to that of a pseudosubstrate inhibitor of the enzyme. A myristoylated scrambled peptide (myr- RLRYRNKRIWRSAYAGR, SEQ ID NO: 3) was used as a control in these experiments.

In order to determine the specificity of the pseudosubstrate inhibitor, 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 Na₃VO₄; complete EDTA-free protease inhibitor cocktail). After migration on a 10% Tris- glycine gel, proteins were transferred to a nitrocellulose membrane (BIO-RAD), which was probed with anti-phospho-PKC (pan) (βll Ser660) (Cell Signaling Technology) or anti-phospho- aPKC (Thr555/PKCι; Thr560/PKCζ) (Upstate) plus HRP-conjugated anti-rabbit (Amersham). The membrane was developed with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

To further evaluate the specificity of the pseudosubstrate inhibitor towards PKCζ versus PKCi, Huh7 cells were transfected (Lipofectamine 2000, Invitrogen) with plasmids encoding

GFP-PKCζ 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-PKCζ and GST-PKCi were determined by probing the membrane with anti-aPKCζ (C20, Santa Cruz Biotechnology), which recognizes the two isoenzymes. The % of inhibition of PKCζ versus PKCi was calculated from the anti-phospho-aPKC signals. All signals were normalized to those of actin.

Results

The cell-based assay described above in Example 1 was used to test the effects of a myristoylated PKCζ pseudosubstrate (myr-SIYRRGARRWRKLYRAN, SEQ ID NO: 2), previously characterized as a specific PKCζ inhibitor (PKCζlnh) (Laudanna et al., 1998; Standaert et al., 1997), on P. berghei infection. A scrambled myristolated peptide was used as control in all PKCζ inhibition experiments (Laudanna et al, 1998). Treatment of cells with PKCζlnh had no obvious effects on nuclear or cell morphology and as well as on the cell number and confluency (Figure 3A-C), as observed for cells transfected with siRNA oligonucleotides targeting PKCζ (Example 1). Still, treatment of cells with PKCζlnh had a significant effect in the level of cell infection by P. berghei sporozoites, as quantified using qRT-PCR-based measurements of Plasmodium 18S rRNA levels found within Huh7 cells (Figure 3D) and mouse primary hepatocyte extracts (Figure 3E) harvested 24 and 48 h after P. berghei sporozoite addition, respectively. These results show that a 20 μM concentration of PKCζlnh leads to a -80% and -60% reduction in P. berghei infection rates in Huh7 hepatoma cells and primary hepatocytes, respectively (Figure 3D and E; p < 0.01 and p < 0.05), offering a RNAi- independent confirmation of the findings on the role of PKCζ in P. berghei infection.

Example 3: Inhibition of PKCζ impairs invasion of host cells by Plasmodium sporozoites

In order to gain a better insight on the possible role of PKCζ in the infection process, the effects of PKCζlnh on different periods of hepatocyte infection were examined by fluorescence activated cell sorting (FACS) analysis of host cells infected with GFP-expressing P. berghei parasites, measuring the proportion of GFP⁺ cells (Prudencio et al, 2007). Indeed, FACS analysis of cells infected with GFP-expressing parasites enables discerning whether the observed effect on infection is due to a decrease in the number of infected cells or to an impairment of Plasmodium development inside host cells (Prudencio et al , 2007).

Materials and Methods

Green fluorescent protein (GFP) expressing P. berghei (parasite line 259cl2) sporozoites (Franke-Fayard et al, 2004) 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).

For PKCζ mRNA level determination by qRT-PCT, PKCζ-specific primers were used (RT2 qPCR Primer Assay for Mouse Prkcz, SuperArray Bioscience Corporation).

Results Treatment of Huh7 cells with PKCζlnh 1 h prior to addition of GFP-expressing P. berghei sporozoites led to a marked, dose-dependent decrease in infection rate, as measured by the proportion of infected cells relative to control samples 24 h after sporozoite addition (Figure 4A; p < 0.05 for PKCζlnh > 5μM). Treatment with PKCζlnh did not affect Plasmodium development, as shown by the similar GFP intensities of treated and control cells (Figure 4B). Next, we sought to determine whether the decrease in the number of infected cells observed at 24 h after sporozoite addition was due to a decrease in invasion rate or to the disappearance of infected cells throughout infection. Since, in the infection assay employed, >95% of invasion events are known to take place within the first 2 h after sporozoite addition (Prudencio et al, 2007), any effects on invasion can be quantified by analyzing cells at this timepoint. As shown in Figure 4C, the effect of PKCζlnh in cells analyzed 2 h after sporozoite addition is closely comparable to that seen with the full 24 h treatment, indicating that PKCζ likely plays a role during host cell invasion by P. berghei sporozoites (p < 0.05 for PKCζlnh > 5μM). In addition, when PKCζlnh was added 2 h after sporozoite addition, no significant effect was observed in infection rate measured at 24 h (Figure 4D), not only showing that the effect observed on the early steps of infection is not due to PKCζlnh toxicity to host cells but also strengthening the notion that PKCζ influences Plasmodium infection by playing a role during cell invasion. Importantly, infection rates were not affected by pre-incubation of Plasmodium sporozoites with PKCζlnh for 1 hour prior to their addition to hepatoma cells, showing that PKCζlnh has no direct effect on sporozoite viability (Figure 4E). Further confirmation of the involvement of PKCζ in sporozoite invasion of Huh7 cells, but not on the parasite's intracellular development was obtained by employing qRT-PCR to quantify infection 24 h after infection of cells incubated with PKCζlnh either during the invasion or the development periods, exclusively (Figure 4F). These results show that a marked decrease in intracellular parasite numbers is observed when cells are incubated with the inhibitor during the first 2 hours after sporozoite addition (p < 0.001), whereas no effect is observed when the compound is added after invasion is completed. Together, these data confirm the physiological relevance of PKCζ, identified in the RNAi screen, and show that the latter plays a role during the invasion of hepatoma cells by P. berghei sporozoites.

