Labyrinthopeptins as anti-viral agents

Abstract

The present invention relates to novel labyrinthopeptin derivatives. These labyrinthopeptin derivatives are useful for the treatment of infectious diseases, such as an infectious disease caused by an infection with human respiratory syncytial virus (RSV), Kaposi sarcoma-associated herpesvirus (KSHV), cytomegalovirus (CMV/HCMV), dengue virus (DENV), chikungunya virus (CHIKV), tick-borne encephalitis virus (TBEV; FSME virus), vesicular stomatitis Indiana virus (VSV), zika virus (ZIKV) and/or hepatitis C virus (HCV). Said labyrinthopeptin derivatives are also useful for analyzing the mode of action of labyrinthopeptins. Also encompassed by the present invention are labyrinthopeptins for use in treating an infectious disease, in particular an infectious disease caused by an infection with any one of the viruses selected from RSV, KSHV, CMV, CHIKV, TBEV, VSV, ZIKV and HCV. The invention further relates to a combination of labyrinthopeptin A1 and A2 for use as a medicament, e.g. for treating an infectious disease caused by an infection with RSV, KSHV, CMV, DENV, CHIKV, TBEV, VSV, ZIKV and/or HCV.

Claims

1. A method of treating and/or preventing an infection with any one of the viruses selected from the group consisting of respiratory syncytial virus (RSV), Kaposi sarcoma-associated herpesvirus (KSHV), cytomegalovirus (CMV), chikungunya virus (CHIKV), tick-borne encephalitis virus (TBEV), vesicular stomatitis Indiana virus (VSV), and zika virus (ZIKV) in a subject in need thereof, wherein the method comprises administering an effective dose of a peptide to the subject, and wherein said peptide comprises the amino acid sequence ##STR00011## wherein: Lab is labionin; X.sub.1 is Asn or Asp; X.sub.2 is Ala or Trp; X.sub.3 is Val or Leu; X.sub.4 is Trp; X.sub.5 is Glu; X.sub.6 is Thr; X.sub.7 is Gly; and X.sub.8 consists of the amino acid sequence Trp-Val-Pro-Phe-dehydrobutyrine (SEQ ID NO:8), or the amino acid sequence Leu-Phe-Ala.

2. The method of claim 1, wherein said peptide is maximal 30 amino acids in length.

3. The method of claim 1, wherein X.sub.1 is Asn; X.sub.2 is Ala; X.sub.3 is Val; X.sub.4 is Trp; X.sub.5 is Glu; X.sub.6 is Thr; X.sub.7 is Gly; and X.sub.8 consists of the amino acid sequence Trp-Val-Pro-Phe-dehydrobutyrine (SEQ ID NO:8).

4. The method of claim 1, wherein X.sub.1 is Asp; X.sub.2 is Trp; X.sub.3 is Leu; X.sub.4 is Trp; X.sub.5 is Glu; X.sub.6 is Thr; X.sub.7 is Gly; and X.sub.8 consists of the amino acid sequence Leu-Phe-Ala.

5. The method of claim 1, wherein the method further comprises co-administration with at least one other active agent.

6. The method of claim 5, wherein the other active agent is a CMV inhibitor, a KSHV inhibitor, a RSV inhibitor, a dengue virus (DENV) inhibitor, a CHIKV inhibitor, a TBEV inhibitor, a VSV inhibitor, a ZIKV inhibitor, or a HCV inhibitor.

7. The method of claim 1, wherein the peptide is administered orally, intravenously, subcutaneously or intramuscularly.

8. The method of claim 1, wherein the virus is RSV.

9. The method of claim 3, wherein the method further comprises administering to the subject and effective dose of a second peptide comprising the amino acid sequence ##STR00012## wherein: X.sub.1 is Asp; X.sub.2 is Trp; X.sub.3 is Leu; X.sub.4 is Trp; X.sub.5 is Glu; X.sub.6 is Thr; X.sub.7 is Gly; and X.sub.8 consists of the amino acid sequence Leu-Phe-Ala.

10. The method of claim 9, wherein the method further comprises co-administration with at least one other active agent.

11. The method of claim 10, wherein the other active agent is a CMV inhibitor, a KSHV inhibitor, a RSV inhibitor, a DENV inhibitor, a CHIKV inhibitor, a TBEV inhibitor, a VSV inhibitor, a ZIKV inhibitor, or a HCV inhibitor.

12. The method of claim 9, wherein the combination is administered orally, intravenously, subcutaneously or intramuscularly.

13. The method of claim 9, wherein the virus is RSV.

Description

(1) The Figures show:

(2) FIG. 1. Chemical structure of (A) lanthionin and (B) labionin

(3) FIG. 2. Chemical structure of dehydrobutyrine

(4) FIG. 3. Chemical structure of LabyA1 (FIG. 3A) and LabyA2 (FIG. 3B) showing their stereochemistry

(5) FIG. 4. Chemical structures of LabyA1, LabyA2, LabyA1-Hexyn and LabyA2-Hexyn

(6) FIG. 5. Dose-dependent inhibition of CMV infection by Labyrinthopeptins. GFP signals (relative units +/−SD) of cultures of NHDF cells (triplicates) infected with a GFP-expressing CMV (i.e. HCMV) strain and treated with the indicated concentrations of Labyrinthopeptins A1 and A2 were measure at day 4 post infection. The IC50 values of the substances were calculated using Graphpad Prism nonlinear fit log (inhibitor) vs. response (three parameters) analysis. One representative result of two experiments is shown.

