EP1319185A1 - Method for identifying compounds with anti-herpes activity - Google Patents

Method for identifying compounds with anti-herpes activity

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Publication number
EP1319185A1
EP1319185A1 EP00945756A EP00945756A EP1319185A1 EP 1319185 A1 EP1319185 A1 EP 1319185A1 EP 00945756 A EP00945756 A EP 00945756A EP 00945756 A EP00945756 A EP 00945756A EP 1319185 A1 EP1319185 A1 EP 1319185A1
Authority
EP
European Patent Office
Prior art keywords
compounds
helicase
primase
heφes
mutation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00945756A
Other languages
German (de)
French (fr)
Inventor
Gerald Kleymann
Ulrich Betz
Rüdiger Fischer
Martin Hendrix
Guy Hewlett
Veniamin Pevzner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
KLEYMANN, GERALD
Bayer AG
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Bayer AG
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Filing date
Publication date
Application filed by Bayer AG filed Critical Bayer AG
Publication of EP1319185A1 publication Critical patent/EP1319185A1/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/54—Nitrogen and either oxygen or sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/035—Herpes simplex virus I or II

Definitions

  • This invention relates to a method for identifying compounds with anti-herpes activity and to medicaments for the treatment of herpes infections in a mammal by inhibiting the herpes helicase-primase enzyme complex.
  • the goal of inventions in the field of the pharmaceutical area is to supply medications or therapies to treat a patient in a tolerable way that a desired therapeutic effect is achieved.
  • a compound that inhibits two essential targets in the life cycle of a given pathogen simultaneously may be more active due to cumulative inhibitory effects as demonstrated by the analogous case of combination therapy in the past which is often superior to a (single compound chemo) mono-therapy where the drug targets only one relevant binding pocket on a single target protein or on one subunit of a complex.
  • a stronger binding may also be observed based on avidity effects.
  • the better binding characteristics and or the cumulative inhibitory effects may result in a superior therapeutic treatment ideally accompanied by a better tolerability which may result from a lower dosage or pill burden during treatment.
  • He ⁇ esviruses are enveloped double stranded DNA viruses which infect cells that carry negative charged structures such as heparansulfate and or glycosaminoglycans in addition to a he ⁇ es viral entry mediator on their surface.
  • One key feature of these viruses is their ability to remain latent in their host for life after primary infection and to reactivate more or less frequently from a pool of latent infected cells upon diverse internal and external stimuli.
  • D ⁇ A replication proteins defining novel antiviral targets, 1993, Antiviral Research,
  • viruses were grouped in 3 sub- families, namely ⁇ -(HHVl to 3), ⁇ -(HHN5 to 7), and ⁇ -(HHN4 and HHN8) he ⁇ esviruses. Common names were derived from the clinical symptoms they cause or historical reasons e.g.
  • He ⁇ es simplex virus 1 or 2 (HSN-1 or -2 the cause of he ⁇ es labialis and genitalis) is used for HHN1 or 2
  • the Naricella-zoster virus (NZN causing chickenpox or zoster) is synonym to HHN3
  • Epstein-Barr virus (EBN) and Cytomegalo virus (HCMN) are synonyms for HHN4 and 5, respectively.
  • Incidence and prevalence data range from below 10 to more than 90 % of the population being infected with one or more he ⁇ esvirases depending on the HHN involved and the age, sex, social status and geographical aspects of the host.
  • he ⁇ esviruses are ubiquitous human pathogens causing a variety of disease ranging from benign illness interfering with normal day activities to life or sight threatening disease especially in immunocompromised patients (keratitis, disseminated disease or retinitis in AIDS patients), pregnancy (abortion, deafness) and newborns (hepatitis, encephalitis), there is a strong medical need for a safe and efficient treatment and much effort has been expended in the search for effective antiviral agents .
  • Acyclovir a selective and specific inhibitor of viral replication, was a true milestone in the development of antiviral drugs in the late 1970s. Newer nucleosidic drags that are similar to acyclovir such as penciclovir or the more convenient pharma- cokinetically optimized pro-drugs like valacyclovir and famciclovir were launched in the late 1990s. Nucleosides indeed became the treatment of choice in the he ⁇ es indication, however, nucleosides are pro-drugs and have to be phosphorylated by the viral thymidine kinase (TK) and subsequently by cellular kinases for activity upon the viral DNA-polymerase. If the virus does not express a functional TK e.g.
  • TK viral thymidine kinase
  • HHV1 strain or TK negative viruses such as HCMN
  • the D ⁇ A-Pol has not the optimal primary structure
  • the potency of the drug diminishes
  • the selectivity index is significantly smaller
  • higher doses have to be administrated
  • adverse effects are more likely to be associated with treatment.
  • nucleosides are obligate or non-obligate chain terminators of D ⁇ A-polymerization they are potentially mutagenic which is well documented for ganciclovir.
  • broad spectrum anti- he ⁇ esvirus activity, efficacy especially upon delayed treatment, safety and resistance are goals for the next generation of drugs directed against novel antiviral targets.
  • WO 97/24343 describes a method to identify inhibitors with anti-he ⁇ es properties by selecting compounds which, upon binding to the D ⁇ A-helicase-primase complex, stabilize the latter.
  • a disadvantage of this method is that often compounds are identi- fied that have not a suitable selectivity index in vitro or the necessary tolerability in vivo, and thus, are often not suitable for treatment of he ⁇ es infections due to side effects.
  • the goal of this invention is therefore to provide a method to identify alternative or more active compounds which have a better selectivity index and/or tolerability.
  • the goal of the present invention is to provide a method for identifying compounds which are active against acyclovir-resistant viral mutants.
  • the present invention solves the abovementioned problems by providing a method for identifying compounds with antiviral activity, characterized in that those compounds are selected in the presence of which resistant viral mutants can be selected, wherein at least one selected viral mutant carries at least one mutation in the primase gene (e.g. UL52 in the case of HSV) and another selected viral mutant carries at least one mutation in the hehcase gene (e.g. UL5 in the case of HSV) or a selected viral mutant carries at least one mutation in the helicase and at least one mutation in the primase gene.
  • the primase gene e.g. UL52 in the case of HSV
  • another selected viral mutant carries at least one mutation in the hehcase gene (e.g. UL5 in the case of HSV)
  • a selected viral mutant carries at least one mutation in the helicase and at least one mutation in the primase gene.
  • the method is further characterized in that the primase and helicase are at least 50% homologue to the primase and helicase of he ⁇ es simplex virus 1 based on the nucleotide sequence or the amino acid sequence, whichever is higher.
  • the method is further characterized in that the virus is a he ⁇ es virus. In another preferred embodiment, the method is further characterized in that before determining the generation of resistant viral mutants in the presence of said compounds,
  • the compounds are tested for their ability to inhibit the helicase and b) the compounds are tested for their ability to inhibit the primase c) such compounds are selected, that inhibit the helicase and the primase
  • the method is further characterized in that such compounds are selected, that bind simultaneously to the helicase and primase subunits of the helicase-primase complex.
  • compounds bind at the interface of the helicase and primase subunits of the helicase-primase complex of a he ⁇ es simplex virus (and homologous regions of other he ⁇ es virus helicase primase enzymes) and thereby cause the inhibition of essential enzymatic activities of the helicase-primase complex (either the minimal heterodimeric complex UL5/UL52 or the native heterotrimeric complex UL5/UL8 and UL52 in the case of a he ⁇ es simplex virus) possibly by blocking the ATP binding site competitively.
