Branched peptide for antiviral use

Abstract

The present invention relates to a branched peptide specific for heparan sulphate proteoglycans, for antiviral use.

Description

[0001] BRANCHED PEPTIDE FOR ANTIVIRAL USE

[0002] The present invention relates to a branched peptide for antiviral use. In particular, the invention relates to a branched peptide specific for heparan sulphate proteoglycans, for antiviral use.

[0003] It is known that viral infections represent a problem for human health, due to a combination of characteristics dramatically highlighted by the COVID 19 pandemic, generated by the SARS-COV-2 virus. In particular, viruses are highly infective and in a global world such as today’s they easily generate pandemics that spread very rapidly and are very difficult to control. Furthermore, viruses mutate very easily, thus they can easily escape therapies and vaccines. The experience of COVID 19 has clearly shown that the rate of spread of viral infections requires the availability of ready-to-use broad-spectrum therapies which, even if unable to eliminate the infection, may decrease virulence and the severity of symptoms.

[0004] It is known that many viruses specifically bind heparan sulphate proteoglycans (HSPGs), using them as cellular receptors or coreceptors

[0001] ,

[0005] Heparan sulphate proteoglycans are long-chain, linear conjugated proteins of glycosaminoglycans (GAGs), generally heparan sulphate and less frequently chondroitin sulphate. GAGs can be sulphated in different positions and to a varying extent.

[0006] HSPGs are present on the cell membrane and in the extracellular matrix [2], The HSPGs present on the cell membrane comprise glypicans, whose polypeptide chain is anchored to the phosphatidylinositols of the cell membrane by means of a GPI bond, and syndecans, which instead have a protein core that crosses the cell membrane, with intracellular domains.

[0007] HSPGs can bind various ligands, including growth factors, morphogens, cytokines and matrix proteins, together indicated as “heparin-binding proteins”. The interaction between HSPGs and ligands is mediated essentially by GAG chains, in particular by electrostatic interactions between the sulphate groups of the GAGs and positively charged residues present on the ligands of the HSPGs [3], HSPGs essentially act as coreceptors for their numerous ligands, even though many recent scientific data point to a more autonomous role thereof in various intracellular signalling functions tied to cell proliferation, the organisation of the cytoskeleton, cell migration and the epithelial-mesenchymal transition (EMT). Furthermore, HSPGs are crucial for the regulation of endocytosis and exocytosis, as well as the generation and turnover of extracellular vesicles [4],

[0008] In addition to endogenous ligands, as mentioned above, heparan sulphate proteoglycans are also specifically bound by many viruses, which use HSPGs as cellular receptors or coreceptors

[0001] , Given the role of membrane HSPGs in endocytosis and in the transport of cellular vesicles, viruses could use membrane HSPGs not only to increase the virus concentration present on the cell membrane, but also to facilitate the internalisation of viral particles.

[0009] Among the viruses that use membrane HSPGs as receptors we find Flavivirus, Herpes Virus, Alphavirus, HIV, HBV and also coronaviruses such as SARS-CoV, MERS-CoV and SARS-CoV-2 [5,6], For some of these viruses, binding to HSPGs has been reported in viral isolates, whereas for many others binding to HSPGs has been detected only in strains derived from in vitro cultures. This circumstance indicates that viruses tend to adapt to binding with HSPGs, which is apparently positively selected in their evolution and may have also occurred in the evolutionary adaptation of SARS-Cov-2.

[0010] Antiviral peptides binding HSPGs are known in the prior art. For example, peptides capable of inhibiting infection caused respectively by human cytomegalovirus and papillomavirus by binding heparan sulphate on the cell surface are described in Luganini et al., 2010 [7] and in Donalisio et al., 2010 [8] .

[0011] Patent application WO2012037498 also discloses a group of peptides targeting heparan sulphate and capable of blocking infection by the herpes simplex virus. Finally, United States patent applications US2017267722 and US2005004020 disclose heparin-binding or heparan sulphate-binding peptides having an antiviral activity.

[0012] However, the ability of peptides to bind heparin is not a direct guarantee of their ability to inhibit infection by viruses that use HSPGs as receptors or coreceptors. The tetra-branched M33 peptide, for example, is known, and described in patent application W02006006195 for its antibacterial activity. The M33 peptide comprises the same peptide sequence as described in Luganini et al. 2010 [7] and Donalisio et al. 2010 [8], As shown further below in Example 1 , the tetra-branched M33 peptide was used as a control in the tests on the inhibition of cell infection by SARS-CoV-2. As may be observed from Figure 7, though the tetra-branched M33 peptide is a ‘heparin-binding peptide’, it is totally ineffective in inhibiting viral infection.

