WO2004083462A2

WO2004083462A2 - Replication assay for discovering antiviral substances using a high throughput screening (hts) method

Info

Publication number: WO2004083462A2
Application number: PCT/EP2004/002176
Authority: WO
Inventors: Arnold Paessens
Original assignee: Bayer Healthcare Ag
Priority date: 2003-03-17
Filing date: 2004-03-04
Publication date: 2004-09-30
Also published as: WO2004083462A3; DE10311563A1

Abstract

The invention relates to an high throughput screening (HTS) testing method based on the virus of the bovine viral diarrhea (BVDV), which is analogous to the hepatitis C virus (HCV), in an in-vitro replication test with infectious BVDV on bovine cells and provided in the form of a one-pot method. The test method is suited for the simple and rapid testing of large substance banks with regard to the discovery and characterization of novel inhibitors for flaviruses.

Description

Replication assay for the detection of antiviral substances using HTS methods

The search for new, better-acting and better-tolerated active ingredients that can be used as therapeutic agents for the treatment of viral infections, in particular the infection with hepatitis C virus (HCV), is an urgent task of pharmaceutical research, in the context of which hundreds of thousands of test compounds are tested for theirs Effectiveness must be examined.

For this purpose, the effect on virus-specific polymerases, helicases or proteases is usually examined in the enzymatic test, or the effect of the test substance on the native, unchanged virus and its behavior on suitable target cells is examined in the in vitro model.

The large number of samples to be tested causes considerable logistical problems that can only be solved by automating the test processes.

This is the state of the art in the case of enzymatic tests, where large sample quantities can be examined using the so-called high-throughput screening method (HTS method). Such tests can and will also be carried out in the case of the enzymes of HCV (Journal of Virology (1997) 71, 11, 8416-8428; V. Lohmann et al.) Or J. Virology (1993) 67, 3835 - 3844, RL Bartenschlager et al.).

Tests based on proteins and enzymes are particularly suitable for carrying out in HTS, because the available read-outs have exact and fast evaluation options, for example as an optical method by measuring the fluorescence. A good signal-to-noise ratio can be achieved by selecting suitable color indicators. However, the basic disadvantage of the enzymatic test is that it only detects a small part of all viral and or cellular systems involved in the replication cycle and that the optimal conditions (cofactors, ionic conditions, pH value, etc.) are insufficiently reflected in the natural environment of the host cell.

In contrast, the investigation of the replication of viruses in cell culture allows the simultaneous checking of all steps involved in virus replication under largely natural conditions (replication assay).

Replication assays are therefore particularly suitable for mapping the known enzymatic processes under physiological conditions and for detecting known and unknown, virus-coded or host-specific processes which are essential for replication and which, in principle, cannot be reproduced in the enzymatic test. With regard to HCV, these are, for example, the attachment to and the penetration into the cell to be infected, as well as the effect of virus-specific regulatory mechanisms or processes in which virus and cell-specific factors interact or the HCV-specific process steps within the replication system (see below) that are not shown virus replication.

In general, the virus-specific steps, which run in vitro - if at all - only with the addition of extremely complex reaction additives, are reproduced in suitable host cells in the replication assay and work e.g. optimal within the replication process - after a relevant experimental infection system, a cell virus replication system has the highest relevance for an infection.

A replication assay not only provides information on the antiviral effects of test compounds but also information on the cytotoxicity / cell tolerance (selectivity index) of the test compounds and thus a first measure for assessing the potential of the active compound. In addition, only those active compounds will be noticed as antiviral that are also cell-compatible.

Against this background, the replication test is particularly suitable for finding new, more effective and better tolerated inhibitors of HCV.

There is no in vitro replication test procedure for HCV comparable to natural infection; Infectious HCV viruses cannot be propagated in vitro in such a way that such systems can be used for test purposes.

However, it was possible to generate a so-called replicon system, in which subgenomic RNA elements of the HCV are used, and which can be multiplied in human hepatoma cells under selection pressure (Journal of Virology (2001) 75.3, 1252-1264; T. Pietschmann et al . or Science (1999) 285, 110-113; V. Lohmann et al. or Science (2000) 290, 1972-1974; KJ Blight et al.). This technology based on the HCV replicon system can be used as a screening for the detection and evaluation of HCV inhibitors.