  • the compound may either be bound in a ternary complex involving residues of the helicase and the primase subunits and possibly stabilized by avidity effects or the compounds bind independently to at least one site on the helicase and the primase subunits.
  • “To bind simultaneously” here describes a state, where the very same molecule is bound to both UL5 and UL52 gene products at the same time.
  • the method is further characterized in that the compounds inhibit the DNA-dependent NTPase activity of a he ⁇ es helicase- primase.
  • One additional assay that may be ran determines the ability of test compounds to inhibit helicase-primase-associated DNA-independent NTPase activity.
  • the compounds useful in this invention do not inhibit this activity, in the way that competitively-acting nucleoside analogues inhibit this activity.
  • the method is further characterized in that the compounds inhibit the replication of a he ⁇ es viras.
  • the method is further characterized in that such compounds are selected, that have the ability to inhibit replication of a he ⁇ es virus in cell culture by at least 50 % at a concentration of less than about 10 ⁇ M.
  • the method is further characterized in that such compounds are selected, that have the ability to inhibit replication of a he ⁇ es viras in cell culture by at least 50 % at a concentration of less than about 500 nM.
  • the method is further characterized in that the he ⁇ es viras is HSV.
  • the method is further characterized in that the he ⁇ es virus is PRV or BHN.
  • the invention relates to a compound identified by the abovementioned method.
  • the invention relates to a compound which is not a nucleoside or nucleotide.
  • the present invention also relates to the use of such a compound for the preparation of a medicament for the treatment and prevention of he ⁇ es infections and to a pharmaceutical composition comprising such a compound.
  • the present invention also relates to a method for treating he ⁇ es infection in a mammal comprising the step of administering to a mammal in need of such treatment a therapeutically effective amount of the abovementioned pharmaceutical composition.
  • the present invention also relates to a method for identifying compounds, characterized in that
  • the compounds are tested for their ability to bind to at least two therapeutically relevant targets simultaneously and b) the compounds are tested for their ability to inhibit and/or activate these targets upon binding c) the compounds are tested for reduction of their ability to inhibit and/or activate these targets in the presence of at least one mutation in one target and at least one mutation in the other target or at least one mutation in either target.
  • the present invention also relates to a compound with antiviral activity, characterized in that those compounds are selected in the presence of which resistant viral mutants can be selected, wherein at least one selected viral mutant carries at least one mutation in the primase gene and another selected viral mutant carries at least one mutation in the helicase gene or a selected mutant carries at least one mutation in the helicase and at least one mutation in the primase gene.
  • the present invention also relates to a compound capable of binding simultaneously to the helicase and primase subunits of the helicase-primase complex.
  • the invention described herein overcomes the above-mentioned limitations and satisfies the medical needs by providing a method for identifying non-nucleosidic inhibitors that act directly by interfering with the essential enzymatic activity helicase-primase in a novel way and consequently inhibits he ⁇ es virus DNA repli- cation and he ⁇ es virus replication in vitro and in vivo. Furthermore, since the he ⁇ es virus helicase-primase enzyme is conserved across the he ⁇ es viridae, the inventors could demonstrate broad anti-he ⁇ es virus spectrum activity (see also table 1). The selective action of these inhibitors against he ⁇ es viruses and especially against acyclovir-resistant he ⁇ es viruses, combined with a wide margin of safety, renders the compounds as desirable agents for combating he ⁇ es infections.
  • he ⁇ es refers to any virus in the he ⁇ es family of viruses and particularly, to those he ⁇ es viruses that encode a he ⁇ es helicase-primase homologous to the he ⁇ es helicase-primase of HSV- 1.
  • the he ⁇ es family of viruses includes, but is not limited to HHV-1 to HHV-8, EHN, BHN, PRN etc.
  • He ⁇ es simplex virus 1 and 2 refers to a virus that is characterized by specific monoclonal antibodies (serotype 1 or 2).
  • helicase-primase refers to the helicase-primase complex involved in D ⁇ A replication.
  • the he ⁇ es simplex viras it is composed of the UL5, UL8 and the UL52 or the least (necessary or essential) UL5 and UL52 gene products, in the case of other he ⁇ es viruses of the corresponding homologues.
  • helicase refers to the helicase subunit of the helicase-primase complex involved in D ⁇ A replication. In the case of the he ⁇ es simplex viras it is the UL5 gene product, in the case of other he ⁇ es viruses it is the corresponding homologue.
  • primase refers to the primase subunit of the helicase-primase complex involved in D ⁇ A replication. In the case of the he ⁇ es simplex viras it is the UL52 gene product, in the case of other he ⁇ es viruses it is the corresponding homologue.
  • selectivity index refers to the quotient of the concentration were the compound reduces the viability of the cell by 50 % and the EC 50 or IC 50 (CC 5 o/IC 50 ).
  • therapeutic index refers to the quotient of the dose (lethal dose) were 50 % of the animals die and the dose (effective dose) were 50 % of the animals survive the infection (LD 50 /ED50).
  • pharmaceutically acceptable carrier or veterinarily acceptable as used herein means a non-toxic, generally inert vehicle for the active ingredient which does not adversely affect the ingredient.
  • target describes a gene product involved in the manifestation of diseases.
  • mutation in one target describes a mutation in a gene that leads to an altered amino acid sequence of the respective protein.
  • reduction describes a loss of activating or inhibiting activity in the order of at least a factor of two, preferably of one order of magnitude.
  • Compounds useful for inhibiting a he ⁇ es helicase-primase according to the above mechanism may either be identified by assaying a test compound's ability to bind to the helicase (subunit) and the primase (subunit) of the helicase-primase complex or more preferably to inhibit enzyme-associated single stranded DNA-dependent
  • NTPase activity of a he ⁇ es helicase-primase advantageously in a high throughput (HTS) assay or by assaying a test compound's ability to inhibit viral replication in a cell-based viral replication assay followed by selecting resistant viruses in the presence of compound and sequencing the helicase-primase genes of the selected mutants.
  • HTS high throughput
  • the enzymatic assay is easy to perform, but the enzyme has to be purified from he ⁇ es infected cell culture or the genes have to be cloned, expressed and produced and purified using an expression system and protein purification techniques.
  • the cell-based viral replication assay is easy to perform and possibly more sensitive than the enzymatic assay, since it mimics the real situation of a viral infection more closely, but mutant viruses have to be generated and analyzed subsequently by sequencing or by a complementation analysis to confirm the viral target helicase primase.
  • Compounds effective in either the enzymatic assay or the cell-based viral replication assay may be further assayed to determine their he ⁇ es helicase-primase binding specificity or compounds effective in a binding assay have to be evaluated in either the enzymatic assay or the cell-based viral replication assay.
  • assays are described in one particular sequence, it should be understood that not all of these assays need to be performed for successful identification of he ⁇ es helicase- primase inhibitors. In addition, the exact order of assay may be altered, if desired. These and other procedural options can be considered by those of ordinary skill in the art. Other assays measure a test compound's ability to inhibit enzyme-mediated RNA primer biosynthesis or a test compound's ability to inhibit helicase activity.
  • Any DNA but preferably single stranded DNA with a primase consensus site for the primase assay or any DNA substrate designed to model a replication fork-like structure may be used for the helicase assay.
  • Several options are outlined in the cited review by Boehmer PE & Lehmann IR 1997 or more recently in the paper entitled High-Throughput Screening Assay for Helicase Enzymes, 1998, Analytical Biochemistry, M Sivaraja, H Giordano, MG Peterson, 265, 22-27.
  • the non-nucleoside compound is preferably further characterized by an ability to inhibit he ⁇ es helicase-primase mediated RNA primer biosynthesis or helicase activity.
