DE102004044556A1 - Use of kinase inhibitors (e.g. serine/threonine kinase inhibitors) for treating viral infections caused by human cytomegalo virus - Google Patents

Abstract

Use of kinase inhibitors (I), which are involved in cellular stress reactions, for the production of medicaments to treat viral infections. An independent claim is also included for a serine/threonine kinase inhibitor for treating viral infections. ACTIVITY : Virucide. MECHANISM OF ACTION : Protein kinase inhibitor; Ser/Thr kinase inhibitor.

Description

at Many viral infections can eliminate the source of infection (infected cells) with currently available antiviral agents not done, because viruses have different efficient survival strategies in the Host organism, e.g. Latency or integration into the host cell genome, have developed the point of attack of currently available antiviral Usually, however, means the proliferating virus or the infection process is.

Serine / threonine Protein kinases are in cellular Integrated signal paths, the u.a. gene expression and cell proliferation regulate (Su & Karin, Curr. Opinion Immunol. 1996, 8,402; Kolch Biochem. J. 2000, 351. 289). Some serine / threonine protein kinases, cyclin-dependent kinases (cdk), regulate transitional steps in the cell cycle (Meyerson et al., EMBO J. 1992, 11, 2909). Are active these kinases, when specific to certain proteins that contribute to Belong to family Cycline, are bound.

virus can replicate only in cells. It could be shown experimentally that the inhibition of protein kinases via an intervention in the life cycle the cell may show promising antiviral effects (Schang Antimicrob Chemother. 2002, 50. 779).

The selective elimination of infected cells through the use of cellular stress mechanisms currently not the subject of antiviral therapies. It should be a possibility are found to selectively destroy virus-infected cells and on the one hand to end the infection and on the other hand with the Therapy possible to be low in side effects.

virus bring with the infection of cells with their nucleic acids. Due to the structure of these nucleic acids, different from the eukaryotic Distinguish cells, a so-called DNA damaging signal is triggered (Raj et al. 2001 Nature, 142, 914). The cell reacts with a stress response, i.e. Mechanisms of cell cycle control and DNA repair mechanisms. By fault This stress response drives the cell into apoptosis. component the mentioned Stress response are various kinases, i.a. Serine / threonine kinases.

The inhibition of serine / threonine kinase "stress" ( 1 ) leads to apoptosis in cells.

By using a DNA-damaging substance (adriamycin) it could be shown that cells in which a DNA damaging signal was triggered are significantly more sensitive to an inhibition of "stress" by means of a specific "stress" inhibitor "A" than not stressed cells ( 2 ). The structure of "A" is in 5a shown.

It could be shown that the expression of serine / threonine kinase "stress" on exposure of cells with DNA-damaging UV light, DNA intercalating substances or with a viral Vector is induced (unpublished).

Connection "B" (structure in 5b ), another specific inhibitor of "stress", demonstrated antiviral effects in NHDF cells infected with human cytomegalovirus ( 3 ).

Depending on the virus concentration used, a selectivity index of up to about 300 between infected and uninfected cells could be worked out ( 4 ).

In comparison, the relatively nonspecific kinase inhibitor "C" ( 5c ) also in uninfected cells marked cytopathic effects. The selectivity index is about 1.

Consequently was surprisingly found that inhibitors of "stress" have antiviral effects, presumably over the selective triggering of apoptosis in virus-infected cells.

The sequence 1 shows the amino acid sequence of the serine / threonine kinase "stress." The enzyme was published under US 10/410764 and under WO 01/81589 Furthermore, a patent application of the company Sugen under the number PCT / US01 / 02337 was published, in which a similar kinase (1 amino acid change dert) is mentioned.

Description of the pictures:

1 : When human cells infected with human cytomegalovirus are treated with the "stress" inhibitor "B", antiviral effects can be observed. The density of the cell lawn increases with increasing substance concentration. At comparable concentrations, the substance does not appear cytostatic on uninfected cells.

2a : Compound "A" (N- (6-quinolinyl) -N- [2- (6-quinolinylamino) -5- (trifluoromethyl) -4-pyrimidinylamine] WO 03/030909 (Publication date: 17 April 2003)

2 B Compound "B" (5-bromo-N- (6-quinolinyl) -N- [2- (6-quinolinylamino) -4-pyrimidinylamine] WO 03/030909 (Publication date: 17 April 2003)

2c Compound "C" (N- [5-bromo-2- (1H-indazol-5-ylamino) -4-pyrimidinyl] -N- (1H-indazol-5-yl) amine) WO 03/030909 (Publication date : April 17, 2003)

Examples

Example 1: Combination therapy Adriamycin and inhibitor "A"

H460 lung cancer cells (ATCC No. HTB-177) were cultured under standard conditions (DMEM, 10% FBS, 10 mM HEPES, 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin at 37 ° C in 5% CO2 in a wet incubator) The cells were dissolved using trypsin from the culture bottles and seeded in a density of about 3000 cells / well in a total of 100 L L og medium / well in a 96-well plate. Adriamycin (Doxorubicin HCL Injection USP, Ben Venue Laboratories, Inc. Bedford, USA) was added 24 hours after seeding at the concentrations described 2 ). After inhibitor "A" was added at various concentrations, the cell count was evaluated using a commercially available MTS assay (Promgega CellTiter 96 Aqueous One Solution Cell Proliferation Assay) 72 hours later according to the manufacturer's instructions provided. Inhibitor "A" was dissolved in 100% DMSO and a 10 mM stock solution was prepared. This solution was further diluted until a 40 μM working solution in 0.4% DMSO was prepared.