(7) FIG. 6. Labyrinthopeptins inhibit CMV infection in an early phase. NHDF cells were infected with a GFP-expressing CMV strain and test compounds were added at the indicated time points. At 96 hpi the GFP expression in cells of infected cultures (triplicates) was measured. The concentrations of Laby A1 and A2 were 3 μM and 7 μM, respectively. Data are means (+/−SD) of values measured in triplicate cultures. Representative data from one of two experiments are shown.

(8) FIG. 7. Mode of action of Labyrinthopeptins. CMV particles or NHDF cells were preincubated with Labyrinthopeptins, PAA or DMSO for 1 h or remained untreated. The media of the samples were then diluted 16-fold, below the concentration of Labyrinthopeptins found to be effective in previous experiment, followed by CMV inoculation for 3 h and finally replacement with new medium. For control cultures were permanently treated with an effective dose of the substances or 16-fold diluted concentrations. EGFP expression of the cells (means from five replicates) was measured at 4 d p.i. The experiment was repeated 3 times with similar results.

(9) FIG. 8. Scheme depicting the design of the TOA experiment.

(10) FIG. 9. Outline of the Mode of action assay.

(11) FIG. 10. Synergistic effects of Labyrinthopeptins A1 and A2 on DENV infection. Huh-7 cells were incubated in the presence of varying concentrations of Laby and after 30 min subjected to DENV. 48 h post-infection the number of DENV-positive cells was determined by evaluating DENV-envelope protein expression (AlexaFluor™488-immunostained) via high-content fluorescence imaging. The number of total cells was determined by evaluating DAPI-stained nuclei. IC.sub.50-values indicate that LabyA1 (IC.sub.50=3.7 μg/ml) is a more potent inhibitor of viral infection than LabyA2 (IC.sub.50=15.4 μg/ml). When LabyA1 and LabyA2 were applied in a 1:1 combination, their anti-viral activity is further improved (IC.sub.50=2.6 μg/ml). (Values are ±SEM; n=5)

(12) FIG. 11. Laby-Hexyn derivatives retain the anti-DENV activity. Huh-7 cells were incubated in the presence of varying concentrations of Laby-Hexyn derivatives and after 30 min subjected to DENV. 48 h post-infection the number of DENV-positive cells was determined by evaluating DENV-envelope protein expression (AlexaFluor™488-immunostained) via high-content fluorescence imaging. The number of total cells was determined by evaluating DAPI-stained nuclei. IC.sub.50-values indicate that Laby-Hexyn derivatives retain their anti-DENV activity: IC.sub.50 (LabyA1-Hexyn)=3.7 μg/ml, IC.sub.50 (LabyA2-Hexyn)=14.2 μg/ml, IC.sub.50 (1:1 combination of LabyA1-Hexyn/LabyA2-Hexyn)=2.0 μg/ml. (values are ±SEM; n=3)

(13) FIG. 12. Dipolar cycloaddition of Biotin-Azide to immobilized Laby-Hexyn derivatives in vitro. 2 μg of Laby-derivatives were immobilized on a Nunc Maxisorp 96 well plate. After blocking with 1% BSA in PBS reaction mix for dipolar cycloaddition with or without Biotin-azide was applied for 2 h. Subsequently success of the reaction was checked by incubating wells with Avidin-labeled HRP for 1 h followed by a color reaction induced by addition of substrate solution C [BioLegend]. Color reaction was stopped by addition of 50 μl H.sub.3PO.sub.4 (1 M) and OD.sub.450nm was monitored. (Values are ±SEM; n=2)

(14) FIG. 13. Schematic representation of the screening system of Example 4.

(15) FIG. 14. Assay validation of the screening system of Example 4. Cell survival is assessed by staining with crystal violet.

(16) FIG. 15. Assay validation of the screening system of Example 4. Cell survival in the presence of RSV and ribavirin is shown.

(17) FIG. 16. Labyrinthopeptins inhibit RSV induced cell death and RSV infection. A: Cell survival in the presence of RSV and LabyA1; B: cell survival in the presence of RSV and LabyA2. C: Labyrinthopeptins inhibit RSV infection.

(18) FIG. 17. A: Characterization of the susceptibility of the chikungunya virus glycoprotein-mediated and the VSV-glycoprotein-mediated cell entry process to pharmacological and immunological inhibition and cellular restriction. B: Labyrinthopeptin A1.

(19) FIG. 18. Effects of labyrinthopeptins on TBEV infection.

(20) FIG. 19. Effects of labyrinthopeptins on ZIKV infection.

(21) FIG. 20. Effects of labyrinthopeptins on HCV infection. A: Labyrinthopeptin A1 antiviral activity against HCV JcR2a. B: Labyrinthopeptin A2 antiviral activity against HCV JcR2a.

(22) FIG. 21. Cytotoxic concentration (CC50) of Labyrinthopeptin A1 and A2.

(23) The Examples illustrate the invention.