  • preferred non-nucleoside inhibitors of this invention are further characterized by an ability to inhibit replication of a he ⁇ es virus in cell culture by at least about 50 % at a concentration of less than about 10 ⁇ M more preferably at a concentration as low as possible e.g. in the nM range or even less.
  • compositions and methods of this invention may be used against nucleoside non-responsive and nucleoside resistant he ⁇ es infections.
  • This aspect of the invention involves a method for treating acyclovir-resistant he ⁇ es infections in a mammal which comprises administering to the mammal an anti- acyclovir-resistant he ⁇ es effective amount of a compound as defined herein, or a therapeutically acceptable acid addition salt thereof.
  • a compound as defined herein or a therapeutically acceptable acid addition salt thereof.
  • the antiviral activity of the compounds can be demonstrated by biochemical, microbiological and biological procedures showing the inhibitory effect of the com- pounds on the replication of he ⁇ es viruses e.g. HSN-1, HSV-2, BHN etc.
  • Biochemical assay A biochemical procedure for demonstrating anti-he ⁇ es activity for compounds is described below under Biochemical assay.
  • This ATPase assay is one among numerous possible assays that are described for instance in High-Throughput Screening Assay for Helicase Enzymes, 1998, M. Sivaraja, H. Giordano and M.G.
  • the therapeutic effect of the compounds can be demonstrated in vivo in a lethal challenge model as shown in section lethal challenge in vivo animal model. Description of the Table
  • Table 1 siimmarizes the ICso or EC 50 of wild type or resistant HSN strains, the ED 50 of wild type, the relevant mutations and cross resistance patterns.
  • Example compounds inhibit acyclovir resistant HSN-1 (F) mutants and wild type HSN-1 (F) with nearly identical
  • IC 50 values Broad Spectrum activity is exemplified by examples 1, 4, 5, 6 and 7, which inhibit as different viruses as the human he ⁇ es simplex virus as well as porcine (PRN) and bovine (BHN) animal viruses. These compounds are also active on all clinical isolates tested so far (39 clinical isolates (HSN-1) and 19 clinical isolates (HSN-2)). It demonstrates for the first time that compounds that qualify the above mentioned characteristics outperform the antiviral activity of the state of the art by at least one order of magnitude.
  • HSN-1 D ⁇ A-dependent ATPase assay in vitro assay based on the inhibition of HSN-1 helicase-primase.
  • HSN-1 helicase-primase heterodimer was produced in doubly infected Sf9 (Spodoptera frugiperda) cells using recombinant baculovirases expressing the UL5 and the UL52 helicase-primase subunits.
  • the genes were amplified by PCR from HSN-1 (F) (American Tissue Culture Collection ATCC NR-733) and cloned into the baculovirus expression system (UL5 - > pFASTBACl; UL52 -> pFASTHTb) according to the Instruction Manual BAC- TO-BAC Baculovirus Expression Systems, Life-Technologies.
  • the heterodimeric enzyme was purified by IMAC-Chromatography as described in "Inhibition of He ⁇ es Simplex Virus Replication by a 2-Amino Thiazole via interactions with the Helicase Component of the UL-5-UL8-UL52 Complex", 1998, F.C. Spector, L. Liang, H. Giordano, M. Sivaraja and M.G. Peterson.
  • the released inorganic phosphate was detected colorirnetrically as described previously in "An improved assay for nanomole amounts of inorganic phosphate", 1979, P.A. Lanzetta, IJ. Alvarez, P.S. Reinach and O.A. Candia, Analytical Biochemistry, 100:95-97.
  • DNA-dependent ATPase activity was calculated from the net absorbance change in the presence and absence of inhibition.
  • Figure 1 shows a dose dependent inhibition/titration of the enzymatic ATPase activity of the HSV-1 helicase-primase with representative example 7.
  • ATPase activity of the helicase-primase heterodimer release of inorganic phosphate (Pi)
  • Pi inorganic phosphate
  • HSV-1 Walki, HSV-1F or HSV-2G were routinely propagated on African green monkey kidney cells (Nero cells; ATCC CCL-81), however, many tissues-culture lines can be used for the growth and quantification of
  • HSN He ⁇ es Simplex Virus Protocols, 1998, Ed. S.M. Brown & A.R. MacLean,
  • the titer of the virus stock was determined in a plaque assay. Briefly, Vero cells were seeded at a density of 4x10 5 cells per well of a 24 well tissue culture plate. After an incubation period of 24 hours (37°C, 5% CO 2 ) cells were infected with dilutions of the viras stock ranging from 10 " to 10A infection volume was 100 ⁇ l per well. It was removed after 1 hour incubation at 37°C, 5 % CO 2 and the cells were gently covered with 1 ml overlay medium (0.5% methyl cellulose, 0.225 % sodium bicarbonate, 2 mM glutamine, 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin, 5%
  • Viras stocks used in the experiments described here were HSV-1 F (ATCC VR-733, stock 5xl0 7 PFU/ml) and HSV-2 G (ATCC VR-
  • Antiviral activity was measured with a microtiter plate screening test system employing cells of diverse origin such as neuronal, lymphoid and epithelial lineages e.g. Vero cells (african green monkey kidney cells), MEF (murine embryonic fibroblasts), HELF (human embryonic lung Fibroblasts), NT2 (human neuronal cells) or Jurkat (humane lymphoid T-cell line).
  • Vero cells african green monkey kidney cells
  • MEF murine embryonic fibroblasts
  • HELF human embryonic lung Fibroblasts
  • NT2 human neuronal cells
  • Jurkat humane lymphoid T-cell line
  • the inhibition of the compounds on the growth, spread and resulting cytopathic effect of the viruses was analyzed in direct comparison to uninfected cells, uninfected but treated cells, infected but untreated cells and infected cells in the presence of example compounds or the reference compound Acyclovir-Natrium (Zovirax R ), a generic nucleosidic anti-He ⁇ es-drug.
  • a cell suspension was added (lxl 0 4 cells/well of a 96 well MTP) e.g. Vero-cells in Ml 99 (Medium 199) complemented with 5 % FCS, 2 mM L-glutamine and optional 100 IU/ml penicillin and 100 ⁇ g/ml streptomycin or MEF-cells in EMEM (Eagle's Minimum Essential Medium) complemented with 10 % FCS, 2 mM L-glutamine and optional 100 IU/ml penicillin and 100 ⁇ g/ml streptomycin, or HELF-cells in EMEM complemented with 10 % FCS, 2 mM L-glutamine and optional 100 IU/ml penicillin and 100 ⁇ g/ml Streptomycin, or NT2- and Jurkat-cells in DMEM (4,5 mg/1 Glucose plus Pyridoxine) complemented with 10 %
  • HSV-1 F or HSV-2 G with an m.o.i (multiplicity of infection) of 0,0025 for HELF, Vero and MEF-cells and an m.o.i. of 0J for NT2- and Jurkat-cells).
  • the plates were then incubated at 37 °C in a CO 2 - incubator (5 % CO 2 ) for several days (preferably 5 days). After the incubation period the cells in compound free wells, starting from 25 infectious centers, are completely lysed or destroyed by the cytophathic effect of replicating He ⁇ es viruses (100 % CPE).
  • the plates were first briefly analyzed under a microscope and then the read out was generated using a quenched fluorescent dye which is cleaved by the enzymatic esterase activity of viable cells.