The replicon system can be used to screen for HCV inhibitors. However, as mentioned, it is artificial and does not depict the natural infection of the HCV (for example, no infectious viruses are formed which can be used for further serial infection). In particular, the use of the complete genome of the HCV by means of a replicon system is very inefficient and therefore practically cannot be used for a HTS. Viruses related to HCV and belonging to the family of flaviviruses can be used alternatively and efficiently for HTS due to their properties for in vitro reproduction. This was principally confirmed in the work of SG Scott et al. shown using the example of the virus of bovine viral diarrhea (BVDV) (Proc. of the Nat. Acad. of Sciences (2000) 97, 14, 7981-7986). BVDV is assigned to the pestiviruses within the flavivirus family. The basic suitability of the BVDV as a surrogate model for HCV for the detection and evaluation of antiviral agents was recently described by VE Buckwold et al. (Antiviral Research 60 (2003) 1-15). There are two isolates for in viti-o cultivation, which can in principle be used for testing for HCV inhibitors; here the BVDV strain NADL is in the foreground, which induces cytopathic properties on Madi Darby bovine kidney cells (MDBK). Surprisingly, it was found that BVDV on MDBK cells is suitable for HTS in the emtopf process.

The basic disadvantage of the replication assay is that the endpoint, namely the assessment of the behavior of the cells exposed to the virus, has not yet been able to be determined using the HTS method. Although it is known to use genetically modified HCV variants with suitable indicator systems such as, for example, the luciferase gene, variants of the HCV which have been modified by cloning reporter genes do not necessarily behave like unchanged strains of the HCV.

Test methods that are used in vitro on the basis of the HCV replicon system are time and resource intensive and cannot be automated as would be necessary for the investigation of a large number of test substances, so they are not in the previously known form for testing large substance libraries suitable.

This is mainly due to the behavior of the HCV replicons in vitro. The replicons do not induce a characteristic cytopathic effect on the target cells, preferably on liver cells.

So-called secondary tests, such as the measurement of virus-specific RNA (e.g. via PCR) or color reactions, which can be made visible via a coupled test cascade using indicator genes, are a method for evaluating HCV-specific changes in replicon-transfected cells.

However, all of these methods are extremely resource-intensive and therefore only insufficiently suitable for testing large substance banks.

It is therefore the task of establishing a replication assay suitable for high-throughput screening (HTS), which uses infectious viruses and suitable ones Host cells, can be performed as a primary test using an optical read-out with good signal / noise ratio.

According to the invention, suitable replication assays are to be understood as those assays which can be carried out using host cells as a one-pot assay without further work-up steps.

It has now surprisingly been found within the scope of the invention that BVDV and in particular the NADL isolate from BVDV also multiply in the presence of significantly lower serum concentrations that were previously used in the prior art. Up to now common growth conditions for host cells of the BVDV were growth media that contain at least 10% fetal calf serum (FCS).

This is of particular importance because high serum concentrations in cell culture media from viral replication systems on suspension cells are unsuitable for optical detection methods based on fluorescence or absorption measurement methods, which is due to the presence of disruptive proteins in the fetal calf serum (FCS) and the enzymatic originating from them Activity. The use of optical detection methods has therefore hitherto been limited to cell culture systems with adherent cells in which the proteins in the supernatant of the culture, which disrupt the optical test, can easily be pipetted off.

The present studies have now shown that significantly lower serum concentrations are sufficient to propagate BVDV and in particular the NADL isolate from BVDV. Surprisingly, it has now been found that BVDV increases in suspension cells even at low FCS serum concentrations such as 0.2 to 2%, a concentration at which the interference proteins contained in the FCS no longer interfere with some optical detection methods.

The low serum concentrations also have the advantage that they have a favorable influence on the physico-chemical properties of the test compounds. A low serum concentration, compared to a higher serum concentration, can lead to test compounds having different binding properties or cell penetration properties.

Based on this observation, the present invention provides an (HTS) screening method for fast, inexpensive and infection-relevant testing of large ones

Substance banks with more than 500,000 test compounds or even larger substance libraries with more than 1 million test compounds with the aim of finding new BVDV inhibitors presented as an analog model for HCV inhibitors, which is based on the fact that the literature-known replication test for the in vitro multiplication of BVDV, which was previously only used on a small scale for testing active compounds against BVDV, now also after targeted changes in the test conditions is feasible as a HTS.