Example 1: Materials and Methods

(24) Dose Response Assay

(25) The antiviral assay is based on the inhibition of CMV-driven GFP expression in NHDF cells. Briefly, NHDF cells (˜1.6×10.sup.4 per well) were seeded in 96-well plates 1 day prior to infection. Various concentrations of Labyrinthopeptin A1 (final conc. 10, 5, 2, 1 and 0.5 μM) and Labyrinthopeptins A2 (final conc. 15, 10, 5, 2 and 1 μM) were dispensed to the cells to a total volume of 200 μl/well in triplicates. PAA (180 μM) was added as positive control. DMSO was added to either infected cells or uninfected cells with the highest concentration as done with the substances added to cells, as a control for substances which were diluted in DMSO. After 1 h of incubation, the GFP-expressing CMV (i.e. HCMV) strain pHG-1 (Borst, J. Virol. 2005 (79): 3615-26; herein called HT8-GFP), which is based on the CMV (i.e. HCMV) laboratory strain AD169, was added to cells at an MOI of 0.5 and cells were incubated for another 4 days. At 4 dpi the media were removed from the wells and cells were fixed with 3% PFA and washed with PBS followed by adding PBS before measurement. GFP expression from the cells was measured using a BioTek Synergy 2; the protocol for excitation wavelength was set to 485/20 nm and the emission wavelength was 516/20 nm.

(26) Time-of-Drug-Addition (TOA) Experiment

(27) The TOA experiment was performed using 1.6×10.sup.4 NHDF cells per well in 96 well plates. Labyrinthopeptin A1 (3 μM), A2 (7 μM), DMSO (0.1%) or PAA (180 μM) were added to the cultures at −1, 0, 1, 3, 6, 24, 48, 72 hours of addition of the virus. Cells were infected with HT8-GFP at a multiplicity of infection (MOI) of 0.5 at 0 h. At 96 hpi GFP expression of cells was measured above.

(28) Mode of Action Assay

(29) 8×10.sup.3 PFU of the HT8-GFP expressing AD169 strain and 1.6×10.sup.4 NHDF cells/well were pre-incubated with 3 μM and 7 μM of TOA LabyA1 and A2, respectively for 1 h and then diluted 16-fold before adding them to cells or infecting with virus in 5 wells. The media was replaced with new media after 3 h of incubation and for controls, viruses and cells were pre-incubated with DMSO (highest concentration of substances) and PAA (180 μM) for 1 h followed by diluting them 16-fold and adding to wells for 3 hours, and then exchanged with new media.

(30) Dose-Dependent Effects of Labyrinthopeptins on KSHV Infection

(31) 3×10.sup.4 HEK 293 cells were seeded in growth medium (DMEM; 10% FBS) onto a 96 well plate and incubated for 24 h at 37° C. and 5% CO.sub.2. After removal of the medium, 180 μl growth medium together with 20 μl of a solution of KSHV (produced from cell line BJAB-rKSHV (Kati, J Virol. 2013, 87(14):8004-16; Kati, J Virol Methods. 2015; 217:79-86) at an MOI of 0.01 were applied to each well. Simultaneously the indicated amounts of LabyA1 or LabyA2 were added. After 48 h of incubation, the GFP expressing HEK 293 cells were counted under a fluorescence microscope. The data points given in the figures are means of triplicates. The results are shown in Table 4, below.

(32) Dose Response Assay

(33) Huh-7.5 cells (3×10.sup.4 per well) were seeded in black 96-well optical-bottom plates [Nunc] in full growth medium one day prior to infection. After washing with PBS, 40 μl assay medium (5% FBS) was added to cells containing either Laby A1, Laby A2 (final conc. 50, 16.7, 5.56, 1.85, 0.62, 0.21, 0.069 μg/ml) or a combination of Laby A1 and Laby A2 (25, 8.3, 2.8, 0.93, 0.31, 0.10, 0.034 μg/ml each). Treatments were run in doublets. PBS served as a control. After 30 min of incubation, cells were infected with Dengue Virus (Type 2 New Guinea C) at an MOI of 0.5 to give a final volume of 60 μl/well. After 2 h incubation at room temperature cells were washed with PBS and 100 μl assay medium was added per well. Infected cells were incubated for another 48 h. Hereafter media were removed from the wells and cells were fixed with 4% PFA. Fixed cells were washed extensively with PBS and permeabilized with 0.25% TritonX-100 for 5 min. After blocking with 5% FB in PBS primary antibody was applied (Anti-Dengue Virus E glycoprotein antibody [DE1] (ab41349) [Abcam], 1:100 diluted in 5% FBS/PBS) for 2 h. After washing secondary antibody (Alexa Fluor® 488 Goat Anti-Mouse IgG (H+L) [Life Technologies], 1:1000 diluted in 5% FBS/PBS) was applied for 1 h. Finally, cells were stained with DAPI (500 ng/ml in PBS) for 5 min.

(34) Fluorescent cells were analyzed by high-content imaging using the automated microscope ImageXpressMicro [Molecular Devices] and the MetaXpress-software. The excitation wavelengths were set to 360 nm (DAPI) and 485 nm (Alexa Fluor488) and the emission wavelengths were set to was 460 nm (DAPI) and 516 nm (Alexa Fluor488). Images of six sites/well were acquired (2 columns, 89 μm spacing; 3 rows, 67 μm spacing). The number of total cells/site was determined by automatically counting DAPI-stained nuclei. The percentage of DENV-positive cells was calculated by automatically evaluating the number of Alexa Fluor 488-positive cells in relation to the total cell number. Values obtained from the six sites were averaged and plotted onto a semi-logarithmic X/Y-chart. IC.sub.50-values were calculated by non-linear regression.

