WO2017153950A1

WO2017153950A1 - Inhibitors of influenza virus nuclease (polymerase complex)

Info

Publication number: WO2017153950A1
Application number: PCT/IB2017/051397
Authority: WO
Inventors: Katarzyna KAMIŃSKA; Janusz BUJNICKI
Original assignee: Biotech Innovations Spółka Z Ograniczoną Odpowiedzialnością
Priority date: 2016-03-10
Filing date: 2017-03-09
Publication date: 2017-09-14
Also published as: PL416447A1

Abstract

The 4-hydroxyphenyl-2-oxoethyl carboxylic derivatives of the general formula: R1-A- R2, wherein the core A is: Formula (I) and R1 is phenyl substituted with two C1-C4 alkyl, 1,3-benzodioxolyl or bicyclo[3.3.1]nonanyl and R2 is H, hydroxyl, amino group optionally substituted with carbonyl- C1-C4 alkyl or amide optionally substituted by C1-C4 alkyl; as well as their pharmaceutically acceptable salts, solvates, polymorphs, co-drugs, cocrystals, prodrugs, tautomers, racemates, enantiomers or diastereomers, or mixtures thereof; as endonucleolytic activity inhibitors in viruses with segmented negative- sense RNA genome for use as an antiviral drug. The invention further includes the use of the said derivative for manufacturing of a medicine for treatment, alleviation or prophylaxis of viral infection caused by viruses from Orthomyxoviridae family among others, including influenza and its complications, as well as for combination therapy with neuraminidase inhibitor, amantadine, neuraminidase, amantadine or rimantadine, or any other combination thereof.

Description

Title of invention: Inhibitors of influenza virus nuclease (polymerase complex)

The present invention relates to the use of small molecule compounds which block the nucleolytic activity, in the viral polymerase complex, for the prophylaxis and/or treatment of viral infections. Small molecule compounds have an inhibitory activity against the nuclease domain in viruses from the Orthomyxoviridae family, in particular influenza A, B and C viruses, Isavirus and Thogotovirus. These viruses, like the ones from Arenaviridae and Bunyaviridae families, which include viruses from the genus Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus and Tospovirus, have a genome consisting of negative-sense RNA segments. These viruses cause a number of diseases in humans and animals including upper and lower respiratory tract infections characterized by systemic manifestations and complications thereof, as well as respiratory illnesses (e.g. hantavirus pulmonary syndrome), cardiovascular and excretory system illnesses (e.g. hemorrhagic fever with renal syndrome in Poland and Europe) and also digestive and neurological system illnesses. These viruses can also infect numerous animal species (including rodents, poultry, wild birds, cattle, horses, dogs, bats), causing a wide range of diseases with similar characteristics to those observed in humans.

Influenza (flu) is a severe, highly infectious respiratory disease, which can affect virtually entire population. Seasonal flu epidemics are an important (though often underestimated) public health problem. They also generate significant social and economic costs of continental or even global reach. It is estimated that influenza infects between 330 million and 1.8 billion people worldwide and causes over 2 million deaths each year (according to World Health Organization (WHO)). In particular, A/H1N1 influenza virus caused the substantial increase of infections and forced the WHO to declare a global pandemic (Stage 6) on June 11th, 2009. One of the most important problem regarding influenza infections is the emergence of drug-resistant strains. This is due to the ability of the virus to rapidly mutate genes whose translation products are targets for existing drugs. Due to the very wide range of this pathogen and the traumatic effects of its infection all attempts to develop new treatments for this disease are highly desirable.

The influenza virus is characterized by a spherical or filamentous structure with helical symmetry and a diameter of 80-120 nm. The core of the infectious particle is formed by ribonucleoprotein complexes (vRNP) surrounded by membrane proteins. The capsid is wrapped around with the host cell lipid bilayer which anchors the transmembrane proteins and the surface glycoproteins. Influenza viruses are divided into 3 serologically different types (A, B and C) according to differences in antigenicity of preserved viral internal structures. Influenza virus of A type is the main reason of human infections.

The influenza virus genome is built with single-stranded negative-sense ribonucleic acid (vRNA). It occurs in virions in the form of 8 (or 7) segments encoding 11 proteins. Single vRNP consists of RNA molecule, trimeric polymerase (PA, PB1, PB2), nucleoprotein and small amounts of NS2 protein. vRNP molecules are independent entities within which the polymerase complex carries out both the transcription process and the replication of the viral genetic material.

Uncomplicated flu infection is characterized by acute onset of respiratory and systemic symptoms that usually resolve within 3 - 7 days. These symptoms are often associated with acute otitis media and bacterial pneumonia in elderly patients. In critical cases, influenza-associated complications may aggravate chronic heart diseases, respiratory diseases, myositis, myoglobinuria, myocarditis and pericarditis, may be the reason of encephalopathy, Reye syndrome or transverse myelitis. According to WHO data (http://www.who.int/en/) the high-risk group include chronically ill patients, immunocompromised, asthmatics, people over 65, pregnant women, infants and small children. The reason for this high incidence of infections is the easy spread of the influenza virus and its high adaptability. It consists of the continuous mutation process leading to changes in individual amino acid residues and the replacement of whole segments of the virus (between its various variants), which constantly generate new variants with novel pathogenic traits and virulence.

Inhibitors of M2 protein - amantadine and rimantadine (not registered in Poland) - were initially used for treatment of influenza in clinical practice. They inhibit the release of genetic material from the viral nucleocapsid into the cell and subsequent stages of its replication. It has been reported recently however that influenza A virus, which causes the highest number of cases, acquired resistance to aforementioned antiviral drugs. It has been found that insensitive strains appear in as many as one third of patients treated with adamantane derivatives. In the United States resistance to amantadine and rimantadine has been observed in 92% of influenza A virus H3N2 isolates. The loss of susceptibility of the pathogen appears because of single amino acid mutation in the M2 protein, which, due to the aforementioned high mutation rate of the viral genome occurs very frequently. As a result, variants of influenza A virus resistant to these drugs appear after 5 to 7 days of treatment with rimantadine or amantadine.

