WO2022171221A1 - Quinacrine as an inhibitor of viral cysteine proteases and/or serine proteases - Google Patents

Quinacrine as an inhibitor of viral cysteine proteases and/or serine proteases Download PDF

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Publication number
WO2022171221A1
WO2022171221A1 PCT/DE2021/000197 DE2021000197W WO2022171221A1 WO 2022171221 A1 WO2022171221 A1 WO 2022171221A1 DE 2021000197 W DE2021000197 W DE 2021000197W WO 2022171221 A1 WO2022171221 A1 WO 2022171221A1
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virus
quinacrine
proteases
group
viruses
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PCT/DE2021/000197
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German (de)
French (fr)
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Monika Aparecida Coronado
Raphael Josef Eberle
Dieter Willbold
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Forschungszentrum Jülich GmbH
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Publication of WO2022171221A1 publication Critical patent/WO2022171221A1/en

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    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines

Definitions

  • the present invention relates to quinacrine for use as an inhibitor and for the production of an inhibitor of viral cysteine proteases and/or serine proteases, in particular for inhibiting viral nsP2 proteases (nsP2 pro ) and/or NS2B/NS3 proteases (NS2B/NS3 pro ), as well as the use of quinacrine for the production of an inhibitor for viral cysteine proteases and serine proteases.
  • the invention further relates to quinacrine for use in medical therapy.
  • Another aspect of the invention is a pharmaceutical composition comprising at least quinacrine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient thereof.
  • Quinacrine can be used to prevent or treat a viral infection in an organism, in particular an infection with at least one virus from the Flaviviridae and/or Togaviridae family.
  • Quinacrine can be used to treat a viral infection in an organism, in particular to treat a combined infection with viruses from the Flaviviridae and Togaviridae families, and to use quinacrine as a drug in the treatment of viral infections, in particular to treat a combined infection with viruses from the Flaviviridae and Togaviridae families can be used.
  • the invention relates to the use of quinacrine as a therapeutic agent and in the manufacture of a medicament for the treatment of a viral infection in an organism, in particular for the treatment of a combined infection with viruses from the Flaviviridae and Togaviridae families, and also to a Method for treating viral infections, in particular for treating combined infections with viruses from the Flaviviridae and Alphavirus families.
  • Quinacrine is a well-known substance that is already approved as a medicinal product. Quinacrine is used, for example, under the trade names Mepacrine or Atabrine. Quinacrine has historically been used for malaria prophylaxis. A use of quinacrine in gardia and protozoa infections is also known.
  • Quinacrine is also used in the treatment of discoid and subcutaneous lupus erythematosus and is given as prophylaxis to prevent recurrence of pneumothorax in high-risk patients, such as those with cystic fibrosis.
  • An inhibitory effect against Zika and dengue virus cell cultures is described in the literature for quinacrine [1; 2; 3], quinacrine also shows an inhibitory effect against Ebola virus infection in mice [4],
  • Quinacrine's exact mechanism of action is currently unknown, but the molecule appears to inhibit translation and transcription of DNA.
  • Quinacrine is also described as an inhibitor of acid sphingomyelinase and cholinesterase.
  • the object of the invention is to provide an active substance for the treatment of viral infections, in particular with viruses from the family Flaviviridae and Togaviridae, especially with representatives of the genus Flavivirus and Alphavirus, especially infections with Zika, Dengue, Yellow fever, West Nile or Chikungunya viruses and combinations of these viruses, such as Zika dengue, Zika yellow fever, Zika dengue Chikungunya. It is a further object of the invention to provide an active ingredient as a pharmaceutical or therapeutic agent for treating these viral infections.
  • the present invention relates to an active substance for inhibiting cysteine proteases and serine proteases and, associated therewith, to treating infections caused by viruses from the group of Flaviviridae and Togaviridae.
  • the cysteine proteases found in Togaviridae family viruses and/or serine proteases found in Flaviviridae family viruses are essential for the viral replication mechanism in the host.
  • the inventors have surprisingly found that quinacrine can inhibit these two protease classes, in particular the viral cysteine protease nsP2 pro and/or the viral serine proteases NS2B/NS3 pro .
  • quinacrine to inhibit viruses from the group of Flaviviridae and Togaviridae not only individually, but also in combination from these groups.
  • the subject matter of the invention is therefore quinacrine for use in the production of an inhibitor for viral cysteine proteases and/or serine proteases.
  • Another subject matter of the invention is quinacrine for use in the production of an inhibitor for cysteine proteases and/or serine proteases of viruses from the Flaviviridae and/or Togaviridae family.
  • a further aspect of the invention is the use of quinacrine to produce an inhibitor for these viral cysteine proteases and/or serine proteases from the Flaviviridae and/or Togaviridae family, in particular for the serine protease NS2B/NS3 pro and/or cysteine protease nsP2 pr0 .
  • a fourth aspect of the invention is quinacrine for use in medical therapy.
  • a fifth aspect is a pharmaceutical composition
  • a pharmaceutical composition comprising at least quinacrine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient thereof.
  • a sixth aspect of the invention is quinacrine as a pharmaceutical composition for use in medical therapy.
  • a seventh aspect of the invention is quinacrine for use in preventing or treating a viral infection in an organism, in particular an infection with at least one virus from the Flaviviridae and/or Togaviridae family.
  • a further aspect is quinacrine for use in the prevention or treatment of a viral infection in an organism, in particular an infection with at least one virus from the group of the Flaviviridae and/or Togaviridae family.
  • flaviviruses can be mentioned as examples of a viral infection from the Flaviviridae family, but are not limited to these: Zika virus, dengue virus, yellow fever virus, TBE virus, JE virus, SLE virus, louping virus Ill Virus, Usutu Virus, Wesselsbron Virus, West Nile Virus, Gadgets Gully Virus, Langat Virus, Modoc Virus, Kadam Virus, MVEV Virus, POW Virus, Karshi Virus, Sepik Virus, TBE Virus, Yokose Virus.
  • alphaviruses can be mentioned as examples of a viral infection from the Togaviridae family, but are not limited to these: Chikungunya virus, Mayaro virus, Getah virus, O'nyong-nyong virus, Ross River virus, Sindbis virus, Ockelbo virus, Babanki virus, Barmah Forest virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Everglades virus, Mucambo virus, Tonate virus, Eastern equine Encephalitis Virus, Western Equine Encephalitis Virus, Highlands Virus, Middelburg Virus, Rio Negro Virus, Aura Virus, Babanki Virus, Ockelbo Virus, Whataroa Virus.
  • the invention also relates to medicaments containing quinacrine and a pharmaceutically suitable carrier and the use of quinacrine in the production of a medicament for the treatment of infections with at least one virus from the Flaviviridae and/or Togaviridae family in an organism.
  • the invention also relates to a method for inhibiting viral serine proteases and/or cysteine proteases, viruses from the Flavirviridae and/or Togaviridae family, and for treating or preventing a viral infection in an organism, in particular for treating a viral infection by at least one Virus from the group Flaviviridae such as, but not limited to, Zika virus, Dengue virus, Yellow fever virus, TBE virus, JE virus, SLE virus, Louping III virus, Usutu virus , Wesselsbron Virus, West Nile Virus, Gadgets Gully Virus, Langat Virus, Modoc Virus, Kadam Virus, MVEV Virus, POW Virus, Karshi Virus, Sepik Virus, TBE Virus, Yokose virus, and/or for example, but not limited to, for the Togaviridae family, by a Chikungunya virus, Mayaro virus, Getah virus, O'nyong-nyong virus, Ross River virus
  • the invention also includes a method for inhibiting the viral cysteine protease and / or serine proteases, in particular the viral NS2B / NS3 per and / or nsP2 per , in an organism in which administration of a therapeutically effective amount of quinacrine or a pharmaceutical composition comprising quinacrine.
  • the subject is also a method for inhibiting or inactivating viral cysteine proteases and/or serine proteases of viruses from the Flaviviridae and/or Togaviridae family, comprising the step of bringing the cells of the organism into contact with a therapeutically effective amount of quinacrine or a pharmaceutical composition the invention.
  • This method can be carried out both in-vivo and ex-vivo and is also the subject of the invention.
  • cysteine proteases are particularly suitable as targets for the use of quinacrine according to the invention, which are present in human-pathogenic or animal-pathogenic alphaviruses from the group consisting of the Chikungunya virus, Mayaro virus, Getah virus, O'nyong-nyong virus, Ross River virus, Sindbis virus, Ockelbo virus, Babanki virus, Barmah Forest virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Everglades virus, Mucambo virus, Tonate virus, Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, Highlands Virus, Middelburg Virus, Rio Negro Virus, Aura Virus, Babanki Virus, Ockelbo Virus, Whataroa Virus Occurrence.
  • Serine proteases as a target for the use of quinacrine according to the invention preferably come in human-pathogenic or animal-pathogenic flaviviruses from the group of Zika virus, dengue virus, yellow fever virus, TBE virus, JE virus, SLE virus, Louping III virus , Usutu Virus, Wesselsbron Virus, West Nile Virus, Gadgets Gully Virus, Langat Virus, Modoc Virus, Kadam Virus, MVEV Virus, POW Virus, Karshi Virus, Sepik Virus, TBE virus, Yokose virus.
  • human-pathogenic or animal-pathogenic flaviviruses from the group of Zika virus, dengue virus, yellow fever virus, TBE virus, JE virus, SLE virus, Louping III virus , Usutu Virus, Wesselsbron Virus, West Nile Virus, Gadgets Gully Virus, Langat Virus, Modoc Virus, Kadam Virus, MVEV Virus, POW Virus, Karshi
  • Quinacrine can therefore be used as a broad spectrum inhibitor against single viral infections and/or co-infections, since it can inhibit the essential viral proteases, particularly the cysteine and serine proteases, of these different virus families.
  • quinacrine means a compound of the following structural formula (I), which consists of a 1:1 mixture of an R form (above) and an S form (below):
  • quinacrine can be used, for example, in the form of its hydrolyzates or other commercially available forms. Within the scope of the invention it was found that quinacrine can efficiently inhibit viral cysteine proteases and/or serine proteases.
  • Flaviviridae such as Zika Virus (ZIKV), Dengue Virus (DENV), Yellow Fever Virus (YFV), West Nile Virus (WNV), but also Efficiently inhibits proteases of viruses belonging to the Togaviridae viruses, specifically the alphaviruses, such as the Chikungunya virus (CHIKV).
  • ZIKV Zika Virus
  • DEV Dengue Virus
  • YFV Yellow Fever Virus
  • WNV West Nile Virus
  • CHIKV Chikungunya virus
  • quinacrine is a competitive inhibitor for serine proteases and cysteine proteases, in particular NS2B/NS3 pro and nsP2 pro (see FIG. 4).
  • quinacrine competes with the substrate and thus binds to the active center of the proteases.
  • the pharmaceutical composition according to the invention which comprises at least quinacrine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient thereof, can comprise, for example but not limited to, salts or complexes thereof, the base addition salts, formed by the reaction of quinacrine with organic or inorganic bases such as hydroxide, carbonate or bicarbonate of a metal cation selected from the group consisting of alkali metals (sodium, potassium or lithium), alkaline earth metals (e.g. calcium or Magnesium). Salts formed from acid addition salts formed with inorganic acids (e.g.
  • hydrochloric acid hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like
  • organic acids such as e.g. B. acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, fumaric acid, maleic acid, ascorbic acid, benzoic acid, benzenesulfonic acid, methanesulfonic acid, tannic acid, palmoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid and polygalacturonic acid are formed.
  • organic acids such as e.g. B. acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, fumaric acid, maleic acid, ascorbic acid, benzoic acid, benzenesulfonic acid, methanesulfonic acid, tannic acid, palmoic acid, alginic acid, poly
  • This pharmaceutical composition comprising quinacrine
  • Quinacrine or the pharmaceutical composition of the present invention may be presented together with a commonly used adjuvant, carrier, diluent or excipient in the form of pharmaceutical compositions and unit dosages thereof, and in this form may be used as solids such as tablets or filled capsules or in the form of liquids such as solutions, suspensions, emulsions, elixirs or capsules filled therewith, all for oral use or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.
  • quinacrine or the pharmaceutical composition of the invention can be administered to the organism orally, intravenously or by inhalation.
  • Quinacrine or the pharmaceutical composition of the present invention may further comprise one or more pharmaceutically acceptable additional ingredients such as alum, stabilizers, antimicrobial agents, buffers, colors, flavors, adjuvants and the like.
  • Quinacrine or the pharmaceutical composition of quinacrine or the drug containing quinacrine is particularly useful in the prevention, anti-viral therapy or treatment of a viral infection in an organism, particularly viral infection by family viruses the Flaviviridae, in particular human and animal pathogenic viruses from the group of flaviviruses, such as, but not limited to, a Zika virus, dengue virus, yellow fever virus, TBE virus, JE virus, SLE virus , Louping Ill Virus, Usutu Virus, Wesselsbron Virus, West Nile Virus, Gadgets Gully Virus, Langat Virus, Modoc Virus, Kadam Virus, MVEV Virus, POW Virus, Karshi Virus , Sepik virus, TBE virus, Yokose virus and/or viruses from the Togaviridae family, in particular human and animal pathogenic viruses from the group of alpha viruses, such as, but not limited to, by a Chikungunya virus, Mayaro virus, Ge tah virus, O'
  • Quinacrine, its pharmaceutical composition or the medicament containing quinacrine can therefore advantageously be used for the treatment of viral infections, both a single infection and a combined infection by flaviviruses and alphaviruses, in a human or animal.
  • treatment and “treating” as used in the context of the invention mean to obtain a desired pharmacological and physiological effect in general. This effect can be prophylactic in order to prevent or partially alleviate a disease, symptom or condition thereof prevent, and/or may be therapeutic to partially or fully cure a disease, condition, symptom or adverse effect attributed to the disease.
  • treatment includes any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease, but still has not been diagnosed; (b) inhibition of the disease, ie stopping its development; or alleviation of the disease.
  • the term "effective amount” includes both a preventive amount and a "treatment effective amount”.
  • the preventively effective amount refers to a concentration of quinacrine effective to inhibit, reduce or prevent the likelihood of infection by Flaviviridae and/or Togaviridae, or to delay the onset of disease by viruses from the group of Prevent Flaviviridae and/or Togaviridae when administered prior to infection, i.e. before, during and/or shortly after the period of exposure to said viruses.