[0013] Furthermore, there are also known peptides in an unbranched form which, however, do not offer guarantees of a possible clinical development, since peptides in linear form are extremely unstable in vivo, whereas branched peptides have a much longer average life (9,10).

[0014] As regards the human immunodeficiency virus (HIV), this virus is responsible for acquired immunodeficiency syndrome (AIDS), a condition characterised by T CD4+ cell depletion and consequent susceptibility to opportunistic infections

[0015] , Combination Antiretroviral Therapy (cART), which consists in a combination of three drugs directed against at least two molecular targets, is widely accepted as the most efficient way to control viraemia due to HIV infection

[0016] ,

[0015] However, though it effectively suppresses viral replication, cART therapy may have severe adverse effects, such as anaemia, hepatitis, kidney failure, pancreatitis, and glucose intolerance.

[0016] It is thus necessary to develop new therapeutic options which may replace or complement existing drugs, are possibly capable of inhibiting infection, and may also be used against resistant strains.

[0017] In the light of the foregoing, it appears evident that there is a need to provide new products for treating viral infections, such as SARS-CoV-2 and HIV infections, which overcome the disadvantages of the known products.

[0018] The solution according to the present invention, which aims to provide a branched peptide having antiviral activity, fits into this context. Such activity, according to the present invention, is obtained by acting not on the virus, but rather on the receptors thereof on the target cells, thereby inhibiting infection and viral internalisation. In particular, the peptide according to the present invention is specific for binding to sulphated GAGs of HSPGs and is capable of inhibiting the cellular internalisation of viruses that use HSPGs as receptors or coreceptors. More particularly, according to the present invention, use was made of the branched NT4 peptide, already known and characterised for its antitumour activity [11 ,12,13,14],

[0019] As shown in the experiments reported further below, according to the present invention it was surprisingly found that the branched NT4 peptide is capable of inhibiting the infection of cells by SARS-CoV-2 and HIV.

[0020] In particular, as shown by the results described further below, according to the present invention it was observed that cells infected with the Omicron and Delta variants of SARS-CoV-2 and treated with the NT4 peptide showed a significant reduction in viral infection, particularly in the case of the Omicron variants. As described above, in the same tests use was also made of the tetra-branched M33 peptide, which also binds heparin, but proved to be completely ineffective. Similarly, it was surprisingly observed that the NT4 peptide according to the present invention inhibits HIV infection by inhibiting, in a dose-dependent manner, the binding of NT4 onto MT-2 cells.

[0021] It is therefore a specific object of the present invention a branched peptide molecule, or a pharmaceutical composition comprising said branched peptide molecule together with one or more pharmaceutically acceptable excipients and / or adjuvants, said branched peptide molecule having the following formula wherein R is selected from a) the sequence PyrLYENKPRRPYIL (SEQ ID N0:1 ), c) , preferably b); wherein R1 is selected in the group consisting of

[0022] -CH(CH3)C00H (which, together with NH, forms the amino acid alanine (Ala)), - CH2COOH (which, together with NH, forms the amino acid glycine (Gly)), - CH2CH2COOH (which, together with NH, forms the amino acid beta-alanine (betaAla)), polyethylene glycol (PEG), lipid, preferably -CH2CH2COOH (beta-alanine); and wherein n is a number ranging from 1 to 20, preferably from 2 to 10, more preferably from 3 to 5, even more preferably equal to 4, for use as an antiviral. In the sequence PyrLYENKPRRPYIL (SEQ ID NO:1 ), Pyr stands for pyroglutamic acid, which is bound to leucine (L) located at the N-terminal of the sequence in position 1 .

[0023] The two R groups of formula (I) can be identical to or differ from each other; they are preferably identical to each other. Preferably, in the above-described branched peptide molecule n is equal, i.e. is the same value, both in formula (I) and in the R groups.

[0024] According to one embodiment of the present invention, both R groups are a) SEQ ID NO:1 and n=4. Therefore, in this case, said branched peptide is a dimer having the following formula:

[0025] According to a further embodiment of the present invention, both R groups are formula b) and n=4. Therefore, in this case, said branched peptide is a tetramer having the following formula:

[0026] According to a further embodiment of the present invention, both R groups are formula c) and n=4. Therefore, in this case, said branched peptide is an octamer.