The second group of drugs are the neuraminidase inhibitors: oseltamivir (trade name Tamiflu) and zanamivir (trade name Relenza). They prevent the aggregation of individual virions and thus prevent the release of virions from the infected cell. In addition, neuraminidase inhibitors reduce the mobility of the virus in the mucins covering the respiratory epithelium. Unfortunately, analyses of clinical isolates indicate an increasing incidence of non-susceptible strains of neuraminidase inhibitors. According to World Health Organization data, oseltamivir resistance is estimated to occur in 1% of adult patients and in 4 - 6% of children. Even higher levels of drug resistance (up to 18%) have been reported in Asian countries (e.g. in Japan) where oseltamivir was introduced in 2003 as the primary drug in SARS and avian influenza (H5N1) therapies. In January 2008, there were reports of oseltamivir resistance in certain strains of the H1N1 virus circulating in Europe in the 2007/2008 season.

The efficacy of the abovementioned anti-influenza drugs is further limited by the fact that virus replication, resulting in the formation of about 1000 progeny virions in one cell, occurs over a period of 6 - 12 hours. This means that administration of the available drugs is only effective 24 - 30 hours after the infection, when influenza virus replication reaches maximum activity in the respiratory tract.

The current epidemiological situation of influenza drug resistance to available pharmacological agents indicates the very serious need to start the process of seeking new influenza drugs that will provide much wider clinical applications, such as the ability to cure patients also at advanced infection levels and protect against rapid onset of insensitive pathogens.

The key step in influenza virus infection cycle is the synthesis of its genomic RNA. The RNA-dependent RNA polymerase which is encoded by the genome of this pathogen plays the major role in this process. Polymerase complex consists of three subunits: PA, PB1 and PB2, catalyzing both viral RNA transcription (vRNA -> mRNA) and replication (vRNA -> cRNA -> vRNA). PA protein performs several functions in this complex. In particular, the N-terminal domain (hereinafter PA_N) harbors the endonuclease activity which initiates the transcription of the viral genetic material and the emergence of progeny virions. The postulated mechanism of viral mRNA synthesis occurs in several stages. First, PB2 protein binds 5' cap structure of host pre-mRNA, while PA_N endonuclease cuts off 10 - 13 nucleotide-long (nt-long) fragments. In this way, capped RNA fragments derived from cell transcripts are used as a primers by the viral polymerase to synthesize its own mRNA. RNA polymerase activity is located in the PB1 subunit of this protein complex. In addition to endonucleolytic properties, PA_N protein also binds to the RNA polymerase promoter and stabilizes whole complex.

In 2009, the X-ray crystal structures of PA_N have been reported in parallel by two research groups: A/H5N1 (avian influenza) (Yuan, P., Bartlam, M., Lou, Z., Chen, S., Zhou, J., He, X., Lv, Z., Ge, R., Li, X., Deng, T., Fodor, E., Rao, Z. and Liu, Y. (2009) Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site. Nature, 458, 909-913.) and A/H3N2 (human flu) (Dias, A., Bouvier, D., Crepin, T., McCarthy, A.A., Hart, D.J., Baudin, F., Cusack, S. and Ruigrok, R.W. (2009) The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature, 458, 914-918.).

The similarity of both domains is very high. It corresponds to 85% identity at the sequence level (including also a loop missing in one of the structures) and 0.88 A at the structure level (average square deviation of C-a atoms, RMSD). In order to illustrate substrate binding mechanism a series of PA_N X-ray structures in complex with adenosine, uridine and thymidine monophosphates have been recently determined (Zhao, C, Lou, Z., Guo, Y., Ma, M., Chen, Y., Liang, S., Zhang, L., Chen, S., Li, X., Liu, Y., Bartlam, M. and Rao, Z. (2009) Nucleoside Monophosphate Complex Structures of the Endonuclease Domain from the Influenza Polymerase PA Subunit Reveal The Substrate Binding Site inside the Catalytic Center. J Virol. 83 (18): 9024- 30.).

PA_N domain exhibits structural similarity and evolutionary relatedness to PD-(D/E)XK domain typical for most of the restriction enzymes (Bujnicki, J.M. and Rychlewski, L. (2001) Grouping together highly diverged PD-(D/E)XK nucleases and identification of novel superfamily members using structure-guided alignment of sequence profiles. J MolMicrobiolBiotechnol, 3, 69-72.; Kosinski, J., Feder, M. and Bujnicki, J.M. (2005) The PD-(D/E)XK superfamily revisited: identification of new members among proteins involved in DNA metabolism and functional predictions for domains of (hitherto) unknown function. BMC Bioinformatics, 6, 172.). The PD-(D/E)XK domain retains spatial architecture of the α/β type. It consists of five β-strands (β1-β5) forming a twisted β-sheet surrounded by several a-helices. The structure of influenza virus nuclease domain is strongly conserved. Amino acid residues that coordinate the bivalent metal ions are located in the negatively charged active site formed by α2-α5 helices and β3 strand. In the influenza virus nuclease structures in complex with nucleoside monophosphates there are two Mn²⁺ ions. The first metal ion (Me²⁺ I) is coordinated by two acidic residues E80 and D108, 3x water molecules stabilized by residues H41 and E119, as well as carbonyl oxygens of K106 and P107. Second metal ion (Me²⁺ II) is coordinated by amino acid residues H41, D108, E119 and carbonyl oxygen of 120. In these systems, both cations adopt an octahedral geometry coordinated by the amino acid residue D108. All the amino acid residues whose side chains are involved in metal ions coordination are conserved in the influenza A, B and C nuclease domain sequences.

