WO2018178935A1

WO2018178935A1 - Pad2 for use in preventing and/or treating or diagnosing infections caused by viruses of the herpesviridae family

Info

Publication number: WO2018178935A1
Application number: PCT/IB2018/052204
Authority: WO
Inventors: Santo Landolfo; Francesca Gugliesi; Valentina DELL'OSTE; Sara PAUTASSO; Marco DE ANDREA; Gloria GRIFFANTE; Matteo BIOLATTI
Original assignee: Universita' Degli Studi Di Torino
Priority date: 2017-03-29
Filing date: 2018-03-29
Publication date: 2018-10-04
Also published as: EP3600386A1; IT201700034630A1

Abstract

The present invention relates to PAD2 (peptidyl arginine deiminase, type 2) for use in preventing and/or treating or diagnosing infections caused by viruses of the Herpesviridae family. The present invention also relates to an inhibitor or antagonist of PAD2 and related pharmaceutical compositions for use as antiviral agents against viruses of the Herpesviridae family.

Description

" PAD 2 FOR USE IN PREVENTING AND/OR TREATING OR DIAGNOSING INFECTIONS CAUSED BY VIRUSES OF THE HERPESVIRIDAE FAMILY"

Priority claim

This application claims priority from Italian Patent Application No. 102017000034630 filed on March 29, 2017, the disclosure of which is incorporated by reference.

Technical field

The present invention relates to PAD2 (peptidyl arginine deiminase, type 2) for use in preventing and/or treating or diagnosing infections caused by viruses of the Herpesviridae family.

Background of the invention

Herpesviruses are double-stranded DNA icosahedral viruses belonging to the Herpesviridae family. Various Herpesviruses are responsible for infections, which can even be severe, in human beings. Eight human Herpesviruses are currently known: Herpes Simplex Virus type 1 (HSV-1), Herpes Simplex Virus type 2 (HSV-2), Varicella-Zoster Virus (VZV) , Cytomegalovirus (CMV) , Human Herpesvirus type 6, Human Herpesvirus type 7, Epstein-Barr Virus (EBV) , Human Herpesvirus type 8 (or Kaposi's sarcoma-associated herpesvirus, KSHV) .

The elected antiviral drugs for herpes infections envisage the use of nucleoside analogues (e.g. ganciclovir) .

Nucleoside analogues can however have high toxicity for the host. In particular, the use of ganciclovir or similar nucleoside derivatives in children (congenital infections or bone marrow transplant) due to the high myelotoxicity can have severe side effects which significantly limit their use .

Furthermore, interfering with the viral DNA synthesis phase, nucleoside analogues frequently induce the appearance of resistance-associated mutations to the genes that code for viral enzymes (e.g. UL54 or UL97 of HCMV) used for the synthesis of DNA itself.

In the field of transplants, for example, the availability of drugs such as ganciclovir and its derivatives has led to remarkable progress in the control of the rejection caused by infections sustained by HCMV, but the frequent appearance of resistant viral clones substantially limits their use in the initial stages of the post-transplant period .

To prevent the onset of resistant viral variants, it would be desirable to identify cellular proteins in the host as targets for inhibiting the replication of viruses. In fact, to replicate, viruses exploit components and transduction pathways of the host cell, which are much less subject to mutations. The identification of a cellular protein to be used as a target for blocking viral replication would allow avoiding the occurrence of genome mutations.

A need is therefore felt to provide new targets for preventing and/or treating or diagnosing infections caused by viruses of the Herpesviridae family.

Disclosure of invention

The object of the present invention is to provide a protein of the host cell involved in the viral replication of viruses of the Herpesviridae family, whose activity can be modulated so as to inhibit viral replication.

According to the present invention, such object is achieved through the use of PAD2 (peptidyl arginine deiminase, type 2) according to claim 1. Inhibitors, antagonists and related compositions for use as antiviral agents against viruses of the Herpesviridae family are also provided .