(35) Synthesis of Laby A1-Hexyn Derivative

(36) To a solution of 2-chloro-4,6-dimethoxy-1,3,5-triazine (1 mg, 0.006 mmol) in dimethylformamide (1 ml), n-methylmorpholine (2 μL, 0.015 mmol) was added at room temperature. After 1 h stirring at room temperature, 5-Hexynoic acid (0.5 μL, 0.004 mmol) was added. After 30 minutes of stirring, Labyrinthopeptin A1 (6 mg, 0.003 mmol) was added to the reaction mixture and allowed to stir for 16 h at room temperature. The reaction mixture was purified by reversed-phase HPLC using a Gemini 5μ C18 column (dimension: 250 mm×20 mm) with acetonitrile in water (10-90%) with 0.1% HCOOH as eluents. Peaks were fractionated based on the UV detection at 220 nm. Collected desired compound was lyophilized to yield 2 mg (31.7%). The product was characterized by high resolution mass spectrometry.

(37) HRMS (Q-tof): Calculated for C.sub.98H.sub.126N.sub.23O.sub.26S.sub.4 [M+H].sup.+ 2168.8127, found 2168.7821

(38) Synthesis of Laby A2-Hexyn Derivative

(39) To a solution of 2-chloro-4,6-dimethoxy-1,3,5-triazine (1 mg, 0.006 mmol) in dimethylformamide (1 ml), n-methylmorpholine (2 μL, 0.015 mmol) was added at room temperature. After 1 h at room temperature 5-Hexynoic acid (0.5 μL, 0.004 mmol) was added. After 30 minutes of stirring, Labyrinthopeptin A2 (6 mg, 0.003 mmol) was added to the reaction mixture and allowed to stir for 16 h at room temperature. The reaction mixture was purified by reversed-phase HPLC using a Gemini 5μ C18 column (dimension: 250 mm×20 mm) with acetonitrile in water (10-90%) with 0.1% HCOOH as eluents. Peaks were fractionated based on the UV detection at 220 nm. Collected desired compound was lyophilized to yield 1.5 mg (23.8%). The product was characterized by high resolution mass spectrometry.

(40) HRMS (Q-tof): Calculated for C.sub.91H.sub.117N.sub.20O.sub.25S.sub.4 [M+H].sup.+ 2017.7381, found 2017.7376

(41) Dipolar Cycloaddition of Biotin-Azide to Immobilized Laby-Hexyn Derivatives

(42) All subsequent washing steps were performed with three times with 200 μl of PBS. 2 μg of either LabyA1-Hexyn, LabyA2-Hexyn or LabyA1 were diluted in Coating Buffer A [BioLegend] and adsorbed in triplets for 16 h at 4° C. to a 96 well plate (Nunc Maxisorp). After washing with PBS wells were blocked with 1% BSA (w/v) in PBS (blocking buffer) for 1 h at room temperature. Wells were again washed with PBS and 100 μl cycloaddition reaction mix (2 mM CuSO.sub.4, 5 mM Sodium ascorbate, 100 μM Biotin-azide [Azide-PEG3-Biotin conjugate, Sigma], diluted in blocking buffer) were applied. Cycloaddition reaction mix without Biotin-Azide served as a control. The reaction was performed for 2 h at room temperature. After washing with PBS, wells were incubated with 100 μl blocking solution containing Avidin-labeled Horse radish peroxidase (HRP) [BioLegend] for 1 h. Finally wells were washed with PBS and 100 μl substrate solution C [BioLegend] was applied to each well. Color reaction was stopped after 15 min by addition of 50 μl H.sub.3PO.sub.4 (1 M). OD.sub.450nm was determined using an automated plate reader [Biotek].

(43) Chikungunya Virus Assay

(44) HEK293T cells were seeded into a 96-well plate (2×10.sup.4 cells/well) and cultured under standard conditions. On the next day VSV-G- and CHIKV gp-pseudotyped lentiviral particles encoding luciferase were treated with indicated concentrations of Labyrinthopeptin A1 or DMSO. Cells were transduced with Labyrinthopeptin A1-treated or DMSO-treated vector particles and cultured for 48 h. Afterwards, luciferase activity in transduced cells was measured luminometrically. IC50 values were calculated using GraphPad Prism 5; see Example 5 and FIG. 17.

Example 2: Effect of Labyrinthopeptins A1 and A2 on CMV Infection

(45) Dose-Dependent Effects of Labyrinthopeptins on CMV Infection

(46) To determine a potential effect of Labyrinthopeptins on CMV (i.e. HCMV) infection, cultures of normal human dermal fibroblasts (NHDF) were treated with various concentrations of Labyrinthopeptins A1 and A2 one hour before infection with a GFP-expression CMV strain at a multiplicity of infection of 0.5 PFU/cell. 4 days later GFP expression of the cells was measured as readout for viral gene expression. DMSO-treated cells (DMSO is the diluent for the Labyrinthopeptins) and untreated cells served as positive controls for the infection, and non-infected cells as negative control. Treatment of infected cell cultures with phosphonoacetic acid (PAA) was used a positive control for inhibition of viral replication.

(47) The Labyrinthopeptins inhibited virus-driven GFP expression in cells inoculated with CMV in a dose-dependent manner (FIG. 5). The IC50 values for Labyrinthopeptins A1 and A2 were approximately 1.3 and 5.4 μM, respectively.

(48) Time Point of the Inhibitory Effect of Labyrinthopeptins During the Life Cycle of CMV

(49) To get an idea on which phase of the CMV infection cycle the Labyrinthopeptins exert their effect, a time-of-addition experiment was performed. Labyrinthopeptins A1 and A2 (at a final concentration of 3 and 7 μM, respectively) were added to the cell cultures, either before, concomitantly or subsequently to inoculation with CMV (see FIG. 6). GFP expression as a readout for successful viral infection was measured at day 4 p.i. DMSO-treated cultures served as controls.