To date, several specific viral endonuclease inhibitors have been described (Hastings, JC, Selnick, H., Wolanski, B. and Tomassini, JE (1996) Anti-influenza virus activities of 4-substituted 2,4-dioxobutanoic acid inhibitors. Tomassini, J., Selnick, H., Davies, ME, Armstrong, ME, Baldwin, J., Bourgeois, M., Hastings, J., Hazuda, D., Lewis, J. McClements, W. et al. (1994) Inhibition of cap (m7GpppXm)-dependent endonuclease of influenza virus by 4-substituted 2,4-dioxobutanoic acid compounds Antimicrob Agents Chemother, 38, 2827-2837; Tomassini, JE Davies, ME, Hastings, JC, Lingham, R., Moena, M., Raghoobar, SL, Singh, SB., Weaver, JS; and Goetz, MA. (1996) A novel antiviral agent which inhibits the endonuclease of influenza viruses. Agents Chemother, 40, 1189-1193.) including also compounds identified with computational methods (QSAR, i.e. without prior knowledge of receptor structure) (Kim, J., Lee, C. and Chong, Y. (2009) Identification of potential influenza virus endonuclease inhibitors through virtual screening based on the 3D-QSAR model. SAR QSAR Environ Res, 20, 103-118.; Parkes, K.E., Ermert, P., Fassler, J., Ives, J., Martin, J.A., Merrett, J.H., Obrecht, D., Williams, G. and Klumpp, K. (2003) Use of a pharmacophore model to discover a new class of influenza endonuclease inhibitors. J Med Chem, 46, 1153-1164.). The most widely recognized and tested inhibitor is 2,4-dioxo-4-phenylbutanoic acid (DPBA), however no antiviral activity has been demonstrated for this compound against influenza-infected cell lines (Dias, A., Bouvier, D., Crepin, T., McCarthy, AA, Hart, DJ, Baudin, F., Cusack, S. and Ruigrok, RW (2009) The cap-snatching endonuclease of the influenza virus polymerase resides in the subunit PA, 458, 914-918.). None of the research attempts taken so far however has led to the emergence of effective anti-flu drugs.

The object of the present invention is the use of known small molecule compounds as a novel type inhibitors of nucleolytic activity in viruses with segmented negative- sense RNA genome that block the action of the viral polymerase for treatment, alleviation or prophylaxis of viral diseases caused by viruses with segmented negative-sense RNA genome in humans or animals.

According to the present invention, the 4-hydroxyphenyl-2-oxoethylcarboxylic derivative represented by the general formula: R1-A-R2, wherein the core A is:

Rl is phenyl substituted with two C1-C4 alkyl, 1,3-benzodioxolyl or bicyclo[3.3.1]nonanyl and R2 is H, hydroxyl, amino group optionally substituted with carbonyl- C1-C4 alkyl or amide optionally substituted by C1-C4 alkyl;

and their pharmaceutically acceptable salts, solvates, polymorphs, co-drugs, cocrystals, prodrugs, tautomers, racemates, enantiomers or diastereomers, or mixtures thereof;

as endonucleolytic activity inhibitors in viruses with segmented negative-sense RNA genome for use as an antiviral drug.

Preferably, Rl is phenyl substituted with methyl in position 2 and 5, 1,3- benzodioxolyl or bicyclo[3.3.1]nonanyl and R2 is hydroxyl at position 2 or ethanamide (acetamide) group at position 3.

Most preferably, the derivative of the invention comprises:

(a) [2-(2,4-dihydroxyphenyl)-2-oxoethyl] l,3-benzodioxole-5-carboxylate (FI5)

(b) [2-(3-acetamido-4-hydroxyphenyl)-2-oxoethyl] 2,5-dimethylbenzoate (FI25)

(c) [2-(2,4-dihydroxyphenyl)-2-oxoethyl] 9-oxobicyclo[3.3.1]nonane-3-carboxylate (FI29)

as well as their pharmaceutically acceptable salts, solvates, polymorphs, co-drugs, co-crystals, prodrugs, tautomers, racemates, enantiomers or diastereomers, or mixtures thereof.

In addition, the present invention includes the use of said derivative for manufacturing of a medicine for treatment, alleviation or prophylaxis of viral diseases, preferably caused by viruses with segmented negative-sense RNA genome, belonging to: Poxyiridae, Herpesviridae, Adenoviridae, Papillomaviridae, Polyomaviridae, Parvoviridae, hepadnaviridae, Retroviridae, Reoviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Coronaviridae, Picornaviridae, Hepeviridae, Caliciviridae, Astroviridae, Togaviridae, Flaviviridae, Deltavirus, Bornaviridae, and prions, preferably Herpesviridae, Retroviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Orthomyxoviridae Bunyaviridae families, including viruses of the genus Hantavirus, Nairovirus, Orthobunyavirus or Phlebovirus, Arenaviridae, Coronaviridae, Picornaviridae, Togaviridae, Flaviviridae, and especially viruses from the Orthomyxoviridae family. Preferably, Orthomyxoviridae viruses include influenza A, B and C viruses, Isavirus and Thogotovirus include bird influenza virus, swine influenza virus and/or human influenza virus, in particular A/H5N1 or A/H3N2 viruses.

In a preferred application, viral infections include influenza and its complications with acute otitis media and bacterial pneumonia in elderly patients, upper and lower respiratory tract infections and their complications, respiratory disorders including hantavirus pulmonary syndrome, cardiovascular diseases, vascular and excretory disorders including haemorrhagic fever with renal syndrome, gastrointestinal disorders, neurological disorders as well as myositis, myoglobinuria, myocarditis and pericarditis and/or encephalopathy, Reye syndrome or transverse myelitis.

In another preferred application, viral infections caused by viruses from Orthomyxoviridae, Arenaviridae and Bunyaviridae family, including viruses of the genus Hantavirus, Nairovirus, Orthobunyavirus or Phlebovirus, in birds and mammals including rodents, poultry, wild birds, cattle, pigs, horses, dogs, bats, are a wide range of diseases with similar characteristics to those observed in humans.

Preferably, said derivative is used in combination therapy with neuraminidase inhibitors, either amantadine or rimantadine, or any other combination thereof. Preferably, said derivative can be administered to a patient, including human or animal patient, orally, buccally, sublingually, intranasally, via pulmonary routes such as by inhalation, via rectal routes, or parenterally, intracavernosally, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intra-urethrally intrasternally, intracranially, intramuscularly, or subcutaneously, they may be administered by infusion or needleless injection techniques.

Preferably, said derivative is used in unit dosages of 1 to 1000 mg/m², preferably 5 to 150 mg/m².