  • the cell culture supernatant in the MTP was aspirated, the remaining cells or debri were washed once with 200 ⁇ l PBS (phosphate buffered saline) and 200 ⁇ l Fluorescein-diacetate dye containing solution were added (10 ⁇ g/ml in PBS). After an incubation of 30-90 min at room temperature the fluorescence of the MTP was recorded in a Fluorescent Ascent (Labsystems) at 485 nm excitation and 538 nm emission wavelength.
  • the viral replication is inhibited by 50 % as compared to the non infected cell control or the read out of the fluorescence based assay reaches 50 % of the signal as compared to the cell control.
  • Naturally occurring, resistant viral mutants were selected in the presence of 1 ⁇ M example 7 or of at least 100 times the IC 5 0 concentration as listed in table 1 using the cell based viral replication assay.
  • 10 000 Vero cell were seeded in 96 well MTPs as described above and incubated over night. Compound was added to the final concentration and the cells were infected with 1000 plaque forming units (m.o.i. 0,1) per well. Mutants were identified at a frequency of 1-5 per million HSV-1 F under a microscope after 3-5 days or by storing replica samples of 10 ⁇ l of each well and analyzing the 20-40 MTP with the fluorescence dye fluorescein diacetate as described above.
  • the fluorescence read out decreases at least by a factor of 3 as compared to mutant free wells.
  • Mutant positive supernatants or stored samples were used to produce stocks, the titer was determined, the DNA prepared by the method found in "He ⁇ es Simplex Viras Protocols, 1998, Ed. S.M. Brown & A.R. MacLean, Humana Press, Totowa, New
  • Compounds as herein provided can be used as drugs for treatment and prophylaxis of diseases caused by He ⁇ es viruses, especially he ⁇ es simplex viruses.
  • mice 6 (six) week old female mice strain BALB/cABom, purchased from Bomholtgard Breeding and Research Centre Ltd., were anaesthetized in a sealed glass vessel with diethylether (Merck). 50 ⁇ l of a diluted virus stock solution (infectious dose 5xl0 4 Pfu (plaque forming units)) were used for intranasal infection of the anaesthetized ammals. This dose leads to the death of 90-100% of the infected animals within 5-8 days. The infected animals show symptoms of a generalized infection such as respiratory or central nervous system symptoms.
  • a diluted virus stock solution infectious dose 5xl0 4 Pfu (plaque forming units)
  • Example 1 to 7 were synthesized according to the following general figures (figure 2 for thiazolyl amides and figure 3 for thiazolyl urea derivatives).

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Abstract

The invention relates to a method for identifying compounds with antiviral activity, characterized in that those compounds are selected in the presence of which resistant viral mutants can be selected, wherein at least one selected viral mutant carries at least one mutation in the primase gene and another selected viral mutant carries at least one mutation in the helicase gene or a selected viral mutant carries at least one mutation in the helicase and at least one mutation in the primase gene.

Description

Method for identifying compounds with anti-herpes activity
This invention relates to a method for identifying compounds with anti-herpes activity and to medicaments for the treatment of herpes infections in a mammal by inhibiting the herpes helicase-primase enzyme complex.
The goal of inventions in the field of the pharmaceutical area is to supply medications or therapies to treat a patient in a tolerable way that a desired therapeutic effect is achieved.
Numerous medications and therapies were invented and more recently, since mono- therapy fails in some cases to show a profound therapeutic benefit for the patient, these methods are often combined to increase the therapeutic efficacy. However, in many cases of combination therapy not only the desired potency of treatment is amplified, but also more side-effects occur and thus, the tolerability is decreased. A classic example is the combination therapy in the field of AIDS treatment.
In the field of anti-infectives, for example, a compound that inhibits two essential targets in the life cycle of a given pathogen simultaneously may be more active due to cumulative inhibitory effects as demonstrated by the analogous case of combination therapy in the past which is often superior to a (single compound chemo) mono-therapy where the drug targets only one relevant binding pocket on a single target protein or on one subunit of a complex. In the case where the compound binds at the interface of two targets a stronger binding may also be observed based on avidity effects. The better binding characteristics and or the cumulative inhibitory effects may result in a superior therapeutic treatment ideally accompanied by a better tolerability which may result from a lower dosage or pill burden during treatment.
Herpesviridae evolved over millions of years and are highly disseminated in nature. Members of the family were identified in humans, non-human primates and most other mammals and vertebrates (Virology, 1996, Fields et al. Lippincott-Raven Publishers, Philadelphia, PA 19106, USA). Heφesviruses are enveloped double stranded DNA viruses which infect cells that carry negative charged structures such as heparansulfate and or glycosaminoglycans in addition to a heφes viral entry mediator on their surface. One key feature of these viruses is their ability to remain latent in their host for life after primary infection and to reactivate more or less frequently from a pool of latent infected cells upon diverse internal and external stimuli.
Eight human heφesviruses (ΗHV1 to HHN8) have been identified so far and their genomes (~ 125-230 kbp in size) were sequenced and published in the internet
(GeneBank, EMBL database etc.). High sequence homologies were identified between the genomes especially in the case of the helicase and primase coding genes, thus, the replication machinery is conserved among the heφes viridae but is clearly different from eukaryotic DΝA replication. The genomes code for more than 50 genes which are essential for the viral replication cycle in vitro and or in vivo and many publications discussed the more or less relevance of these targets for antiviral therapy. The function of the helicase-primase complex in the viral life cycle and its qualification as a target for antiviral (chemo) therapy of an infected host are published (Heφes Simplex Virus Replication, 1997, Annual review of Biochemistry, Boehmer PE & Lehmann IR, 66, 347-384; The Structure and function of the HSN
DΝA replication proteins: defining novel antiviral targets, 1993, Antiviral Research,
Matthews JT, Terry BJ Field AK, 20, 89-114).
Based on their similarity in biological properties the viruses were grouped in 3 sub- families, namely α-(HHVl to 3), β-(HHN5 to 7), and γ-(HHN4 and HHN8) heφesviruses. Common names were derived from the clinical symptoms they cause or historical reasons e.g. Heφes simplex virus 1 or 2 (HSN-1 or -2 the cause of heφes labialis and genitalis) is used for HHN1 or 2, the Naricella-zoster virus (NZN causing chickenpox or zoster) is synonym to HHN3 and Epstein-Barr virus (EBN) and Cytomegalo virus (HCMN) are synonyms for HHN4 and 5, respectively. Incidence and prevalence data range from below 10 to more than 90 % of the population being infected with one or more heφesvirases depending on the HHN involved and the age, sex, social status and geographical aspects of the host. Since some heφesviruses are ubiquitous human pathogens causing a variety of disease ranging from benign illness interfering with normal day activities to life or sight threatening disease especially in immunocompromised patients (keratitis, disseminated disease or retinitis in AIDS patients), pregnancy (abortion, deafness) and newborns (hepatitis, encephalitis), there is a strong medical need for a safe and efficient treatment and much effort has been expended in the search for effective antiviral agents .
Acyclovir, a selective and specific inhibitor of viral replication, was a true milestone in the development of antiviral drugs in the late 1970s. Newer nucleosidic drags that are similar to acyclovir such as penciclovir or the more convenient pharma- cokinetically optimized pro-drugs like valacyclovir and famciclovir were launched in the late 1990s. Nucleosides indeed became the treatment of choice in the heφes indication, however, nucleosides are pro-drugs and have to be phosphorylated by the viral thymidine kinase (TK) and subsequently by cellular kinases for activity upon the viral DNA-polymerase. If the virus does not express a functional TK e.g. a resistant HHV1 strain or TK negative viruses such as HCMN, or the DΝA-Pol has not the optimal primary structure, the potency of the drug diminishes, the selectivity index is significantly smaller, higher doses have to be administrated and adverse effects are more likely to be associated with treatment. Since nucleosides are obligate or non-obligate chain terminators of DΝA-polymerization they are potentially mutagenic which is well documented for ganciclovir. Thus, broad spectrum anti- heφesvirus activity, efficacy especially upon delayed treatment, safety and resistance are goals for the next generation of drugs directed against novel antiviral targets.