An isolate to be used according to the invention in connection with low FCS concentrations is, for example, the NADL isolate of the BVDV, which changes MDBK cells in such a way that viral infections can be visualized by means of color reagents via changed biochemical processes in the optical test, since the infection of MDBK cells with the NADL isolate in the presence of low FCS concentrations leads to the fact that the infected cells are brought into a physiological state, which enables the discrimination of protected virus-destroyed cells using color reagents.

Surprisingly, XTT, MTT, neutral red and in particular alamar-Blue (alamar-Blue ™, Laboserv GmbH, Giessen, Germany; USP 5501959) or fluorescent dyes such as e.g. Fluorodiacetate found.

The prerequisite for this type of detection is the surprising finding that some infection-relevant strains of BVDV, such as the NADL isolate, also multiply in cells whose culture medium contains only small amounts of fetal calf serum (FCS) as an essential growth factor. This finding is an essential prerequisite for the method according to the invention, since the higher concentrations of FCS which were previously considered essential disrupt the use of color reagents and therefore preclude them.

The cytopathic and cytotoxic effects induced by the NADL isolate can, as was surprisingly found, by adding trypsin (in concentrations of 0.5 to 2 μg / ml, preferably 2 μg / ml) at the FCS concentrations according to the invention in conjunction with the concentrations according to the invention Color reagents significantly strengthen, and thereby facilitate a HTS to find active substances against the BVDV HCV.

It was further found that alamar blue and fluorescent dyes such as e.g. Fluorodiacetate can be used, which can be measured using optical methods using the HTS technique.

It was further found that the combination according to the invention consisting of natural BVDV isolate, cell culture medium with a low FCS concentration according to the invention, in the presence of 2μg / ml trypsin, detection dye according to the invention and MDBK cells in one HTS can be configured as a one-step process so that this can be carried out in 384-well or 1536-well plates.

However, the test method according to the invention can preferably be used for HCV to test large substance libraries and thus to find new substance classes with an effect against BVDV. These new substance classes can also be used for other viral infections analogous to HCV, in particular infections caused by the West Nile Virus, infections caused by the early summer Meningoencephalitis Virus (TBE), infections caused by Dengue viruses, infections caused by the virus of the Japanese encephalitis and infections caused by the yellow fever virus.

The test methods according to the invention are understood in particular to mean those test methods in which target cells sensitive to native viruses, in particular BVDV viruses, in particular MDCK cells in a cell culture medium, for example Earle's MEM, with the addition of only small amounts of fetal calf serum and of trypsin in multi-well Plates together with said viruses and a test or reference substance for a period of 2-20 days, preferably 5-10 days, in which saturation moisture resulting for the respective temperature is cultivated, then a colorant, preferably alamar, as read-out. Blue or fluorescein diacetate are added, then incubated again and subsequently measured optically in the HTS method.

It was further found that the combination according to the invention consisting of natural BVDV isolate, cell culture medium with a FCS concentration according to the invention of 2 μg / ml trypsin, detection dye according to the invention and MDBK cells can be configured in a HTS as a one-step process so that it can be carried out quickly , and inexpensive discovery of new active substances against HCV can be used.

New active substances in the sense of the invention are preferably small-molecular active substances which intervene in the replication of the BVDV / HCV virus and specifically inhibit its multiplication, with small-molecular compounds in the context of the invention having a molecular weight in the range from about 100 to 500 or 100 to 1000 and be understood beyond.

However, high-molecular compounds, such as. B. Antibodies that can be produced in large numbers in libraries come into question.

The test is also suitable for using the inhibitors identified therein as inhibitors of BVDV in the indication area viral bovine diarrhea in veterinary medicine. The invention relates to methods for finding inhibitors of viral replication (replication assay), in particular inhibitors of HCV, characterized in that an analog virus isolate is incubated together with sensitive target cells in a cell culture medium (incubation time preferably 2-20 days), which fetal calf serum in contains a concentration of less than 5%, and a detection dye is added and, after subsequent incubation, is measured optically.

A method according to the invention is a method according to the method described above, the medium additionally containing trypsin in a concentration of 2 μg / ml.

Also according to the invention is a method as described above, wherein a cell culture medium containing fetal calf serum is used in a concentration of 1-2 vol%.

The invention relates to a method according to the method described above, wherein an isolate of BVDV is used as the natural virus isolate and MDBK cells are used as sensitive target cells.

The invention also relates to a method according to the method described above, the analog virus isolate being the NADL isolate from BVDV, and, as a cell culture medium, a medium which contains fetal calf serum in a concentration of 0.1-5% by volume, preferably 1 Contains -2 vol%.