The invention will now be further illustrated in a preferred embodiment, with respect to the accompanying figures, where:

Fig. 1 Nucleolytic activity of N-terminal domain of PA protein against single- stranded RNA. Denaturing polyacrylamide gel image of substrate degradation stained with ethidium bromide. The endonuclease activity was tested by incubation at 37 °C of 12 μΜ enzyme with 10 μΜ of 46-nt long fragment of viral ph-RNA for 60 minutes. The reactions were stopped by adding EDTA to a final concentration of 20 mM. Lane 1: untreated substrate, lane 5: substrate incubated with enzyme in the presence of 1 mM Mn²⁺ ions. The control reactions were carried out: without the addition of Mn²⁺ ions (lane 2), at a final EDTA concentration of 20 mM (lane 3), and in the presence of a PA_N variant with a catalytic substitution of D108A amino acid residue (lane 4).

Fig. 2 Nucleolytic activity of N-terminal domain of PA protein against ribosomal

RNA. Agarose gel image of substrate degradation stained with ethidium bromide. The endonuclease activity was tested by incubation at 37 °C of 12 μΜ enzyme with 7 μg/μl of rRNA for 60 minutes. The reactions were stopped by adding EDTA to a final concentration of 20 mM. Lane 1: untreated substrate, lane 2: substrate incubated with enzyme in the presence of 1 mM Mn²⁺ ions. The control reactions were carried out: without the addition of Mn²⁺ ions (lane 3), at a final EDTA concentration of 20 mM (lane 4) and in the presence of a PA_N variant with a catalytic substitution of D108A amino acid residue (lane 5). The "M" symbol corresponds to the molecular-weight size marker. Small (16S rRNA) and large (23S rRNA) subunits of bacterial rRNA were labeled with arrows.

Fig. 3 Nucleolytic activity of N-terminal domain of PA protein against double- stranded φ6 phage genomic RNA. Agarose gel image of substrate degradation stained with ethidium bromide. The endonuclease activity was tested by incubation at 37 °C of 12 μΜ enzyme with 10 μΜ of dsRNA for 60 minutes. The reactions were stopped by adding EDTA to a final concentration of 20 mM. Lane 1: untreated substrate, lane 5: substrate incubated with enzyme in the presence of 1 mM Mn²⁺ ions. The control reactions were carried out: without the addition of Mn²⁺ ions (lane 2), at a final EDTA concentration of 20mM (lane 3) and in the presence of a PA_N variant with a catalytic substitution of D108A amino acid residue (lane 4). The "M" symbol corresponds to the molecular-weight size marker.

Fig. 4 Nucleolytic activity of N-terminal domain of PA protein against single- stranded M13mpl8 DNA in time dependence. Agarose gel image of substrate degradation stained with ethidium bromide. The endonuclease activity was tested by incubation at 37 °C of 12 μΜ enzyme with 50 μg/μl of DNA for 60 minutes. The reactions were stopped by adding EDTA to a final concentration of 20mM at various time points: 15 minutes (lane 5), 30 minutes (lane 6), 45 minutes (lane 7), 60 minutes (lane 8), 120 minutes (lane 9). Lane 1: untreated substrate. The control reactions were carried out: without the addition of Mn²⁺ ions (lane 2), at a final EDTA concentration of 20mM (lane 3) and in the presence of a PA_N variant with a catalytic substitution of D108A amino acid residue (lane 4). The "M" symbol corresponds to the molecular-weight size marker.

Fig. 5 Effect of divalent ions on the nucleolytic activity of N-terminal domain of PA protein. Denaturing polyacrylamide gel image of substrate degradation stained with ethidium bromide. The endonuclease activity was tested by incubation at 37 °C of 12 μΜ enzyme with 10 μΜ of DNA-32 substrate for 60 minutes, without the addition of ions ("DNA-32") and in the presence of Mn²⁺, Ca²⁺, Co²⁺, Ni²⁺ and Mg²⁺ ions. The reactions were stopped by adding EDTA to a final concentration of 20mM.

Fig. 6 Influence of new influenza virus nuclease inhibitors FI29, FI5 and FI25 on the nucleolytic activity of enzymes from PD-(D/E)XK superfamily. Agarose gel image of substrate degradation. Reactions were performed in the presence of FI29, FI5 and FI25 inhibitors at a concentration of 1000 μΜ at 37 °C for 120 minutes. (A) phage λ (dam-, dcm-) DNA digested with EcoRV enzyme, (B) phage λ (dam-, dcm-) DNA digested with Mval enzyme, (C) pBR322 plasmid DNA digested with Dpnl enzyme. The "λ" and "pBR322" symbols (Fig. 6 A, B, C lane 2) correspond to untreated substrate while "+" symbol (Fig. 6 A, B, C lane 3) to substrate digested with enzyme in the absence of said chemical compounds.

Fig. 7 Comparison of the morphology of untreated HEK293 cells and HEK293 cells exposed on various concentrations of FI29 inhibitor. Cellular examinations were carried out under the contrast microscope at lOx magnification. The left panel ("kontrola") shows control untreated HEK293 cells, while the right panel, shows HEK293 cells incubated for 24 hours with increasing concentrations of FI29 inhibitor (A) 125 μΜ, (B) 250 μΜ, (C) 500 μΜ and (D) 1000 μΜ.

Preferred embodiments of the invention

The invention is illustrated by the following, non-limiting embodiments.

The following examples are provided solely for the purpose of illustrating the invention and for explaining its particular aspects, and not for limiting it, thus the scope of the invention has been determined by the appended claims, rather than by the examples given.

In the below examples, unless indicated otherwise, standard materials and methods described in Sambrook J. et al., "Molecular Cloning: A Laboratory Manual" 2nd edition. 1989. Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory Press have been used or manufacturer's recommendations for specific materials and methods have been followed.

As used herein, unless indicated otherwise, standard abbreviations are used for the names of amino acids and nucleotides or ribonucleotides.

The FI5, FI25 and FI29 chemical compounds were purchased from the eMolecules catalog.

Example 1. Examination of the ability of chemical compounds FI5, FI25, FI29 to block the endonucleolvtic activity of viruses with segmented negative-sense RNA genome, exemplified by the A/H5N1 influenza virus: Enzymatic activity and substrate requirements of viral nuclease

In order to examine the inhibitory activity of the FI5, FI25, FI29 chemical compounds, the N-terminal domain of the PA protein, including amino acid residues 1 - 259, from A/H5N1 avian influenza has been used (sequence number deposited in the PDB database in 3EBJ record and non-redundant protein sequence database (NCBI) Gl 223365853 (A/goose/Guangdong/1/1996 (H5N1)). The C-terminus polyhistidine- tagged protein was overproduced in bacterial cells and further purified for high homogeneity of the sample.