WO 97/24343 describes a method to identify inhibitors with anti-heφes properties by selecting compounds which, upon binding to the DΝA-helicase-primase complex, stabilize the latter. A disadvantage of this method is that often compounds are identi- fied that have not a suitable selectivity index in vitro or the necessary tolerability in vivo, and thus, are often not suitable for treatment of heφes infections due to side effects.
WO 99/42455 and Spector, F.C., Journal of Virology 1998, pages 6979-6987 describe compounds that exclusively bind to the UL5 subunit of the complex, which is extensively confirmed by analysis of at least 25 different UL5 mutants.
The goal of this invention is therefore to provide a method to identify alternative or more active compounds which have a better selectivity index and/or tolerability.
In another aspect the goal of the present invention is to provide a method for identifying compounds which are active against acyclovir-resistant viral mutants.
The present invention solves the abovementioned problems by providing a method for identifying compounds with antiviral activity, characterized in that those compounds are selected in the presence of which resistant viral mutants can be selected, wherein at least one selected viral mutant carries at least one mutation in the primase gene (e.g. UL52 in the case of HSV) and another selected viral mutant carries at least one mutation in the hehcase gene (e.g. UL5 in the case of HSV) or a selected viral mutant carries at least one mutation in the helicase and at least one mutation in the primase gene.
In a preferred embodiment, the method is further characterized in that the primase and helicase are at least 50% homologue to the primase and helicase of heφes simplex virus 1 based on the nucleotide sequence or the amino acid sequence, whichever is higher.
In another preferred embodiment, the method is further characterized in that the virus is a heφes virus. In another preferred embodiment, the method is further characterized in that before determining the generation of resistant viral mutants in the presence of said compounds,
a) the compounds are tested for their ability to inhibit the helicase and b) the compounds are tested for their ability to inhibit the primase c) such compounds are selected, that inhibit the helicase and the primase
In more preferred embodiment, the method is further characterized in that such compounds are selected, that bind simultaneously to the helicase and primase subunits of the helicase-primase complex. Without being bound by theory, it is believed that compounds bind at the interface of the helicase and primase subunits of the helicase-primase complex of a heφes simplex virus (and homologous regions of other heφes virus helicase primase enzymes) and thereby cause the inhibition of essential enzymatic activities of the helicase-primase complex (either the minimal heterodimeric complex UL5/UL52 or the native heterotrimeric complex UL5/UL8 and UL52 in the case of a heφes simplex virus) possibly by blocking the ATP binding site competitively. The compound may either be bound in a ternary complex involving residues of the helicase and the primase subunits and possibly stabilized by avidity effects or the compounds bind independently to at least one site on the helicase and the primase subunits. "To bind simultaneously" here describes a state, where the very same molecule is bound to both UL5 and UL52 gene products at the same time.
In another more preferred embodiment, the method is further characterized in that the compounds inhibit the DNA-dependent NTPase activity of a heφes helicase- primase. One additional assay that may be ran determines the ability of test compounds to inhibit helicase-primase-associated DNA-independent NTPase activity. The compounds useful in this invention do not inhibit this activity, in the way that competitively-acting nucleoside analogues inhibit this activity. In another preferred embodiment, the method is further characterized in that the compounds inhibit the replication of a heφes viras.
In another more preferred embodiment, the method is further characterized in that such compounds are selected, that have the ability to inhibit replication of a heφes virus in cell culture by at least 50 % at a concentration of less than about 10 μM.
In another more preferred embodiment, the method is further characterized in that such compounds are selected, that have the ability to inhibit replication of a heφes viras in cell culture by at least 50 % at a concentration of less than about 500 nM.
In another more preferred embodiment, the method is further characterized in that the heφes viras is HSV.
In another more preferred embodiment, the method is further characterized in that the heφes virus is PRV or BHN.
In another embodiment, the invention relates to a compound identified by the abovementioned method.
In a more preferred embodiment, the invention relates to a compound which is not a nucleoside or nucleotide.
The present invention also relates to the use of such a compound for the preparation of a medicament for the treatment and prevention of heφes infections and to a pharmaceutical composition comprising such a compound.
The present invention also relates to a method for treating heφes infection in a mammal comprising the step of administering to a mammal in need of such treatment a therapeutically effective amount of the abovementioned pharmaceutical composition. The present invention also relates to a method for identifying compounds, characterized in that
a) the compounds are tested for their ability to bind to at least two therapeutically relevant targets simultaneously and b) the compounds are tested for their ability to inhibit and/or activate these targets upon binding c) the compounds are tested for reduction of their ability to inhibit and/or activate these targets in the presence of at least one mutation in one target and at least one mutation in the other target or at least one mutation in either target.
The present invention also relates to a compound with antiviral activity, characterized in that those compounds are selected in the presence of which resistant viral mutants can be selected, wherein at least one selected viral mutant carries at least one mutation in the primase gene and another selected viral mutant carries at least one mutation in the helicase gene or a selected mutant carries at least one mutation in the helicase and at least one mutation in the primase gene.
The present invention also relates to a compound capable of binding simultaneously to the helicase and primase subunits of the helicase-primase complex.
Compounds with antiviral activity are described in PCT/00/01498. They are not preferred in the context of the present invention.
The invention described herein overcomes the above-mentioned limitations and satisfies the medical needs by providing a method for identifying non-nucleosidic inhibitors that act directly by interfering with the essential enzymatic activity helicase-primase in a novel way and consequently inhibits heφes virus DNA repli- cation and heφes virus replication in vitro and in vivo. Furthermore, since the heφes virus helicase-primase enzyme is conserved across the heφes viridae, the inventors could demonstrate broad anti-heφes virus spectrum activity (see also table 1). The selective action of these inhibitors against heφes viruses and especially against acyclovir-resistant heφes viruses, combined with a wide margin of safety, renders the compounds as desirable agents for combating heφes infections.
The term "heφes" as used herein refers to any virus in the heφes family of viruses and particularly, to those heφes viruses that encode a heφes helicase-primase homologous to the heφes helicase-primase of HSV- 1. The heφes family of viruses includes, but is not limited to HHV-1 to HHV-8, EHN, BHN, PRN etc. Preferred are heφes simplex viruses type 1 and 2. Heφes simplex virus 1 and 2 refers to a virus that is characterized by specific monoclonal antibodies (serotype 1 or 2).
The term helicase-primase refers to the helicase-primase complex involved in DΝA replication. In the case of the heφes simplex viras it is composed of the UL5, UL8 and the UL52 or the least (necessary or essential) UL5 and UL52 gene products, in the case of other heφes viruses of the corresponding homologues.
The term helicase refers to the helicase subunit of the helicase-primase complex involved in DΝA replication. In the case of the heφes simplex viras it is the UL5 gene product, in the case of other heφes viruses it is the corresponding homologue.
The term primase refers to the primase subunit of the helicase-primase complex involved in DΝA replication. In the case of the heφes simplex viras it is the UL52 gene product, in the case of other heφes viruses it is the corresponding homologue.
The term selectivity index refers to the quotient of the concentration were the compound reduces the viability of the cell by 50 % and the EC50 or IC50 (CC5o/IC50).