  • the term “effective amount” can refer to a concentration of quinacrine which, in the treatment of an infection caused by the viruses mentioned, leads, for example, to a reduction in the virus concentration.
  • organism should preferably be understood to mean mammals, in particular humans.
  • quinacrine is based on the fact that, by inhibiting the viral cysteine proteases and/or serine proteases, the polyprotein processing of the translated viral genome in the host cell is disrupted or prevented, as a result of which the virus becomes unable to replicate.
  • a method for treating a viral infection or a virus from the family Flaviviridae and Togaviridae is provided which is for viral cysteine protease inhibition and/or serine protease inhibition, in particular viral NS2B/NS3 pro inhibition and/or nsP2 pra - inhibition, in an organism, including administering to said organism a therapeutically effective amount of quinacrine or a pharmaceutically acceptable salt, solvate or hydrate thereof, said organism preferably being a mammal, in particular a human or can be an animal.
  • the administration of quinacrine, the pharmaceutical composition or the medicament of the invention can also be carried out in conjunction with other therapeutic agents which are useful in the treatment of viral infections.
  • a method for inhibiting the viral cysteine protease and/or serine proteases, in particular the viral NS2B/NS3 pr0 and/or the nsP2 pro , in a mammal comprises administering a therapeutically effective amount of quinacrine or a pharmaceutically acceptable salt, solvate or hydrate thereof to the mammal.
  • the mammal is a human or an animal.
  • Quinacrine can be administered orally, intravenously or by inhalation.
  • Figure 1 Primary inhibition tests of quinacrine against NS2B/NS3 pr0 from ZIKV, YFV, DENV and WNV (20 ⁇ mol/L quinacrine) and nsP2 pro from CHIKV (5 ⁇ mol/L quinacrine)
  • FIG. 2 Quinacrine titration against various flavivirus and alphavirus proteases; Axis Y: normalized protease activity (black bars) or inhibition (grey bars) in [%] of flavivirus and alphavirus proteases X axis: molar concentration of quinacrine [ ⁇ mol/L]
  • C quinacrine and WNV NS2B/NS3 per
  • D quinacrine and YFV NS2B/NS3 per
  • E quinacrine and CHIKV nsP2 per virus proteases
  • A Lineweaver-Burk plot of ZIKV NS2B/NS3 pr0 inhibition by quinacrine.
  • B Lineweaver-Burk plot of DENV NS2B/NS3 pro inhibition by quinacrine
  • Inhibition assays were performed to demonstrate the effect of quinacrine on viral cysteine protease activity and/or serine protease activity.
  • the influence of quinacrine on the protease enzyme activity of the NS2B/NS3 pro of the Flaviviridae and nsP2 pro of the Togaviridae was examined as an example.
  • the activity tests of the Flaviviridae proteases were performed using the fluorogenic substrate BOC-GRR-AMC (Bachem, Switzerland) in 20 mmol/L Tris pH 8.5, 10% glycerol, 0.01% Triton X-100 [1-3].
  • Togaviridae protease (CHIKV nsP2 pr0 ) activity assays were performed using a fluorogenic substrate DABCYL-KTSAVLQjSGFRKME-EDANS (Bachem, Switzerland) in a buffer containing 20 mmol/L Tris pH 7.2, 200 mmol/L NaCl, 1 mmol /L EDTA and 1 mmol/L TCEP.
  • reaction mixtures were pipetted into a Corning 96-well plate (Sigma Aldrich) containing 0.5 ⁇ mol/L NS2B/NS3 per proteinase protein or nsP2 per proteinase protein, and the assay was performed with the addition of the respective ones above mentioned substrates in a final concentration of 50 ⁇ mol/L.
  • nsP2 pro protease 0.5 ⁇ mol/L of the NS2B/NS3 pro or nsP2 pro protein was incubated with 20 ⁇ mol/L quinacrine for the NS2B/NS3 pro protease and 5 ⁇ mol/L quinacrine for the nsP2 pro protease. The mixtures were incubated at RT for 30 minutes. After addition of the substrate with a final concentration of 50 ⁇ mol/L, the increase/change in fluorescence intensities was measured at 60 s intervals over 30 minutes using an Infinite 200 PRO plate reader (Tecan, Switzerland). The temperature was set at 37°C.
  • the excitation and emission wavelengths were 360 nm and 460 nm for nsP2 pro of the CHIKV and 380 nm and 465 nm for NS2B/NS3 per of the ZIKV, DENV, YFV and WNV.
  • the inhibition tests were carried out in triplicate. The results are shown in Figure 1. Different experimental approaches (ZIKV, YFV, DENV, WNV and CHIKV) with the different virus proteases are shown on the abscissa (X-axis). Experimental approach "Control” shows results without the addition of quinacrine or inhibitor (black bar).
  • ZI KV shows results of the serine protease activity from the Zika virus after the addition of 20 mGhoI/I-quinacrine.
  • IC50 value could be determined (see Figure 2 and Table 1 ).
  • ZIKV NS2B/NS3 per 2.5 ⁇ 0.7 ⁇ mol/L DENV NS2B/NS3 per : 2.8 ⁇ 1.0 ⁇ mol/L YFV NS2B/NS3 per : 1.6 ⁇ 0.4 ⁇ mol/L WNV NS2B /NS3 per : 1.3 ⁇ 0.5 ⁇ mol/L CHIKV nsP2 per : 0.46 ⁇ 0.1 ⁇ mol/L
  • Table 1 shows the results of the measurements of the protease activity of different viruses with different added molar concentrations of quinacrine.
  • Table 1 Decrease in the tested virus protease activity of different viruses as a function of the quinacrine concentration added
  • IC 50 determination To determine the value of the half-maximal incubation concentration (IC5 0 value) for quinacrine, the initial rate of the enzymatic reaction was plotted against various concentrations of quinacrine using a dose-response curve in the GraphPad Prism5 software.
  • FIG. 3 the normalized activity [%] of flavivirus and alphavirus proteases is plotted on the ordinate (Y-axis) against the logarithm of the molar concentration in mihoI/L (abscissa X-axis) of quinacrine.
  • Table 2 shows the IC5 0 and K values obtained from the dose-response curves presented above.
  • Table 2 IC50 values and K values of quinacrine as an inhibitor against proteases of different representatives of flaviviruses and alphaviruses
  • the reaction speed of the NS2B/NS3 protease and the nsP" protease was determined at different final concentrations of the quinacrine inhibitor and the aforementioned substrate. 0.5 ⁇ mol/L of these proteases was mixed with quinacrine in various Concentrations incubated for 30 minutes at room temperature (RT). The reaction was then initiated by adding the appropriate concentration series of the substrate and the reaction rate was determined by determining the fluorescence intensity in accordance with the general test conditions described above under I). The data analysis was carried out using a Lineweaver-Burk plot. The reciprocal of the reaction rate (1/V) was plotted against the reciprocal of the substrate concentration (1/[S]) [5, 6],
  • the Ki value indicates the affinity of the enzyme or protease for the respective inhibitor.
  • the ICso value was determined as previously described.
  • the KM value was obtained from a database (BRENDA database) and [S] is the concentration of the substrate used in the test batches [7],
  • the abscissa (X-axis) shows the reciprocal of the substrate concentration [1/S].
  • the reciprocal value of the reaction rate [1/V] is plotted on the ordinate (Y-axis).
  • quinacrine is a competitive inhibitor for the viral cysteine proteases and/or serine proteases, in particular viral NS2B/NS3 and/or nsP2 proteases.

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Abstract

The present invention relates to an active ingredient for inhibiting cysteine proteases and serine proteases and, consequently, to a treatment of infections caused by viruses from the group of Flaviviridae and Togaviridae. The invention therefore relates to quinacrine for use for the production of an inhibitor for viral cysteine proteases and/or serine proteases. The invention also relates to quinacrine for use for the production of an inhibitor for cysteine proteases and/or serine proteases of viruses from the family of Flaviviridae and/or Togaviridae.

Description

Beschreibung description
Quinacrin als Inhibitor viraler Cysteinproteasen und/oder Serinproteasen Quinacrine as an inhibitor of viral cysteine proteases and/or serine proteases
Die vorliegende Erfindung bezieht sich auf Quinacrin zur Verwendung als Inhibitor und für die Herstellung eines Inhibitors von viralen Cysteinproteasen und/oder Serinproteasen, insbeson- dere zur Inhibierung von viralen nsP2 Proteasen (nsP2pro) und/oder NS2B/NS3 Proteasen (NS2B/NS3pro), sowie die Verwendung von Quinacrin zur Herstellung eines Inhibitors für virale Cysteinproteasen und Serinproteasen. The present invention relates to quinacrine for use as an inhibitor and for the production of an inhibitor of viral cysteine proteases and/or serine proteases, in particular for inhibiting viral nsP2 proteases (nsP2 pro ) and/or NS2B/NS3 proteases (NS2B/NS3 pro ), as well as the use of quinacrine for the production of an inhibitor for viral cysteine proteases and serine proteases.
Die Erfindung bezieht sich weiterhin auf Quinacrin zur Verwendung in der medizinischen The- rapie. Ein weiterer Aspekt der Erfindung ist eine pharmazeutische Zusammensetzung, die min- destens Quinacrin oder ein pharmazeutisch geeignetes Salz davon und ein/einen pharmazeu- tisch geeigneten/geeignetes Träger, Verdünnungsmittel oder Arzneiträger davon umfasst. Quinacrin kann zur Verwendung bei der Vorbeugung oder Behandlung einer viralen Infektion in einem Organismus, insbesondere einer Infektion mit wenigstens einem Virus aus der Fami- lie der Flaviviridae und/oder Togaviridae dienen. Quinacrin kann dabei zur Behandlung einer viralen Infektion in einem Organismus, insbesondere zur Behandlung einer kombinierten In- fektion mit Viren aus der Familie der Flaviviridae und Togaviridae, sowie auf Quinacrin als Arzneimittel in der Behandlung von viralen Infektionen, insbesondere zur Behandlung einer kombinierten Infektion mit Viren aus der Familie der Flaviviridae und Togaviridae verwendet werden. The invention further relates to quinacrine for use in medical therapy. Another aspect of the invention is a pharmaceutical composition comprising at least quinacrine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient thereof. Quinacrine can be used to prevent or treat a viral infection in an organism, in particular an infection with at least one virus from the Flaviviridae and/or Togaviridae family. Quinacrine can be used to treat a viral infection in an organism, in particular to treat a combined infection with viruses from the Flaviviridae and Togaviridae families, and to use quinacrine as a drug in the treatment of viral infections, in particular to treat a combined infection with viruses from the Flaviviridae and Togaviridae families can be used.
Darüber hinaus betrifft die Erfindung die Verwendung von Quinacrin als therapeutisches Mittel, sowie in der Herstellung eines Arzneimittels zur Behandlung einer viralen Infektion in einem Organismus, insbesondere zur Behandlung einer kombinierten Infektion mit Viren aus der Fa- milie der Flaviviridae und Togaviridae, und weiterhin auf ein Verfahren zur Behandlung von viralen Infektionen, insbesondere zur Behandlung von kombinierten Infektionen mit Viren aus der Familie der Flaviviridae und Alphaviren. In addition, the invention relates to the use of quinacrine as a therapeutic agent and in the manufacture of a medicament for the treatment of a viral infection in an organism, in particular for the treatment of a combined infection with viruses from the Flaviviridae and Togaviridae families, and also to a Method for treating viral infections, in particular for treating combined infections with viruses from the Flaviviridae and Alphavirus families.
Stand der Technik State of the art
Quinacrin ist eine bekannte Substanz, die bereits als Arzneistoff zugelassen ist. Quinacrin wird beispielsweise unter dem Handelsnamen Mepacrin oder Atabrine eingesetzt. Quinacrin wurde in der Vergangenheit zur Malariaprophylaxe verwendet. Ein Einsatz von Quinacrin bei Gardia- und Protozoen Infektionen ist ebenfalls bekannt. Quinacrine is a well-known substance that is already approved as a medicinal product. Quinacrine is used, for example, under the trade names Mepacrine or Atabrine. Quinacrine has historically been used for malaria prophylaxis. A use of quinacrine in gardia and protozoa infections is also known.
Quinacrin wird weiterhin bei der Behandlung von discoidem und subkutanem Lupus erythe- matodes verwendet und wird als Prophylaxe zur Verhinderung des Wiederauftretens von Pneumothorax bei Patienten mit hohem Risiko, zum Beispiel bei Mukoviszidose-Patienten, gegeben. Zu Quinacrin wird in der Literatur ein inhibierender Effekt gegen Zika und Dengue Virus Zellkul- turen beschrieben [1; 2; 3], Quinacrin zeigt weiterhin eine hemmende Wirkung gegen eine Ebola Virus Infektion in Mäusen [4], Quinacrine is also used in the treatment of discoid and subcutaneous lupus erythematosus and is given as prophylaxis to prevent recurrence of pneumothorax in high-risk patients, such as those with cystic fibrosis. An inhibitory effect against Zika and dengue virus cell cultures is described in the literature for quinacrine [1; 2; 3], quinacrine also shows an inhibitory effect against Ebola virus infection in mice [4],
Der genaue Wirkungsmechanismus von Quinacrin ist derzeit nicht bekannt, aber das Molekül scheint Translation und Transkription von DNA zu hemmen. Quinacrin wird auch als Inhibitor der sauren Sphingomyelinase und Cholinesterase beschrieben. Quinacrine's exact mechanism of action is currently unknown, but the molecule appears to inhibit translation and transcription of DNA. Quinacrine is also described as an inhibitor of acid sphingomyelinase and cholinesterase.
Aufgabe und Lösung task and solution
Die Aufgabe der Erfindung ist es, einen Wirkstoff zur Behandlung von viralen Infektionen, ins- besondere mit Viren aus der Familie der Flaviviridae und Togaviridae, vor allem mit Vertretern der Gattung der Flaviviren und Alphaviren, bereitzustellen, ganz besonders von Infektionen mit Zika, Dengue, Gelbfieber, West-Nil oder Chikungunya Viren und Kombinationen dieser Viren, wie beispielsweise Zika-Dengue, Zika-Gelbfieber, Zika-Dengue-Chikungunya. Es ist weiterhin Aufgabe der Erfindung einen Wirkstoff als Arzneimittel oder therapeutisches Mittel zur Behandlung dieser viralen Infektionen bereitzustellen. The object of the invention is to provide an active substance for the treatment of viral infections, in particular with viruses from the family Flaviviridae and Togaviridae, especially with representatives of the genus Flavivirus and Alphavirus, especially infections with Zika, Dengue, Yellow fever, West Nile or Chikungunya viruses and combinations of these viruses, such as Zika dengue, Zika yellow fever, Zika dengue Chikungunya. It is a further object of the invention to provide an active ingredient as a pharmaceutical or therapeutic agent for treating these viral infections.