[0027] According to a particularly preferred embodiment of the present invention, the peptide molecule has the above-described formula (III), wherein both R groups are formula b) and n=4, and R1 is -CH2CH2COOH (which, together with NH, forms the amino acid beta-alanine (beta-Ala)).

[0028] According to the present invention, the use as an antiviral can be against a virus that uses heparan sulphate proteoglycans (HSPGs) as cellular receptors or coreceptors to mediate the infection. In particular, according to the present invention, the use as an antiviral agent can be against a virus that uses heparan sulphate proteoglycans (HSPGs) as cellular receptors or coreceptors.

[0029] In particular, according to the present invention, said virus can be selected in the group consisting of Adenovirus, Flavivirus, Herpes Virus, Alphavirus, Papilloma virus, Hepatitis B virus (HBV), Hepatitis Delta virus, Hepatitis C Virus, Coronavirus, Lentivirus.

[0030] More in particular, said Lentivirus can be the HIV virus. Furthermore, according to the present invention, said Coronavirus can be selected from SARS- CoV-2, preferably the SARS-CoV-2 Omega variant, SARS-CoV, and MERS-CoV. According to the present invention, said pharmaceutical composition can further comprise a drug other than the peptide molecule as defined above, for example an antiviral drug.

[0031] The present invention also relates to a combination comprising

[0032] A) a branched peptide molecule, or a pharmaceutical composition comprising said branched peptide molecule together with one or more pharmaceutically acceptable excipients and / or adjuvants, said branched peptide molecule having the following formula wherein R is selected from a) the sequence PyrLYENKPRRPYIL (SEQ ID NO:1 ), b)

[0033] c) , preferably b), wherein R1 is selected in the group consisting of

[0034] -CH(CH3)C00H (which, together with NH, forms the amino acid alanine (Ala)), - CH2COOH (which, together with NH, forms the amino acid glycine (Gly)), - CH2CH2COOH (which, together with NH, forms the amino acid beta-alanine (betaAla)), polyethylene glycol (PEG), lipid, preferably -CH2CH2COOH (beta-alanine), and wherein n is a number ranging from 1 to 20, preferably from 2 to 10, more preferably from 3 to 5, even more preferably equal to 4; and

[0035] B) a drug other than A), for example an antiviral drug, for separate or sequential use as an antiviral.

[0036] As mentioned above, the two R groups of formula (I) can be identical to or different from each other; they are preferably identical to each other. Furthermore, as mentioned above, in the above-described branched peptide molecule n is equal, i.e. is the same value, both in formula (I) and in the R groups.

[0037] According to one embodiment of the combination of the present invention, in A) both R groups are SEQ ID NO:1 and n=4. Therefore, in this case, said branched peptide is a dimer having the above-described formula (II).

[0038] According to a further embodiment of the combination of the present invention, in A) both R groups are formula b) and n=4. Therefore, in this case, said branched peptide is a tetramer having the above-described formula (III).

[0039] According to a further embodiment of the combination of the present invention, in A) both R groups are formula c) and n=4. Therefore, in this case, said branched peptide is an octamer.

[0040] Furthermore, according to the combination of the present invention, the use as an antiviral can be against a virus that uses heparan sulphate proteoglycans (HSPGs) as cellular receptors or coreceptors to mediate the infection. In particular, according to the present invention, the use as an antiviral agent can be against a virus that uses heparan sulphate proteoglycans (HSPGs) as cellular receptors or coreceptors to increase the viral concentration present on the cell membrane and / or to facilitate the internalisation of the viral particles within the cell.

[0041] According to the invention, said virus can be selected in the group consisting of Adenovirus, Flavivirus, Herpes Virus, Alphavirus, Papilloma virus, Hepatitis B virus (HBV), Hepatitis Delta virus, Hepatitis C Virus, Coronavirus, and Lentivirus. More particularly, said Lentivirus can be the HIV virus. Furthermore, according to the present invention, said Coronavirus can be selected from SARS-CoV-2, preferably the SARS-CoV-2 Omega variant, SARS-CoV, and MERS-CoV.