Example 4: PKCζ knock-down in mouse livers confirms the physiological relevance of PKCζ role in malaria infection in vivo

Furthermore, the in vivo physiological relevance of the above cell-based findings was tested more thoroughly by using systemically-delivered, liposome-formulated siRNAs designed to specifically silence PKCζ expression in adult mice, and infecting these with P. berghei sporozoites. In vivo RNAi treatments using the same systemic administration of siRNAs including the same liposome formulation used here have previously been shown to yield potent gene-specific knock-downs in adult mice without major toxicity, nor any detectable disruption of the endogenous microRNA pathway (Akinc et al, 2008; Rodrigues et al, 2008; John et al, 2007).

Materials and Methods In vivo RNAi

C57B1/6 mice (male, 6-8 weeks) were treated with a single intravenous (i.v.) administration of 5 mg/kg of siRNA formulated in liposomal nanoparticles (Alnylam). Three different modified siRNAs targeting PKCζ were used: 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

2'OMe nucleotides, dT refers to deoxythymidine, and "s" represents phosphorothioate linkage.

36 h after siRNA administration mice were infected by i.v. injection of 2 x 10⁴ P. berghei sporozoites. Remaining PKCζ 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.

Infection of mice treated with one PKCζ siRNA was allowed to proceed onto the blood stage and parasitemia (% of infected red blood cells) was measured daily. The PKCζ protein level in the liver of siRNA-treated mice was determined by Western blot.

Quantification of host PKCC protein expression in the liver

PKCζ protein level in the liver of mice treated with a PKCζ siRNA was quantified by Western blot using the primary antibody (rabbit anti-PKCζ (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).

Results

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. In order to address the risk of sequence-dependent off-target effects, three distinct siRNA sequences targeting PKCζ 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. Under these conditions, no toxicity was observed and PKCζ expression was reduced in adult mouse livers when using each of the 3 distinct PKCζ-specific siRNAs, yielding an average of -56-73% remaining PKCζ mRNA, as measured by qRT-PCR of liver extracts taken 76 h after siRNA treatment, relative to the controls (Figure 5A; p < 0.05). This silencing was accompanied, for all three PKCζ-specific siRNAs, by significant reductions in liver infection, yielding an average per siRNA of -9-40% of control infection loads, as measured by qRT-PCR of P. berghei 18S rRNA in liver extracts taken 76 h after siRNA treatment, as described above (Figure 5A, p < 0.05). The reductions in liver infection load showed a broad correlation with the level of PKCζ silencing achieved by the siRNAs with siRNA #1, which leads to the most significant reduction in PKCζ, showing the most striking difference in infection (Figure 5 A; p < 0.01). In a further, parallel experiment, semi-quantitative Western blotting analysis of liver extracts taken 76 h after siRNA treatment, from mice injected with the PKCζ siRNA yielding the strongest reduction in liver infection, confirmed that PKCζ expression was significantly reduced at the protein level in these mice (-55%; p < 0.01). Additionally, another 3 independent groups of mice treated with the same 3 distinct PKCζ siRNAs showed a decrease in blood parasitaemia (percentage of infected erythrocytes), relative to control mice (Figure 5B). In fact, while by day 4 after sporozoite injection all 5 mice in the control group were positive for blood stages, none of the 6 mice in the group pre-treated with the strongest PKCζ-specific siRNA were (Figure 5B; p < 0.001). Although less striking, both other siRNAs also led to a delay in the appearance of parasites in the blood and siRNA #2 led to a significant reduction in average blood parasitaemia (Figure 5B; p < 0.05). Together, these data demonstrate that PKCζ is a physiologically important host factor needed for the liver stage of Plasmodium infection both in cultured cells in vitro and in animals in vivo.