Die Aufgaben der Erfindung werden gelöst durch Quinacrin mit den Merkmalen des Hauptan- spruchs sowie durch die Verfahren zur Behandlung gemäß nebengeordneter Ansprüche. The objects of the invention are achieved by quinacrine having the features of the main claim and by the methods of treatment according to the independent claims.
Vorteilhafte Ausgestaltungen der Verwendung von Quinacrin und der Verfahren ergeben sich aus den darauf rückbezogenen Ansprüchen. Advantageous configurations of the use of quinacrine and of the methods result from the claims referring back thereto.
Gegenstand der Erfindung: Subject of the invention:
Die vorliegende Erfindung richtet sich auf einen Wirkstoff zur Inhibierung von Cysteinpro- teasen und Serinproteasen und damit verbunden auf eine Behandlung von Infektionen durch Viren aus der Gruppe der Flaviviridae und Togaviridae. Die Cysteinproteasen, die in Viren aus der Familie der Togaviridae Vorkommen, und/oder Serinproteasen, die in Viren aus der Familie der Flaviviridae Vorkommen, sind essentiell für den viralen Replikationsmechanismus im Wirt. Die Erfinder haben in überraschender Weise herausgefunden, dass Quinacrin diese beiden Proteaseklassen, insbesondere die virale Cysteinprotease nsP2pro, und/oder die vi- rale Serinproteasen NS2B/NS3pro hemmen kann. Es ist daher möglich, mit Quinacrin Viren aus der Gruppe der Flaviviridae und Togaviridae nicht nur einzeln, sondern auch in Kombi- nation aus diesen Gruppen zu hemmen. Gegenstand der Erfindung ist daher Quinacrin zur Verwendung für die Herstellung eines In- hibitors für virale Cysteinproteasen und/oder Serinproteasen. The present invention relates to an active substance for inhibiting cysteine proteases and serine proteases and, associated therewith, to treating infections caused by viruses from the group of Flaviviridae and Togaviridae. The cysteine proteases found in Togaviridae family viruses and/or serine proteases found in Flaviviridae family viruses are essential for the viral replication mechanism in the host. The inventors have surprisingly found that quinacrine can inhibit these two protease classes, in particular the viral cysteine protease nsP2 pro and/or the viral serine proteases NS2B/NS3 pro . It is therefore possible to use quinacrine to inhibit viruses from the group of Flaviviridae and Togaviridae not only individually, but also in combination from these groups. The subject matter of the invention is therefore quinacrine for use in the production of an inhibitor for viral cysteine proteases and/or serine proteases.
Ein weiterer Gegenstand der Erfindung ist Quinacrin zur Verwendung für die Herstellung ei- nes Inhibitors für Cysteinproteasen und/oder Serinproteasen von Viren aus der Familie der Flaviviridae und/oder Togaviridae. Another subject matter of the invention is quinacrine for use in the production of an inhibitor for cysteine proteases and/or serine proteases of viruses from the Flaviviridae and/or Togaviridae family.
Damit verbunden ist insbesondere Quinacrin zur Verwendung für die Herstellung eines Inhi- bitors für die Serinprotease NS2B/NS3pr0 und/oder Cysteinprotease nsP2pro. This is associated in particular with quinacrine for use in the production of an inhibitor for the serine protease NS2B/NS3 pr0 and/or cysteine protease nsP2 pro .
Ein weiterer Aspekt der Erfindung ist die Verwendung von Quinacrin zur Herstellung eines Inhibitors für diese viralen Cysteinproteasen und/oder Serinproteasen aus der Familie der Flaviviridae und/oder Togaviridae, insbesondere für die Serinprotease NS2B/NS3pro und/oder Cysteinprotease nsP2pr0. A further aspect of the invention is the use of quinacrine to produce an inhibitor for these viral cysteine proteases and/or serine proteases from the Flaviviridae and/or Togaviridae family, in particular for the serine protease NS2B/NS3 pro and/or cysteine protease nsP2 pr0 .
Ein vierter Aspekt der Erfindung ist Quinacrin zur Verwendung in der medizinischen Thera- pie. A fourth aspect of the invention is quinacrine for use in medical therapy.
Ein fünfter Aspekt ist eine pharmazeutische Zusammensetzung, die mindestens Quinacrin oder ein pharmazeutisch geeignetes Salz davon und ein/einen pharmazeutisch geeigne- ten/geeignetes Träger, Verdünnungsmittel oder Arzneiträger davon umfasst. A fifth aspect is a pharmaceutical composition comprising at least quinacrine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient thereof.
Ein sechster Aspekt der Erfindung ist Quinacrin als pharmazeutische Zusammensetzung zur Verwendung in der medizinischen Therapie. A sixth aspect of the invention is quinacrine as a pharmaceutical composition for use in medical therapy.
Ein siebter Aspekt der Erfindung ist Quinacrin zur Verwendung bei der Vorbeugung oder Be- handlung einer viralen Infektion in einem Organismus, insbesondere einer Infektion mit we- nigstens einem Virus aus der Familie der Flaviviridae und/oder Togaviridae. A seventh aspect of the invention is quinacrine for use in preventing or treating a viral infection in an organism, in particular an infection with at least one virus from the Flaviviridae and/or Togaviridae family.
Ein weiterer Aspekt ist Quinacrin zur Verwendung bei der Vorbeugung oder Behandlung ei- ner viralen Infektion in einem Organismus, insbesondere einer Infektion mit wenigstens ei- nem Virus aus der Gruppe der Familie der Flaviviridae und/oder Togaviridae. Zu einer viralen Infektion aus der Familie der Flaviviridae können beispielhaft, jedoch nicht darauf be- schränkt, folgende Flaviviren genannt werden: Zika-Virus, Dengue-Virus, Gelbfieber-Virus, FSME-Virus, JE-Virus, SLE-Virus, Louping-Ill-Virus, Usutu-Virus, Wesselsbron-Virus, West- Nil-Virus, Gadgets-Gully-Virus, Langat-Virus, Modoc-Virus, Kadam-Virus, MVEV-Virus, POW-Virus, Karshi-Virus, Sepik-Virus, TBE-Virus, Yokose-Virus. Zu einer viralen Infektion aus der Familie der Togaviridae können beispielhaft, jedoch nicht darauf beschränkt, fol- gende Alphaviren genannt werden: Chikungunya-Virus, Mayaro-Virus, Getah-Virus, O’nyong-nyong-Virus, Ross-River-Virus, Sindbis-Virus, Ockelbo-Virus, Babanki-Virus, Bar- mah-Forest-Virus, Semliki-Forest-Virus, Venezuelan-Equine-Encephalitis-Virus, Everglades- Virus, Mucambo-Virus, Tonate-Virus, Eastern-Equine-Encephalitis-Virus, Western-Equine- Encephalitis-Virus, Highlands-Virus, Middelburg-Virus, Rio Negro-Virus, Aura Virus, Babanki Virus, Ockelbo Virus, Whataroa Virus. Gegenstand der Erfindung sind auch Arzneimittel enthaltend Quinacrin sowie einen pharma- zeutisch geeigneten Träger und die Verwendung von Quinacrin in der Herstellung eines Arz- neimittels zur Behandlung von Infektionen mit wenigstens einem Virus aus der Familie der Flaviviridae und/oder Togaviridae in einem Organismus. A further aspect is quinacrine for use in the prevention or treatment of a viral infection in an organism, in particular an infection with at least one virus from the group of the Flaviviridae and/or Togaviridae family. The following flaviviruses can be mentioned as examples of a viral infection from the Flaviviridae family, but are not limited to these: Zika virus, dengue virus, yellow fever virus, TBE virus, JE virus, SLE virus, louping virus Ill Virus, Usutu Virus, Wesselsbron Virus, West Nile Virus, Gadgets Gully Virus, Langat Virus, Modoc Virus, Kadam Virus, MVEV Virus, POW Virus, Karshi Virus, Sepik Virus, TBE Virus, Yokose Virus. The following alphaviruses can be mentioned as examples of a viral infection from the Togaviridae family, but are not limited to these: Chikungunya virus, Mayaro virus, Getah virus, O'nyong-nyong virus, Ross River virus, Sindbis virus, Ockelbo virus, Babanki virus, Barmah Forest virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Everglades virus, Mucambo virus, Tonate virus, Eastern equine Encephalitis Virus, Western Equine Encephalitis Virus, Highlands Virus, Middelburg Virus, Rio Negro Virus, Aura Virus, Babanki Virus, Ockelbo Virus, Whataroa Virus. The invention also relates to medicaments containing quinacrine and a pharmaceutically suitable carrier and the use of quinacrine in the production of a medicament for the treatment of infections with at least one virus from the Flaviviridae and/or Togaviridae family in an organism.
Ebenfalls Gegenstand der Erfindung ist ein Verfahren zur Inhibierung viraler Serinproteasen und/oder Cysteinproteasen, von Viren aus der Familie der Flavirviridae und/oder Toga- viridae, sowie zur Behandlung oder Verhinderung einer Virusinfektion in einem Organismus, insbesondere zur Behandlung einer viralen Infektion durch wenigstens einen Virus aus der Gruppe der Flaviviridae, wie beispielsweise, jedoch nicht darauf beschränkt, durch einen Zika-Virus, Dengue-Virus, Gelbfieber-Virus, FSME-Virus, JE-Virus, SLE-Virus, Louping-Ill- Virus, Usutu-Virus, Wesselsbron-Virus, West-Nil-Virus, Gadgets-Gully-Virus, Langat-Virus, Modoc-Virus, Kadam-Virus, MVEV-Virus, POW-Virus, Karshi-Virus, Sepik-Virus, TBE-Virus, Yokose-Virus, und/oder beispielsweise, jedoch nicht darauf beschränkt, für die Familie der Togaviridae, durch einen Chikungunya-Virus, Mayaro-Virus, Getah-Virus, O’nyong-nyong- Virus, Ross-River-Virus, Sindbis-Virus, Ockelbo-Virus, Babanki-Virus, Barmah-Forest-Virus, Semliki-Forest-Virus, Venezuelan-Equine-Encephalitis-Virus, Everglades-Virus, Mucambo- Virus, Tonate-Virus, Eastern-Equine-Encephalitis-Virus, Western-Equine-Encephalitis-Virus, Highlands-Virus, Middelburg-Virus, Rio Negro-Virus, Aura Virus, Babanki Virus, Ockelbo Vi- rus, Whataroa Virus, bei dem Quinacrin oder eine pharmazeutische Zusammensetzung um- fassend Quinacrin gemäß zuvor genannter Merkmale in einer therapeutisch wirksamen Menge zugegeben wird. The invention also relates to a method for inhibiting viral serine proteases and/or cysteine proteases, viruses from the Flavirviridae and/or Togaviridae family, and for treating or preventing a viral infection in an organism, in particular for treating a viral infection by at least one Virus from the group Flaviviridae such as, but not limited to, Zika virus, Dengue virus, Yellow fever virus, TBE virus, JE virus, SLE virus, Louping III virus, Usutu virus , Wesselsbron Virus, West Nile Virus, Gadgets Gully Virus, Langat Virus, Modoc Virus, Kadam Virus, MVEV Virus, POW Virus, Karshi Virus, Sepik Virus, TBE Virus, Yokose virus, and/or for example, but not limited to, for the Togaviridae family, by a Chikungunya virus, Mayaro virus, Getah virus, O'nyong-nyong virus, Ross River virus, Sindbis virus , Ockelbo virus, Babanki virus, Barmah forest virus, Semliki forest virus s, Venezuelan equine encephalitis virus, Everglades virus, Mucambo virus, Tonate virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Highlands virus, Middelburg virus, Rio Negro virus , Aura virus, Babanki virus, Ockelbo virus, Whataroa virus, in which quinacrine or a pharmaceutical composition comprising quinacrine according to the aforementioned features is added in a therapeutically effective amount.
Darüber hinaus umfasst die Erfindung auch ein Verfahren zur Inhibierung der viralen Cys- teinprotease und/oder Serinproteasen, insbesondere der viralen NS2B/NS3pro und/oder der nsP2pro, in einem Organismus, bei dem eine Verabreichung einer therapeutisch wirksamen Menge von Quinacrin oder einer pharmazeutischen Zusammensetzung, umfassend Quinacrin, erfolgt. In addition, the invention also includes a method for inhibiting the viral cysteine protease and / or serine proteases, in particular the viral NS2B / NS3 per and / or nsP2 per , in an organism in which administration of a therapeutically effective amount of quinacrine or a pharmaceutical composition comprising quinacrine.
Auch ein Verfahren zur Inhibierung oder zur Inaktivierung viraler Cysteinproteasen und/oder Serinproteasen von Viren aus der Familie der Flaviviridae und/oder Togaviridae, umfassend den Schritt des Inkontaktbringens der Zellen des Organismus mit einer therapeutisch wirksa- men Menge von Quinacrin oder einer pharmazeutischen Zusammensetzung ist Gegenstand der Erfindung. Dieses Verfahren kann sowohl in-vivo als auch ex-vivo durchgeführt werden und ist ebenfalls Gegenstand der Erfindung. Erfindungsgemäß sind insbesondere Cysteinproteasen als Target für die erfindungsgemäße Verwendung von Quinacrin geeignet, die in humanpathogenen oder tierpathogenen Alphavi- ren aus der Gruppe der Chikungunya-Virus, Mayaro-Virus, Getah-Virus, O’nyong-nyong-Vi- rus, Ross-River-Virus, Sindbis-Virus, Ockelbo-Virus, Babanki-Virus, Barmah-Forest-Virus, Semliki-Forest-Virus, Venezuelan-Equine-Encephalitis-Virus, Everglades-Virus, Mucambo- Virus, Tonate-Virus, Eastern-Equine-Encephalitis-Virus, Western-Equine-Encephalitis-Virus, Highlands-Virus, Middelburg-Virus, Rio Negro-Virus, Aura Virus, Babanki Virus, Ockelbo Vi- rus, Whataroa Virus Vorkommen. The subject is also a method for inhibiting or inactivating viral cysteine proteases and/or serine proteases of viruses from the Flaviviridae and/or Togaviridae family, comprising the step of bringing the cells of the organism into contact with a therapeutically effective amount of quinacrine or a pharmaceutical composition the invention. This method can be carried out both in-vivo and ex-vivo and is also the subject of the invention. According to the invention, cysteine proteases are particularly suitable as targets for the use of quinacrine according to the invention, which are present in human-pathogenic or animal-pathogenic alphaviruses from the group consisting of the Chikungunya virus, Mayaro virus, Getah virus, O'nyong-nyong virus, Ross River virus, Sindbis virus, Ockelbo virus, Babanki virus, Barmah Forest virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Everglades virus, Mucambo virus, Tonate virus, Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, Highlands Virus, Middelburg Virus, Rio Negro Virus, Aura Virus, Babanki Virus, Ockelbo Virus, Whataroa Virus Occurrence.