In order to confirm the purity and lack of nuclease contamination additional protein variant containing substitution of catalytic residue D108 to alanine has been designed. It was also intended to serve as a negative control in PA_N nuclease activity assays. Within 60 minutes (under any of the conditions used in the assay) no enzyme activity was observed for the D108A variant. It has been therefore selected for further tests.

The endonuclease activity assays (Tab. 1) were performed by incubation of 12 μΜ of PA_N with selected substrates at a suitable O buffer concentration (50 mM Tris-HCI (pH 7.5 at 37 °C), 0.1 mg/ml BSA) in the presence of 1 mM Mn²⁺ ions for 60 to 120 minutes at 37 °C. The effect of divalent ions on enzyme activity were carried out in the presence of Mn²⁺, Mg²⁺, Ca²⁺, Ni²⁺ and Co²⁺ at 1 mM concentration and at 7.5 pH. The reactions were stopped by adding EDTA to a final concentration of 20 mM. In all cases the simultaneous control reactions were carried out without the addition of Mn²⁺ ions, at a final EDTA concentration of 20mM as well as in the presence of a PA_N variant with the catalytic substitution of D108A amino acid residue. The reaction products were loaded on denaturing polyacrylamide or agarose gel and visualized in UV light after staining with ethidium bromide.

Tab. 1 DNA oligonucleotide sequences

The nucleolytic activity of the resulting protein sample was tested using a set of RNA and DNA substrates of varying length, nucleotide sequence and secondary structure. First, the native biological activity of the PA_N domain, i.e. the ability to hydrolyse phosphodiester bonds in RNA, was verified (Klumpp, K., Doan, L, Roberts, NA and Handa, B. (2000). RNA and DNA hydrolysis are catalyzed by the influenza virus endonuclease J BiolChem, 275, 6181-6188.). To this end a fragment from one out of eight segments of influenza virus genomic RNA (vRNA) was selected. An 81-nt long vRNA fragment of the so-called ph-RNA (panhandle-RNA) (Baudin, F. Bach, C. Cusack, S. Ruigrok, RW. (1994) Structure of influenza virus RNP I . Influenza virus nucleoprotein secondary structure in RNA pandemic and exposes the bases EMBO J, 1994 Jul 1; 13 (13): 3158-65.) was used to design a substrate comprising an unstructured 46 nt-long DNA fragment of the domain I II in ph-RNA. The PA_N domain was capable to completely degrade such substrate (Fig. 1, lane 5).

I n the next step, the nucleolytic activity of the viral nuclease domain against ribosomal RNA (rRNA) subunits isolated from E. coli cells was examined. The aim of the experiment was to investigate the ability of PA_N nuclease to hydrolyse phosphodiester bonds in larger, partially structured substrates. After incubation with the enzyme for 60 minutes the substrate degraded to lower mass fragments (Fig. 2, lane 2).

Due to incomplete rRNA degradation that could arise from specificity of the enzyme for single-stranded substrates, an attempt to verify the activity of the PA_N nuclease against double-stranded φ6 phage genomic RNA was made. This substrate was resistant to degradation (Fig. 3).

Although the major role of nucleases in the influenza virus polymerase is the cleavage of short, capped fragments of host host mRNA during initiation of viral RNA transcription, the enzyme also has the ability to digest single stranded DNA without specific sequential preference (Klumpp K, Doan L, Roberts NA, Handa B. RNA and DNA hydrolysis are catalyzed by the influenza virus endonuclease. J Biol Chem. 2000 Mar 3;275(9):6181-8.). I n order to test the actual ability of purified protein sample to degrade a single stranded DNA, digestion tests using single-stranded circular bacteriophage M13mpl8 DNA were performed. The results indicate that the substrate undergoes partial degradation over time to almost complete degradation after 120 minutes. In an analogous experiment using phage λ (dam-, dcm-) double- stranded DNA substrate degradation was not observed, similarly as with double- stranded φ6 phage genomic RNA.

In the final step, the effect of divalent ions on the nucleolytic activity of the N- terminal domain of the PA protein has been determined. The study was conducted in the presence of Mn²⁺, Ca²⁺, Co²⁺, Ni²⁺ and Mg²⁺ at a 1 mM concentration at 7.5 pH. The enzyme was active in the presence of Mg²⁺ and Mn²⁺ ions, however the activity was higher for the latter. In the presence of Ca²⁺, Co²⁺ and Ni²⁺ ions no change compared to control without the addition of ions was observed (Fig. 5).

Example 2. Investigation of the ability of chemical compounds FI5, FI25, FI29 to block the endonucleolvtic activity of viruses with segmented negative-sense RNA genome, exemplified by the A/H5N1 influenza virus: The inhibitory activity of FI5, FI25, FI29 determined with FRET technique

The ability of chemical compounds to block the activity of PA_N viral nuclease was investigated. To this end the FRET method based on the Forster theory was used (Zheng, J. (2006)), Shapiro, Mark S. Ion Channels: Methods and Protocols. Methods in Molecular Biology, Volume 337. Totowa, NJ: Humana Press pp. 65-77.). The FRET technique allows for measurement of the fluorescence signal growth that occurs as a result of reporter and quencher separation caused by nucleolytic cleavage of molecular beacon. The resulting time-dependent fluorescence curve illustrates an increase in fluorescence of the acceptor in real-time. It also enables determination of the IC₅₀ value which corresponds to the concentration of the inhibitor causing 50% inhibition of enzyme activity.

The molecular beacon was designed using a single-stranded 46-nt long DNA oligonucleotide with a reporter fluorescent label at one end and a quencher at the other end of the molecule. An 32 nt-long enzyme recognition sequence was placed between two complementary DNA fragments (7 nucleotides at the 5' and 3' ends) to form a hairpin structure. The DY-510XL fluorophore with an excitation wavelength of 509 nm and 590 nm emission was attached at the 5' end of the molecular beacon while non-fluorescent BHQ-2 chromophore with a maximum absorption at 579 nm and a quenching range of 559 - 650 nm was attached at the 3' end. The primary structure of the double-stranded stem of the molecular beacon was adjusted to the required melting point of the duplex which was 30 - 32 °C.