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SEQUENCE LISTING - FREE TEXT INFORMATION SEQ ID NO: 1 : Inhibitor of PKC zeta, non-myristoylated

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: 9: 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: 22 siRNA# 1 targeting WNKl , sense 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, antisense strand

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, antisense strand

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

Claims

1. 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 (PKCζ);

(b) Serine/threonine-protein kinase WNK 1 (PRKWNK 1 );

(c) Serine/threonine-protein kinase Sgk2 (SGK2); and

(d) Serine/threonine-protein kinase 35 (STK35).

2. The use according to claim 1, wherein the inhibitor of the protein kinase is a small interfering RNA (siRNA) capable of inhibiting expression of said protein kinase.

3. The use according to claim 2, wherein the protein kinase is (a) PKCζ and the siRNA is a duplex comprising: 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: 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; (c) SGK2 and the siRNA is a duplex comprising: 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

(d) STK35 and the siRNA is a duplex comprising: 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.

4. The use according to claim 1 , wherein the inhibitor of the protein kinase is an antibody specifically binding to said protein kinase.

5. The use according to claim 1, wherein the inhibitor is a peptide.

6. The use of claim 5, wherein the protein kinase is PKCζ and the peptide is selected from

(a) SIYRRGARRWRKLYRAN (SEQ ID NO: 1); or

(b) a peptide comprising 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).

7. The use of claim 6, wherein the peptide is acylated and/or miristoylated at the N-terminal amino acid.

8. The use according to claim 1, wherein the inhibitor of the protein kinase is a small molecule.

9. The use according to claim 8, wherein the protein kinase is SGK2 and the small molecule is a compound of formula (I) or a pharmaceutically acceptable salt thereof

(I) wherein R₃ is

wherein

X and Y are each independently CR^la or N;

Z is NR, O or S;

A, B, D, and R^la are each independently hydrogen; OR; CN; halogen; CO₂R; CONRiR₂;

NRiR₂; NR₃R₄; aryl; heteroaryl; (Ci_-3)alkyl-NRiR₂; (C,_-6)alkyl, or (Ci_-6)haloalkyl; wherein R_b is

(P)

wherein

M is independently hydrogen; (Ci_-3)alkyl-NRiR₂; (Ci_-3)alkyl-OR; halogen; CO₂R; OR; NRiR₂; (COaIlCyI-NR₃R₄; CONR,R₂; (C_1-6)alkyl-CONR,R₂; CHO; (C,. 6)alkylCO₂R; (Ci_-6)alkyl; Or NR₃R₄;

M' is independently hydrogen; (Ci_-3)alkyl-NRiR₂; (Ci_-3)alkyl-OR; halogen; CO₂R; OR; NR₁R₂; (C,_-3)alkyl-NR₃R4; CONR₁R₂; (Ci_-6)alkyl-CONR,R₂; CHO; (C_1- ₆)alkylCO₂R; NR₃R₄; (C_1-6)alkyl; or phenyl;

P, Q, T, U, V and W are each independently hydrogen; halogen; (C_1-6)alkyl; (C_1-3)alkylOR; (C_1-6)haloalkyl; CO₂R; CHO; (C_1-6)alkyl-CO₂R; (C_1-3)alkyl-NR₁R₂; OR; NR₁R₂; NR₃R₄; CONRiR₂; (C_1-6)alkyl-CONRiR₂; aryl; or heteroaryl;

M" and M'" are independently at each occurrence hydrogen; (C_1-3)alkylaryl; (C₁. 3)alkylheteroaryl; (C_1-6)alkyl; or (Ci_-6)haloalkyl;

Ri and R₂ are independently at each occurrence hydrogen; (C_1-6)alkyl; (C_1-3)alkylNRR⁽; (Ci_-3)alkylOR; (Ci_-6)cyanoalkyl; NRR'; (Ci_-3)alkylaryl; (Ci_-3)alkylheteroaryl; (C_1- ₆)haloalkyl; or together with the nitrogen that they are attached to form a 4, 5, 6, or 7 member non-aromatic ring, said ring optionally containing up to 2 additional heteroatoms selected from the group consisting of NR; O; or S(O)_n; and said ring is unsubstituted or substituted with from 1-3 substituents selected from the group consisting of halogen; (Ci_-6)alkyl; OR; NRR'; CN; halogen; (Ci^haloalkyl; phenyl; heteroaryl and heterocyclyl; n is independently at each occurrence 0, 1 or 2;

R₃ is independently at each occurrence hydrogen; (Ci_-6)alkyl; or (Ci_-6)haloalkyl; R₄ is C(=O)(C,.₆)alkyl; C(=O)(C_1-3)alkyl-NRR'; or C(=O)— (C,_-3)alkylaryl wherein said aryl is unsubstituted or substituted with 1-3 substituents selected from the group consisting of halogen, (Ci_-3)alkyl and (Ci_-3)alkoxy); C(=O) — (Ci_-3)alkylheteroaryl; C(=O)phenyl wherein the phenyl group is unsubstituted or substituted with 1-3 substituents selected from the group consisting of halogen, (Ci_-6)alkyl and OR.