[0042] The present invention further relates to a branched peptide molecule, or a pharmaceutical composition comprising said branched peptide molecule together with one or more pharmaceutically acceptable excipients and / or adjuvants, said branched peptide molecule having the following formula wherein R is selected from a) the sequence PyrLYENKPRRPYIL (SEQ ID NO:1 ), wherein R1 is selected in the group consisting of

[0043] -CH(CH3)COOH (which, together with NH, forms the amino acid alanine (Ala)), - CH2COOH (which, together with NH, forms the amino acid glycine (Gly)), - CH2CH2COOH (which, together with NH, forms the amino acid beta-alanine (betaAla)), polyethylene glycol (PEG), lipid, preferably -CH2CH2COOH (beta-alanine); wherein n is a number ranging from 1 to 20, preferably from 2 to 10, more preferably from 3 to 5, even more preferably equal to 4; and wherein, when both R groups are b), n is other than 4 and / or R1 is other than -CH2CH2COOH (which, together with NH, forms the amino acid beta-alanine).

[0044] According to one embodiment of the present invention, R is selected from a) and c).

[0045] The two R groups of formula (I) can be identical to or different from each other; they are preferably identical to each other.

[0046] Preferably, in the above-described branched peptide molecule n is equal, i.e. is the same value, both in formula (I) and in the R groups.

[0047] According to one embodiment of the molecule according to the present invention, both R groups are a) SEQ ID NO:1 and n=4. Therefore, in this case, said branched peptide is a dimer having the above-described formula (II).

[0048] According to a further embodiment of the molecule according to the present invention, both R groups are formula c) and n=4. Therefore, in this case, said branched peptide is an octamer.

[0049] According to the present invention, the aforesaid pharmaceutical composition can further comprise a drug other than the peptide molecule as defined above, for example an antiviral drug.

[0050] The present invention also relates to a kit of parts comprising

[0051] A) a branched peptide molecule, or a pharmaceutical composition comprising said branched peptide molecule together with one or more pharmaceutically acceptable excipients and / or adjuvants, said branched peptide molecule having the following formula wherein R is selected from a) the sequence PyrLYENKPRRPYIL (SEQ ID NO:1 ), b) c) , preferably b), wherein R1 is selected in the group consisting of

[0052] -CH(CH3)COOH (which, together with NH, forms the amino acid alanine (Ala)), - CH2COOH (which, together with NH, forms the amino acid glycine (Gly)), - CH2CH2COOH (which, together with NH, forms the amino acid beta-alanine (betaAla), polyethylene glycol (PEG), lipid, preferably -CH2CH2COOH (beta-alanine), wherein n is a number ranging from 1 to 20, preferably from 2 to 10, more preferably from 3 to 5, even more preferably equal to 4; and wherein, when both R groups are b), n is other than 4 and / or R1 is other than -CH2CH2COOH (which, together with NH, forms the amino acid beta-alanine); and

[0053] B) is a drug other than A), for example an antiviral drug.

[0054] According to one embodiment of the kit of the present invention, R is selected from a) and c).

[0055] The two R groups of formula (I) can be identical to or different from each other; they are preferably identical to each other.

[0056] Preferably, in the above-described branched peptide molecule, n is equal, i.e. is the same value, both in formula (I) and in the R groups.

[0057] According to one embodiment of the kit according to the invention, both R groups are a) SEQ ID NO:1 and n=4. Therefore, in this case, said branched peptide is a dimer having the above-described formula (II).

[0058] According to a further embodiment of the kit according to the invention, both R groups are formula c) and n=4. Therefore, in this case, said branched peptide is an octamer.

[0059] The present invention will now be described, by an illustrative but non-limiting way, according to a preferred embodiment thereof, with particular reference to the examples and the figures in the appended drawings, wherein:

[0060] - Figure 1 shows the binding of the NT4 peptide to human Caco-2 cells, analysed by means of a cytofluorometry assay. A-B) Binding of NT4, C-D) inhibition of the binding of NT4 by heparin, E-F) binding of NT4 onto Caco-2 cells treated with heparinase I. *** p<0.001 , ** p<0.01 calculated using one-way ANOVA with Dunnett’s multiple comparisons test versus the control (D), calculated using two- tailed unpaired t-tests (F) with GraphPad PRISM software version 10.

[0061] - Figure 2 shows the cytotoxicity assay of the NT4 peptide on Caco-2 cells.

[0062] - Figure 3 shows the antiviral activity of the NT4 peptide against the Omicron BA.1 and BA.5 strains and against the Delta strain of SARS-CoV-2 in the Caco-2 cell line. The logarithm of the micromolar peptide concentration is indicated on the x-axis. The horizontal dashed line indicates the IC50 concentration of NT4 peptide corresponding to 50% of the viral replication calculated using GraphPad PRISM software version 10 (La Jolla, CA, USA).