Brief description of the figures

For a better understanding of the present invention, it will also be described with reference to the attached figures, which illustrate the following:

- Figure 1 represents an SDS-page of protein extracts labelled with the Rh-PG probe (MW = molecular weights; hpi = hours post-infection) ;

- Figure 2 represents a graph of the mRNA expression levels of the enzymes of the PAD family in cells infected or not (mock) by HCMV;

- Figure 3A represents a graph of the enzyme activity of PAD2 in cells infected or not (mock) by HCMV;

- Figure 3B represents a graph of the measurement of the activity of scalar concentrations of the recombinant enzyme PAD2 (OD, optical density) ;

- Figure 4A represents a graph of the viral titration of the supernatants obtained from cells treated with Cl- amidine or an equal volume of solvent and infected with HCMV (strain Merlin MOI=0.1);

- Figure 4B represents a graph of the viral titration of the supernatants obtained from cells treated with Cl- amidine or an equal volume of solvent (DMSO) and infected with HCMV (strain Merlin MOI=0.1);

- Figure 5 represents a graph of the viral titration of the supernatants obtained from cells electroporated with PAD2 siRNA or with a control siRNA (ctrl) and infected with HCMV (strain Merlin MOI=0.1);

- Figure 6A represents a graph of the viral titration of the supernatants obtained from cells treated with Cl- amidine or an equal volume of solvent (DMSO) and infected with HSV-1 (MOI= 0.1);

- Figure 6B represents a graph of the viral titration of the supernatants obtained from cells treated with CI- amidine or an equal volume of solvent (DMSO) and infected with HSV-1 (MOI=0.1);

- Figure 6C represents a graph of the viral titration of the supernatants obtained from cells treated with Cl- amidine or an equal volume of solvent (DMSO) and infected with HSV-2 (MOI=0.1);

- Figure 6D represents a graph of the viral titration of the supernatants obtained from cells treated with Cl- amidine or an equal volume of solvent (DMSO) and infected with HSV-2 (MOI=0.1) .

Detailed description of the invention

According to the present invention, the PAD2 (peptidyl arginine deiminase, type 2) protein is used in preventing and/or treating or in diagnosing infections caused by viruses of the Herpesviridae family.

PADs (peptidyl arginine deiminases) are a family of cellular enzymes that catalyse the citrullination reaction that consists of the deimination of arginine to citrulline. The family of human and murine PADs is composed of five members, PAD 1-4 and PAD 6 that have peculiar tissue expression characteristics and substrate specificities. PAD2 is expressed in different cell districts, such as muscle, brain, mammary glands, PAD4 mainly in hematopoietic cells such as neutrophils, monocytes and macrophages, PAD1 in the epidermis and the uterus, PAD3 in the pilifer follicles and PAD 6 at the ovules (Darrah et al . 2012; Wang and Wang 2013) .

An aberrant activation of the citrullination process can lead to the onset of antibodies against the citrullinated proteins as can be observed during the course of various inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, neoplasia and degenerative diseases (Bicker et al.2013; Nguyen H et al . 2016) . In these diseases antibodies appear against the citrullinated proteins, as well as a high deposit of immune complexes especially affecting the renal apparatus and the joints with severely compromised function of the organ.

Unlike the previous diseases, prior to the present invention no citrullination studies had been performed during viral infections, therefore it was not known whether the viruses were also able to stimulate the citrullination of viral or cellular proteins and which role these citrullinated proteins could perform in the pathogenesis of the viral infection.

The sequence of the PAD2 protein corresponds to the sequence SEQ ID NO: 2. The nucleotide sequence of PAD2 corresponds to the sequence SEQ ID NO:l.

Preferably, the viruses of the Herpesviridae family are Cytomegalovirus, Herpes simplex Virus 1 or Herpes simplex Virus 2.

According to the present invention, an inhibitor or antagonist of PAD2 is also used as an antiviral agent against viruses of the Herpesviridae family.

The inhibitor or antagonist is preferably selected from the group comprising Cl-amidine ( C16H20C I F3N4O4 , trifluoroacetate) , BB-Cl-amidine ( C26H26C IN5O ) , F-amidine ( C14H19 FN4O2 · C F3COOH , trifluoroacetate) , streptonigrin (C25H₂2N₄08) , YW3-56 ( C27H33C IN5O2 ) , TDFA ( Thr-Asp-F-amidine ) , TDCA ( Thr-Asp-Cl-amidine ) and derivatives thereof.