The term therapeutic index (tolerability) refers to the quotient of the dose (lethal dose) were 50 % of the animals die and the dose (effective dose) were 50 % of the animals survive the infection (LD50/ED50). The term pharmaceutically acceptable carrier or veterinarily acceptable as used herein means a non-toxic, generally inert vehicle for the active ingredient which does not adversely affect the ingredient.
The term inhibit, when used in connection with enzymatic activity, refers generally to inhibiting the enzymatic activity by at least about 50% at a concentration of about IC50 = 100 μM or less and preferably at a concentration as low as possible in a conventional in vitro assay for enzymatic inhibition.
The term inhibit, when used in connection with in an in vitro cell-based viral replication assay, refers generally to inhibiting viral replication by at least about 50% at a concentration of about IC50 = 10 μM or less and preferably at a concentration as low as possible in the in vitro cell-based viral replication assay presented here.
The term target describes a gene product involved in the manifestation of diseases.
The term mutation in one target describes a mutation in a gene that leads to an altered amino acid sequence of the respective protein.
The term reduction describes a loss of activating or inhibiting activity in the order of at least a factor of two, preferably of one order of magnitude.
The compounds of this patent are new and can be clearly differentiated structurally from WO 97/24343 , WO 99/42455 and any other publication to date.
Compounds useful for inhibiting a heφes helicase-primase according to the above mechanism may either be identified by assaying a test compound's ability to bind to the helicase (subunit) and the primase (subunit) of the helicase-primase complex or more preferably to inhibit enzyme-associated single stranded DNA-dependent
NTPase activity of a heφes helicase-primase (such as the helicase-primase of heφes simplex viruses) advantageously in a high throughput (HTS) assay or by assaying a test compound's ability to inhibit viral replication in a cell-based viral replication assay followed by selecting resistant viruses in the presence of compound and sequencing the helicase-primase genes of the selected mutants.
Numerous assays are described to directly determine binding of compounds to proteins, however, several of these assays require purified or at least partially purified protein. In addition to that, not all binders interfere with the function of the enzyme or protein.
The enzymatic assay is easy to perform, but the enzyme has to be purified from heφes infected cell culture or the genes have to be cloned, expressed and produced and purified using an expression system and protein purification techniques.
The cell-based viral replication assay is easy to perform and possibly more sensitive than the enzymatic assay, since it mimics the real situation of a viral infection more closely, but mutant viruses have to be generated and analyzed subsequently by sequencing or by a complementation analysis to confirm the viral target helicase primase.
Compounds effective in either the enzymatic assay or the cell-based viral replication assay may be further assayed to determine their heφes helicase-primase binding specificity or compounds effective in a binding assay have to be evaluated in either the enzymatic assay or the cell-based viral replication assay. Although the following assays are described in one particular sequence, it should be understood that not all of these assays need to be performed for successful identification of heφes helicase- primase inhibitors. In addition, the exact order of assay may be altered, if desired. These and other procedural options can be considered by those of ordinary skill in the art. Other assays measure a test compound's ability to inhibit enzyme-mediated RNA primer biosynthesis or a test compound's ability to inhibit helicase activity.
Any DNA but preferably single stranded DNA with a primase consensus site for the primase assay or any DNA substrate designed to model a replication fork-like structure may be used for the helicase assay. Several options are outlined in the cited review by Boehmer PE & Lehmann IR 1997 or more recently in the paper entitled High-Throughput Screening Assay for Helicase Enzymes, 1998, Analytical Biochemistry, M Sivaraja, H Giordano, MG Peterson, 265, 22-27.
Compounds useful in this invention do not intercalate into, nor otherwise bind directly, double stranded DNA and are not positive in a cytogenetic or Ames Test.
In all of the above-methods, the non-nucleoside compound is preferably further characterized by an ability to inhibit heφes helicase-primase mediated RNA primer biosynthesis or helicase activity. In addition, preferred non-nucleoside inhibitors of this invention are further characterized by an ability to inhibit replication of a heφes virus in cell culture by at least about 50 % at a concentration of less than about 10 μM more preferably at a concentration as low as possible e.g. in the nM range or even less.
It is important to recognize that the compounds, compositions and methods of this invention may be used against nucleoside non-responsive and nucleoside resistant heφes infections.
This aspect of the invention involves a method for treating acyclovir-resistant heφes infections in a mammal which comprises administering to the mammal an anti- acyclovir-resistant heφes effective amount of a compound as defined herein, or a therapeutically acceptable acid addition salt thereof. Using the above noted screening methodologies, several compounds were identified as inhibitors of heφes helicase-primase. The selective action of these compounds against these viruses, combined with a wide margin of safety, renders the compounds as desirable agents for combating heφes infections.
Anti-heφes Activity
The antiviral activity of the compounds can be demonstrated by biochemical, microbiological and biological procedures showing the inhibitory effect of the com- pounds on the replication of heφes viruses e.g. HSN-1, HSV-2, BHN etc.
A biochemical procedure for demonstrating anti-heφes activity for compounds is described below under Biochemical assay. This ATPase assay is one among numerous possible assays that are described for instance in High-Throughput Screening Assay for Helicase Enzymes, 1998, M. Sivaraja, H. Giordano and M.G.
Peterson, Analytical Biochemistry, 265, 22-27. Other assays cited there measure the helicase or the primase activity of the helicase-primase complex with diverse assay configuration or methods.
Methods for demonstrating the inhibitory effect of compounds on heφes viral replication involving in vitro and cell culture techniques are described in cell based viral replication assay.
The therapeutic effect of the compounds can be demonstrated in vivo in a lethal challenge model as shown in section lethal challenge in vivo animal model. Description of the Table
Table 1 siimmarizes the ICso or EC50 of wild type or resistant HSN strains, the ED50 of wild type, the relevant mutations and cross resistance patterns.
A direct comparison of the in vitro cell culture IC50 values shows that some of the example compounds are at least one order of magmtude more potent than Acyclovir/Zovirax. The in vivo data demonstrate that this antiviral activity translates to a superior potency and efficacy in animal models. Example compounds inhibit acyclovir resistant HSN-1 (F) mutants and wild type HSN-1 (F) with nearly identical
IC50 values. Broad Spectrum activity is exemplified by examples 1, 4, 5, 6 and 7, which inhibit as different viruses as the human heφes simplex virus as well as porcine (PRN) and bovine (BHN) animal viruses. These compounds are also active on all clinical isolates tested so far (39 clinical isolates (HSN-1) and 19 clinical isolates (HSN-2)). It demonstrates for the first time that compounds that qualify the above mentioned characteristics outperform the antiviral activity of the state of the art by at least one order of magnitude.
Assay
HSN-1 DΝA-dependent ATPase assay (in vitro assay based on the inhibition of HSN-1 helicase-primase).
a) Preparation of the enzyme
HSN-1 helicase-primase heterodimer was produced in doubly infected Sf9 (Spodoptera frugiperda) cells using recombinant baculovirases expressing the UL5 and the UL52 helicase-primase subunits.