(50) The strongest inhibition of viral gene expression (almost complete inhibition) was observed when the substances were added before or concomitantly with virus inoculation of the cultures. About 50% inhibition occurred when the compounds were added 1 h post inoculation and approximately 25% inhibition upon addition at 3 and 6 h p.i. At later times points viral infection could be inhibited only minimally.

(51) These results suggest that the labyrinthopeptins act on an early step of viral infection or possibly directly on viral particles. One has to point out that in the experiment shown, entry of CMV into the cells is not synchronized. Although a substantial portion of the inoculated virus has entered the cells one hour p.i., other particles could remain attached to the cell surface and enter the cells only subsequently.

(52) Putative Effect of Labyrinthopeptins A1 and A2 on Virus Particles

(53) To learn whether the substances act primarily on the virus particles or on cells, pretreatment was performed using concentrations of Labyrinthopeptins A1 and A2 3 μM and 7 μM of LabyA1 and A2, respectively) that in the previous experiments were found to be effective (>98% inhibition of infection).

(54) Following incubation for 1 h the virus sample or the medium in the cell cultures were diluted 16-fold, resulting in a concentration of the compounds which according to the dose-response curve (cf. FIG. 5) would not exert an effect. Pretreated virus was then added to cells (FIG. 7, group “virus pretreated”) or untreated virus was added to pretreated cells (FIG. 7, group “cells pretreated”), followed by incubation for 3 h and replacement of the inocula with new media. GFP expression was determined at 96 h p.i. In parallel, cultures were treated with the effective doses of the compounds or 16-fold diluted concentrations concomitantly with CMV inoculation to verify inhibition by the compounds and loss upon 16-fold dilution (FIG. 7, dilution). PAA and DMSO were used as controls.

(55) Upon pretreatment of viruses with Labyrinthopeptins A1 and A2, viral gene expression was inhibited by ˜50% and ˜90%, respectively. Basically no inhibition was observed upon pretreatment of cells. Treatment of infected cells throughout the infection cycle with high concentrations of Labyrinthopeptins A1 and A2 resulted in nearly 100% inhibition as expected (FIG. 7, group “permanently treated, effective dose), whereas only slight inhibition was seen following permanent treatment with the 16-fold diluted concentration (FIG. 7, group “permanently treated, 16-fold dilution”). PAA—as a soluble substance that acts on the viral DNA polymerase at a later stage of infection—exerted inhibition only when permanently present in the culture medium (to a lesser extent when the 16-fold diluted concentration was applied as expected), but not when it was removed after pretreatment of the virus or cells.

(56) The results suggest that the labyrinthopeptins act primarily on the virus particles. The somehow reduced inhibitory effect upon pretreatment with Labyrinthopeptin A1 compared to the effect seen upon permanent treatment could either indicate that the inhibition is partially reversible or that the substance is additionally effective during the cell attachment or entry phase of CMV.

Example 3: Effects of Labyrinthopeptins A1 and A2 on DENV Infection

(57) Synergisitc Effects of Labyrinthopeptins A1 and A2 on DENV Infection

(58) To determine a potential synergistic effect of Labyrinthopeptins A1 and A2 on DENV-infection a dose response assay was performed. Cultures of Huh-7.5 cells (human hepatocarcinoma cell line) were treated with various concentrations of either Laby A1, LabyA2 or an equivalent combination of LabyA1 and LabyA2 for 30 min. Cells were subsequently infected with DENV (Type 2 New Guinea C) at a MOI of 0.5 PFU for 2 h at room temperature. Unbound viral particles were removed and infected cells were incubated for 48 h. Hereafter cells were fixed using 4% PFA in PBS and immunostained for DENV envelope protein expression. For this purpose, a combination of anti-Dengue Virus E glycoprotein antibody and an Alexa Fluor 488-conjugated secondary antibody was applied. Additionally cell nuclei were stained with DAPI. The number of total cells as well as the percentage of Alexa Fluor 488-positive cells (=DENV positive cells) was determined by high-content fluorescence imaging.

(59) IC.sub.50-values indicate that LabyA1 (IC50=3.7 μg/ml) is a more potent inhibitor of viral infection than LabyA2 (IC.sub.50=15.4 μg/ml). When LabyA1 and LabyA2 were applied in a 1:1 combination, their anti-viral activity is further improved (IC.sub.50=2.6 μg/ml) (FIG. 10).

(60) Laby-Hexyn Derivatives Retain the Anti-DENV Activity

(61) To conduct mode of action studies we generated Laby-derivatives carrying an N-terminal Hexyn-group. These can further be derivatized in vitro and in vivo by dipolar cycloaddition e.g. of an azide-labeled fluorophore. To determine the biological activity of Laby-Hexyn derivatives on DENV-infection a dose response assay was performed as depicted above. The IC.sub.50-values obtained are similar to the IC.sub.50-values obtained for uncoupled Laby and thus indicate that Laby-Hexyn derivatives retain their anti-DENV activity: IC.sub.50 (LabyA1-Hexyn)=3.7 μg/ml, IC.sub.50 (LabyA2-Hexyn)=14.2 μg/ml, IC.sub.50 (1:1 combination of LabyA1-Hexyn/LabyA2-Hexyn)=2.0 μg/ml (FIG. 11).