Even more preferably, the inhibitor or antagonist is Cl-amidine.

In an alternative embodiment, the inhibitor or antagonist is preferably selected from the group comprising siRNA (short/small interfering RNA) specific for PAD2, miRNA (microRNA) specific for PAD2 and LNA (locked nucleic acid) specific for PAD2, dominant negative DNA specific for PAD2.

Even more preferably, the inhibitor or antagonist is a siRNA specific for PAD2.

According to the present invention, a pharmaceutical composition comprising at least one inhibitor or antagonist of PAD2 and a pharmaceutical excipient is also used as an antiviral agent against viruses of the Herpesviridae family.

The pharmaceutical composition preferably comprises a further viral agent.

The following examples demonstrate that the replication of Herpesviruses depends on the citrullination of viral proteins and bona fide of cellular proteins, and that it is possible to inhibit the replication of Herpesviruses and, therefore, the progression of the infection inhibiting the activation of the PAD2 enzyme.

In particular, the following examples demonstrate that:

- during in vitro infection from human cytomegalovirus (HCMV) the citrullination of viral and cellular proteins is observed through the activation of the PAD2 enzyme;

- the treatment of cells infected by HCMV with Cl- amidine, a commercial pharmacological inhibitor of PADs, immediately before and during the infection significantly inhibits the replication of the virus;

the expression of PAD2 is necessary for the replication of HCMV;

- the treatment of cells infected by Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) with Cl-amidine immediately before and during the infection significantly inhibits the replication of the virus.

Further experiments, not described in detail, have shown that :

- the HIV virus, a lentivirus, is not sensitive to the antiviral action of inhibitors of PAD2 ;

- with other commercial pharmacological inhibitors of PADs (e.g. BB-C1 amidine) results are obtained which are similar to those with Cl-amidine on the inhibition of the replication of HCMV, HSV-1 and HSV-2.

Thanks to the fact that it has been demonstrated that during in vitro infection from human Cytomegalovirus (HCMV) the citrullination of viral and cellular proteins has been observed through the activation of the PAD2 enzyme, it is possible to use the PAD2 enzyme for diagnosing infections caused by viruses of the Herpesviridae family, in particular HCMV.

Thanks to the fact that it has been demonstrated that PAD2 is necessary for the replication of HCMV and that inhibitors of PAD2 inhibit the replication of Herpesviruses, it is possible to use the PAD2 enzyme for preventing and/or treating infections caused by viruses of the Herpesviridae family .

Examples

Example 1

Cells infected by human cytomegalovirus (HCMV) have an increase in citrullinated proteins.

To assess the citrullination profile induced by infection with human Cytomegalovirus (HCMV) , primary Human Foreskin Fibroblasts (HFF) , isolated from foreskin and cultivated in DMEM (Dulbecco's Modified Eagle Medium) in the presence of 10% Fetal Bovine Serum (FBS) ( SIGMA-ALDRICH) , 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 g/ml streptomycin sulphate (SIGMA-ALDRICH) were used as a study model. The cells were infected with HCMV, strain Merlin, at a multiplicity of infection (MOI) of 1 and collected at different times from the infection (24, 48, 72, 96 hours), or non-infected (mock) as a negative control. The cells were lysed with a denaturing lysis buffer (1% SDS, 5 mM EDTA, SIGMA-ALDRICH) and subsequently quantified with the Lowry method (BIO-RAD Laboratories) . The citrullination of the proteins was analysed through a technique based on the use of the rhodamine-phenylglyoxal probe (Rh-PG, CAYMAN CHEMICAL) which chemoselectively binds the citrulline in acidic pH conditions.