Serinproteasen als Target für die erfindungsgemäße Verwendung von Quinacrin, kommen vorzugsweise in humanpathogenen oder tierpathogenen Flaviviren aus der Gruppe des Zika- Virus, Dengue-Virus, Gelbfieber-Virus, FSME-Virus, JE-Virus, SLE-Virus, Louping-Ill-Virus, Usutu-Virus, Wesselsbron-Virus, West-Nil-Virus, Gadgets-Gully-Virus, Langat-Virus, Modoc- Virus, Kadam-Virus, MVEV-Virus, POW-Virus, Karshi-Virus, Sepik-Virus, TBE-Virus, Yokose- Virus vor. Serine proteases as a target for the use of quinacrine according to the invention preferably come in human-pathogenic or animal-pathogenic flaviviruses from the group of Zika virus, dengue virus, yellow fever virus, TBE virus, JE virus, SLE virus, Louping III virus , Usutu Virus, Wesselsbron Virus, West Nile Virus, Gadgets Gully Virus, Langat Virus, Modoc Virus, Kadam Virus, MVEV Virus, POW Virus, Karshi Virus, Sepik Virus, TBE virus, Yokose virus.
Quinacrin kann daher als Breitband Inhibitor gegen einzelne Virusinfektionen und/oder Ko- Infektionen verwendet werden, da es die essentiellen viralen Proteasen, insbesondere die Cystein- und Serin-Proteasen, dieser verschiedenen Virusfamilien hemmen kann. Quinacrine can therefore be used as a broad spectrum inhibitor against single viral infections and/or co-infections, since it can inhibit the essential viral proteases, particularly the cysteine and serine proteases, of these different virus families.
Unter der Bezeichnung Quinacrin wird im Rahmen der vorliegenden Erfindung eine Verbin- dung der folgenden Strukturformel (I) verstanden, die aus einem 1 :1 Gemisch aus einer R- Form (oben) und einer S-Form (unten) besteht:
Figure imgf000006_0001
In the context of the present invention, the term quinacrine means a compound of the following structural formula (I), which consists of a 1:1 mixture of an R form (above) and an S form (below):
Figure imgf000006_0001
Quinacrin kann im Rahmen der Erfindung beispielsweise in Form seiner Hydrolysate oder anderer im Handel erhältlichen Formen eingesetzt werden. Im Rahmen der Erfindung wurde herausgefunden, dass Quinacrin virale Cysteinproteasen und/oder Serinproteasen, effizient hemmen kann. Within the scope of the invention, quinacrine can be used, for example, in the form of its hydrolyzates or other commercially available forms. Within the scope of the invention it was found that quinacrine can efficiently inhibit viral cysteine proteases and/or serine proteases.
Primäre Inhibitionstests haben gezeigt, dass Quinacrin, Proteasen von Viren, die zu den Fla- viviridae gehören, wie beispielsweise Zika Virus (ZIKV), Dengue Virus (DENV), Gelbfieber Virus (YFV), West-Nile Virus (WNV), aber auch Proteasen von Viren, die zu den Viren der Togaviridae, speziell der Alphaviren gehören, wie beispielsweise den Chikungunya Virus (CHIKV), effizient hemmt. Im Fall der Flaviviridae betrifft dies NS2B/NS3pro und im Fall der Togaviridae, insbesondere Alphaviren, nsP2pro. Primary inhibition tests have shown that quinacrine, proteases of viruses belonging to the Flaviviridae, such as Zika Virus (ZIKV), Dengue Virus (DENV), Yellow Fever Virus (YFV), West Nile Virus (WNV), but also Efficiently inhibits proteases of viruses belonging to the Togaviridae viruses, specifically the alphaviruses, such as the Chikungunya virus (CHIKV). In the case of Flaviviridae, this concerns NS2B/NS3 pro and in the case of Togaviridae, especially alphaviruses, nsP2 pro .
Zusätzliche Aktivitätstests, basierend auf klassischen Michaelis-Menten Annahmen, konnten zeigen, dass Quinacrin ein kompetitiver Inhibitor für Serinproteasen und Cysteinproteasen insbesondere NS2B/NS3pro und nsP2pro ist (siehe Figur 4). Quinacrin als kompetitiver Inhi- bitor tritt dabei in Konkurrenz mit dem Substrat und bindet also im aktiven Zentrum der Pro- teasen. Additional activity tests, based on classic Michaelis-Menten assumptions, were able to show that quinacrine is a competitive inhibitor for serine proteases and cysteine proteases, in particular NS2B/NS3 pro and nsP2 pro (see FIG. 4). As a competitive inhibitor, quinacrine competes with the substrate and thus binds to the active center of the proteases.
Die erfindungsgemäße pharmazeutische Zusammensetzung, die mindestens Quinacrin oder ein pharmazeutisch geeignetes Salz davon und ein/einen pharmazeutisch geeigneten/geeig- netes Träger, Verdünnungsmittel oder Arzneiträger davon umfasst, können beispielsweise, jedoch nicht darauf beschränkt Salze oder Komplexe davon umfassen, die Basenadditions- salze, die durch Reaktion von Quinacrin mit organischen oder anorganischen Basen wie Hydroxid, Carbonat oder Bicarbonat eines Metallkations gebildet werden, wie sie in der Gruppe ausgewählt sind, die aus Alkali besteht Metalle (Natrium, Kalium oder Lithium), Erdalkalimetalle (z. B. Calcium oder Magnesium). Es können weiterhin auch Salze umfasst sein, die aus mit anorganischen Säuren gebildeten Säureadditionssalzen (z. B. Salzsäure, Bromwasserstoffsäure, Schwefelsäure, Phosphorsäure, Salpetersäure und dergleichen) ge- bildet werden, sowie Salze, die mit organischen Säuren wie z. B. Essigsäure, Oxalsäure, Weinsäure, Bernsteinsäure, Äpfelsäure, Fumarsäure, Maleinsäure, Ascorbinsäure, Benzoe- säure, Benzolsulfonsäure, Methansulfonsäure, Gerbsäure, Palmoesäure, Alginsäure, Poly- glutaminsäure, Naphthalinsulfonsäure, Naphthalindisulfonsäure und Polygalacturonsäure ge- bildet werden. The pharmaceutical composition according to the invention, which comprises at least quinacrine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient thereof, can comprise, for example but not limited to, salts or complexes thereof, the base addition salts, formed by the reaction of quinacrine with organic or inorganic bases such as hydroxide, carbonate or bicarbonate of a metal cation selected from the group consisting of alkali metals (sodium, potassium or lithium), alkaline earth metals (e.g. calcium or Magnesium). Salts formed from acid addition salts formed with inorganic acids (e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like) can also be included, as well as salts formed with organic acids such as e.g. B. acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, fumaric acid, maleic acid, ascorbic acid, benzoic acid, benzenesulfonic acid, methanesulfonic acid, tannic acid, palmoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid and polygalacturonic acid are formed.
Dabei kann diese pharmazeutische Zusammensetzung, umfassend Quinacrin, ein oder meh- rere pharmazeutisch geeignete Träger beinhalten. Hier sind alle nach dem Stand der Tech- nik bekannten Arzneiträgerstoffe geeignet. Quinacrin oder die erfindungsgemäße pharmazeutische Zusammensetzung können zusam- men mit einem herkömmlich verwendeten Adjuvans, Träger, Verdünnungsmittel oder Hilfs- stoff in Form von pharmazeutischen Zusammensetzungen und Einheitsdosierungen davon gegeben werden und können in dieser Form als Feststoffe wie Tabletten oder gefüllte Kap- seln eingesetzt werden oder in Form von Flüssigkeiten wie Lösungen, Suspensionen, Emul- sionen, Elixiere oder damit gefüllte Kapseln, alle zur oralen Anwendung oder in Form von sterilen injizierbaren Lösungen zur parenteralen (einschließlich subkutanen) Anwendung. Quinacrin oder die Pharmazeutische Zusammensetzung der Erfindung können dem Organis- mus beispielsweise oral, intravenös oder durch Inhalation verabreicht werden. This pharmaceutical composition, comprising quinacrine, can contain one or more pharmaceutically suitable carriers. All excipients known according to the state of the art are suitable here. Quinacrine or the pharmaceutical composition of the present invention may be presented together with a commonly used adjuvant, carrier, diluent or excipient in the form of pharmaceutical compositions and unit dosages thereof, and in this form may be used as solids such as tablets or filled capsules or in the form of liquids such as solutions, suspensions, emulsions, elixirs or capsules filled therewith, all for oral use or in the form of sterile injectable solutions for parenteral (including subcutaneous) use. For example, quinacrine or the pharmaceutical composition of the invention can be administered to the organism orally, intravenously or by inhalation.
Quinacrin oder die erfindungsgemäße pharmazeutische Zusammensetzung können ferner einen oder mehrere pharmazeutisch verträgliche zusätzliche Bestandteile wie Alaun, Stabi- lisatoren, antimikrobielle Mittel, Puffer, Farbstoffe, Aromastoffe, Adjuvantien und dergleichen umfassen. Quinacrine or the pharmaceutical composition of the present invention may further comprise one or more pharmaceutically acceptable additional ingredients such as alum, stabilizers, antimicrobial agents, buffers, colors, flavors, adjuvants and the like.
Quinacrin oder die pharmazeutische Zusammensetzung von Quinacrin oder das Arzneimittel enthaltend Quinacrin ist insbesondere zur Verwendung bei der Vorbeugung, in der anti-vira- len Therapie oder zur Behandlung einer viralen Infektion in einem Organismus ersetzbar, wobei hier insbesondere die virale Infektion durch Viren aus der Familie der Flaviviridae, ins- besondere human- und tierpathogene Viren aus der Gruppe der Flaviviren, wie beispielhaft, jedoch nicht darauf beschränkt, durch einen Zika-Virus, Dengue-Virus, Gelbfieber-Virus, FSME-Virus, JE-Virus, SLE-Virus, Louping-Ill-Virus, Usutu-Virus, Wesselsbron-Virus, West- Nil-Virus, Gadgets-Gully-Virus, Langat-Virus, Modoc-Virus, Kadam-Virus, MVEV-Virus, POW-Virus, Karshi-Virus, Sepik-Virus, TBE-Virus, Yokose-Virus und/oder Viren aus der Fa- milie der Togaviridae, insbesondere human- und tierpathogene Viren aus der Gruppe der Al- phaviren, wie beispielhaft, jedoch nicht darauf beschränkt, durch einen Chikungunya-Virus, Mayaro-Virus, Getah-Virus, O’nyong-nyong-Virus, Ross-River-Virus, Sindbis-Virus, Ockelbo- Virus, Babanki-Virus, Barmah-Forest-Virus, Semliki-Forest-Virus, Venezuelan-Equine-Ence- phalitis-Virus, Everglades-Virus, Mucambo-Virus, Tonate-Virus, Eastern-Equine-Encephali- tis-Virus, Western-Equine-Encephalitis-Virus, Highlands-Virus, Middelburg-Virus, Rio Negro- Virus, Aura Virus, Babanki Virus, Ockelbo Virus, Whataroa Virus, Anwendung finden. Quinacrin, seine pharmazeutische Zusammensetzung oder das Arzneimittel enthaltend Quinacrin, kann daher vorteilhaft zur Behandlung viraler Infektionen, sowohl einer einzelnen Infektion, als auch einer kombinierten Infektion durch Flaviviren und Alphaviren, in einem Menschen oder Tier eingesetzt werden. Der im Rahmen der Erfindung verwendete Begriff „Behandlung“ und „behandeln“ bedeutet, im Allgemeinen eine gewünschte pharmakologische und physiologische Wirkung zu erhal- ten. Diese Wirkung kann prophylaktisch sein, um eine Krankheit, ein Symptom oder einen Zustand davon zu verhindern oder teilweise zu verhindern, und / oder kann therapeutisch sein, um eine Krankheit, einen Zustand, ein Symptom oder eine nachteilige Wirkung, die der Krankheit zugeschrieben wird, teilweise oder vollständig zu heilen. Der Begriff "Behandlung", wie er hier verwendet wird, umfasst jede Behandlung einer Krankheit bei einem Säugetier, insbesondere einem Menschen, und umfasst: (a) Verhindern des Auftretens der Krankheit bei einem Subjekt, das für die Krankheit prädisponiert sein kann, aber noch nicht diagnosti- ziert wurde; (b) Hemmung der Krankheit, d.h. Stillstand ihrer Entwicklung; oder Linderung der Krankheit. Quinacrine or the pharmaceutical composition of quinacrine or the drug containing quinacrine is particularly useful in the prevention, anti-viral therapy or treatment of a viral infection in an organism, particularly viral infection by family viruses the Flaviviridae, in particular human and animal pathogenic viruses from the group of flaviviruses, such as, but not limited to, a Zika virus, dengue virus, yellow fever virus, TBE virus, JE virus, SLE virus , Louping Ill Virus, Usutu Virus, Wesselsbron Virus, West Nile Virus, Gadgets Gully Virus, Langat Virus, Modoc Virus, Kadam Virus, MVEV Virus, POW Virus, Karshi Virus , Sepik virus, TBE virus, Yokose virus and/or viruses from the Togaviridae family, in particular human and animal pathogenic viruses from the group of alpha viruses, such as, but not limited to, by a Chikungunya virus, Mayaro virus, Ge tah virus, O'nyong-nyong virus, Ross River virus, Sindbis virus, Ockelbo virus, Babanki virus, Barmah Forest virus, Semliki Forest virus, Venezuelan equine encephalitis Virus, Everglades Virus, Mucambo Virus, Tonate Virus, Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, Highlands Virus, Middelburg Virus, Rio Negro Virus, Aura Virus, Babanki Virus , Ockelbo Virus, Whataroa Virus, find application. Quinacrine, its pharmaceutical composition or the medicament containing quinacrine can therefore advantageously be used for the treatment of viral infections, both a single infection and a combined infection by flaviviruses and alphaviruses, in a human or animal. The terms "treatment" and "treating" as used in the context of the invention mean to obtain a desired pharmacological and physiological effect in general. This effect can be prophylactic in order to prevent or partially alleviate a disease, symptom or condition thereof prevent, and/or may be therapeutic to partially or fully cure a disease, condition, symptom or adverse effect attributed to the disease. The term "treatment" as used herein includes any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease, but still has not been diagnosed; (b) inhibition of the disease, ie stopping its development; or alleviation of the disease.