[0063] - Figure 4 shows the antiviral activity of the NT4 peptide on the BA.5 strain of SARS-CoV-2 in the Caco-2 cell line analysed by flow cytofluorometry. A) Cytograms of uninfected, untreated cells (control), cells infected with the virus (BA.5), infected cells treated with NT4 (BA.5 + NT4) and infected cells treated with serum (BA.5 + serum); B) Histograms relating to the quantisation of the infection by the virus. ** p<0.01 , * p<0.05 calculated using one-way ANOVA with Dunnett’s multiple comparisons test versus the control using GraphPad PRISM software version 10.

[0064] - Figure 5 shows the binding of the tetrabranched NT4 peptide at a concentration of 1 pM (black peak) onto the different cell lines MT-2, MT-4, PM-1 and H9 assessed by cytofluorometry (A). Inhibition of the binding of NT4 to MT-2 cells by heparin, assessed by cytofluorimetric analysis (B).

[0065] - Figure 6 shows the assay of the antiviral activity of the NT4 peptide on MT- 2 cells infected with MOI 0.1 of HIV virus. The dash-dotted line on the right indicates the cytotoxicity of the NT4 peptide, again on MT-2 cells.

[0066] - Figure 7 shows a comparison between the M33 peptide (A) having sequence KKIRVRLSA (SEQ ID NO:2) and the NT4 peptide (B) having sequence PyrLYENKPRRPYIL (SEQ ID NO:1 ) for the binding to heparin and their antiviral activity against SARS-CoV-2 and HIV.

[0067] EXAMPLE 1 : Study on the effects of the NT4 peptide (in the tetramer form) according to the present invention against SARS-CoV-2 and HIV viruses.

[0068] Materials and methods

[0069] Peptide synthesis

[0070] The synthesis of the NT4 peptide was carried out using Fmoc chemistry as described by Brunetti J. et al., Sci. Rep. 2016. Once synthesised, the NT4 peptide was purified by HPLC and checked by MALDI spectrometry.

[0071] Cell cultures

[0072] The cell lines used were Caco-2, PM-1 , H9, MT-2 and MT-4. They were purchased from ATCC and their genetic profile was analysed in order to confirm their authenticity (BMR Genomics).

[0073] The cell cultures were grown in the recommended culture medium supplemented with 10% foetal bovine serum (FBS), glutamine (200 pg / ml), streptomycin (100 pg / ml) and penicillin (60 pg / ml). The cell lines were maintained at 37°C with a concentration of CO2 equal to 5%. The products used for the cell cultures were sold by the company Euroclone.

[0074] Cytotoxicity

[0075] Caco-2 and MT-2 cells were plated at a density of 2.5 x 104cells per well, in 96-well microplates. 24 h after plating, different molar concentrations of NT4 peptide (from 300 pM to 0.2 pM) were added and the cells were allowed to grow for 24h in an incubator. Cell viability was evaluated using the CellTiter-Glo kit, by measuring the ATP produced.

[0076] Cytofluorometry

[0077] All experiments were conducted using 2 x 105cells in 96-well Il-bottom plates. All dilutions were carried out in phosphate buffer (PBS) with the addition of 5 mM EDTA and 1 % BSA.

[0078] In order to evaluate the binding of NT4 peptide, the cells were incubated for 30 minutes with different molar concentrations of NT4 peptide conjugated to biotin, in the presence or absence of heparin, and subsequently with streptavidin-FITC.

[0079] For the treatment with heparinase, the cells were incubated for 1 h at 37°C with 0.03IU / ml of heparinase I (Sigma Aldrich) before staining with the NT4 peptide. 10000 events were analysed using Guava FACS (Millipore) and FCS Express 6 flow cytometry software.

[0080] Antiviral activity

[0081] The model of viral infection was constructed with the SARS-CoV-2 Omicron BA.1 , Omicron BA.5 and Delta strains (AOIIS, Le Scotte, Siena) in the Caco-2 intestinal cell line with 1 hour of incubation. The virus was removed, fresh medium was added, and the cells were incubated for 24h. The supernatant containing the virus was transferred to a highly permissive reporter cell line, VERO E6 (monkey kidney cells) (ATCC).

[0082] The TCID50 dose was determined after 24 hours by measuring the expression of the virus nucleocapsid by means of an ELISA assay. The experiments were carried out in quadruplicate, with and without treatment with heparinase.