(62) Dipolar Cycloaddition of Biotin-Azide to Immobilized Laby-Hexyn Derivatives

(63) To test whether the dipolar cycloaddition works in vitro with Laby-Hexyn derivatives, these were immobilized on 96 well plate (Nunc Maxisorp). 2 μg of either LabyA1-Hexyn, LabyA2-Hexyn or LabyA1 were diluted in Coating Buffer A [BioLegend] and adsorbed in triplets to one well of the 96 well plate for 16 h at 4° C. Cycloaddition reaction of Biotin-azide was performed as given in material and methods. Cycloaddition reaction mix without Biotin-Azide served as a control.

(64) The data demonstrate that the dipolar cycloaddition of Biotin-azide to immobilized Laby-Hexyn derivatives works well indicated by the pronounced OD.sub.450nm. In contrast, dipolar cycloaddition does not work with non-alkenylated Laby (FIG. 12).

Example 4: Effects of Labyrinthopeptins A1 and A2 on RSV Induced Cell Death and RSV Infection

(65) Labyrinthopeptins A1 and A2 Inhibit RSV Induced Cell Death

(66) A cell-based screening system was used to determine the antiviral effect of labyrinthopeptin A1 and A2 against the human respiratory syncytial virus (RSV, also called hRSV). A schematic representation of the screening system is depicted in FIG. 13.

(67) HEp-2 cells, which are stably expressing the reporter gene of a firefly luciferase (FF-luc), were seeded in a 96-well plate in 200 μl appropriate media. The incubation time has been set to 72 h at 37° C. with a MOI of 3 to obtain a useful measuring window between uninfected and infected cells. After 72 h the cells have been lysed and the luminescence produced by the FF-luc was measured using a plate luminometer (in RLU). The cells were infected by RSV in the presence of different concentrations of labyrinthopeptin A1 and A2. RSV is a lytic virus and kills infected cells. Therefore, unrestricted infection and spread of RSV will lead to cell death and as a result of this, decrease of luciferase gene expression. The number of surviving cells is indirectly proportional to the virus infection/replication efficiency. In other words, the more cells survive, the less RSV was able to infect cells. Cell survival can be assessed and quantified either by determination of total viable cells (e.g. by staining with crystal violet, which stains remaining cells; compare FIG. 14). Moreover, cell survival is proportional to residual luciferase expression. To validate our assay, we used ribavirin, a guanosine nucleoside analogue, which is known to inhibit RSV replication in cell culture (FIG. 15). The half maximal inhibitory concentration (IC.sub.50) has been calculated for labyrinthopeptin A1 IC.sub.50=3.87 μM in 6 independent experiments (FIG. 16A) and for labyrinthopeptin A2 IC.sub.50=47.93 μM in 3 independent experiments (FIG. 16B).

(68) Labyrinthopeptins A1 and A2 Inhibit RSV Infection

(69) Wild-type RSV (i.e. hRSV) infection and intracellular RSV-P staining was used to determine IC50 of labyrinthopeptin A1 and A2 (see FIG. 16 C).

(70) Therefore, 1×10.sup.5 HEp 2 cells seeded in a 12-well plate were inoculated for 4 h with the wild-type RSV at a multiplicity of infection (MOI) of 1 on a horizontal shaker. Inoculation was done together with different concentrations of labyrinthopeptin A1 or A2. After 4 h the cells were washed with sterile PBS and incubated at 37° C. 18 hours post infection the cells were detached by trypsinization and fixed in fixation buffer (0.5% paraformaldehyde, 1% fetal calf serum [FCS] in phosphate-buffered saline [PBS]) for 30 min at 4° C. Subsequently, the cells were permeabilized with a saponin-containing permeabilization buffer (0.1% saponin, 1% FCS in PBS) for 20 min at 4° C. Afterwards, the cells were stained for 30 min at 4° C. with an RSV-P-specific antibody (26D6G5C6) diluted 1:500 in permeabilization buffer. Subsequently, the cells were washed with PBS, and bound antibodies were detected by incubation for 30 min at 4° C. with mouse-specific Alexa 488 secondary antibodies (Thermo Fisher Scientific) at a 1:200 dilution in permeabilization buffer. The stained cells were washed twice with PBS and analyzed using an Accuri C6 and FlowJo software.

(71) The half maximal inhibitory concentrations (IC50) have been calculated for labyrinthopeptin A1 IC50=0.39 μM and for labyrinthopeptin A2 IC50=4.97 μM (see FIG. 16 C).

Example 5: Characterization of the Susceptibility of the Chikungunya Virus (CHIKV) Glycoprotein-Mediated and Stomatitis Indiana Virus (VSV) Glycoprotein-Mediated Cell Entry Process to Pharmacological and Immunological Inhibition and Cellular Restriction

(72) Here, use is made of lentiviral vectors carrying CHIKV glycoproteins E1 and E2 or VSV glycoprotein on their surface for studying properties of the CHIKV or VSV glycoprotein-mediated entry process and their susceptibility to pharmacological and immunological inhibition, as well as to restriction by cellular antiviral IFITM proteins.