Der verwendete Begriff „wirksame Menge“ umfasst sowohl eine Menge, die vorbeugend wirkt, als auch eine „behandlungswirksame Menge“. Die vorbeugend wirksame Menge be- zieht sich auf eine Konzentration von Quinacrin, die wirksam ist, um die Wahrscheinlichkeit einer Infektion durch Flaviviridae und/oder durch Togaviridae zu hemmen, zu verringern oder zu verhindern oder das verzögerte Auftreten der Krankheit durch Viren aus der Gruppe der Flaviviridae und/oder Togaviridae zu verhindern, wenn vor einer Infektion verabreicht, d.h. vor, während und / oder kurz nach der Expositionsdauer gegenüber den genannten Viren. Ebenso kann der Begriff „wirksame Menge“ sich auf eine Konzentration von Quinacrin bezie- hen, die bei der Behandlung einer Infektion durch die genannten Viren, beispielsweise zu ei- ner Verringerung der Virenkonzentration führt. As used, the term "effective amount" includes both a preventive amount and a "treatment effective amount". The preventively effective amount refers to a concentration of quinacrine effective to inhibit, reduce or prevent the likelihood of infection by Flaviviridae and/or Togaviridae, or to delay the onset of disease by viruses from the group of Prevent Flaviviridae and/or Togaviridae when administered prior to infection, i.e. before, during and/or shortly after the period of exposure to said viruses. Likewise, the term “effective amount” can refer to a concentration of quinacrine which, in the treatment of an infection caused by the viruses mentioned, leads, for example, to a reduction in the virus concentration.
Unter der Bezeichnung Organismus sollen vorzugsweise Säugetiere, insbesondere Men- schen, verstanden werden. The term organism should preferably be understood to mean mammals, in particular humans.
Es wird angenommen, dass die antivirale Wirkung von Quinacrin darauf beruht, dass durch Hemmung der viralen Cysteinproteasen und/oder Serinproteasen, die Polyproteinverarbei- tung des translatierten, viralen Genoms in der Wirtszelle gestört oder verhindert wird, wodurch das Virus replikationsunfähig wird. It is assumed that the antiviral effect of quinacrine is based on the fact that, by inhibiting the viral cysteine proteases and/or serine proteases, the polyprotein processing of the translated viral genome in the host cell is disrupted or prevented, as a result of which the virus becomes unable to replicate.
Dementsprechend ist ein Verfahren zur Behandlung einer Virusinfektion oder eines Virus aus der Familie der Flaviviridae und Togaviridae vorgesehen, das für eine virale Cysteinpro- tease-Inhibierung und/oder Serinprotease-Inhibierung, insbesondere eine virale NS2B/NS3pro-lnhibierung und/oder nsP2pra-lnhibierung, in einem Organismus empfänglich ist, einschließlich der Verabreichung einer therapeutisch wirksamen Menge von Quinacrin oder eines pharmazeutisch annehmbaren Salzes, Solvats oder Hydrats hiervon an den Or- ganismus, wobei der Organismus vorzugsweise ein Säugetier, insbesondere ein Mensch oder ein Tier sein kann. Die Verabreichung von Quinacrin, der pharmazeutischen Zusam- mensetzung oder des Arzneimittels der Erfindung kann dabei auch zusammen mit anderen therapeutischen Mitteln erfolgen, die bei der Behandlung von Infektionen durch Viren nützlich sind. Accordingly, a method for treating a viral infection or a virus from the family Flaviviridae and Togaviridae is provided which is for viral cysteine protease inhibition and/or serine protease inhibition, in particular viral NS2B/NS3 pro inhibition and/or nsP2 pra - inhibition, in an organism, including administering to said organism a therapeutically effective amount of quinacrine or a pharmaceutically acceptable salt, solvate or hydrate thereof, said organism preferably being a mammal, in particular a human or can be an animal. The administration of quinacrine, the pharmaceutical composition or the medicament of the invention can also be carried out in conjunction with other therapeutic agents which are useful in the treatment of viral infections.
In einem anderen Aspekt der vorliegenden Erfindung ist ein Verfahren zur Inhibierung der vi- ralen Cysteinprotease und/oder Serinproteasen, insbesondere der viralen NS2B/NS3pr0 und/oder der nsP2pro, in einem Säugetier vorgesehen, das die Verabreichung einer therapeu- tisch wirksamen Menge von Quinacrin oder eines pharmazeutisch annehmbaren Salzes, Solvats oder Hydrats davon an das Säugetier einschließt. In einer Ausgestaltung ist das Säugetier ein Mensch oder ein Tier. Quinacrin kann dabei oral, intravenös oder durch Inhala- tion verabreicht werden. In another aspect of the present invention, a method for inhibiting the viral cysteine protease and/or serine proteases, in particular the viral NS2B/NS3 pr0 and/or the nsP2 pro , in a mammal is provided, which comprises administering a therapeutically effective amount of quinacrine or a pharmaceutically acceptable salt, solvate or hydrate thereof to the mammal. In one embodiment, the mammal is a human or an animal. Quinacrine can be administered orally, intravenously or by inhalation.
Obwohl die Erfindung in den Figuren und der vorangegangenen Beschreibung ausführlich dargestellt und beschrieben wurde, sind diese Abbildungen und Beschreibungen als illustra- tiv oder exemplarisch und nicht einschränkend zu betrachten. Es versteht sich, dass Ände- rungen und Modifikationen durch gewöhnliche Fertigkeiten im Rahmen der folgenden An- sprüche vorgenommen werden können. Insbesondere umfasst die vorliegende Erfindung weitere Ausführungsformen mit einer beliebigen Kombination von Merkmalen aus verschie- denen Ausführungsformen, die oben und unten beschrieben sind. Although the invention has been shown and described in detail in the figures and the foregoing description, these figures and descriptions are to be considered as illustrative or exemplary and not restrictive. It is understood that changes and modifications can be made through ordinary skill within the scope of the following claims. In particular, the present invention includes further embodiments with any combination of features from different embodiments described above and below.
Spezieller Beschreibunqsteil Special description part
Im Weiteren wird die Erfindung anhand von exemplarischen und experimentellen Untersu- chungsergebnissen und einigen Figuren näher und detaillierter erläutert. Die Erfindung ist nicht auf die hier offenbarten exemplarischen Ausführungsbeispiele beschränkt. Alle be- schriebenen und/oder bebilderten Merkmale können sich einzeln oder auch kombiniert in un- terschiedlichen Ausführungsformen manifestieren. Die Merkmale der unterschiedlichen Aus- führungsformen dieser Erfindung und ihre jeweiligen Vorteile werden beim Lesen der nach- folgend ausgeführten Ausführungsbeispiele in Zusammenhang mit den Figuren offenbart. Dabei zeigen: The invention is explained in greater detail below using exemplary and experimental test results and some figures. The invention is not limited to the exemplary embodiments disclosed herein. All features described and/or illustrated can manifest themselves individually or in combination in different embodiments. The features of the different embodiments of this invention and their respective advantages are disclosed when reading the exemplary embodiments set out below in connection with the figures. show:
Figur 1 : Primäre Inhibitions Tests von Quinacrin gegen NS2B/NS3pr0 aus ZIKV, YFV, DENV and WNV (20 μmol/L Quinacrin) und nsP2pro aus CHIKV (5 μmol/L Quinacrin) Figure 1: Primary inhibition tests of quinacrine against NS2B/NS3 pr0 from ZIKV, YFV, DENV and WNV (20 μmol/L quinacrine) and nsP2 pro from CHIKV (5 μmol/L quinacrine)
Figur 2: Quinacrin Titration gegen verschiedene Flavivirus und Alphavirus Proteasen; Ordinatenachse Y: normalisierte Protease Aktivität (schwarze Balken) bzw. Inhi- bition (graue Balken) in [%] von Flavivirus und Alphavirus Proteasen Abszissenachse X: Stoffmengenkonzentration Quinacrin [μmol/L] FIG. 2: Quinacrine titration against various flavivirus and alphavirus proteases; Axis Y: normalized protease activity (black bars) or inhibition (grey bars) in [%] of flavivirus and alphavirus proteases X axis: molar concentration of quinacrine [μmol/L]
A: Quinacrin und ZIKV NS2B/NS3pro B: Quinacrin und DENN/ NS2B/NS3pro A: Quinacrine and ZIKV NS2B/NS3 pro B: Quinacrine and DENN/ NS2B/NS3 pro
C: Quinacrin und WNV NS2B/NS3pro D: Quinacrin und YFV NS2B/NS3pro E: Quinacrin und CHIKV nsP2pro Figur 3: Dosis-Wirkungs-Kurve zur ICso-Bestimmung Ordinatenachse Y: normalisierte Proteaseaktivität [%] von Flavivirus und Alpha- virus Proteasen C: quinacrine and WNV NS2B/NS3 per D: quinacrine and YFV NS2B/NS3 per E: quinacrine and CHIKV nsP2 per virus proteases
Abszissenachse X: Logarithmus der Stoffmengenkonzentration von Quinacrin [μmol/L] X abscissa axis: logarithm of the molar concentration of quinacrine [μmol/L]
A: Dosis-Wirkungs-Kurve von Quinacrin und ZIKV NS2B/NS3pro B: Dosis-Wirkungs-Kurve von Quinacrin und DENV NS2B/NS3pro A: Dose-response curve of quinacrine and ZIKV NS2B/NS3 pro B: Dose-response curve of quinacrine and DENV NS2B/NS3 pro
C: Dosis-Wirkungs-Kurve von Quinacrin und WNV NS2B/NS3pro D: Dosis-Wirkungs-Kurve von Quinacrin und YFV NS2B/NS3pro E: Dosis-Wirkungs-Kurve von Quinacrin und CHIKV nsP2pro Figur 4: Lineweaver-Burk-Diagramme zur Bestimmung des Inhibitionsmodus von Quinacrin gegen Flavivirus und Alphavirus Proteasen. C: Dose-response curve of quinacrine and WNV NS2B/NS3 per D: Dose-response curve of quinacrine and YFV NS2B/NS3 per E: Dose-response curve of quinacrine and CHIKV nsP2 per Figure 4: Lineweaver-Burk Graphs for determining the mode of inhibition of quinacrine against flavivirus and alphavirus proteases.
Ordinatenachse Y: 1/v mit v = Reaktionsgeschwindigkeit Abszissenachse X: 1/[S] mit [S] =Substratkonzentration Y axis of ordinates: 1/v with v=reaction rate X axis of abscissas: 1/[S] with [S]=substrate concentration
A: Lineweaver-Burk-Diagramm zur ZIKV NS2B/NS3pr0 Hemmung durch Quinacrin B: Lineweaver-Burk-Diagramm zur DENV NS2B/NS3pro Hemmung durch Quinacrin A: Lineweaver-Burk plot of ZIKV NS2B/NS3 pr0 inhibition by quinacrine. B: Lineweaver-Burk plot of DENV NS2B/NS3 pro inhibition by quinacrine
C: Lineweaver-Burk-Diagramm zur WNV NS2B/NS3pro Hemmung durch Quinacrin D: Lineweaver-Burk-Diagramm zur YFV NS2B/NS3pro Hemmung durch Quinacrin E: Lineweaver-Burk-Diagramm zur CHIKV nsP2pro Hemmung durch Quinacrin I) Aktivitätstest und Inhibitionstest C: Lineweaver-Burk plot of WNV NS2B/NS3 per inhibition by quinacrine D: Lineweaver-Burk plot of YFV NS2B/NS3 per inhibition by quinacrine E: Lineweaver-Burk plot of CHIKV nsP2 per inhibition by quinacrine I) activity assay and inhibition test
Zum Nachweis der Wirkung von Quinacrin auf die virale Cysteinproteaseaktivität und/oder Serinproteaseaktivität, wurden Inhibitionstests durchgeführt. Inhibition assays were performed to demonstrate the effect of quinacrine on viral cysteine protease activity and/or serine protease activity.
Dabei wurde beispielhaft der Einfluss von Quinacrin auf die Protease Enzym-Aktivität der NS2B/NS3pro der Flaviviridae und nsP2pro der Togaviridae untersucht. Die Aktivitätstests der Flaviviridae Proteasen (ZIKV, DENN/, YFV und WNV NS2B/NS3pro) wurden unter Verwendung des fluorogenen Substrats BOC-GRR-AMC (Bachem, Schweiz) in 20 mmol/L Tris pH 8,5, 10% Glycerol, 0.01% Triton X-100 [1-3] durchgeführt. The influence of quinacrine on the protease enzyme activity of the NS2B/NS3 pro of the Flaviviridae and nsP2 pro of the Togaviridae was examined as an example. The activity tests of the Flaviviridae proteases (ZIKV, DENN/, YFV and WNV NS2B/NS3 pro ) were performed using the fluorogenic substrate BOC-GRR-AMC (Bachem, Switzerland) in 20 mmol/L Tris pH 8.5, 10% glycerol, 0.01% Triton X-100 [1-3].
Die Aktivitätstests der Togaviridae Protease (CHIKV nsP2pr0) wurden unter Verwendung ei- nes fluorogenen Substrats DABCYL-KTSAVLQjSGFRKME-EDANS (Bachem, Schweiz) in einem Puffer mit 20 mmol/L Tris pH 7,2, 200 mmol/L NaCI, 1 mmol/L EDTA und 1 mmol/L TCEP durchgeführt. Togaviridae protease (CHIKV nsP2 pr0 ) activity assays were performed using a fluorogenic substrate DABCYL-KTSAVLQjSGFRKME-EDANS (Bachem, Switzerland) in a buffer containing 20 mmol/L Tris pH 7.2, 200 mmol/L NaCl, 1 mmol /L EDTA and 1 mmol/L TCEP.