In detail, a reaction mixture was prepared containing: protein extract (10 μg) , 10 μΐ tricarboxylic acid (20% final TCA, SIGMA-ALDRICH), Rh-PG (0.1 mM), PBS (SIGMA-ALDRICH), in a final volume of 30 μΐ . The samples were then incubated for an hour at 37 °C. Subsequently, 3 μΐ of Quenching solution (L-citrulline 100 mM, CAYMAN CHEMICAL) was added and the samples were incubated for 30 minutes in ice. Subsequently, the samples were centrifuged at 13000 rpm for 15 minutes at 4°C and the protein pellet thus obtained was washed with 100% glacial acetone. After washing, the pellet was re- suspended in a 0.2 M (20 μΐ) NaOH solution to which 20 μΐ of Laemmli sample buffer 2X (100 mM Tris-Cl pH 6.8, 4% SDS, 20% glycerol, 4% β-mercaptoethanol ) was added. The mixture thus obtained was heated to 95°C for 5 minutes and 20 μΐ of each sample was separated through polyacrylamide gel electrophoresis (8%) - SDS page. At the end of the run, the gel was analysed using the ChemiDoc (BIO-RAD LABORATORIES) instrument with 532 nm excitation and 580 nm emission to highlight the presence of citrullinated proteins. The results obtained (Figure 1), demonstrate that the infected cells have a different citrullinated protein profile from the mock sample.

Example 2

Expression of the enzymes of the PAD family in cells infected or not infected by HCMV.

To assess the level of transcription (mRNA) of PADs in infected HFF cells (HCMV strain Merlin) or not infected (mock) a RT-qPCR - real-time quantitative reverse transcription-PCR - analysis was performed. The analysis was performed on mock cells or on cells infected with the HCMV virus at MOI 1, 24 hours from infection. The total RNA was extracted from the cells using the NucleoSpin RNA Kit (MACHEREY-NAGEL) . Subsequently 1 \iq of RNA was reverse transcribed using the Revert Aid™ H Minus First Strand cDNA Synthesis Kit ( THERMO-FISHER SCIENTIFIC) . In detail, the sample was placed at 25°C for 5 minutes then 42°C for 60 minutes and finally 70°C for 5 minutes in a PCR Reaction Buffer (250 mM Tris-HCl pH 8.3, 250 mM KC1, 200 mM MgC12, 500 mM DTT) mix containing 1 μΐ random hexamer primer, 0.5 mM dNTP, 20 U/μΙ Ribolock RNase Inhibitor and 200 U/μΙ of RevertAid H Minus M-MuLV Reverse Transcritase in a final volume of 20 μΐ . The RT-PCR real time analysis was performed using the Mx3000P (STRATAGENE) instrument using SYBR Green (THERMO-FISHER SCIENTIFIC) as a non-specific fluorescence marker. Primers used: PAD1 (Fw: TCCAGAGACCCTGAAGCTGT (SEQ ID NO:3); Rev: GTGCAGCTGTCCCTGAAGAT (SEQ ID NO : 4 ) ) ; PAD2 (Fw: ACCTCCTCAGCCTCCCC (SEQ ID NO: 5); Rev: CCTACCTCTGGACCGATGTC (SEQ ID NO: 6) ) ; PAD3 (Fw: GCGTCCCATAGACCTCAAAC (SEQ ID NO : 7 ) ; Rev: CAGAGAATCGTGCGTGTGTC (SEQ ID NO : 8 ) ) ; PAD4 (Fw: CCTGTGGATTTCTTCTTGGC (SEQ ID NO: 9)); Rev:

GGGCACCTTGACTCAGCTT (SEQ ID NO: 10)); PAD6 (Fw: CAAGGTATAGGCGTGCTGGT (SEQ ID NO: 11); Rev:

TCCTCCATACCTCCAAGGAA (SEQ ID NO:12)); GAPDH: Fw 5'- AGTGGGTGTCGCTGTTGAAGT-3 ' (SEQ ID NO:13); Rw 5'- AACGTGTCAGTGGTGGACCTG-3 ' (SEQ ID NO:14)) .

In Figure 2, each bar represents the expression value of each PAD normalised on GADPH (ACt) . The results obtained from the RT-qPCR analysis demonstrate how, in human fibroblasts, the two most frequently expressed isoforms of the PAD family are PAD2 and PAD6.