10. The use according to claim 8 or 9, wherein 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}propanoic acid; f) {3-[5-(2-naphthyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}methanol; g) 4- {5-[3-(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl} benzoic acid; h) 3 -(4- { 5 - [3 -(methyloxy)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } phenyl)propanoic acid; i) 3-{3-[4-(aminomethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; j) 4- { 5- [3-(aminocarbonyl)phenyl]- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } benzoic acid; k) 4- [5 -(3 -cyanophenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl] benzoic acid; 1) 4-{5-[6-(methyloxy)-3-pyridinyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; m) 3-{3-[3-(aminomethyl)phenyl]-l-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; n) 4-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; o) 2-fluoro-4-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; p) 3-amino-5-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; q) 3-{4-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; r) 3-(4-{5-[3-(aminocarbonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl}phenyl)propanoic acid; s) 3-{4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; t) 3 -(4- { 5 - [6-(methyloxy)-3 -pyridinyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 - yl}phenyl)propanoic acid; u) {4-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; v) (4- { 5 - [3 -(aminocarbonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } phenyl)acetic acid; w) {4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; x) (4- { 5- [6-(methyloxy)-3 -pyridinyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } pheny l)acetic acid; y) 3-{4-[5-(3-Methanesulfonylamino-phenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl}- phenyl] -propionic acid; z) {4-[5-(3-Methanesulfonylamino-phenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}- acetic acid; aa) 3-{4-[5-(3-Methanesulfonyl-phenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}- propionic acid; ab) { 4- [5 -(3 -Methanesulfony 1-phenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl] -phenyl } -acetic acid; ac) 3-[3-fluoro-4-(methyloxy)phenyl]-5-phenyl-lH-pyrrolo[2,3-b]pyridine; ad) 5-phenyl-3-pyridin-4-yl-lH-pyrrolo[2,3-b]pyridine; ae) 3-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; af) N- [3 -(5 -phenyl- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl)-phenyl] -acetamide; ag) 4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenylamine; ah) 4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenol; ai) 4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzylamine; aj ) 3 -(5 -phenyl- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl)-phenol ; ak) 5-(3,4-dimethoxyphenyl)-3-pyridin-4-yl-lH-pyrrolo[2,3-b]pyridine; al) 4-[5-(3,4-dimethoxyphenyl)-lH-pyrrolo[2,3-b]pyridin-yl]-phenol; am) 4-[5-(3,4-dimethoxyphenyl)-lH-pyrrolo[2,3-b]pyridin-yl]-phenylamine; an) 4- [5 -(3 ,4-dimethoxyphenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl] -benzoic acid; ao) 4-[5-(4-chlorophenyl)- 1 H-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; ap) N-[4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; aq) 3,5-bis-(4-hydroxyphenyl)-lH-pyrrolo[2,3-b]pyridine; ar) 3,5-bis-(4-carboxyphenyl)- 1 H-pyrrolo[2,3-b]pyridine; as) 4-[5-(4-aminophenyl)-lH-pyπOlo[2,3-b]pyridin-3-yl)-benzylamine; at) 4-[5(4-aminophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; au) 4-[5-(2-fluoro-biphen-4-yl)- 1 H-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; av) N-[3-(5-thiophen-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; aw) 4-(5-thiophen-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; ax) 4-(5-thiophen-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenol; ay) 4-(5-thiophen-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzamide; az) N-[3-(5-pyridin-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; ba) 4-(5-pyridin-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzoic acid; bb) 4-(5-pyridin-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenol; be) 4-(5-thiophen-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzylamine; bd) 3-(lH-indol-5-yl)-5-thiophen-3-yl-lH-pyrrolo[2,3-b]pyridine; be) N-[4-(5-thiophen-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; bf) 5-(3-pyridinyl)-3-(4-pyridinyl)-lH-indole; bg) 4-(5-pyridin-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzamide; bh) 4-[3-(2-fluorobiphenyl-4-yl-lH-pyrrolo[2,3-b]pyridin-5-yl]-benzylamine; bi) 4-(5-pyridin-3-yl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenylamine; bj) {3-[5-(4-methanesulfonylphenyl-lH-pyrrolo[2,3-b]pyridin-3-yl]-phenyl}-acetic acid; bk) N-[3-(3-thiophen-3-yl-l//-pyrrolo[2,3-b]pyridin-5-yl)-phenyl]-acetamide; bl) N-{3-[3-(3-pyridinyl)-lH-pyrrolo[2,3-b]pyridin-5-yl]phenyl}acetamide; bm) 4-[5-(3-acetylaminophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; bn) N-{3-[3-(2,3-difluorophenyl)-lH-pyrrolo[2,3-b]pyridin-5-yl]-phenyl}-acetamide; bo) N- { 3 - [3 -(4-hydroxypheny I)- 1 H-pyrrolo [2,3 -b]pyridin-5 -yl] -phenyl } -acetamide; bp) N- { 3 - [3 -(4-aminomethylphenyl)- 1 H-pyrrolo [2,3 -b]pyridin-5 -yl] -phenyl } - acetamide; bq) N- { 3 - [3 -(4-aminophenyl)- 1 H-pyrrolo [2,3 -b]pyridin-5 -yl] -phenyl } -acetamide; br) N-iS-P-ClH-indol-S-yO-lH-pyrroloP^-bJpyridin-S-ylJ-phenylJ-acetamide; bs) 4-[3-(2-fluorobiphenyl-4-yl)-lH-pyrrolo[2,3-b]pyridin-5-yl]-benzoic acid; bt) N-{3-[3-(4-pyridinyl)-lH-pyrrolo[2,3-b]pyridin-5-yl]phenyl}acetamide; bu) N-IS-tS-CS-fluoropheny^-lH-pyrroloP^-bJpyridin-S-y^-phenyll-acetamide; by) 4-[5-(3-acetylaminophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-benzamide; bw) 4-[S(3-fluorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-benzoic acid; bx) 4-[S(3-fluorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-phenylamine; by) N-{4-[5-(3-fluorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-phenyl} -acetamide; bz) 4-[5-(3-fluorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-benzamide; ca) 2-chloro-N-[4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-benzamide; cb) 2-phenyl-N-[4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-phenyl]-acetamide; cc) 2-chloro-N-[3-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-benzyl] l-benzamide; cd) 2-phenyl-N- [3 -(5 -phenyl- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl)-benzyl] -acetamide; ce) (4-{5-[3-(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; cf) 3-[4-(5-{4-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3- yl)phenyl]propanoic acid; eg) [4-(5-{4-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3- yl)phenyl]acetic acid; ch) (4-{5-[3,4,5-tris(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; 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)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl]phenyl } acetic acid; en) 3-{4-[5-(5-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; co) 3-(4-{5-[6-(methyloxy)-2-naphthalenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl}phenyl)propanoic acid; cp) 3-{4-[5-(3,4-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; c^cύ {4-[5-(3,4-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; cr) {4-[5-(2,3-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; cs) 3-{4-[5-(2,3-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; ct) {4-[5-(2,3-dichlorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; cu) 3-{4-[5-(2,3-dichlorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; cv) {4-[5-(l-benzothien-3-yl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid; cw) [(3 - { 5 - [3 -(methy loxy)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 - yl}phenyl)methyl]amine; ex) 7-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-l,2,3,4- tetrahydroisoquinoline; cy) 2-fluoro-4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; cz) 2-fluoro-4-{5-[3-(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; da) 2-methyl-4- { 5-[3-(methyloxy)phenyl]- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } benzoic acid; db) 5-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-thiophenecarbaldehyde; dc) 5-{5-[3-(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}-2- thiophenecarbaldehyde; dd) 2-methyl-4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; de) (4-{5-[6-(methyloxy)-2-naphthalenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}phenyl)acetic acid; df) 2-fluoro-4-{5-[6-(methyloxy)-2-naphthalenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl} benzoic acid; dg) 2-fluoro-4-[5-(5-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; dh) 4-[5-(5-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; di) 2-methyl-4-[5-(5-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; dj) 4-[5-(3,4-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; dk) 4-[5-(3,4-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; dl) 4-[5-(3,4-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; dm) 4-[5-(2,3-dichlorophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; dn) 4- [5 -(2,3 -dichlorophenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl] -2-methylbenzoic acid ; do) 4-[5-(l-benzothien-3-yl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; dp) 4- [5 -( 1 -benzothien-3 -yl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl] -2-fluorobenzoic acid; dq) 4-[5-(l-benzothien-3-yl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; dr) 6-{3-[4-(ethylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5-yl}quinoline; ds) 4-(5-(3 - [(methylsulfonyl)amino]phenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl)benzoic acid; dt) 3 - [4-(butyloxy)phenyl] -5 - [3 -(methy lsulfony l)phenyl] - 1 H-pyrrolo [2,3 -b]pyridine ; du) N-(3-{3-[4-(aminomethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5- y 1 } pheny l)methanesulfonamide ; dv) N-(3-{3-[3-(aminomethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5- yl}phenyl)methanesulfonamide; dw) 3-amino-5-(5-{3-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3- yl)benzoic acid; dx) 2-fluoro-4-(5-{3-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3- yl)benzoic acid; dy) 3-amino-5-{5-[3-(methylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; dz) 2-fluoro-4- { 5- [3 -(methyl sulfonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3-yl } benzoic acid; ea) [(4- { 5 - [3 -(methy lsulfony l)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 - y 1 } phenyl)methy 1] amine; eb) [(3 - { 5 - [3 -(methylsulfonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 - y 1 } pheny l)methy 1] amine ; ec) 5 - { 5 - [3 -(methylsulfonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl} -2- thiophenecarbaldehyde; ed) 4-{5-[3-(methylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; ee) N-(4-{3-[4-(butyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-5- y 1 } phenyl)methanesulfonamide; ef) 1,1 -dimethylethyl [2-(3 - { 5 - [3 -(methylsulfony l)pheny 1] - 1 H-pyrrolo [2,3 -b] pyridin-3 - yl}phenyl)ethyl]carbamate; eg) 3-amino-5-(5-{4-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3- yl)benzoic acid; eh) 2-fluoro-4-(5 - { 4- [(methy lsulfonyl)amino]phenyl } - 1 H-pyrrolo [2,3 -b] pyridin-3 - yl)benzoic acid; ei) 4-(5-{4-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; ej) N-(4- { 3 - [4-(aminomethy l)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-5 - yl } phenyl)methanesulfbnamide; ek) N-(4-{3-[3-(aminomethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5- yl}phenyl)methanesulfbnamide; el) N-{4-[3-(5-formyl-2-thienyl)-lH-pyrrolo[2,3-b]pyridin-5- y l]phenyl } methanesulfonamide; em) 7-{5-[3-(methylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}-l,2,3,4- tetrahydroisoquinoline; en) N-{3-[3-(l,2,3,4-tetrahydro-7-isoquinolinyl)-lH-pyrrolo[2,3-b]pyridin-5- yl]phenyl}methanesulfonamide; eo) N-{4-[3-(l,2,3,4-tetrahydro-7-isoquinolinyl)-lH-pyrrolo[2,3-b]pyridin-5- yl]pheny 1 } methanesulfonamide ; ep) 2-methyl-4-{5-[3-(methylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; eq) 5 - { 5 - [3 -(methylsulfonyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } -2- thiophenecarboxylic acid; er) 3-{3-[3-(aminomethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; es) 2-fluoro-4- { 5 - [6-(methyloxy)-3 -pyridinyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } benzoic acid; et) 3-[3-(l,2,3,4-tetrahydro-7-isoquinolinyl)-lH-pyrrolo[2,3-b]pyridin-5- yl]benzonitrile; eu) 7-{5-[3,4,5-tris(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}-l,2,3,4- tetrahydroisoquinoline; ev) 4-{5-[3,4,5-tris(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; ew) 2-fluoro-4-{5-[3,4,5-tris(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl} benzoic acid; ex) 2-amino-4-{5-[3,4,5-tris(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl} benzoic acid; ey) 4-[5-(6-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; ez) 4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; fa) 4-[5-(2,3-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; fb) 4-[5-(2,3-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; fc) 2-methyl-4-[5-(6-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; fd) 2-methyl-4-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; fe) 4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; ff) 2-methyl-4- { 5 - [6-(methyloxy)-3 -pyridinyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } benzoic acid; fg) 2-methyl-4-{5-[3,4,5-tris(methyloxy)phenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl} benzoic acid; fh) 2-(l-methylethyl)-4-[5-(l-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; fi) 4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-(l-methylethyl)benzoic acid; fj) 2-(l-methylethyl)-4-[5-(6-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; fk) 4-[5-(2,3-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-methylbenzoic acid; fl) 2-chloro-4-{5-[3-(methylsulfonyl)phenyl]-l-H-pyrrolo-[2,3-b]pyridin-3-yl}benzoic acid; fm) 4-{5-[3-(methylsulfonyl)phenyl]-l-H-pyrrolo-[2,3-b]pyridin-3-yl}-2,6- bis(trifluoromethyl)benzoic acid; fn) methyl 2-(azidomethyl)-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoate; fo) 2-ethyl-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fp) 2-(methylamino)-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fq) 2-(dimethylamino)-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fr) 2-cyclopentyl-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fs) 4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-2-propylbenzoic acid; ft) 2,6-difluoro-4-(5 -phenyl- 1 H-pyrrolo [2,3-b]pyridin-3 -yl)benzoic acid; fti) 2,6-dimethyl-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fv) 2-(2-propyl)-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fw) 6-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)-lH-indazole; fx) 2-(2-methylpropyl)-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fy) 2-methyl-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; fz) 4-[5-(3-hydroxyphenyl)-l-H-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; ga) 3-amino-5-[5-(3-hydroxyphenyl)lH-pyrrolo[2,3-b]-pyridin-3-yl]-benzoic acid; gb) {4-[5-hydroxyphenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-phenyl} acetic acid; gc) 4-[5-(3-aminophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; gd) 3-{4-[5-(3-hydroxyphenyl)-l-H-pyrrolo[2,3-b]pyridine-3-yl]-phenyl}-propionic acid; gd) 3-{4-[5-(3-aminophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; gf) {4- [5 -(3 -aminophenyl)- 1 H-pyrrolo [2,3 -b]pyridin-3 -y ljphenyl } acetic acid; gg) 4-{5-[3-(aminomethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; gh) (4- { 5 - [3 -(aminomethyl)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } phenyl)acetic acid; gi) 4-[5-(3-hydroxyphenyl)-l-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; gj) 2-fluoro-4-[5-(3 -hydro xyphenyl)-l -H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; gk) 5-[5-(3-hydroxyphenyl)-l-H-pyrrolo-[2,3-b]pyridin-3-yl]thiophene-2- carbaldehyde; gl) 3-{3-[3-(2-aminoethyl)phenyl]-l-H-pyrrolo-[2,3-b]pyridin-5-yl}phenol; gm) 3-[3-(l,2,3,4-tetrahydroisoquinolin-7-yl)-l-H-pyrrolo-[2,3-b]pyridin-5-yl]phenol; gn) 3-amino-5-[5-(3-aminophenyl)-l-H-pyrrolo-[2,3-b]pyridin-3-yl]benzoic acid; go) 4-[5-(3-aminophenyl)-l-H-pyrrolo-[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; gp) 2-fluoro-4-(5 -phenyl- 1 H-pyrrolo [2,3 -b]pyridin-3 -yl)phenol ; gq) 2,6-difluoro-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)phenol; gr) 5-phenyl-3-[4-(lH-tetrazol-5-yl)phenyl]-lH-pyrrolo[2,3-b]pyridine; gs) 5-(2-naphthalenyl)-3-[4-(lH-tetrazol-5-yl)phenyl]-lH-pyrrolo[2,3-b]pyridine; gt) 3-[3-fluoro-4-(lH-tetrazol-5-yl)phenyl]-5-phenyl-lH-pyrrolo[2,3-b]pyridine; gu) 2-methyl-2-(4-{5-[3-(methylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl}phenyl)propanoic acid; gv) 2-{4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; gw) 2-(4-{5-[3-(aminocarbonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}phenyl)-2- methylpropanoic acid; gx) 2-{4-[5-(3-cyanophenyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}-2- methylpropanoic acid; gy) 2-methyl-2-{4-[5-(6-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; gz) 2-methyl-2-{4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3- yljphenyl} propanoic acid; ha) 2-methyl-2-[4-(5-{3-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3- yl)phenyl]propanoic acid; hb) 2-methyl-2-[4-(5-{4-[(methylsulfonyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3- yl)phenyl]propanoic acid; he) 2-methyl-2-(4-{5-[6-(methyloxy)-2-naphthalenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl}phenyl)propanoic acid; hd) 2-methyl-2-{4-[5-(5-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; he) 2-methyl-2-{4-[5-(3-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; hf) 2-{4-[5-(6-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; hg) 2-{4-[5-(5-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; hh) 2-{4-[5-(3-quinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]phenyl}propanoic acid; hi) 2-(4-{5-[3-(methylsulfonyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl}phenyl)propanoic acid; hj) 2-(4-{5-[6-(methyloxy)-2-naphthalenyl]-lH-pyrrolo[2,3-b]pyridin-3- yl}phenyl)propanoic acid; hk) 2-methyl-2-[4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)phenyl]propanoic acid; hi) 2-methyl-2-{4-[5-(2-naphthalenyl)-lH-pyrrolo[2,3-b]pyridin-3- yl]phenyl}propanoic acid; hm) 4-[5-(6-amino-3-pyridinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]benzoic acid; hn) 4-{5-[6-(β-alanylamino)-3-pyridinyl]-lH-pyrrolo[2,3-b]pyridin-3-yl}benzoic acid; ho) 4-(5-(6-indolyl)-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; hp) ^-(S-IS-P-CmethylsulfonyOpheny^-lH-pyrroloP^-^pyridin-S- yl}phenyl)ethyl]amine; hq) N-(3-{3-[3-(2-aminoethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5- yl}phenyl)methanesulfonamide; hr) N-(4-{3-[3-(2-aminoethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5- yl}phenyl)methanesulfonamide; hs) 4- { 5 - [3 -(2-aminoethy l)pheny 1] - 1 -H-pyrrolo- [2,3 -b]pyridin-3 -yl } benzoic acid ; ht) 4- { 5 - [3 -(2-aminoethy l)pheny 1] - 1 -H-pyrrolo- [2 ,3 -b]pyridin-3 -yl } -2-methy lbenzoic acid; hu) 4-{5-[3-({[2-(dimethylamino)ethyl]amino)carbonyl)phenyl]-lH-pyrrolo[2,3- b]pyridin-3-yl} benzoic acid; hv) 4-(5-(3-[4-(l , 1 -dimethylethyloxycarbonyl)aminobutanoyl]amino)phenyl- 1 H- pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; hw) 4-(5-(3-[3-(l,l -dimethylethyloxycarbonyl)aminopropanoyl] amino)phenyl- 1 H- pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; hx) 2-(2-propyl)-4-[5-(3-[3-(l,l- dimethylethyloxycarbonyl)aminopropanoyl]amino)phenyl-lH-pyrrolo[2,3- b]pyridin-3-yl]benzoic acid; hy) 4- { 5 - [3 -(β-alany lamino)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } benzoic acid; hz) 4-(5-{3-[(4-aminobutanoyl)amino]phenyl}-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; ia) 4- { 5- [3 -(beta-alanylamino)phenyl] - 1 H-pyrrolo[2,3 -b]pyridin-3 -yl } -2- methylbenzoic acid; ib) 4- { 5 - [3 -(beta-alanylamino)phenyl] - 1 H-pyrrolo [2,3 -b]pyridin-3 -yl } -2-( 1 - methylethyl)benzoic acid; ic) 4-[5-(3-{[(2-aminoethyl)amino]carbonyl}phenyl)-lH-pyrrolo[2,3-b]pyridin-3- yljbenzoic acid; id) 4-[5-(3-{[(3-aminopropyl)amino]carbonyl}phenyl)-lH-pyrrolo[2,3-b]pyridin-3- yljbenzoic acid; ie) S-amino-S-fS^S-aminopheny^-l-H-pyrrolo-PjS-blpyridin-S-ylJbenzoic acid; if) S-amino-S-IS-fS-CaminomethyOpheny^-l-H-pyrrolo-^^-bJpyridin-S-ylJbenzoic acid; ig) 4-{5-[3-(aminomethyl)phenyl]-l-H-pyπOlo-[2,3-b]pyridin-3-yl}-2-fluorobenzoic acid; ih) 2-(aminomethyl)-4-(5-phenyl-lH-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid; ii) 3-{3-[3-(2-aminoethyl)phenyl]-lH-pyrrolo[2,3-b]pyridin-5-yl}benzonitrile; and U) 4-[5-(3-amino-l-isoquinolinyl)-lH-pyrrolo[2,3-b]pyridin-3-yl]-2-fluorobenzoic acid; or a pharmaceutically acceptable salt thereof.