Diese Reaktionsmischungen wurde in eine Corning 96-Well-Platte (Sigma Aldrich), enthal- tend 0,5 μmol/L NS2B/NS3pro Proteinase Protein bzw. nsP2pro Proteinase Proteins, pipettiert, und der Assay wurde mit der Zugabe der jeweiligen zuvor genannten Substrate in einer End- konzentration von 50 μmol/L gestartet. These reaction mixtures were pipetted into a Corning 96-well plate (Sigma Aldrich) containing 0.5 μmol/L NS2B/NS3 per proteinase protein or nsP2 per proteinase protein, and the assay was performed with the addition of the respective ones above mentioned substrates in a final concentration of 50 μmol/L.
Die Inhibierung der Serinprotease bzw. der Cysteinprotease-Aktivität aus unterschiedlichen Viren (ZIKV, YFV, DENV, WNV und CHIKV) durch Quinacrin wurde mit dem oben beschrie- benen Aktivitätsassay untersucht. 10 μmol/L der Proteasen wurden jeweils einzeln für einen ersten Screening-Test verwendet. The inhibition of the serine protease or cysteine protease activity from different viruses (ZIKV, YFV, DENV, WNV and CHIKV) by quinacrine was investigated using the activity assay described above. 10 μmol/L of the proteases were each used individually for a first screening test.
Für die anschließenden Inhibitionstests wurden 0,5 μmol/L des NS2B/NS3pro bzw. des nsP2pro Proteins mit 20 μmol/L Quinacrin für die NS2B/NS3pro Protease und 5 μmol/L Quinacrin für die nsP2pro Protease inkubiert. Die Mischungen wurden 30 Minuten lang bei RT inkubiert. Nach Zugabe des Substrats mit einer Endkonzentration von 50 μmol/L wurde die Zunahme/Änderung der Fluoreszenzintensitäten in 60 s-lntervallen über 30 Minuten mit ei- nem Plattenlesegerät Infinite 200 PRO (Tecan, Männedorf, Schweiz) gemessen. Die Tempe- ratur wurde auf 37 °C eingestellt. Sobald das fluorogene Substrat von der NS2B/NS3pr0 Pro- tease bzw. der nsP2pro Protease gespalten wird, erhöht sich die messbare Fluoreszenz, da der FRET (Förster-Resonanzenergietransfer) Farbstoff des Substrats freigesetzt und damit messbar wird. Also entspricht eine hohe Fluoreszenzintensität einer hohen Proteaseaktivität- Aktivität. Eine Reduktion der Fluoreszenzintensität kann daher auf eine entsprechende Inhi- bierung dieser Protease zurückgeführt werden. For the subsequent inhibition tests, 0.5 μmol/L of the NS2B/NS3 pro or nsP2 pro protein was incubated with 20 μmol/L quinacrine for the NS2B/NS3 pro protease and 5 μmol/L quinacrine for the nsP2 pro protease. The mixtures were incubated at RT for 30 minutes. After addition of the substrate with a final concentration of 50 μmol/L, the increase/change in fluorescence intensities was measured at 60 s intervals over 30 minutes using an Infinite 200 PRO plate reader (Tecan, Männedorf, Switzerland). The temperature was set at 37°C. As soon as the fluorogenic substrate is cleaved by the NS2B/NS3 pr0 protease or the nsP2 pro protease, the measurable fluorescence increases since the FRET (Förster resonance energy transfer) dye of the substrate is released and thus becomes measurable. So a high fluorescence intensity corresponds to a high protease activity. A reduction in the fluorescence intensity can therefore be attributed to a corresponding inhibition of this protease.
Die Anregungs- und Emissionswellenlängen betrugen 360 nm bzw. 460 nm für nsP2pro des CHIKV und 380 nm bzw. 465 nm für NS2B/NS3pro der ZIKV, DENV, YFV und WNV. Die Inhi- bitionstests wurden als Triplikate durchgeführt. Die Ergebnisse sind in Figur 1 dargestellt. Auf der Abszisse (X-Achse) sind unterschiedliche Versuchsansätze (ZIKV, YFV, DENV, WNV und CHIKV) mit den unterschiedlichen Virusproteasen dargestellt. Versuchsansatz „Kontrolle“ zeigt Ergebnisse ohne Zusatz von Quinacrin bzw. Inhibitor (schwarzer Balken). The excitation and emission wavelengths were 360 nm and 460 nm for nsP2 pro of the CHIKV and 380 nm and 465 nm for NS2B/NS3 per of the ZIKV, DENV, YFV and WNV. The inhibition tests were carried out in triplicate. The results are shown in Figure 1. Different experimental approaches (ZIKV, YFV, DENV, WNV and CHIKV) with the different virus proteases are shown on the abscissa (X-axis). Experimental approach "Control" shows results without the addition of quinacrine or inhibitor (black bar).
Versuchsansatz „ZI KV“ zeigt Ergebnisse der Serin-Proteaseaktivität aus dem Zika Virus nach Zusatz von 20 mGhoI/I- Quinacrin. Experimental approach "ZI KV" shows results of the serine protease activity from the Zika virus after the addition of 20 mGhoI/I-quinacrine.
Versuchsansatz „YFV“ zeigt Ergebnisse der Serin-Proteaseaktivität aus dem Gelbfieber Vi- rus nach Zusatz von 20 mGhoI/I_ Quinacrin. Experimental approach "YFV" shows results of the serine protease activity from the yellow fever virus after the addition of 20 mGhoI/I_ quinacrine.
Versuchsansatz „DENV“ zeigt Ergebnisse der Serin-Proteaseaktivität aus dem Dengue Virus nach Zusatz von 20 μmol/L Quinacrin. Experimental approach "DENV" shows results of the serine protease activity from the dengue virus after the addition of 20 μmol/L quinacrine.
Versuchsansatz „WNV“ zeigt Ergebnisse der Serin-Proteaseaktivität aus dem West-Nile Vi- rus nach Zusatz von 20 mihoI/L Quinacrin. Experimental approach "WNV" shows results of the serine protease activity from the West Nile virus after the addition of 20 ml/L quinacrine.
Versuchsansatz „CHIKV“ zeigt Ergebnisse der Cystein-Proteaseaktivität aus dem Chikungunya Virus nach Zusatz von 5 mihoI/L Quinacrin. Experimental approach "CHIKV" shows results of the cysteine protease activity from the Chikungunya virus after the addition of 5 mihoI/L quinacrine.
Jeder Versuchsansatz wurde mit frischem, gereinigtem Protease Protein durchgeführt.Each experimental approach was carried out with fresh, purified protease protein.
Die Hemmung der Protease-Aktivität wurde mit folgender Gleichung (1) berechnet: The inhibition of protease activity was calculated using the following equation (1):
(1) Protease-Aktivität in [%] = Fluoreszenz-Intensität der nach der Inhibition ver- bleibenden Enzymaktivitäts-Intensität / Fluoreszenz-Intensität der Enzymakti- vität ohne Inhibition (1) Protease activity in [%]=fluorescence intensity of the enzyme activity intensity remaining after inhibition/fluorescence intensity of the enzyme activity without inhibition
Die Ergebnisse zeigen, dass eine Zugabe von 20 μmol/L Quinacrin die Aktivität der NS2B/NS3pro Protease der Flaviviridae um mehr als 88% effizient hemmen konnte (siehe Fi- gur 1). The results show that the addition of 20 μmol/L quinacrine was able to efficiently inhibit the activity of the NS2B/ NS3 protease of Flaviviridae by more than 88% (see FIG. 1).
Da bei einer Quinacrin Konzentration von 20 mhioI/I_ die CHIKV nsP2pro Aktivität bereits um 100% gehemmt war, wurde das Experiment für diese Protease noch einmal mit einer gerin- geren Konzentration, und zwar mit einer Konzentration von 5 mhioI/I- Quinacrin wiederholt. Nach Zugabe dieser geringeren Konzentration wurde die CHIKV nsP2pro Protease Aktivität um ca. 95% inhibiert (Siehe Figur 1). Since the CHIKV nsP2 per activity was already inhibited by 100% at a quinacrine concentration of 20 mhl/l_, the experiment for this protease was repeated once more with a lower concentration, namely with a concentration of 5 mhl/l-quinacrine . After addition of this lower concentration, the CHIKV nsP2 pro protease activity was inhibited by about 95% (see FIG. 1).
Die Ergebnisse zeigen insgesamt, dass Quinacrin die Protease Aktivität aller getesteten Vi- rus Proteasen um mehr als 88% hemmen konnte. Overall, the results show that quinacrine was able to inhibit the protease activity of all virus proteases tested by more than 88%.
Anschließend wurden Quinacrin Titrationsexperimente mit allen fünf Virus Proteasen durch- geführt, um den Konzentrationseffekt des Inhibitors auf die Protease Aktivität zu untersuchen (siehe Figur 2 und Tabelle 1). Subsequently, quinacrine titration experiments were carried out with all five virus proteases in order to investigate the concentration effect of the inhibitor on the protease activity (see FIG. 2 and Table 1).
Basierend auf diesen Titrationsexperimenten des erfindungsgemäßen Inhibitors in einem spezifischen Aktivitätstest mit verschiedenen Flavivirus und Alphavirusproteasen (Zika Virus (ZIKV), Dengue Virus (DENV), Gelbfieber Virus (YFV), West-Nil Virus (WNV) und auch Pro- tease des Chikungunya Virus (CHI KV), konnte jeweils ein IC50 Wert bestimmt werden (siehe Figur 2 und Tabelle 1). Based on these titration experiments of the inhibitor according to the invention in a specific activity test with various flavivirus and alphavirus proteases (Zika virus (ZIKV), dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV) and also protease of the chikungunya virus (CHI KV), an IC50 value could be determined (see Figure 2 and Table 1 ).
Basierend auf diesen Quinacrin Titrationsexperimenten konnten die IC50 und K, Werte für diese Serin- und Cystein- Virus Proteasen bestimmt werden. Insgesamt lagen diese IC50 Werte alle < 3 μmol/l: Based on these quinacrine titration experiments, the IC50 and K values for these serine and cysteine virus proteases could be determined. Overall, these IC50 values were all < 3 μmol/l:
ZIKV NS2B/NS3pro: 2,5 ± 0,7 μmol/L DENV NS2B/NS3pro: 2,8 ± 1,0 μmol/L YFV NS2B/NS3pro: 1 ,6 ± 0,4 μmol/L WNV NS2B/NS3pro: 1 ,3 ± 0,5 μmol/L CHIKV nsP2pro: 0,46 ± 0,1 μmol/L ZIKV NS2B/NS3 per : 2.5 ± 0.7 μmol/L DENV NS2B/NS3 per : 2.8 ± 1.0 μmol/L YFV NS2B/NS3 per : 1.6 ± 0.4 μmol/L WNV NS2B /NS3 per : 1.3 ± 0.5 μmol/L CHIKV nsP2 per : 0.46 ± 0.1 μmol/L
(Siehe Figur 3 und Tabelle 2). (See Figure 3 and Table 2).
Die folgende Tabelle 1 zeigt die Ergebnisse der Messungen der Proteaseaktivität unter- schiedlicher Viren bei unterschiedlich zugegebenen Stoffmengenkonzentrationen von Quinacrin. Table 1 below shows the results of the measurements of the protease activity of different viruses with different added molar concentrations of quinacrine.
Tabelle 1 : Abnahme der getesteten Virus Protease Aktivität unterschiedlicher Viren in Abhängigkeit der zugegebenen Quinacrin Konzentration
Figure imgf000014_0001
Table 1: Decrease in the tested virus protease activity of different viruses as a function of the quinacrine concentration added
Figure imgf000014_0001
II) IC50 Bestimmung: Um den Wert der halbmaximalen Inkubationskonzentration (IC50 Wert) für Quinacrin zu be- stimmen, wurde die Anfangsgeschwindigkeit der enzymatischen Reaktion gegen verschie- dene Konzentrationen von Quinacrin mit Hilfe einer Dosis-Wirkungs-Kurve in der Software GraphPad Prism5 aufgetragen. II) IC 50 determination: To determine the value of the half-maximal incubation concentration (IC5 0 value) for quinacrine, the initial rate of the enzymatic reaction was plotted against various concentrations of quinacrine using a dose-response curve in the GraphPad Prism5 software.
In Figur 3 ist auf der Ordinate (Y-Achse) die normalisierte Aktivität [%] von Flavivirus und Al- phavirus Proteasen gegen den Logarithmus der Stoffmengenkonzentration in mihoI/L (Abs- zisse X-Achse) von Quinacrin, aufgetragen. Dabei zeigen die einzelnen Graphen folgendes: Figur 3 Graph A: Dosis-Wirkungs-Kurve von Quinacrin auf ZIKV NS2B/NS3pro Figur 3 Graph B: Dosis-Wirkungs-Kurve von Quinacrin auf DENV NS2B/NS3pro Figur 3 Graph C: Dosis-Wirkungs-Kurve von Quinacrin auf WNV NS2B/NS3pr0 Figur 3 Graph D: Dosis-Wirkungs-Kurve von Quinacrin und YFV NS2B/NS3pro Figur 3 Graph E: Dosis-Wirkungs-Kurve von Quinacrin und CHIKV nsP2pr0 In FIG. 3, the normalized activity [%] of flavivirus and alphavirus proteases is plotted on the ordinate (Y-axis) against the logarithm of the molar concentration in mihoI/L (abscissa X-axis) of quinacrine. The individual graphs show the following: Figure 3 Graph A: dose-response curve of quinacrine on ZIKV NS2B/NS3 per Figure 3 Graph B: dose-response curve of quinacrine on DENV NS2B/NS3 per Figure 3 Graph C: dose- Response curve of quinacrine on WNV NS2B/NS3 pr0 Figure 3 Graph D: Dose-response curve of quinacrine and YFV NS2B/NS3 per Figure 3 Graph E: Dose-response curve of quinacrine and CHIKV nsP2 pr0
Tabelle 2 zeigt die IC50 und K Werte, die aus den zuvor aufgeführten Dosis-Wirkungs-Kurven erhalten wurden. Table 2 shows the IC5 0 and K values obtained from the dose-response curves presented above.
Tabelle 2: ICsoWerte und K Werte von Quinacrin als Inhibitor gegen Proteasen unter- schiedliche Vertreter von Flaviviren und Alphaviren
Figure imgf000015_0001
Table 2: IC50 values and K values of quinacrine as an inhibitor against proteases of different representatives of flaviviruses and alphaviruses
Figure imgf000015_0001
III) Bestimmung von Inhibitionsmodus und Inhibitionskonstante Basierend auf der bekannten Michaelis-Menten-Gleichung/ Kinetik wurden zusätzliche Pro- teaseaktivitätstests durchgeführt, um den Inhibitionsmodus von Quinacrin auf die Protease- aktivität näher zu untersuchen. III) Determination of the inhibition mode and inhibition constant Based on the well-known Michaelis-Menten equation/kinetics, additional protease activity tests were carried out in order to investigate the inhibition mode of quinacrine on the protease activity in more detail.