[0083] In order to determine the antiviral activity, serial dilutions of NT4 peptide, starting from a nontoxic dose, were incubated with a fixed amount of the BA.5, BA.1 and Delta strains (MOI=0.01 ) for 1 hour at 37°C on Caco-2 cells, previously seeded. After incubation, the virus-peptide mixture was removed, fresh medium containing NT4 peptide was added, and the cells were allowed to grow for 24 hours. The supernatant was transferred to the VERO E6 cell line and incubated for 24 hours.

[0084] The IC50 of the NT4 peptide was determined by measuring the expression of the virus nucleocapsid by means of an ELISA assay.

[0085] In order to evaluate the antiviral activity of the NT4 peptide against the HIV virus, different dilutions of NT4 were incubated with a fixed concentration of HIV virus (MOI 0.1 ) for 72 hrs on MT-2 cells.

[0086] Subsequently, 50pl of supernatant was used to infect PZNBL reporter cells for 48 hrs. The activity of luciferase, proportional to the amount of virus present, was calculated using the Bright-GLO kit.

[0087] Results

[0088] SARS-CoV-2

[0089] Binding of NT4 peptide onto Caco-2 cells

[0090] The Caco-2 human colon carcinoma epithelial cell line, permissive to SARS- CoV-2 infection

[0017] , was used in this study to determine: i) the binding of the NT4 peptide onto cells; ii) the level of cytotoxicity of NT4 in the same cells; iii) the antiviral activity using NT4 on the SARS-CoV-2 virus.

[0091] The ability of the NT4 peptide to bind Caco-2 cells (Figures 1 A and 1 B) and its inhibition by heparin (Figure 1 C and 1 D) was evaluated by means of cytofluorometry assays. As may be seen in the figure, the NT4 peptide binds the Caco-2 cells in a dose-dependent manner and the binding thereof is inhibited by heparin.

[0092] For the purpose of confirming the specificity of the binding of the NT4 peptide to heparan sulphate proteoglycans (HSPGs), the binding of NT4 was tested on Caco-2 cells treated with a mixture of heparinase I which removes the heparan sulphate chains from proteoglycans (Figures 1 E and 1 F). The binding of NT4 to cells treated with heparinase proved to be much lower compared to the control cells.

[0093] Cytotoxicity of NT4 peptide in Caco-2 cells

[0094] The cytotoxicity of the NT4 peptide was evaluated by means of an in vitro cytotoxicity assay on Caco-2 cells (Figure 2). Progressive molar concentrations of the NT4 peptide (from 300 pM to 0.2 pM) were incubated with Caco-2 cells for 24h.

[0095] The NT4 peptide, also at high concentrations, showed no effect on cell viability.

[0096] Antiviral activity of NT4 peptide on Caco-2 cells infected with different strains of the SARS-CoV-2 virus.

[0097] The antiviral activity of the NT4 peptide was evaluated in the Caco-2 cell line infected with the Omicron BA.1 , Omicron BA.5 and Delta strains of SARS-CoV-2. Semi-confluent Caco-2 cells were infected with the different strains of SARS-CoV-2 at 0.01 multiplicity of infection (MOI) in the presence of progressive concentrations of NT4 peptide, starting from a nontoxic dose of 100 pM (Figure 3) for 1 h. After incubation, the virus-peptide mixture was removed, medium with NT4 was added, and the cells were allowed to grow for 24 hours. The supernatant was then transferred to the VERO E6 cell line and incubated for 24 hours. The antiviral activity of NT4 was measured by evaluating the expression of the virus N protein in the cell monolayer using a specific antibody and expressed as IC50.

[0098] NT4 is capable of inhibiting the infection of Caco-2 cells by the Omicron BA.1 and BA.5 strains, with an IC50 of 21.5±2.8 and 19.5±1 .7 pM, respectively, whereas it shows a lesser antiviral effect against the Delta variant of SARS-CoV-2, producing a statistically significant reduction of about 10% (Figure 3). This result indicates a crucial role of HSPGs, present on the membrane of the target cells, in the infection of cells by the Omicron variants. The infection of Caco-2 cells by the Delta variant, however, seems to be less dependent on the membrane HSPGs. The determinant role played by the membrane HSPGs of target cells in the infection by Omicron variants was confirmed by experiments conducted on cells which were treated with heparinase in order to remove the heparan sulphate chains from the HSPGs of the cell membrane. Caco-2 cells treated with heparinase did not show to be more permissive to infection by Omicron, whereas a modest reduction (4.6 times) was measured for the Delta variant, as shown in Table 1. In particular, Table 1 shows the antiviral activity of the NT4 peptide against the Omicron BA.1 strain and against the Delta strain of SARS-CoV-2, as measured in Caco-2 cells treated with heparinase, compared to the same cells, not treated.