Hybridization of the molecular probe was carried out with: initial denaturation for 1 minute at 95 °C, then 58 steps 5 seconds each lowering the temperature by 1 °C until final incubation at 37 °C for 10 minutes.

All compounds were dissolved in an organic solvent DMSO and further serially diluted in reaction buffer O (50 mM Tris-HCI (pH 7.5 at 37 ° C), 0.1 mg/ml BSA) with 1 mM Mn²⁺. The final maximum DMSO concentration was 2.5% (v/v) in all test and control reactions. Appropriate concentrations of compounds (over a concentration ranging from 3 to 1000 μΜ) were incubated with PA_N at a 1 μΜ concentration for 30 minutes at room temperature under cover to prevent evaporation. Next, 1 μΜ fluorescence labeled and 2 μΜ unlabelled molecular probe were added to the reactions. The protein to substrate ratio was selected so that the increment of the reaction product relative to the consecutive substrate concentrations was linear. Inhibition assays were performed in the presence and absence of Triton X-100 detergent (at a final concentration of 0.01% (v/v)) due to the aggregation/nonspecific binding (e.g. covalent binding) of test compounds. In all cases the simultaneous control reactions were carried out without the addition of Mn²⁺ ions, at a final EDTA concentration of 20mM as well as in the presence of a PA_N variant with the catalytic substitution of D108A amino acid residue. For the fluorescent measurements, black non-binding, polystyrene, 96-well microtiter plates were used. Measurements were carried out at 521 nm excitation and 594 nm emission wavelengths at 37 °C for 1 hour with an acquisition interval of 1 minute. Fluorescent signal changes were monitored using a Tecan Infinite^® M1000 fluorescence imaging plate reader. The initial reaction velocity (V₀) was determined by measuring 30 minutes for each dilution of the compound under test . The IC₅₀ value, defined as the concentration of the compound under test causing 50% inhibition of an enzymatic reaction, was determined for data from four independent experiments using 4-parametric model whereby positive and negative controls were included to define the top and bottom of the curve.

Two variants of FRET method were used for the measurements: without incubation and allowing 30-minute-long incubation of the enzyme with the compound under test before the substrate was added. In order to eliminate inhibitors that may block the viral nuclease activity in non-specific manner, e.g. by tendency to aggregate or form nonspecific covalent bonds to amino acid residues, all tests were performed both in the presence and absence of Triton X-100 detergent at a final concentration of 0,01%. The potential detergent effect on viral nuclease was excluded in additional control. Due to the fact that initial dilutions of compounds under test were prepared using an organic solvent DMSO, its acceptable concentration was set at 2.5% of the reaction mixture volume (max value). A simultaneous control proved that DMSO at the above concentration did not affect the activity of the influenza virus nuclease. Measurements of inhibition activity of FI5, FI25 and FI29 small molecule compounds using FRET technique were performed in four independent experiments. All obtained results were additionally confirmed by digestion tests that were visualized on denaturing polyacrylamide gels.

FI5, FI25 and FI29 chemical compounds blocked PA_N nuclease activity completely at the initial concentration (1000 μΜ). None of the inhibitors in this group exhibited time-dependent inhibitory activity. No inhibitor was observed to decrease activity after adding the detergent to the reaction mixture. None of the inhibitors demonstrated elevated (abnormal) increase in fluorescence signal. Tab. 2 Inhibitory activity (IC₅₀) of the chemical compounds FI5, FI25, FI29 against influenza virus nuclease determined using the FRET technique

In the sequences of the influenza A, B and C virus nuclease domain, and possibly other viruses with segmented negative-sense RNA genome, amino acid residues essential for enzyme activity are strongly conserved. Therefore, the FI5, FI25 and FI29 chemical compounds should have the same effect on the other nuclease domains in the polymerase complexes of all types of viruses.

Example 3. Investigation of the effect of chemical compound FI29 on substrate binding, exemplified by influenza A/H5N1 virus

The effect of inhibitor FI29 on substrate binding was investigated. Measurements of the dissociation constant (K_D) and relative substrate binding affinity were evaluated by nitrocellulose filter binding method. The DNA substrate was radiolabeled using the bacteriophage T4 polynucleotide kinase (PNK). The reaction was carried out in a buffer suggested by the manufacturer using 10 pmol substrate, 10 units of kinase and 10 μθϊ [γ-33Ρ]-ΑΤΡ at 37 °C for an hour. Next, the enzyme was thermally inactivated by incubation at 70 °C for 10 minutes. To remove unincorporated ATP the product was purified using Sephadex G-25 .

Dissociation constant (K_D) measurements: appropriate concentrations of PA_N were incubated with radiolabeled DNA-46 substrate (1 nM) (reaction mixture volume 60 μΙ) in Hepes-Ca buffer at room temperature for 30 minutes.

Relative binding affinity measurements: the protein (at a concentration equal to twice the dissociation constant) was incubated with labeled (1 nM) and unlabeled DNA-46 substrate (100 nM) (reaction mixture volume 60 μΙ) in the presence of increasing concentrations of FI29 (½xlC50, lxlC50, 2xlC50). The reaction was conducted in Hepes-Ca buffer at room temperature for 30 minutes. Measurements were done in triplicates.

The nitrocellulose membrane was rinsed in Hepes-Ca buffer without BSA and in the presence of Ca²⁺ at room temperature for 1 hour. After incubation, 50 μΙ of the sample was filtered through the Optitran BA-S 83 nitrile membrane (with a cutoff of 0.2 μιη and a binding capacity of 75 - 90 μg/cm², Whatman TM, GE Healthcare) using Bio-dot Apparatus connected to the vacuum pump and washed with 400 μΙ of Hepes-Ca buffer. The membrane was dried at room temperature, exposed to the Storage Phosphor screen, and finally visualized using a Typhoon scanner. ImageQuant software was used to analyse the intensity of an autoradiographic image. Dissociation constant was calculated using simple ligand binding model, one site saturation.