(73) Treatment of 293T cells with Labyrinthopeptin A1 resulted in dose-dependent reduction of transduction efficiency upon challenge with CHIKV glycoprotein-expressing pseudotypes, yielding an IC.sub.50 value between 0.5-1.7 μM; see FIG. 17. Introduction of sublineage-specific mutations in CHIKV glycoproteins did not grossly modulate the entry efficiency of lentiviral vectors, and all variants remained susceptible to neutralization by a monoclonal antibody targeting CHIKV E2. The capability of cellular IFITM proteins to restrict CHIKV glycoprotein-mediated cell entry of lentiviral vectors was interrogated in 293T cell lines stably expressing individual C-terminal HA-tagged human IFITM proteins. Interestingly, expression of IFITM1-HA, IFITM2-HA and IFITM3-HA on target cells resulted in a 2-fold reduction of transduction efficiency, respectively, as compared to vector-expressing cells. The necessity of appropriate post-translational modification of IFITM proteins, including palmitoylation and ubiquitination of conserved residues, for their antiviral activity, as well as the species-specificity of IFITM proteins' antiviral capacity are currently investigated. Interestingly, selected CHIKV glycoprotein variants seem to display increased susceptibility to IFITM protein-mediated restriction.

(74) The VSV glycoprotein-mediated entry process as well as its susceptibility to inhibition by Labyrinthopeptin A1 was analyzed in the same manner as described for CHIKV, above. Treatment of 293T cells with Labyrinthopeptin A1 resulted in dose-dependent reduction of transduction efficiency upon challenge with VSV glycoprotein-expressing pseudotypes, yielding an IC.sub.50 value as shown in FIG. 17.

Example 6: Effects of Labyrinthopeptins on TBEV Infection

(75) 1.5×10.sup.4 Vero-B4 cells were seeded in growth medium (DMEM; 10% FBS) onto a 96 well plate and incubated for 24 h at 37° C. and 5% CO.sub.2. After removal of the medium, LabyA1, Laby A2 or a 1:1 mixture of LabyA1/LabyA2 was added to the cells in 26.5 μl medium. After 30 min of incubation at 37° C., cells were infected with TBEV (Toro isolate) at a MOI of 0.01 for 1 h. In the resulting total volume of 64 μl per well, top concentration of Laby was 50 μg/ml with serial 3-fold dilutions down to 0.07 μg/ml being applied. After removal of the inoculum, infected cells were cultivated for 3 days in a mixture of Avicel and DMEM. Eventually, infected cells were fixed with 6% Formaldehyde. After permaebilization with TritonX, TBEV-envelope protein was detected by respective primary and HRP-linked secondary antibodies. The enzymatic reaction was performed using TrueBlue Peroxidase substrate. Pictures of cell culture plates were taken with the ChemiDoc Imaging System [BioRad]; see FIG. 18. IC.sub.50 values were estimated by visual inspection of the wells. In the picture, areas of infection appear black and non-infected areas appear white; see FIG. 18.

(76) The IC.sub.50 values for LabyA1 were 24.10 μM and 50 μg/ml, respectively; the IC.sub.50 values for LabyA2 were >25.99 μM and >50 μg/ml, respectively; and the IC.sub.50 values for the combination of LabyA1 and LabyA2 were 8.34 μM and 16.67 μg/ml, respectively; see Table 4.

Example 7: Effects of Labyrinthopeptins A1 and A2 on ZIKV Infection

(77) Cultures of Huh-7.5 cells (human hepatocarcinoma cell line) were treated with various concentrations of either LabyA1, LabyA2 or an equivalent combination of LabyA1 and LabyA2 for 30 min. Cells were subsequently infected with ZIKV (Strain MR766-NIID) at a MOI of 0.5 PFU for 2 h at room temperature. Unbound viral particles were removed and infected cells were incubated for 48 h. Hereafter viral RNA was isolated from the cell culture supernatant (150 μl) using the NucleoSpin® 96 Virus Kit [Macherey-Nagel] according to the vendor's manual. RNA was quantified by absorbance and 2.5 μg were reversely transcribed via RevertAid Reverse Transcriptase [Thermo] with RT-Primer [5′-GGTTTCCCAGCTTCTCCTGG-3′] (SEQ ID NO:5). 100 ng of reverse transcribed RNA were subjected to SYBR-green based quantitative RT-PCR using the LightCycler®480 with LightCycler®480 SYBR Green I Master [Roche] and a ZIKV-specific forward and reverse primer pair (5′-AAAAACCCCATGTGGAGAGG-3′ (SEQ ID NO:6) and 5′-CATTCCTTCAGTGTGTCACC-3 (SEQ ID NO:7)′, respectively). The absolute number of ZIKV-genome copy equivalents (GCE) was determined via standard curves generated from plasmids with known concentrations carrying the respective amplified fragment of the ZIKV genome. Values are ±SEM; n=4. LabyA1 (IC.sub.50=4.6 μg/ml); LabyA2 (IC.sub.50=5.4 μg/ml); combination LabyA1 and LabyA2 (IC.sub.50=3.5 μg/ml).

Example 8: Effects of Labyrinthopeptins A1 and A2 on HCV Infection

(78) Huh 7.5 Firefly luciferase expressing cells (Huh 7.5 Fluc) cells were seeded in 96 well plates (10*10.sup.3 cells/well) and incubated overnight (18 hours approx) at 37° C. with 5% CO.sub.2 supply. Huh 7.5 Fluc cells are a hepatoma cell line stably expressing firefly luciferase protein which is used to measure cell viability.