The genes were amplified by PCR from HSN-1 (F) (American Tissue Culture Collection ATCC NR-733) and cloned into the baculovirus expression system (UL5 - > pFASTBACl; UL52 -> pFASTHTb) according to the Instruction Manual BAC- TO-BAC Baculovirus Expression Systems, Life-Technologies. The heterodimeric enzyme was purified by IMAC-Chromatography as described in "Inhibition of Heφes Simplex Virus Replication by a 2-Amino Thiazole via interactions with the Helicase Component of the UL-5-UL8-UL52 Complex", 1998, F.C. Spector, L. Liang, H. Giordano, M. Sivaraja and M.G. Peterson.
b) ATPase Assay
Purified heterodimeric helicase-primase complex (200 ng) was incubated with 1 μg DΝA (Sigma D3287 or D8681) in 20 mM HEPES (pH 7.6; 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid), 5 mM MgCl2, 0,5-5 mM ATP, 100 μg BSA (bovine serum albumin) per ml, 10 % glycerol, 1 mM DTT (DL-dithiothreitiol) and for 60 min at 37 °C. The released inorganic phosphate was detected colorirnetrically as described previously in "An improved assay for nanomole amounts of inorganic phosphate", 1979, P.A. Lanzetta, IJ. Alvarez, P.S. Reinach and O.A. Candia, Analytical Biochemistry, 100:95-97. DNA-dependent ATPase activity was calculated from the net absorbance change in the presence and absence of inhibition.
Figure 1 shows a dose dependent inhibition/titration of the enzymatic ATPase activity of the HSV-1 helicase-primase with representative example 7. For their puφose, ATPase activity of the helicase-primase heterodimer (release of inorganic phosphate (Pi)) was measured in the presence of saturated DNA concentrations and varying ATP and inhibitor concentrations as described under ATPase Assay. Inhibition of the ATPase activity by compound example 7 is dose dependent and in addition dependent on the ATP concentration (e.g. 50 % inhibition of Pi release at
5 mM ATP and ~ 10 μM of the compound of example 7 versus 50 % inhibition of Pi release at -100 nM of the compound of example 7 (ATP control : without enzyme and without DNA)
Cell based viral replication assay
a) Growth of HSV and preparation of viras stocks
Heφes simplex viruses (HSV-1 Walki, HSV-1F or HSV-2G) were routinely propagated on African green monkey kidney cells (Nero cells; ATCC CCL-81), however, many tissues-culture lines can be used for the growth and quantification of
HSN (Heφes Simplex Virus Protocols, 1998, Ed. S.M. Brown & A.R. MacLean,
Humana Press, Totowa, New Jersey). These cells were grown in Ml 99 media supplemented with 5 % foetal calf serum (FCS), 2 mM glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin in cell culture flasks (e.g. 175 cm2 / 25-50 ml) at
37°C in the presence of 5 % CO2. Cells were split 1:4 twice a week (cell viability
> 95%). For viras infection the medium was removed, the cells were washed with
Hank's solution, trypsinized with 0.05% trypsin, 0.02% EDTA (Seromed, L2143) and seeded at a density of 4xl05 cells per ml in cell culture flasks and incubated for 24 hours at 37°C in the presence of 5 % CO2 . The medium was removed and viras suspension was added at a multiplicity of infection (m.o.i) of < 0.05 in a volume of 2 ml per 175 cm2 surface area. The flask was allowed to stand 1 hour at 37°C in the presence of 5 % CO2 with occasional agitation to distribute the viras evenly over the cells. Then medium was added to a final volume of 50 ml per 175 cm2 tissue culture flask. The cultures were incubated until all cells showed signs of infection (approximately 2-3 days). To harvest the viras, flasks were frozen at -80°C and thawed at 37°C (in a water bath) two times. Cell debris was removed by centri- fugation (300 g, 10 min, 4°C) and the supernatant was stored in aliquots at -80°C.
The titer of the virus stock was determined in a plaque assay. Briefly, Vero cells were seeded at a density of 4x105 cells per well of a 24 well tissue culture plate. After an incubation period of 24 hours (37°C, 5% CO2) cells were infected with dilutions of the viras stock ranging from 10" to 10A infection volume was 100 μl per well. It was removed after 1 hour incubation at 37°C, 5 % CO2 and the cells were gently covered with 1 ml overlay medium (0.5% methyl cellulose, 0.225 % sodium bicarbonate, 2 mM glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 5%
FCS in MEME (MEM-Eagle with Earle's salt)) and allowed to stand for 3 days at
37°C in the presence of 5 % CO2. Cells were fixed with 4% formaline for at least 30 minutes, washed with water, stained with giemsa solution (Merck, Darmstadt) for 30 minutes and washed again. After drying the plates, plaques were counted and the titer of the viras stock was calculated. Viras stocks used in the experiments described here were HSV-1 F (ATCC VR-733, stock 5xl07 PFU/ml) and HSV-2 G (ATCC VR-
734; stock 3.5x10° PFU/ml).
b) Measurement of antiviral activity of test compounds in vitro
Antiviral activity was measured with a microtiter plate screening test system employing cells of diverse origin such as neuronal, lymphoid and epithelial lineages e.g. Vero cells (african green monkey kidney cells), MEF (murine embryonic fibroblasts), HELF (human embryonic lung Fibroblasts), NT2 (human neuronal cells) or Jurkat (humane lymphoid T-cell line). The inhibition of the compounds on the growth, spread and resulting cytopathic effect of the viruses was analyzed in direct comparison to uninfected cells, uninfected but treated cells, infected but untreated cells and infected cells in the presence of example compounds or the reference compound Acyclovir-Natrium (ZoviraxR), a generic nucleosidic anti-Heφes-drug.
Compounds were dissolved in DMSO (Dimethyl Sulfoxide) to a stock concentration of 50 mM and analyzed on microtiter plates (e.g. 96-Well MTP or higher formats such as 384 or 1536 well plates) at final concentration of 250 - 0,5 μM (micro molar) in duplicates (e.g. 4 compounds/96 well MTP). In the case of potent compounds the serial 1 :2 dilutions were continued over several plates to the pM (pico molar) range. Precipitation of compounds at higher concentrations as well as toxic and cytostatic side effects were recorded in parallel by microscopic evaluation. After the serial i dilution (1 :2) of compounds in media on the MTP a cell suspension was added (lxl 04 cells/well of a 96 well MTP) e.g. Vero-cells in Ml 99 (Medium 199) complemented with 5 % FCS, 2 mM L-glutamine and optional 100 IU/ml penicillin and 100 μg/ml streptomycin or MEF-cells in EMEM (Eagle's Minimum Essential Medium) complemented with 10 % FCS, 2 mM L-glutamine and optional 100 IU/ml penicillin and 100 μg/ml streptomycin, or HELF-cells in EMEM complemented with 10 % FCS, 2 mM L-glutamine and optional 100 IU/ml penicillin and 100 μg/ml Streptomycin, or NT2- and Jurkat-cells in DMEM (4,5 mg/1 Glucose plus Pyridoxine) complemented with 10 % FCS, 2 mM L-glutamine, 1 mM Natrium- Pyruvate, non essential amino acids and optional 100 IU/ml penicillin and 100 μg/ml streptomycin to each well. The cells in the relevant wells were than infected with the viras under investigation as follows: HSV-1 F or HSV-2 G with an m.o.i (multiplicity of infection) of 0,0025 for HELF, Vero and MEF-cells and an m.o.i. of 0J for NT2- and Jurkat-cells). The plates were then incubated at 37 °C in a CO2- incubator (5 % CO2) for several days (preferably 5 days). After the incubation period the cells in compound free wells, starting from 25 infectious centers, are completely lysed or destroyed by the cytophathic effect of replicating Heφes viruses (100 % CPE). The plates were first briefly analyzed under a microscope and then the read out was generated using a quenched fluorescent dye which is cleaved by the enzymatic esterase activity of viable cells. The cell culture supernatant in the MTP was aspirated, the remaining cells or debri were washed once with 200 μl PBS (phosphate buffered saline) and 200 μl Fluorescein-diacetate dye containing solution were added (10 μg/ml in PBS). After an incubation of 30-90 min at room temperature the fluorescence of the MTP was recorded in a Fluorescent Ascent (Labsystems) at 485 nm excitation and 538 nm emission wavelength.