Relative binding affinity measurements were performed using ³³P radiolabeled at the 5' end substrate for which the K_D equal to 3.36 μΜ (± 0.8) was determined. Substrate with identical nucleotide sequence was previously used to measure inhibitory activity with FRET method (DNA-46, Tab. 1). At substrate concentrations above twice the K_D value and in the presence of FI29 compound at a concentration corresponding to IC₅₀ (80 μΜ), its half (40 μΜ) and its double value (160 μΜ), the relative binding affinity was reduced about 50% (Tab. 3).

Tab. 3 The effect of FI29 viral nuclease inhibitor on the relative binding affinity of the enzyme to the substrate. The substrate binding level in the presence of the compound under test with respect to the binding of the PA_N domain to the substrate without an inhibitor. K_D of ³³P radiolabeled DNA-46 in the presence of increasing FI29 concentration. Values were obtained after normalization with respect to PA_N domain substrate binding level without inhibitor (wt). The standard deviation was determined in three independent measurements.

Example 4. Examination of the receptor specificity of chemical compounds FI5, FI25, FI29 exemplified by enzymes from the PD-(D/E)XK superfamily: EcoRV, Mval and Dpnl

The receptor specificity of the influenza virus nuclease inhibitors FI5, FI25, Fi29 was determined based on their activity against enzymes from the PD-(D/E)XK superfamily.

The PA_N domain exhibits evolutionary relatedness and structural similarity to PD- (D/E)XK domain typical for restriction enzymes, but also many enzymes responsible for key functions in the human body. In order to investigate how a new inhibitors of influenza virus nuclease affect the activity of other enzymes from PD-(D/E)XK superfamily, a series of digestion assays using EcoRV, Mval and Dpnl enzymes were performed.

DNA digestion with PD-(D/E)XK superfamily was performed using commercially available EcoRV, Mval and Dpnl (Fermentas) protein samples. The reactions were performed in buffers suggested by the manufacturer. For each restriction digestion optimized conditions were used varying with: time, temperature and relative ratio of enzyme units to 1 μg of DNA. The reaction products were loaded on agarose gel, separated and subsequently isolated from the gel using Gel-out kit.

Chemical compounds were added to the reaction mixture in geometric series of concentrations at a range from 125 to 1000 μΜ. Furthermore, in order to prevent possible aggregation of compounds under test, the reactions were carried out in the presence of detergent (Triton X-100 at a final concentration of 0.01% (v/v)). For each enzyme, suitable substrates were selected: DNA phage λ (dam-, dcm-) for EcoRV and Mval, and DNA plasmid pBR322 for Dpnl.

The choice of substrates was driven by the possibility of comparing separation patterns of enzyme digestion products with the DNA digest profile provided by the manufacturer. This allowed for the elimination of possible star activity of the enzymes, which could be misinterpreted as a result of the action of the test compound.

EcoRV, Mval and Dpnl enzymes were selected for the study because they belong to the PD-(D/E)XK protein superfamily same as PA_N nuclease. EcoRV nuclease is a homodimer that recognizes and specifically cleaves palindromic DNA sequence 5'- GAT ATC-3' (where corresponds to the cleavage site) (Yang W. (2011) Nucleases: diversity of structure, function and mechanism. Epub 2010 Sep 21. Review.). In addition to EcoRV, a series of digestion tests were carried out using Mval and Dpnl proteins. The selection of enzymes was done in order to analyze the effects of inhibitors on various mechanisms of catalyzed reactions, modes of interaction and structure, substrate preferences of PD-(D/E)XK superfamily enzymes, as well as specific features i.e. sensitivity to substrate DNA modification. The second enzyme selected for the study - Mval - is a monomeric protein recognizing the pseudo- palindromic DNA sequence 5'-CC WGG-3' (where W corresponds to A or T). This enzyme is also insensitive to substrate modification (Yang W. (2011) Nucleases: diversity of structure, function and mechanism. Review.). Dpnl, on the other hand, is a monomer recognizing and digesting methylated DNA 5'-GM6A TC-3' sequence (Yang W. (2011) Nucleases: diversity of structure, function and mechanism. Epub 2010 Sep 21. Review.).

Digestion tests were performed for a single concentration of compounds that was previously proven to inhibit the activity of PA_N domain. The receptor specificity of chemical compounds FI5, FI25, FI29 has been shown in the Fig. 6. For none of the inhibitors, no changes in substrate degradation were observed, nor did they result in differences in DNA separation pattern (Fig. 6).

Example 5. Examination of the cytotoxicity of chemical compounds FI5, FI25, FI29 against human cell lines; exemplified by human embryonic kidney cells HEK293 and human fibroblast cell line

The MTT assay was used to evaluate the impact of FI5, FI25, FI29 on the functioning of human cells. It is a colorimetric method for assessing cell metabolic activity based on NAD(P)H-dependent mitochondrial oxidoreductase enzymes. These enzymes are capable of reducing the yellow water-soluble tetrazolium dye MTT 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan. In a cell culture incubated in the presence of the compound under test, the formazan forms purple colored precipitate which is then dissolved in dimethyl sulfoxide (DMSO). The resulting amount of product reflects the number of viable/ proliferating cells and can be quantified by spectrophotometric methods (Carmichael J, DeGraff W, Gazdar AF (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay., 936-942.). The MTT assay is a widely used method in in vitro toxicology studies. In damaged, metabolically impaired or deformed cells, formazan is formed in smaller amounts, or not formed at all, which determines the percentage of functional cells and the effects of the compounds under test on cell viability (e.g. proliferation).

MTT tests were performed on human embryonic kidney HEK293 and human fibroblast cell lines. Eukaryotic cell cultures were seeded in sterile (single- and multi- well) adherent plates until full confluence. Cell lines were passaged using lx concentrated phosphate buffer saline (PBS) and trypsin-EDTA solution 0.25%. Cultures were incubated in a sterile incubator at 37 °C and 5% C0₂. Cellular manipulations were carried out inside a laminar flow hood to ensure adequate levels of septicity.