Table 1

Treatment of H460 cells with suboptimal concentrations of adriamycin (1 nM) does not result in cytopathic effects. While the "stress" kinase inhibitor "A" has an IC ₅₀ of approximately 1 μM upon exposure of H460 cells, an IC ₅₀ of approximately 60 nM is observed in pretreatment (damage to the DNA and induction of a repair response). H460 IC50 [M] no preincubation, "A" alone 1.27E-06 24 hours incubation with 1 nM adriamycin (no "A") inactive 24 hours preincubation with 1 nM adriamycin + "A" 5.89E-08 4 days incubation with 1 nM adriamycin (no "A") inactive 4 days preincubation with 1 nM adriamycin + "A" 5.94E-07 24 hours incubation with 10 nM adriamycin (no "A") 40% inh 24 hours preincubation with 10 nM Adriamycin + "A" <1.65E-08 4 days incubation with 10 nM adriamycin (no "A") 40% inh 4 days preincubation with 10 nM Adriamycin + "A" <1.65E-08 24 hours incubation with 100 nM adriamycin (no "A") 100% inh 24 hours preincubation with 100 nM adriamycin + "A" <1.65E-08 4 days incubation with 100 nM adriamycin (no "A") 100% inh 4 days preincubation with 100 nM adriamycin + "A" <1.65E-08 24 hours incubation with 1 μM adriamycin (no "A") 100% inh 24 hours preincubation with 1 μM adriamycin + "A" <1.65E-08 4 days incubation with 1 μM adriamycin (no "A") 100% inh 4 days preincubation with 1 μM adriamycin + "A" <1.65E-08

Example 2: Anti-HCMV (Anti-Human Cytomegalovirus) Cytopathogenicity tests

The test compounds are used as 50 millimolar (mM) solutions in dimethylsulfoxide (DMSO). After the addition of 2 .mu.l each of the 50, 5, 0.5 and 0.05 mM DMSO stock solutions of 98 .mu.l cell culture medium in the series 2 AH in duplicate, 1: 2 dilutions with 50 ul medium to 11 row of 96-well Plate. The wells in rows 1 and 12 each contain 50 μl of medium. 150 μl of a suspension of 1 × 10 ⁴ cells (human foreskin fibroblasts [NHDF]) are then pipetted into the wells (row 1 = cell control) or into the rows 2-12 a mixture of HCMV-infected and noninfected NHDF cells ( MOI = 0.001 - 0.002), ie 1-2 infected cells per 1000 non-infected cells. The series 12 (without substance) serves as a virus control. The final test concentrations are 250 - 0.0005 μM. The plates are incubated for 6 days at 37 ° C / 5% CO2, ie until all cells are infected in the virus controls (100% cytopathogenic effect [CPE]). The wells are then fixed and stained by adding a mixture of formalin and Giemsa's dye (30 minutes), with double-distilled water. washed and dried in a drying oven at 50 ° C. Thereafter, the plates are visually evaluated with an overhead microscope (Plaque Multiplier from Technomara).

The following data can be determined from the test plates:
CC50 (NHDF) = substance concentration in μM, in which no visible cytostatic effects on the cells are recognizable compared to the untreated cell control;
EC50 (HCMV) = substance concentration in μM, which inhibits the CPE (cytopathic effect) by 50% compared to the untreated virus control;
SI (selectivity index) = CC50 (NHDF) / EC50 (HCMV).

Table 2 shows the selectivity of infected cells vs. uninfected cells are mediated by specific inhibition of "stress." HCMV (+) = 1: 900 infected and uninfected cells, HCMV (-) = uninfected cells only, CC50 = concentration (μM) Cell growth is 50% limited, EC50 = concentration (μM) at which virus replication is 50% inhibited, SI = selectivity index (quotient from CC50 and EC50). Table 2

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (7)

Use of kinase inhibitors, which in cellular Stress reactions are involved in the production of drugs for the treatment of viral infections. Use according to claim 1, wherein the viral infection caused by HCMV. Use according to claim 1 or 2, wherein the kinase Inhibitors are serine / threonine kinase inhibitors. Use according to claims 1-3, wherein the kinase has an amino acid sequence as in 1 having. Use according to claims 1-3, wherein the kinase has an amino acid sequence which is at least 80% identical to the sequence in 1 has. A serine / threonine kinase inhibitor for treatment viral infections according to the methods of claims 1-5. Use of a serine / threonine kinase inhibitor according to Claim 6 for the preparation of a medicament for the treatment of viral Infections.