Example 3

Infection from HCMV causes an increase in the enzyme activity of the PAD2 enzyme. To assess the capacity of HCMV to stimulate the enzyme activity of the PAD2 enzyme, an ELISA assay was prepared, based on the capacity of PAD2 to citrullinate the histone H3 substrate. In detail: the HFF cells were infected with HCMV (strain Merlin, MOI=3) or not infected (mock) and treated, where indicated, with 100 μΜ Cl-amidine or an equal volume of solvent, dimethyl sulfoxide (DMSO, SIGMA ALDRICH) , an hour before infection and for the entire duration of the infection. Subsequently, the cells were collected 15, 24, 48 and 72 hours after infection and lysed with a lysis buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 20μ1/πΛ protease inhibitor cocktail -SIGMA) for 30 minutes in ice. The samples were centrifuged subsequently for 30 min at 13000 rpm at 4°C to remove the cellular debris. The proteins with molecular weight less than 30 kDa were removed through the use of concentrators (Pierce™ Protein Concentrators PES, 30K MWCO, 0.5 mL) ; then, for every protein sample, 50 μg of extract, diluted 1:2 in a buffer for deimination without calcium (2 mM Dithiothreitol , 50 mM NaCl, 100 mM Tris-HCl pH 7.5), were incubated for about 20 hours at 37°C in a well where the histone H3 had been previously immobilized. The results illustrated in Figure 3A show how there is a greater activation of the PAD2 enzyme in infected cells than in mock cells and, furthermore, that such activity is drastically reduced following treatment with the inhibitor Cl-amidine. As an internal control, scalar concentrations of recombinant human PAD2 (from 10 to 2.5 mU) , diluted in an equal volume of lysis buffer of the samples and an equal volume of buffer for deimination with calcium (10 mM CaCl2, 2 mM dithiothreitol, 50 mM NaCl, 100 mM Tris-HCl pH 7.5), were incubated in the presence or absence of 100 μΜ Cl- amidine or in the presence of the same volume of solvent, DMSO. The PAD2 activity was calculated through an ELISA (Enzyme-Linked Immunosorbent Assay) , that analyses the capacity of the same enzymes to citrullinate the histone H3, known substrate of PAD2. The absorbance value detected for each sample represents a measurement of the citrullinated histone H3 from which the blank was taken. As can be seen in Figure 3B, each point represents the value obtained from the untreated sample (triangle) , in the presence of Cl-amidine (circle) or DMSO (square), respectively.

Example 4

Cl-amidine inhibits the replication of HCMV.

To assess the activity of Cl-amidine on the replication of HCMV, HFF cells were infected with HCMV (strain TR, MOI=0.1) or not infected (mock) and treated with 100 μΜ Cl- amidine, a commercial pharmacological inhibitor of PADs, or an equal volume of solvent, DMSO, an hour before infection and for the entire duration of the infection. The titration of the virus of the supernatants of the samples treated as previously described was performed 144 hours after infection through plaque assay. Specifically, the supernatant of the samples being examined and the cells were collected and lysed through three nitrogen/37 °C cycles and subsequently centrifuged at 1500 rpm for 10 min to remove the cellular debris. The supernatant thus obtained, containing the viral particles, was analysed through plaque assay, to determine the viral titre. For that purpose, HFF cells were cultivated in plates with 96 wells in 100 μΐ of DMEM (SIGMA) with 10% of FBS, infected with serial dilutions of the virus and centrifuged at 2000 rpm for 30 minutes to promote the adsorption thereof. After 2 hours of incubation at 37°C 5% CO2 the viral inoculum was removed and replaced with 100 μΐ of 0.8% methylcellulose (SIGMA), 1% FBS (SIGMA) . After one week the methylcellulose was removed and replaced with a solution of 0.1% crystal violet (SIGMA) diluted in 10% ethanol (SIGMA) for 30 minutes in the dark. Subsequently, the plate was decolourised with running water and the infectious plaques were counted under the inverted optical microscope (DM IL LED, LEICA) . The viral titre was expressed as plaque-forming units per ml (PFU/ml) . The results are illustrated in Figure 4A.