Cells were plated out into 96-well plates at 1:3, 1:6, 1:8 ratio and incubated for 48 hours. After this time, serial dilutions of compounds (at a concentration range from 125 to 1000 μΜ) in culture medium were prepared. Old medium was replaced and plates were incubated for another 24 hours. All dilutions were subjected to PVDF membrane filtration (Millipore). On the last day of culture, MTT reagent at the final concentration of 0.5 mg/ml was added to each well and further incubated for 3 hours. After removing the culture medium, 100 μΙ of DMSO was added to each well in a plate and shaken for 5 minutes. Absorption measurements were performed at 595 nm wavelength . For each compound under test, three independent measurements were performed. Based on the analysis of the obtained results 50% cytotoxic concentration (CC₅₀) values were determined. The CC₅₀ was defined as the extract concentration that reduced the cell viability by 50% when compared to untreated controls. Examination of morphological changes in control cells (without inhibitor addition) and cells exposed to increasing concentrations of compounds under test was performed using an Olympus 1X70 contrast-phase microscope with a lOx magnification lens.

In order to analyse the potential impact of different cell monolayer densities on the observed cytotoxicity (which could arise from e.g. differences of proliferation rate, increased/decreased resistance to damage, etc.) MTT tests were performed with decreasing cell density at plating: at 1:3, 1:6 and 1:8 ratio.

The results of the tests showed that chemical compounds FI5, FI25 and FI29, in the analyzed concentration range, did not adversely affect the functioning of the HEK293 and human fibroblast cells relative to controls without the addition of an inhibitor. For any of the inhibitors, no difference in MTT results was observed for different cell densities.

Tab. 3 Cytotoxicity/cytostatic activity of new viral nuclease inhibitors ¥129, ¥15, FS25 against human HEK293 and human fibroblast cell lines

The MTT assay allows for measurement of energy conversion in mitochondria which can provide information about the functional state of the cell but does not indicate potential changes in cell morphology i.e. changes in cell features, intracellular vacuole formation and/or cell monolayer violation, in particular do not distinguish the cytostatic effect from cell death. In order to determine the effect of FI29, FI5 and FI25 on the morphology of HEK293 cells, a microscopic analysis was performed in which cells exposed to increasing concentrations of FI29 (Fig. 7, right panel, results for FI29) were compared with control without addition of inhibitor (Fig. 7, left panel, results for FI29). According to the results obtained in the MTT tests, no changes in morphology of cells treated and untreated with FI29, FI5 and FI25 was observed.

Claims

1. The 4-hydroxyphenyl-2-oxoethylcarboxylic derivative represented by the general formula: R1-A-R2, wherein the core A is:

Rl is phenyl substituted with two C1-C4 alkyl, 1,3-benzodioxolyl or bicyclo[3.3.1]nonanyl and R2 is H, hydroxyl, amino group optionally substituted with carbonyl- C1-C4 alkyl or amide optionally substituted by Cl- C4 alkyl;

as endonucleolytic activity inhibitors in viruses with segmented negative- sense RNA genome for use as an antiviral drug.

The derivative according to claim 1, characterized in that Rl is phenyl substituted with methyl in position 2 and 5, 1,3-benzodioxolyl or bicyclo[3.3.1]nonanyl and R2 is hydroxyl at position 2 or ethanamide (acetamide) group at position 3.

The derivative according to claim 1 or 2, characterized in that it comprises:

(a) [2-(2,4-dihydroxyphenyl)-2-oxoethyl] l,3-benzodioxole-5-carboxylate (FI5) 28

as well as their pharmaceutically acceptable salts, solvates, polymorphs, codrugs, cocrystals, prodrugs, tautomers, racemates, enantiomers or diastereomers, or mixtures thereof.

4. Use of the derivative of any of claims from 1 to 3 for manufacturing of a medicine for treatment, alleviation or prophylaxis of viral diseases, preferably caused by viruses with segmented negative-sense RNA genome, belonging to family: Poxyiridae, Herpesviridae, Adenoviridae, Papillomaviridae, Polyomaviridae, Parvoviridae, hepadnaviridae, Retroviridae, Reoviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Coronaviridae, Picornaviridae, Hepeviridae, Caliciviridae, Astroviridae, Togaviridae, Flaviviridae, Deltavirus, Bornaviridae, and prions, preferably Herpesviridae, Retroviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Orthomyxoviridae Bunyaviridae families, including viruses of the genus Hantavirus, Nairovirus, Orthobunyavirus or Phlebovirus, Arenaviridae, Coronaviridae, Picornaviridae, Togaviridae, Flaviviridae, and especially viruses from the Orthomyxoviridae family.

5. Use according to claim 4, characterized in that Orthomyxoviridae viruses include influenza A, B and C viruses, and Isavirus and Thogotovirus include bird influenza virus, swine influenza virus and/or human influenza virus, in particular A/H5N1 or A/H3N2 viruses.

6. Use according to claim 4 or 5, characterized in that viral infections include influenza and its complications with acute otitis media and bacterial pneumonia in elderly patients, upper and lower respiratory tract infections and their complications, respiratory disorders including hantavirus pulmonary syndrome, cardiovascular diseases, vascular and excretory disorders including haemorrhagic fever with renal syndrome, gastrointestinal disorders, neurological disorders as well as myositis, myoglobinuria, myocarditis and pericarditis and/or encephalopathy, Reye syndrome or transverse myelitis.

7. Use according to claim 4 or 5, characterized in that viral infections caused by viruses from Orthomyxoviridae family, Arenaviridae family and Bunyaviridae family, including viruses of the genus Hantavirus, Nairovirus, Orthobunyavirus or Phlebovirus, in birds and mammals including rodents, poultry, wild birds, cattle, pigs, horses, dogs, bats, are a wide range of diseases with similar characteristics to those observed in humans.

8. Use according to any one of the preceding claims 4 - 7, in combination therapy with neuraminidase inhibitors, or amantadine, or rimantadine, or any other combination thereof.

9. Use according to any one of the preceding claims. 4 - 8, in which said derivative can be administered to a patient, including human or animal patient, orally, buccally, sublingually, intranasally, via pulmonary routes such as by inhalation, via rectal routes, or parenterally, intracavernosally, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intra-urethrally intrasternally, intracranially, intramuscularly, or subcutaneously, they may be administered by infusion or needleless injection techniques.

10. Use according to claim 9, in which said derivative is used in unit dosages of 1 to 1000 mg/m², preferably 5 to 150 mg/m², according to the particular application and the potency of the said active component.