Dazu wurde bei verschiedenen Endkonzentrationen des Inhibitors Quinacrin und des zuvor genannten Substrats die Reaktionsgeschwindigkeit der NS2B/NS3 Protease bzw. der nsP“ Protease bestimmt. 0,5 μmol/L dieser Proteasen wurde dazu mit Quinacrin in verschiedenen Konzentrationen 30 Minuten lang bei Raumtemperatur (RT) inkubiert. Anschließend wurde die Reaktion durch Zugabe der entsprechenden Konzentrationsreihe des Substrats eingelei- tet und die Reaktionsgeschwindigkeit durch Bestimmung der Fluoreszenzintensität gemäß den zuvor unter I) beschriebenen allgemeinen Versuchsbedingungen bestimmt. Die Daten- analyse wurde mit Hilfe eines Lineweaver-Burk-Plots durchgeführt. Dabei wurde der Kehr- wert der Reaktionsgeschwindigkeit (1/V) gegen den Kehrwert der Substratkonzentration (1/[S]) aufgetragen [5, 6], For this purpose, the reaction speed of the NS2B/NS3 protease and the nsP" protease was determined at different final concentrations of the quinacrine inhibitor and the aforementioned substrate. 0.5 μmol/L of these proteases was mixed with quinacrine in various Concentrations incubated for 30 minutes at room temperature (RT). The reaction was then initiated by adding the appropriate concentration series of the substrate and the reaction rate was determined by determining the fluorescence intensity in accordance with the general test conditions described above under I). The data analysis was carried out using a Lineweaver-Burk plot. The reciprocal of the reaction rate (1/V) was plotted against the reciprocal of the substrate concentration (1/[S]) [5, 6],
Alle Messungen wurden in dreifacher Ausfertigung durchgeführt und die Daten als Mittelwert ± Standardabweichung dargestellt. Die Inhibitionskonstante (K) wurde mit der folgenden Gleichung (2) ermittelt: All measurements were performed in triplicate and data presented as mean ± standard deviation. The inhibition constant (K) was determined using the following equation (2):
(2) Ki = IC50/(([S]/KM)+1)) (2) K i = IC 50 /(([S]/KM)+1))
Der Ki Wert gibt die Affinität des Enzyms bzw. der Protease zum jeweiligen Inhibitor an.The Ki value indicates the affinity of the enzyme or protease for the respective inhibitor.
Der ICsoWert wurde wie zuvor beschrieben bestimmt. Der KM Wert wurde aus einer Daten- bank (BRENDA-Datenbank) gewonnen und [S] ist die Konzentration des in den Versuchsan- sätzen verwendeten Substrats [7], The ICso value was determined as previously described. The KM value was obtained from a database (BRENDA database) and [S] is the concentration of the substrate used in the test batches [7],
In Figur 4 ist auf der Abszisse (X-Achse) der Kehrwert der Substratkonzentration [1/S] ange- geben. Auf der Ordinate (Y-Achse) ist der Kehrwert der Reaktionsgeschwindigkeit [1/V] auf- getragen. In FIG. 4, the abscissa (X-axis) shows the reciprocal of the substrate concentration [1/S]. The reciprocal value of the reaction rate [1/V] is plotted on the ordinate (Y-axis).
Die Graphen A bis E in Figur 4 zeigen: Graphs A to E in Figure 4 show:
A: Lineweaver-Burk-Diagramm zur ZIKV NS2B/NS3pr0 Hemmung durch Quinacrin bei unter- schiedlichen Endkonzentrationen von Quinacrin und zwar 0; 0,25; 0,5; und 1 ,0 μmol/L. Die Ergebnisse bei den unterschiedlich eingestellten Endkonzentrationen von Quinacrin und die dazugehörigen Werte der Reaktionsgeschwindigkeiten sind in Graph A durch unterschiedli- che Symbole (●, ■, ♦ ,▲) dargestellt. A: Lineweaver-Burk plot of ZIKV NS2B/NS3 pr0 inhibition by quinacrine at different final concentrations of quinacrine, specifically 0; 0.25; 0.5; and 1.0 µmol/L. The results for the differently adjusted end concentrations of quinacrine and the associated values of the reaction rates are shown in graph A by different symbols (●, ■, ♦ ,▲).
B: Lineweaver-Burk-Diagramm zur DENV NS2B/NS3pro Hemmung durch Suramin bei unter- schiedlichen Endkonzentrationen von Quinacrin und zwar 0; 0,5; 0,75; 1 ,0 μmol/L. Die Er- gebnisse bei den unterschiedlich eingestellten Endkonzentrationen von Quinacrin und die dazugehörigen Werte der Reaktionsgeschwindigkeiten sind in Graph B durch unterschiedli- che Symbole (●, ■, ♦ ,▲) dargestellt. B: Lineweaver-Burk plot of DENV NS2B/NS3 per inhibition by suramin at different final concentrations of quinacrine, namely 0; 0.5; 0.75; 1.0 μmol/L. The results for the differently adjusted final concentrations of quinacrine and the associated values of the reaction rates are shown in graph B by different symbols (●, ■, ♦ ,▲).
C: Lineweaver-Burk-Diagramm zur WNV NS2B/NS3pr0 Hemmung durch Quinacrin bei unter- schiedlichen Endkonzentrationen von Quinacrin und zwar 0; 0,5; 0,75; und 1,0 μmol/L. Die Ergebnisse bei den unterschiedlich eingestellten Endkonzentrationen von Quinacrin und die dazugehörigen Werte der Reaktionsgeschwindigkeiten sind in Graph A durch unterschiedli- che Symbole (●, ■, ♦ ,▲) dargestellt. C: Lineweaver-Burk plot of WNV NS2B/NS3 pr0 inhibition by quinacrine at different final concentrations of quinacrine, specifically 0; 0.5; 0.75; and 1.0 µmol/L. The results for the differently adjusted end concentrations of quinacrine and the associated values of the reaction rates are shown in graph A by different symbols (●, ■, ♦ ,▲).
D: Lineweaver-Burk-Diagramm zur YFV NS2B/NS3pr0 Hemmung durch Quinacrin bei unter- schiedlichen Endkonzentrationen von Quinacrin und zwar 0; 0,5; 0,75; und 1 ,0 μmol/L. Die Ergebnisse bei den unterschiedlich eingestellten Endkonzentrationen von Quinacrin und die dazugehörigen Werte der Reaktionsgeschwindigkeiten sind in Graph A durch unterschiedli- che Symbole (●, ■, ♦ ,▲) dargestellt. D: Lineweaver-Burk plot of YFV NS2B/NS3 pr0 inhibition by quinacrine at different final concentrations of quinacrine, specifically 0; 0.5; 0.75; and 1.0 µmol/L. The results at the differently adjusted end concentrations of quinacrine and the The associated values of the reaction speeds are shown in graph A by different symbols (●, ■, ♦ ,▲).
E: Lineweaver-Burk-Diagramm zur CHIKV NS2B/NS3pro Hemmung durch Quinacrin bei un- terschiedlichen Endkonzentrationen von Quinacrin und zwar 0; 0,1; 0,25; und 0,5 μmol/L. Die Ergebnisse bei den unterschiedlich eingestellten Endkonzentrationen von Quinacrin und die dazugehörigen Werte der Reaktionsgeschwindigkeiten sind in Graph A durch unterschiedli- che Symbole (●, ■, ♦ ,▲) dargestellt. E: Lineweaver-Burk plot of CHIKV NS2B/NS3 per inhibition by quinacrine at different final concentrations of quinacrine, namely 0; 0.1; 0.25; and 0.5 µmol/L. The results for the differently adjusted end concentrations of quinacrine and the associated values of the reaction rates are shown in graph A by different symbols (●, ■, ♦ ,▲).
Die in dieser Darstellung erhaltenen Ergebnisse deuten darauf hin, dass Quinacrin ein kom- petitiver Inhibitor für die viralen Cysteinproteasen und/oder Serinproteasen, insbesondere vi- rale NS2B/NS3 und/oder nsP2 Proteasen ist. The results obtained in this representation indicate that quinacrine is a competitive inhibitor for the viral cysteine proteases and/or serine proteases, in particular viral NS2B/NS3 and/or nsP2 proteases.
Während die Erfindung anhand von besonderen Ausführungsbeispielen im vorangehenden Teil der Anmeldung eingehend beschrieben und bebildert wurde, sollen diese Beschreibung und die Figuren lediglich als Beispiel gelten, ohne dadurch einschränkend zu wirken. Es ist davon auszugehen, dass ein Fachmann im Rahmen seines Fachwissens selbst weitere Än- derungen und Modifikationen der nachfolgenden Ansprüche vornehmen würde und könnte, die durch den Schutzbereich der Ansprüche mit abgedeckt sind. Insbesondere sind im Rah- men der Erfindung weitere Ausführungsformen mit jeder Art von Kombinationen der erwähn- ten Merkmale einzelner Ausführungsbeispiele mit umfasst. While the invention has been described and illustrated in detail in the preceding part of the application on the basis of particular exemplary embodiments, this description and the figures should only apply as an example without having any restrictive effect. It is to be assumed that a person skilled in the art would and could make further changes and modifications to the following claims within the scope of his or her specialist knowledge, which are also covered by the scope of protection of the claims. In particular, further embodiments with any type of combination of the mentioned features of individual exemplary embodiments are included within the scope of the invention.
Literatur: Literature:
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[2] High-content assay to identify Inhibitors of dengue virus infection. Shum et al. 2010, As- say and drug development technologies, 8(5), 553-570. DOI: 10.1089/adt.2010.0321 [2] High-content assay to identify inhibitors of dengue virus infection. Shum et al. 2010, Assay and drug development technologies, 8(5), 553-570. DOI: 10.1089/adt.2010.0321
[3] Antimalarial drugs and their metabolites are potent Zika virus Inhibitors. Han et al. 2019, Journal of medical virology, 91(7), 1182-1190. DOI: 10.1002/jmv.25440 [3] Antimalarial drugs and their metabolites are potent Zika virus inhibitors. Han et al. 2019, Journal of medical virology, 91(7), 1182-1190. DOI: 10.1002/jmv.25440
[4] Repurposing quinacrine against Ebola virus infection in vivo. Lane et al. 2019, Antimicro- bial agents and chemotherapy, 63(9), e01142-19. DOI: 10.1128/AAC.01142-19 [4] Repurposing quinacrine against Ebola virus infection in vivo. Lane et al. 2019, Antimicrobial agents and chemotherapy, 63(9), e01142-19. DOI: 10.1128/AAC.01142-19
[5] Roy A, Lim L, Srivastava S, Lu Y, Song J, Solution conformations of Zika NS2B-NS3pro and its Inhibition by natural products from edible plants. PloS one 2017;12:e0180632 [6] Motulsky H, Christopoulos A, Fitting models to biological data using linear and nonlinear regression: a practical guide to curve fitting. Oxford University Press. 2004 [7] Wu QY, Jiang LL, Yang SG, Zuo Y, Wang ZF, Xi Z, et al. Hexahydrophthalimide-benzo- thiazole hybrids as a new dass of protoporphyrinogen oxidase Inhibitors: synthesis, struc- ture-activity relationship, and DFT calculations. New J. Chem. 2014;38(9): 4510-4518 [5] Roy A, Lim L, Srivastava S, Lu Y, Song J, Solution conformations of Zika NS2B-NS3pro and its Inhibition by natural products from edible plants. Plos one 2017;12:e0180632 [6] Motulsky H, Christopoulos A, Fitting models to biological data using linear and nonlinear regression: a practical guide to curve fitting. Oxford University Press. 2004 [7] Wu QY, Jiang LL, Yang SG, Zuo Y, Wang ZF, Xi Z, et al. Hexahydrophthalimide-benzothiazole hybrids as a new that of protoporphyrinogen oxidase inhibitors: synthesis, structure-activity relationship, and DFT calculations. New J Chem 2014;38(9):4510-4518

Claims

Patentansprüche patent claims
1. Quinacrin zur Verwendung für die Herstellung eines Inhibitors für virale Cysteinpro- teasen und/oder Serinproteasen. 1. Quinacrine for use in the manufacture of an inhibitor for viral cysteine proteases and/or serine proteases.
2. Quinacrin zur Verwendung für die Herstellung eines Inhibitors für Cysteinproteasen und/oder Serinproteasen von Viren aus der Familie der Togaviridae und/oder Flavi- viridae. 2. Quinacrine for use in the manufacture of an inhibitor for cysteine proteases and/or serine proteases of viruses from the Togaviridae and/or Flaviviridae family.
3. Quinacrin zur Verwendung für die Herstellung eines Inhibitors für Cysteinproteasen und/oder Serinproteasen von Viren aus der Gruppe der Alphaviren, insbesondere von wenigstens einem Virus aus der Gruppe des Chikungunya-Virus, Mayaro-Virus, Getah- Virus, O’nyong-nyong-Virus, Ross-River-Virus, Sindbis-Virus, Ockelbo-Virus, Babanki- Virus, Barmah-Forest-Virus, Semliki-Forest-Virus, Venezuelan-Equine-Encephalitis-Vi- rus, Everglades-Virus, Mucambo-Virus, Tonate-Virus, Eastern-Equine-Encephalitis-Vi- rus, Western-Equine-Encephalitis-Virus, Highlands-Virus, Middelburg-Virus, Rio Negro- Virus, Aura Virus, Babanki Virus, Ockelbo Virus, Whataroa Virus und/oder aus der Gruppe der Flaviviren, insbesondere von wenigstens einem Virus aus der Gruppe des Zika-Virus, Dengue-Virus, Gelbfieber-Virus, FSME-Virus, JE-Virus, SLE-Virus, Lou- ping-lll-Virus, Usutu-Virus, Wesselsbron-Virus, West-Nil-Virus, Gadgets-Gully-Virus, Langat-Virus, Modoc-Virus, Kadam-Virus, MVEV-Virus, POW-Virus, Karshi-Virus, Se- pik-Virus, TBE-Virus, Yokose-Virus. 3. Quinacrine for use in the production of an inhibitor for cysteine proteases and/or serine proteases of viruses from the group of alphaviruses, in particular of at least one virus from the group of Chikungunya virus, Mayaro virus, Getah virus, O'nyong-nyong virus, Ross River virus, Sindbis virus, Ockelbo virus, Babanki virus, Barmah Forest virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Everglades virus, Mucambo virus , Tonate Virus, Eastern Equine Encephalitis Virus, Western Equine Encephalitis Virus, Highlands Virus, Middelburg Virus, Rio Negro Virus, Aura Virus, Babanki Virus, Ockelbo Virus, Whataroa Virus and/or from the group of flaviviruses, in particular at least one virus from the group of Zika virus, dengue virus, yellow fever virus, TBE virus, JE virus, SLE virus, Louping III virus, Usutu virus , Wesselsbron Virus, West Nile Virus, Gadgets Gully Virus, Langat Virus, Modoc Virus, Kadam Virus, MVEV Virus, POW Virus, Karshi Virus, Se- pik virus, TBE virus, Yokose virus.