The number of plaques was expressed as a function of the concentration of Cl-amidine used for the treatment and the IC50 (concentration of Cl-amidine able to reduce the formation of plaques by 50% with respect to the virus not treated with the inhibitor) was calculated. The cell toxicity of the Cl-amidine was assessed through the MTT colorimetric assay (3- (4, 5-dimethylthiazol-2-yl ) -2, 5-diphenyltetrazolium bromide; SIGMA ALDRICH) . For that purpose, the cells were exposed to increasing concentrations of Cl-amidine and after 144 hours of incubation, the supernatant was removed and after washing the well with PBS1X (SIGMA) , the percentage of viable cells was determined by incubating the samples for 2 hours at 37 °C and 5% of C0₂ with DMEM with 500 g/ml of MTT solution added. At the end of the incubation, the supernatant was removed, the cells solubilised with 300 μΐ of DMSO of which 100 μΐ were used to measure the absorbance at the 570 nm wavelength on the Victor3 spectrophotometer multi-plate reader (PERKIN ELMER) . The absorbance value obtained from the untreated sample was considered as 100% viability and the other values were related to it.

Example 5

The silencing of the expression of the PAD2 enzyme in human fibroblasts inhibits the replication of HCMV.

To confirm that the expression of PAD2 was necessary for the replication of HCMV, the HFF cells were electroporated with a specific siRNA for PAD2 or with a control siRNA (OREGENE) and 24 hours after electroporation the cells were infected with HCMV (MOI=0.1) or not infected (mock) . 144 hours after infection, the supernatant of the samples treated as previously described was titrated using the plaque assay method as explained in the previous paragraph (Figure 5) .

Example 6

Cl-amidine inhibits the replication of Herpes Simplex Virus type 1 and 2.

To check whether the activity of the PAD2 enzyme was also necessary for the replication of other viruses belonging to the Herpesviridae family, VERO epithelial cells, isolated from African green monkey kidneys, were infected with Herpes Simplex Virus type 1 and 2 (HSV-1 and HSV-2) at MOI 1 or not infected (mock) and treated with 100 μΜ Cl-amidine, or an equal volume of DMSO, an hour before infection and for the entire duration of the infection. The titration of the virus on the supernatants of the samples treated as previously described was performed 48 hours after infection through plaque assay as illustrated in the paragraph related to Figure 4A (Figure 6A for HSV-1 and Figure 6C for HSV-2) . The IC50 and cell toxicity were calculated according to the methods described in the paragraph related to Figure 4B (Figure 6B for HSV-1 and Figure 6D for HSV-2) .

Claims

1. PAD2 (peptidyl arginine deiminase, type 2) for use in preventing and/or treating infections caused by viruses of the Herpesviridae family.

2. PAD2 for use in diagnosing infections caused by viruses of the Herpesviridae family.

3. PAD2 for use according to claim 1 or 2, wherein the viruses of the Herpesviridae family are cytomegalovirus, herpes simplex virus 1 or herpes simplex virus 2.

4. An inhibitor or antagonist of PAD2 for use as antiviral agent against viruses of the Herpesviridae family.

5. The inhibitor or antagonist for use according to claim 4, selected from the group consisting of Cl-amidine (C16H20CIF3N4O4, trifluoroacetate) , BB-Cl-amidine (C26H26CIN5O) , F-amidine (C14H19FN4O2 · CF3COOH, trifluoroacetate) streptonigrin (C25H22N4O8) , YW3-56 (C27H33CIN5O2) , TDFA (Thr-Asp-F-amidine) , TDCA (Thr-Asp-Cl- amidine) and derivatives thereof.

6. The inhibitor or antagonist for use according to claim 4, selected from the group consisting of siRNA

(short/small interfering RNA) specific for PAD2, miRNA (microRNA) specific for PAD2, LNA (locked nucleic acid) specific for PAD2, dominant negative DNA specific for PAD2.

7. The inhibitor or antagonist for use according to any of claims 4 to 6, wherein the viruses of the Herpesviridae family are cytomegalovirus, herpes simplex virus 1 or herpes simplex virus 2.

8. A pharmaceutical composition comprising at least one inhibitor or antagonist of PAD2 and a pharmaceutical excipient for use as antiviral agent against viruses of the Herpesviridae family.

9. The pharmaceutical composition for use according to claim 8, comprising another viral agent.