The invention also relates to a method according to the method described above, alamar blue or fluorescein diacetate being used as the detection dye.

The invention relates to a method according to the method described above, wherein the BVDV isolate, in particular the NADL isolate from BVDV, is used as an analog virus for HCV.

One use according to the invention is the use of the replication assays described here for the detection of antivirally active inhibitors, in particular inhibitors of BVDV as an analog model for HCV in high throughput assay systems.

Another use according to the invention is the use of inhibitors of viral replication, which are found using one of the replication assays described here, for the production of a medicament for the prevention and treatment of infections.

The use of inhibitors of viral replication of BVDV / HCV, in particular the NADL isolate of BVDV, which are associated with one of the described methods for the manufacture of a medicament for the prevention and treatment of infections by HCV.

The invention also relates to the use of inhibitors of viral replication of BVDV / HCV, in particular the NADL isolate of BVDV, which were found using one of the methods described above, for the manufacture of a medicament for the prevention and treatment of infections caused by the West Nile Virus , Infections caused by the early summer meningoencephalitis virus (TBE), infections caused by dengue viruses, infections caused by the Japanese encephalitis virus and infections caused by the yellow fever virus.

According to the invention is also a method for inhibiting the replication of HCV by active substances which are found by one of the methods described here.

The invention also relates to medicaments which are produced using the active compounds found using the processes described here.

The present invention furthermore relates to medicaments which are produced using the active compounds found using the processes described here, preferably together with one or more pharmacologically acceptable auxiliaries or excipients, and their use for the abovementioned purposes.

The active substance can act systemically and / or locally. For this purpose it can be applied in a suitable manner, e.g. oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, transdermal, conjunctival, otic or as an implant.

The active ingredient can be administered in suitable administration forms for these administration routes.

Known application forms which deliver the active ingredient quickly and / or modified, such as e.g. Tablets (non-coated and coated tablets, e.g. tablets or film-coated tablets provided with enteric coatings), capsules, dragees, granules, pellets, powders, emulsions, suspensions, solutions and aerosols.

Parenteral administration can be done bypassing a resorption step

(intravenous, intraarterial, intracardial, intraspinal or intralumbal) or with the involvement of one

Absorption (intramuscular, subcutaneous, intracutaneous, percutaneous, or intraperitoneal). Application forms suitable for parenteral administration include injection and muscular preparations in the form of solutions, suspensions, emulsions, lyophilisates and sterile powders.

For the other application routes, e.g. Inhalation forms (including powder irmalators, nebulizers), nasal drops / solutions, sprays; tablets or capsules to be administered lingually, sublingually or buccally, suppositories, ear and eye preparations, vaginal capsules, aqueous suspensions (lotions, shaking mixes), lipophilic suspensions, ointments, creams, milk, pastes, powder or implants.

The active compounds can be converted into the administration forms mentioned in a manner known per se. This is done using inert, non-toxic, pharmaceutically suitable excipients. These include Carriers (e.g. microcrystalline cellulose), solvents (e.g. liquid polyethylene glycols), emulsifiers (e.g. sodium dodecyl sulfate), dispersants (e.g. polyvinylpyrrolidone), synthetic and natural biopolymers (e.g. albumin), stabilizers (e.g. antioxidants such as ascorbic acid), dyes (e.g. inorganic pigments such as iron oxides) ) or taste and / or smell.

In general, it has proven to be advantageous to administer amounts of approximately 0.001 to 10 mg / kg, preferably approximately 0.01 to 5 mg / kg body weight in the case of parenteral administration in order to achieve effective results. In the case of oral administration, the amount is approximately 0.01 to 25 mg / kg, preferably approximately 0.1 to 10 mg / kg body weight.

Nevertheless, it may be necessary to deviate from the amounts mentioned, depending on body weight, route of administration, individual behavior towards the active ingredient, type of preparation and time or interval at which the application is carried out. In some cases it may be sufficient to make do with less than the aforementioned minimum quantity, while in other cases the above upper limit must be exceeded. In the case of application of larger quantities, it may be advisable to distribute them in several individual doses over the day. example 1

In each case 1.4 × 10 ³ MDBK cells infected with BVDV are per well of a 384-well microtiter plate in the presence of 0.5 to 10% FCS (preferably 2%) and 0.5 to 10 μg / ml trypsin (preferably 2 μg / ml) sown, the wells containing suitable dilutions of the test substances or reference substance.