4. Quinacrin zur Verwendung für die Herstellung eines Inhibitors für die Cysteinprotease nsP2 und/oder die Serinprotease NS2B/NS3. 4. Quinacrine for use in the manufacture of an inhibitor for the cysteine protease nsP2 and/or the serine protease NS2B/NS3.
5. Verwendung von Quinacrin zur Herstellung eines Inhibitors für virale Cysteinproteasen und/oder Serinproteasen. 5. Use of quinacrine for the production of an inhibitor for viral cysteine proteases and/or serine proteases.
6. Verwendung von Quinacrin zur Herstellung eines Inhibitors für Cysteinproteasen und/oder Serinproteasen von Viren aus der Familie der Togaviridae und/oder Flavi- viridae. 6. Use of quinacrine for producing an inhibitor for cysteine proteases and/or serine proteases of viruses from the Togaviridae family and/or Flaviviridae.
7. Verwendung von Quinacrin zur Herstellung eines Inhibitors für Cysteinproteasen und/oder Serinproteasen von Viren aus der Gruppe der Alphaviren, insbesondere von wenigstens einem Virus aus der Gruppe des Chikungunya-Virus, Mayaro-Virus, Getah- Virus, O’nyong-nyong-Virus, Ross-River-Virus, Sindbis-Virus, Ockelbo-Virus, Babanki- Virus, Barmah-Forest-Virus, Semliki-Forest-Virus, Venezuelan-Equine-Encephalitis-Vi- rus, Everglades-Virus, Mucambo-Virus, Tonate-Virus, Eastern-Equine-Encephalitis-Vi- rus, Western-Equine-Encephalitis-Virus, Highlands-Virus, Middelburg-Virus, Rio Negro- Virus, Aura Virus, Babanki Virus, Ockelbo Virus, Whataroa Virus und/oder aus der Gruppe der Flaviviren, insbesondere von wenigstens einem Virus aus der Gruppe des Zika-Virus, Dengue-Virus, Gelbfieber-Virus, FSME-Virus, JE-Virus, SLE-Virus, Lou- ping-lll-Virus, Usutu-Virus, Wesselsbron-Virus, West-Nil-Virus, Gadgets-Gully-Virus, Langat-Virus, Modoc-Virus, Kadam-Virus, MVEV-Virus, POW-Virus, Karshi-Virus, Se- pik-Virus, TBE-Virus, Yokose-Virus. 7. Use of quinacrine for the production of an inhibitor for cysteine proteases and/or serine proteases of viruses from the group of alphaviruses, in particular of at least one virus from the group of chikungunya virus, Mayaro virus, Getah virus, O'nyong-nyong virus, Ross River virus, Sindbis virus, Ockelbo virus, Babanki Virus, Barmah Forest Virus, Semliki Forest Virus, Venezuelan Equine Encephalitis Virus, Everglades Virus, Mucambo Virus, Tonate Virus, Eastern Equine Encephalitis Virus, Western Equine Encephalitis virus, Highlands virus, Middelburg virus, Rio Negro virus, Aura virus, Babanki virus, Ockelbo virus, Whataroa virus and/or from the group of flaviviruses, in particular at least one virus from the group of Zika viruses, Dengue virus, yellow fever virus, TBE virus, JE virus, SLE virus, Louping III virus, Usutu virus, Wesselsbron virus, West Nile virus, Gadgets Gully virus, Langat virus Virus, Modoc virus, Kadam virus, MVEV virus, POW virus, Karshi virus, Sepik virus, TBE virus, Yokose virus.
8. Verwendung von Quinacrin zur Herstellung eines Inhibitors für die Cysteinprotease nsP2 und/oder die Serinprotease NS2B/NS3. 8. Use of quinacrine for the production of an inhibitor for the cysteine protease nsP2 and/or the serine protease NS2B/NS3.
9. Quinacrin zur Verwendung in der medizinischen Therapie. 9. Quinacrine for use in medical therapy.
10. Pharmazeutische Zusammensetzung, die mindestens Quinacrin oder ein pharmazeu- tisch geeignetes Salz davon und ein/einen pharmazeutisch geeigneten/geeignetes Trä- ger, Verdünnungsmittel oder Arzneiträger davon umfasst. 10. A pharmaceutical composition comprising at least quinacrine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient thereof.
11. Quinacrin als pharmazeutische Zusammensetzung gemäß vorhergehendem Anspruch zur Verwendung in der medizinischen Therapie. 11. Quinacrine as a pharmaceutical composition according to the preceding claim for use in medical therapy.
12. Quinacrin zur Verwendung bei der Vorbeugung oder Behandlung einer viralen Infektion in einem Organismus, insbesondere einer Infektion mit wenigstens einem Virus aus der Familie der Flaviviridae und/oder Togaviridae. 12. Quinacrine for use in the prevention or treatment of a viral infection in an organism, in particular an infection with at least one virus from the Flaviviridae and/or Togaviridae family.
13. Quinacrin zur Verwendung bei der Vorbeugung oder Behandlung einer viralen Infektion in einem Organismus, insbesondere einer Infektion mit wenigstens einem Virus aus der Gruppe der Flaviviren, insbesondere einer Infektion mit einem Virus aus der Gruppe des Zika-Virus, Dengue-Virus, Gelbfieber-Virus, FSME-Virus, JE-Virus, SLE-Virus, Louping-Ill-Virus, Usutu-Virus, Wesselsbron-Virus, West-Nil-Virus, Gadgets-Gully-Vi- rus, Langat-Virus, Modoc-Virus, Kadam-Virus, MVEV-Virus, POW-Virus, Karshi-Virus, Sepik-Virus, TBE-Virus, Yokose-Virus und/oder mit wenigstens einen Virus aus der Gruppe der Alphaviren, insbesondere einer Infektion mit einem Virus aus der Gruppe des Chikungunya-Virus, Mayaro-Virus, Getah-Virus, O’nyong-nyong-Virus, Ross-River- Virus, Sindbis-Virus, Ockelbo-Virus, Babanki-Virus, Barmah-Forest-Virus, Semliki-Fo- rest-Virus, Venezuelan-Equine-Encephalitis-Virus, Everglades-Virus, Mucambo-Virus,13. Quinacrine for use in the prevention or treatment of a viral infection in an organism, in particular an infection with at least one virus from the group of flaviviruses, in particular an infection with a virus from the group of the Zika virus, dengue virus, yellow fever Virus, TBE virus, JE virus, SLE virus, Louping Ill virus, Usutu virus, Wesselsbron virus, West Nile virus, Gadgets Gully virus, Langat virus, Modoc virus, Kadam virus, MVEV virus, POW virus, Karshi virus, Sepik virus, TBE virus, Yokose virus and/or with at least one virus from the group of alphaviruses, in particular an infection with a virus from the group of Chikungunya virus, Mayaro virus, Getah virus, O'nyong-nyong virus, Ross River virus, Sindbis virus, Ockelbo virus, Babanki virus, Barmah Forest virus, Semliki Forest Virus, Venezuelan Equine Encephalitis Virus, Everglades Virus, Mucambo Virus,
T onate-Virus, Eastern-Equine-Encephalitis-Virus, Western-Equine-Encephalitis-Virus, Highlands-Virus, Middelburg-Virus, Rio Negro-Virus, Aura Virus, Babanki Virus, O- ckelbo Virus, Whataroa Virus. tonate virus, eastern equine encephalitis virus, western equine encephalitis virus, Highlands Virus, Middelburg Virus, Rio Negro Virus, Aura Virus, Babanki Virus, Ockelbo Virus, Whataroa Virus.
14. Arzneimittel enthaltend Quinacrin sowie einen pharmazeutisch geeigneten Träger und die Verwendung von Quinacrin in der Herstellung eines Arzneimittels zur Behandlung von Infektionen mit wenigstens einem Virus aus der Familie der Flaviviridae und/oder Togaviridae in einem Organismus. 14. Medicaments containing quinacrine and a pharmaceutically suitable carrier and the use of quinacrine in the manufacture of a medicament for the treatment of infections with at least one virus from the Flaviviridae and/or Togaviridae family in an organism.
15. Verfahren zur Inhibierung viraler Cysteinproteasen und/oder Serinproteasen, insbeson- dere Cysteinproteasen von Viren aus der Familie der Flavirviridae und/oder Toga- viridae, sowie zur Behandlung oder Verhinderung einer Virusinfektion in einem Orga- nismus, insbesondere zur Behandlung einer viralen Infektion durch wenigstens einen Virus aus der Gruppe der Flaviviren, insbesondere durch einen Virus aus der Gruppe des Zika-Virus, Dengue-Virus, Gelbfieber-Virus, FSME-Virus, JE-Virus, SLE-Virus, Louping-Ill-Virus, Usutu-Virus, Wesselsbron-Virus, West-Nil-Virus, Gadgets-Gully-Vi- rus, Langat-Virus, Modoc-Virus, Kadam-Virus, MVEV-Virus, POW-Virus, Karshi-Virus, Sepik-Virus, TBE-Virus, Yokose-Virus und/oder durch wenigstens einen Virus aus der Gruppe der Alphaviren, insbesondere durch einen Virus aus der Gruppe des Chikungunya-Virus, Mayaro-Virus, Getah-Virus, O’nyong-nyong-Virus, Ross-River-Vi- rus, Sindbis-Virus, Ockelbo-Virus, Babanki-Virus, Barmah-Forest-Virus, Semliki-Forest- Virus, Venezuelan-Equine-Encephalitis-Virus, Everglades-Virus, Mucambo-Virus, Ton- ate-Virus, Eastern-Equine-Encephalitis-Virus, Western-Equine-Encephalitis-Virus, Highlands-Virus, Middelburg-Virus, Rio Negro-Virus, Aura Virus, Babanki Virus, O- ckelbo Virus, Whataroa Virus, bei dem Quinacrin oder eine pharmazeutische Zusam- mensetzung umfassend Quinacrin gemäß Anspruch 10 in einer therapeutisch wirksa- men Menge zugegeben wird. 15. Method for inhibiting viral cysteine proteases and/or serine proteases, in particular cysteine proteases of viruses from the Flavirviridae and/or Togaviridae family, and for treating or preventing a viral infection in an organism, in particular for treating a viral infection by at least one virus from the group of flaviviruses, in particular by a virus from the group of Zika virus, dengue virus, yellow fever virus, TBE virus, JE virus, SLE virus, Louping III virus, Usutu virus , Wesselsbron Virus, West Nile Virus, Gadgets Gully Virus, Langat Virus, Modoc Virus, Kadam Virus, MVEV Virus, POW Virus, Karshi Virus, Sepik Virus, TBE Virus , Yokose virus and/or by at least one virus from the group of alphaviruses, in particular by a virus from the group of Chikungunya virus, Mayaro virus, Getah virus, O'nyong-nyong virus, Ross River Vi - rus, Sindbis virus, Ockelbo virus, Babanki virus, Barmah Forest virus, Semliki Forest virus, Venezuelan equine encephalitis virus, Everglades virus, Mucambo virus, Tonate virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Highlands virus, Middelburg virus, Rio Negro virus , Aura virus, Babanki virus, O- ckelbo virus, Whataroa virus, in which quinacrine or a pharmaceutical composition comprising quinacrine according to claim 10 is added in a therapeutically effective amount.
16. Verfahren zur Inhibierung oder zur Inaktivierung einer viralen Cysteinprotease und/oder Serinproteasen, insbesondere der viralen NS2B/NS3pro und/oder der nsP2pro, in einem Organismus, bei dem eine Verabreichung einer therapeutisch wirksamen Menge von Quinacrin oder einer pharmazeutischen Zusammensetzung umfassend Quinacrin ge- mäß Anspruch 10 erfolgt. 16. A method for inhibiting or inactivating a viral cysteine protease and/or serine proteases, in particular the viral NS2B/NS3 pro and/or the nsP2 pro , in an organism in which administration of a therapeutically effective amount of quinacrine or a pharmaceutical composition comprising quinacrine according to claim 10 takes place.
17. Verfahren zur Inhibierung oder zur Inaktivierung einer viraler Cysteinproteasen und/o- der Serinproteasen, insbesondere Cysteinproteasen von Viren aus der Familie der To- gaviridae und/oder Serinproteasen von Viren aus der Familie der Flaviviridae, umfas- send den Schritt des Inkontaktbringens der Zellen des Organismus mit einer therapeu- tisch wirksamen Menge von Quinacrin oder einer pharmazeutischen Zusammenset- zung gemäß Anspruch 10. 17. A method for inhibiting or inactivating a viral cysteine protease and/or serine proteases, in particular cysteine proteases from viruses from the Togaviridae family and/or serine proteases from viruses from the Flaviviridae family, comprehend and the step of contacting the cells of the organism with a therapeutically effective amount of quinacrine or a pharmaceutical composition according to claim 10.
18. Ex-Vivo Verfahren zur Inhibierung oder zur Inaktivierung einer viraler Cysteinproteasen und/oder Serinproteasen, insbesondere Cysteinproteasen von Viren aus der Familie der Togaviridae und/oder Serinproteasen von Viren aus der Familie der Flaviviridae, umfassend den Schritt des ex-vivo Inkontaktbringens der Zellen des Organismus mit einer wirksamen Menge von Quinacrin oder einer pharmazeutischen Zusammenset- zung gemäß Anspruch 10. 18. Ex vivo method for inhibiting or inactivating a viral cysteine proteases and/or serine proteases, in particular cysteine proteases of viruses from the Togaviridae family and/or serine proteases of viruses from the Flaviviridae family, comprising the step of bringing the cells into contact ex vivo of the organism with an effective amount of quinacrine or a pharmaceutical composition according to claim 10.
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