1 1. The use according to claim 8 or 9, wherein 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.

12. The use according to claim 8, wherein the protein kinase is PKCζ and the small molecule is a compound of formula (II) or a pharmaceutically acceptable salt thereof:

(H) wherein R₅ is aryl or heteroaryl, optionally substituted once, twice or three times.

13. The use according to claim 12, wherein R₅ is a phenyl group that is optionally substituted once, twice or three times.

14. The use according to claim 12 or 13, wherein R₅ is

(U) wherein

R₆ is hydrogen, -OH, or -NH₂; preferably -OH or -NH₂; most preferably-NH₂; R₇ is hydrogen, methoxy, or F; preferably hydrogen or F, most preferably hydrogen:

R₈ is hydrogen or methoxy; preferably hydrogen.

15. The use according to claim 14, wherein R₆ is -NH₂, R₇ is hydrogen and R₈ is hydrogen.

16. The use according to claim 8, wherein the protein kinase is PKCζ and the small molecule is a compound of formula (III) or a pharmaceutically acceptable salt thereof:

(III) wherein 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; R₉ is hydrogen or Ci-C₅ alkyl or C₃-C₅ cycloalkyl; and Rio is hydrogen or Ci-C₅ alkyl or C₃-C₅ cycloalkyl;

17. The use according to claim 16, wherein Ring(A) is

(V) (w)

18. The use according to claim 16 or 17, wherein Ring(B) is

(X) (y) (Z)

(aa)

19. The use according to any one of claims 16 to 18, wherein one of R₉ and Rio is hydrogen and the other one is selected from the group consisting of hydrogen, Ci-C₅ alkyl, and C₃-C₅ cycloalkyl.

20. The use according to any of claims 1 to 19, wherein the protozoal infection is an infection with a protozoa 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.

21. 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%.

22. The method of claim 21, wherein the protein kinase is selected from the group consisting of:

(a) protein kinase C zeta (PKCζ);

(b) Serine/threonine-protein kinase WNKl (PRKWNKl); (c) Serine/threonine-protein kinase Sgk2 (SGK2); and

(d) Serine/threonine-protein kinase 35 (STK35).

23. The method of any one of claims 21 or 22, further comprising the step of formulating the test compound selected in step (iv) with pharmaceutically acceptable additives and/or auxiliary substances.

24. Use of a test compound selected in step (iv) of the method of any one of claims 21 to 22 for the production of a medicament for the therapy and/or prophylaxis of infectious diseases, which involve infection of liver and/or hematopoietic cells.

25. Test compound selected in step (iv) of the method of any one of claims 21 to 22 for use in therapy and/or prophylaxis of infectious diseases, which involve infection of liver and/or hematopoietic cells.

26. Pharmaceutical composition comprising a compound usable according to claims 1 to 20 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.

27. 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.

28. The method of claim 27, wherein the protein kinase is selected from the group consisting of: (a) protein kinase C zeta (PKCζ);

(b) Serine/threonine-protein kinase WNKl (PRKWNKl);

(c) Serine/threonine-protein kinase Sgk2 (SGK2); and

(d) Serine/threonine-protein kinase 35 (STK35).