With suitable dilution in the sense of the invention, test substance concentrations between 0.1 to 50 μM are used, test substance concentrations from 1 to 20 μM are preferably used.

The test plates are subsequently incubated for 7 days at 37 ° C. under 5% CO ₂ and saturated moisture in the incubator. Subsequently, to determine the anti-viral effects or the cell tolerance of the test substances as detection dye Alarmar Blue (Biosource International) (or fluorescein diacetate (200 µg / ml in PBS) in an amount of 1/10 volume part of the well content is added In the case of alamar-Blue, incubated for a further 5-7 hours under the conditions mentioned above, the photometric evaluation is then carried out by measuring absorption or emission, and in the case of fluorescein diacetate, the fluorescence emission is measured after just 20-60 minutes of incubation.

Under the test conditions 2% FCS and 2 μg / ml trypsin in the culture medium, an optical density of 1.9 extinction units was measured for the cell control, while the virus control had an optical density of 0.02 extinction units. This astonishingly favorable signal / noise ratio of approximately 1: 100 is an optimal prerequisite for carrying out an HTS assay with BVDV in the sense of the task at hand.

In contrast, when using the high FCS concentrations previously used in this technology in the BVDV-infected cell culture (10% by volume FCS), extinction values for cell control amounting to 1.5 extinction units and 0.4 extinction units in virus control are found.

This low signal / noise ratio of about 1: 4 is not sufficient to carry out a sensitive HTS.

literature

1) Journal of Virology (1997) 71, 11, 8416-8428, V. Loh ann et al. 2) J. Virology (1993) 67, 3835-3844, RL Bartenschlager et al 3) Antiviral Research 47 (2000) 139-148, Yves Pommier et al. 4) J Virol. 71, 7005-7011, Hazuda et al.

5) Antiviral Chem. Chemother. 10, 63-70, Hazuda et al.

6) Journal of Virology (2001) 75.3, 1252-1264, T. Pietschmann et al.

7) Science (1999) 285, 110-113, V. Lohmann et al. > 8) Science (2000) 290, 1972 - 1974, K.J. Blight et al.

9) Proc. ofthe Nat. Acad. of Sciences (2000) 97, 14, 7981-7986), S.G. Scott et al.

10) Antiviral Research (2003) 60, 1-15, V.E. Buckwold et al.

Claims

claims

1. A method for the detection of inhibitors of viral replication (replication assay), in particular of HCV inhibitors, characterized in that

i) an analog virus isolate is incubated together with sensitive target cells in a cell culture medium which contains fetal calf serum in a concentration of less than 5% by volume,

such as

ii) a detection dye is added and optically measured after subsequent incubation.

2. The method according to claim 1, wherein the medium additionally contains trypsin in a concentration of 2 μg / ml.

3. The method according to claims 1 or 2, wherein an isolate of BVDV is used as the natural virus isolate, and MDBK cells are used as sensitive target cells.

4. The method according to claim 3, wherein a cell culture medium containing fetal calf serum is used in a concentration of 1-2 vol%.

5. The method according to claims 3 and 4, wherein the analog virus isolate is the NADL isolate from BVDV.

6. The method according to claims 1-5, wherein alamar blue or fluorescein diacetate is used as the detection dye.

7. The method according to claims 3-6, wherein the BVDV isolate, in particular the NADL

Isolate from BVDV is used as an analog virus for HCV.

8. Use of replication assays of claims 1-7 for the detection of antivirally active inhibitors, in particular inhibitors of BVDV as an analog model for HCV in high throughput assay systems.

9. Use of inhibitors of viral replication, which are found by a method according to claims 1 to 8, for the manufacture of a medicament for the prevention and treatment of infections.

10. Use of inhibitors of viral replication of BVDV / HCV, in particular the NADL isolate of BVDV, which were found by a method according to claims 1-8, for the manufacture of a medicament for the prevention and treatment of infections by HCV.

11. Use of inhibitors of viral replication of BVDV / HCV, in particular the NADL isolate of BVDV, which have been found by a method according to claims 1-8, for the manufacture of a medicament for the prevention and treatment of infections caused by the West Nile Virus, Infections caused by the early summer meningoencephalitis virus (TBE), infections caused by dengue viruses, infections caused by the Japanese encephalitis virus and infections caused by the yellow fever virus.

12. A method for inhibiting the replication of HCV by active substances which have been found by a method of claims 1-8.

13. Medicament produced according to one of claims 9 to 11.