[0099] Table 1

[0100] The antiviral activity of the NT4 peptide was also investigated by cytofluorometry.

[0101] Caco-2 cells infected with the Omicron BA.5 strain of SARS-CoV-2 at an MOI of 0.05 in the presence of the NT4 peptide at a concentration of 40 and 20 pM were analysed by cytofluorometry; the presence of the virus N protein was detected using a specific commercially available antibody (Figure 4). For the purpose of evaluating the system’s sensitivity and the significance of the results in terms of the inhibition of cell infection obtained with the NT4 peptide, these results were compared with those regarding the inhibition of cell infection produced by a neutralising human serum specific for the same viral variant, under the same experimental conditions.

[0102] As may be observed in figure 4, cells infected with the Omicron BA.5 variant of SARS-CoV-2 and treated with the NT4 peptide show a significant dosedependent reduction in the fluorescence signal due to the presence of the virus.

[0103] HIV

[0104] Binding of the NT4 peptide

[0105] The binding of the NT4 peptide was tested by cytofluorometry on the cell lines PM-1 , H9, MT-2 and MT-4, which express the coreceptor CCR5 or CXCR-4 (Figure 5A).

[0106] NT4 shows to be capable of binding all the cells tested, both the cells which express the coreceptor CCR5 and those with CXCR-4.

[0107] In the MT-2 cells, used in subsequent assays to evaluate antiviral activity, the ability of heparin to inhibit the binding of the NT4 peptide to cells was assessed by cytofluorometry (Figure 5B). As may be observed in the figure, heparin inhibits the binding of NT4 to MT-2 cells in a dose-dependent manner.

[0108] Antiviral activity of NT4 peptide in MT-2 cells infected with the HIV virus

[0109] In order to succeed in evaluating antiviral activity, different dilutions of the tetra-branched peptide NT4 were incubated with a fixed concentration of the HIV virus (MOI 0.1 ) for 72 hrs in MT-2 cells.

[0110] The maximum concentration used was about 10 times lower than the TD50 (toxic dose 50) previously calculated for the same MT-2 cell line (Figure 6, dash- dotted curve on the right). Subsequently, 50pl of supernatant was used to infect PZNBL reporter cells for 48hrs. The activity of luciferase, proportional to the amount of virus present, was calculated using the Bright-GLO kit.

[0111] The NT4 peptide inhibits HIV infection with an IC50 of 0.9 pM.