At an IC50 concentration the viral replication is inhibited by 50 % as compared to the non infected cell control or the read out of the fluorescence based assay reaches 50 % of the signal as compared to the cell control.
The relevant data and results are summarized in Table 1.
c) Generation and sequencing of resistant viral mutants
Naturally occurring, resistant viral mutants were selected in the presence of 1 μM example 7 or of at least 100 times the IC50 concentration as listed in table 1 using the cell based viral replication assay. 10 000 Vero cell were seeded in 96 well MTPs as described above and incubated over night. Compound was added to the final concentration and the cells were infected with 1000 plaque forming units (m.o.i. 0,1) per well. Mutants were identified at a frequency of 1-5 per million HSV-1 F under a microscope after 3-5 days or by storing replica samples of 10 μl of each well and analyzing the 20-40 MTP with the fluorescence dye fluorescein diacetate as described above. If a resistant virus is present in an individual well, the fluorescence read out decreases at least by a factor of 3 as compared to mutant free wells. Mutant positive supernatants or stored samples were used to produce stocks, the titer was determined, the DNA prepared by the method found in "Heφes Simplex Viras Protocols, 1998, Ed. S.M. Brown & A.R. MacLean, Humana Press, Totowa, New
Jersey" and sequenced according to "DNA sequencing with chain-terminating inhibitors, 1977, Sanger F, Nicklen S, Coulson AR, PNAS, 74, 5463-5467" using the ABI PRISM Big Dye Terminator Kit from Applied Biosystems or the DYEnamic ET Terminator Cycle Sequencing Kit from Amersham (apbiotech).
The mutations are summarized in Table 1. Table 1 Pharmacological profile of helicase primase inhibitors
Preferred are compounds with an IC50 (HSV-1 F/Nero) obtained with the above described in vitro cell based viral replication screening test system using the dye read out of about or less than 50 μM, more preferred with an IC50 of about 25 μM or less and ideally with an IC50 below 10 μM.
Compounds as herein provided can be used as drugs for treatment and prophylaxis of diseases caused by Heφes viruses, especially heφes simplex viruses.
The following indications are listed as examples:
1) Treatment and prophylaxis of heφes infections, especially Heφes simplex infections in patients with disease symptoms such as heφes labialis, heφes genitalis, and keratitis, encephalitis, pneumonia, hepatitis etc. caused by heφes viruses
2) Treatment and prophylaxis of heφes infections, especially heφes simplex infections in immunocompromized patients (e.g. AIDS patients, cancer patients, patients with genetically based immundeficiency, transplant patients)
3) Treatment and prophylaxis of heφes infections, especially heφes simplex infections in newborns, babies, infants and children.
4) Treatment and prophylaxis of heφes infections, especially heφes simplex infections and Heφes-, especially heφes simplex positive patients for maintenance or suppression therapy of recurrent heφes disease
d) In vivo- Activity
6 (six) week old female mice strain BALB/cABom, purchased from Bomholtgard Breeding and Research Centre Ltd., were anaesthetized in a sealed glass vessel with diethylether (Merck). 50μl of a diluted virus stock solution (infectious dose 5xl04 Pfu (plaque forming units)) were used for intranasal infection of the anaesthetized ammals. This dose leads to the death of 90-100% of the infected animals within 5-8 days. The infected animals show symptoms of a generalized infection such as respiratory or central nervous system symptoms.
Treatment and Analysis of Potency and Efficacy:
6 hours after infection animals were treated with doses of 0,1-100 mg/kg body weight 3 times daily (7.00 h, 14.00 h and 19:00 h) over a period of 5 days. The compounds were dissolved in DMSO and resuspended in Tylose/PBS(Hoechst)
(final concentration 1,5% DMSO, 0,5 % Tylose in PBS).
After the final application animals were observed and the time of death was recorded.
Data are summarized in Table 1 e.g. an ED50 of 0,7 mg/kg means that 50 % of the infected animals survived at this dose.
The following test compounds Example 1 to 7 were synthesized according to the following general figures (figure 2 for thiazolyl amides and figure 3 for thiazolyl urea derivatives).

Claims

Claims
A method for identifying compounds with antiviral activity, characterized in that those compounds are selected in the presence of which resistant viral mutants can be selected, wherein at least one selected viral mutant carries at least one mutation in the primase gene and another selected viral mutant carries at least one mutation in the helicase gene or a selected viral mutant carries at least one mutation in the helicase and at least one mutation in the primase gene.
A method according to claim 1, characterized in that the primase and helicase are at least 50% homologue to the primase and helicase of heφes simplex virus 1 based on the nucleotide sequence or the amino acid sequence, whichever is higher.
A method according to claim 1 or 2, characterized in that the viras is a heφes virus.
4. A method according to claim 1, 2 or 3, characterized in that, before determining the generation of resistant viral mutants in the presence of said compounds,
a) the compounds are tested for their ability to inhibit the helicase and b) the compounds are tested for their ability to inhibit the primase c) such compounds are selected, that inhibit the helicase and the primase
5. A method according to claim 4, characterized in that such compounds are selected, that bind simultaneously to the helicase and primase subunits of the helicase-primase complex.
6. A method according to any one of claims 1 to 5, characterized in that the compounds inhibit the DNA-dependent NTPase activity of a heφes helicase- primase.
7. A method according to any one of claims 1 to 6, characterized in that the compounds inhibit the replication of a heφes viras.
8. A method according to any one of claims 1 to 7, characterized in that such compounds are selected, that have the ability to inhibit replication of a heφes viras in cell culture by at least 50 % at a concentration of less than about 10 μM.
9. A method according to claim 8, characterized in that such compounds are selected, that have the ability to inhibit replication of a heφes virus in cell culture by at least 50 % at a concentration of less than about 500 nM.
10. A method according to any one of claims 1 to 9 wherein the heφes virus is HSV.
11. A method according to any one of claims 1 to 9 wherein the heφes viras is
PRV or BHV.
12. A compound identified by the method according to any one of claims 1 to 11.
13. A compound according to claim 12, characterized in that the compound is not a nucleoside or nucleotide.
14. Use of a compound according to claim 12 or 13 for the preparation of a medicament for the treatment and prevention of heφes infections.
15. A pharmaceutical composition comprising a compound according to claim 12 or 13.
16. A method for treating heφes infection in a mammal comprising the step of administering to a mammal in need of such treatment a therapeutically effective amount of the pharmaceutical composition according to claim 15.
17. A method for identifying compounds, characterized in that
a) the compounds are tested for their ability to bind to at least two therapeutically relevant targets simultaneously and b) the compounds are tested for their ability to inhibit and/or activate these targets upon binding c) the compounds are tested for reduction of their ability to inhibit and/or activate these targets in the presence of at least one mutation in one target and at least one mutation in the other target or at least one mutation in either target.
18. A compound with antiviral activity, characterized in that those compounds are selected in the presence of which resistant viral mutants can be selected, wherein at least one selected viral mutant carries at least one mutation in the primase gene and another selected viral mutant carries at least one mutation in the helicase gene or a selected mutant carries at least one mutation in the helicase and at least one mutation in the primase gene.
19. A compound capable of binding simultaneously to the helicase and primase subunits of the helicase-primase complex.
EP00945756A 2000-06-15 2000-06-15 Method for identifying compounds with anti-herpes activity Withdrawn EP1319185A1 (en)

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