(79) The following day, labyrinthopeptin at various concentrations was added to medium containing HCV JcR2a generated in culture (445.5 μL medium containing virus was transferred to 8 eppendorfs, each of which contained 4.5 μL compound at various concentrations to achieve desired final concentrations; i.e 50 μM, 25 μM etc) the 9th eppendorf, a positive control, contained DMSO solvent. A hepatitis C virus JcR2a reporter construct was used. (A genotype 2, starin A, chemiric construct which has a Renilla luciferase gene fused in frame its open reading frame. Viral genome translation and replication is relative to Renilla luciferase expression).

(80) The virus/compound preparation was then inoculated to the cells, in duplicates, and incubated for 4 hours at 37° C. with 5% CO.sub.2 supply. After 4 hours the medium containing virus/compound was aspirated from the cells following which cells were washed with sterile PBS, 200 μL per well, to removal any residual virus or compound.

(81) The cells were replenished with 200 μL/well DMEM and incubated for 48 hours at 37° C. with 5% CO.sub.2 supply. After 48 hours, the medium was aspirated from the wells and then the cells were washed twice with 200 μL PBS. Cells were then lysed with 1× passive lysis buffer and the lysate was analysed on a Berthold plate luminometer.

(82) Virus entry into the cells was quantified by measuring Renilla luciferase expression. The read-outs from wells that were treated with compounds were normalized to the read-out from wells that were treated with DMSO control. A decrease in Renilla luciferase expression, with stable expression of firefly luciferase, is relative to ability of the compound to inhibit virus entry into the cells.

(83) As can be seen from FIG. 20A, LabyA1 inhibits HCV virus entry with an IC50 of 1.05 μM and an IC90 of 9.163 μM. FIG. 20B shows that LabyA2 inhibits HCV virus entry with an IC50 of 1.728 μM and an IC90 of 24.9 μM. No cytotoxicity was observed at the tested LabyA1 and LabyA2 concentrations.

Example 9: Cell-Based High-Throughput Screening (HTS) System

(84) A cell-based screening system was used to determine the cytotoxic concentration (CC50) of labyrinthopeptin A1 and A2.

(85) 1×10.sup.4 HEp-2 cells, which are stably expressing the reporter gene of a firefly luciferase (FF-luc), were seeded in a 96-well plate in 200 μl appropriate media. After 72 h of incubation at 37° C. in the presence of increasing concentrations of labyrinthopeptin A1 or A2 up to 100 μM, the cells have been lysed in 35 μl lysis buffer and the extinction of the FF-luc (RLU) was measured using a plate luminometer (Berthold). The number of surviving cells is indirectly proportional to residual luciferase expression.

(86) The half maximal cytotoxic concentration (CC50) has been calculated for labyrinthopeptin A1 CC50=79.70 μM. There was no cytotoxic affect for labyrinthopeptin A2 detectable up to a concentration of 100 μM.

Example 10: Summary of Anti-Viral Effects of Labyrinthopeptins

(87) Table 4 shows the IC50 values of the anti-viral activities of LabyA1, LabyA2, the combination of LabyA1 and LabyA2 (LabyA1/A2), the LabyA1 derivative “LabyA1-hexyn” (herein also called “LabyA1-Hexyn”), the LabyA2 derivative “LabyA2-hexyn” (herein also called “LabyA2-Hexyn”), as well as the combination of LabyA1-hexyn and LabyA2-hexyn (LabyA1/A2-hexyn).

(88) TABLE-US-00004 TABLE 4 IC50 values (in μM and μg/ml) of the anti-viral activity of labyrinthopeptins, combinations of labyrinthopeptins and labyrinthopeptin derivatives. LabyA1/A2 - LabyA1 LabyA2 LabyA1/A2 LabyA1-hexyn LabyA2-hexyn hexyn μM μg/ml μM μg/ml μM μg/ml μM μg/ml μM μg/m μM μg/ml DENV 1.78 3.7 8.00 15.4 1.30 2.6 1.70 3.7 7.03 14.2 0.96 2.0 RSV 0.39.sup.1 0.8.sup.1 4.97.sup.1 9.56.sup.1 — — — — — — — — KSHV 2 4.2 15 28.9 — — — — — — — — TBEV 24.10 50 >25.99 >50 8.34 16.67 — — — — — — CMV 1.3 2.7 5.4 μM 10.4 — — — — — — — — CHIKV 0.5-1.7 1.0-3.5 — — — — — — — — — — VSV 1.1-3.7 2.3-7.7 — — — — — — — — — — ZIKV 2.22 4.6 2.81 5.4 1.75 3.5 — — — — — — HCV 1.05 2.18 1.728 3.32 — — — — — — — — LabyA1 = LabyA2 = LabyA1-hexyn = LabyA2-hexyn = 2075.33 g/mol 1924.16 g/mol 2170.45 g/mol 2019.28 g/mol .sup.1These values are measured by intracellular RSV-P staining (see FIG. 16C).

(89) The present invention refers to the following amino acid sequences:

(90) SEQ ID NO: 1: Amino acid sequence of the labyrinthopeptin of the present invention

(91) SEQ ID NO: 2: Amino acid sequence of the labyrinthopeptin derivative of the present invention

(92) SEQ ID NO: 3: Amino acid sequence of LabyA1

(93) SEQ ID NO: 4: Amino acid sequence of LabyA2

(94) TABLE-US-00005 SEQ ID NO: 5: RT-Primer 5′-GGTTTCCCAGCTTCTCCTGG-3′ SEQ ID NO: 6: ZIKV-specific forward primer 5′-AAAAACCCCATGTGGAGAGG-3′ SEQ ID NO: 7: ZIKV-specific reverse primer 5′-CATTCCTTCAGTGTGTCACC-3′