[0112] References

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Claims

CLAIMS1 ) Branched peptide molecule, or pharmaceutical composition comprising said branched peptide molecule together with one or more excipients and / or adjuvants, said branched peptide molecule having the following formulawherein R is selected from a) the sequence PyrLYENKPRRPYIL (SEQ ID NO: 1 ),, preferably b); wherein R1 is selected in the group consisting of-CH(CH3)COOH, -CH2COOH,-CH2CH2COOH, polyethylene glycol (PEG), lipid, preferably -CH2CH2COOH;and wherein n is a number ranging from 1 to 20, preferably from 2 to 10, more preferably from 3 to 5, for use as an antiviral.2) Peptide molecule or pharmaceutical composition according to claim 1 , for use according to claim 1 , wherein both R groups are a) SEQ ID NO:1 and n=4.3) Peptide molecule or pharmaceutical composition according to claim 1 , for use according to claim 1 , wherein both R groups are formula b) and n=4.4) Peptide molecule or pharmaceutical composition according to claim 1 , for use according to claim 1 , wherein both R groups are formula c) and n=4.5) Peptide molecule or pharmaceutical composition according to any one of the preceding claims, for use according to any one of the preceding claims, wherein the use as an antiviral is against a virus that uses heparan sulphate proteoglycans (HSPGs) as cellular receptors or coreceptors to mediate the infection.6) Peptide molecule or pharmaceutical composition according to claim 5, for use according to claim 5, wherein said virus is selected in the group consisting of Adenovirus, Flavivirus, Herpes Virus, Alphavirus, Papilloma virus, Hepatitis B virus (HBV), Hepatitis Delta virus, Hepatitis C Virus, Coronavirus, Lentivirus.7) Peptide molecule or pharmaceutical composition according to claim 6, for use according to claim 6, wherein said Lentivirus is the HIV virus.8) Peptide molecule or pharmaceutical composition according to claim 6, for use according to claim 6, wherein said Coronavirus is selected from SARS-CoV-2, preferably the SARS-CoV-2 Omega variant, SARS-CoV, and MERS-CoV.9) Pharmaceutical composition according to any one of the preceding claims, for use according to any one of the preceding claims, said pharmaceutical composition further comprising a drug other than the peptide molecule as defined in claims 1 -8, for example an antiviral drug.10) Combination comprisingA) a branched peptide molecule, or pharmaceutical composition comprising said branched peptide molecule together with one or more excipients and / or adjuvants, said branched peptide molecule having the following formulawherein R is selected from a) the sequence PyrLYENKPRRPYIL (SEQ ID NO:1 ),, preferably b), wherein R1 is selected in the group consisting of -CH(CH3)COOH, -CH2COOH, -CH2CH2COOH, polyethylene glycol (PEG), lipid, preferably -CH2CH2COOH, and wherein n is a number ranging from 1 to 20, preferably from 2 to 10, more preferably from 3 to 5; andB) a drug other than A), for example an antiviral drug, for separate or sequential use as an antiviral.11 ) Combination according to claim 10, for use according to claim 10, wherein both R groups are SEQ ID NO:1 and n=4. 12) Combination according to claim 10, for use according to claim 10, whereinboth R groups are formula b) and n=4.13) Combination according to claim 10, for use according to claim 10, wherein both R groups are formula c) and n=4.14) Combination according to any one of claims 10-13, for use according to any one of claims 10-13, wherein the use as an antiviral is against a virus that uses heparan sulphate proteoglycans (HSPGs) as cellular receptors or coreceptors to mediate the infection.15) Combination according to claim 14, for use according to claim 14, wherein said virus is selected in the group consisting of Adenovirus, Flavivirus, Herpes Virus, Alphavirus, Papilloma virus, Hepatitis B virus (HBV), Hepatitis Delta virus, Hepatitis C Virus, Coronavirus, and Lentivirus.16) Combination according to claim 15, for use according to claim 15, wherein said Lentivirus is the HIV virus.17) Combination according to claim 15, for use according to claim 15, wherein said Coronavirus is selected from SARS-CoV-2, preferably the SARS-CoV-2 Omega variant, SARS-CoV, and MERS-CoV.18) Branched peptide molecule, or pharmaceutical composition comprising said branched peptide molecule together with one or more excipients and / or adjuvants, said branched peptide molecule having the following formulawherein R is selected from a) the sequence PyrLYENKPRRPYIL (SEQ ID NO:1 ),wherein R1 is selected in the group consisting of -CH(CH3)COOH, -CH2COOH, -CH2CH2COOH, polyethylene glycol (PEG), lipid, preferably - CH2CH2COOH; wherein n is a number ranging from 1 to 20, preferably from 2 to 10, more preferably from 3 to 5; and wherein, when both R groups are b), n is other than 4 and / or R1 is different from -CH2CH2COOH.19) Peptide molecule or pharmaceutical composition according to claim 18, wherein both R groups are a) SEQ ID NO:1 and n=4.20) Peptide molecule or pharmaceutical composition according to claim 18, wherein both R groups are formula c) and n=4.21 ) Pharmaceutical composition according to any one of claims 18-20, said pharmaceutical composition further comprising a drug other than the peptide molecule as defined in claims 18-20, for example an antiviral drug.22) Kit of parts comprisingA) a branched peptide molecule, or pharmaceutical composition comprising said branched peptide molecule together with one or more excipients and / or adjuvants, said branched peptide molecule having the following formulawherein R is selected from a) the sequence PyrLYENKPRRPYIL (SEQ ID N0:1 ),wherein R1 is selected in the group consisting of- CH(CH3)COOH, -CH2COOH,-CH2CH2COOH, polyethylene glycol (PEG), lipid, preferably -CH2CH2COOH, wherein n is a number ranging from 1 to 20, preferably from 2 to 10, more preferably from 3 to 5, and wherein, when both R groups are b), n is other than 4 and / or R1 is different from -CH2CH2COOH; andB) a drug other than A), for example an antiviral drug.23) Kit according to claim 22, wherein both R groups are a) SEQ ID NO:1and n 4.24) Kit according to claim 22, wherein both R groups are formula c) and n=4.

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