WO2016115616A1

WO2016115616A1 - Inhibition of cellular proteases as treatment for influenza

Info

Publication number: WO2016115616A1
Application number: PCT/CA2015/050038
Authority: WO
Inventors: Darwyn Kobasa; Mable HAGAN
Original assignee: Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health
Priority date: 2015-01-21
Filing date: 2015-01-21
Publication date: 2016-07-28

Abstract

Described herein is a method of treating an influenza virus infection, wherein an effective amount of a MASP1 (mannan-binding lectin serine protease 1) inhibitor or a PRSS33 (protease, serine, 33) inhibitor is administered to an individual in need of such treatment.

Description

INHIBITION OF CELLULAR PROTEASES AS TREATMENT FOR INFLUENZA

BACKGROUND OF THE INVENTION

Influenza can infect as much as 5-15% of the world population, resulting in 3-5 million cases of severe illness and up to 500,000 deaths per year. In the US alone, flu epidemics lead to approximately 300,000 influenza-related hospital admissions and 36,000 influenza related deaths annually in addition to an estimated cost of $12 billion per year. Current seasonal influenza vaccines are produced with strains recommended by the World Health Organization about 9-12 months ahead of the targeted season. The vaccines typically contain two type A influenza strains and one type B influenza strain, which are predicted to be the most likely strains to cause the upcoming flu epidemic.

The influenza virus is a member of the Orthomyxovihdae family and has an enveloped, ssRNA genome comprised of 8 segments which encode up to 1 proteins including the polymerases (PB1 , PB2, PA), HA (subtypes 1-18), NA (subtypes 1-1 1), NP, M1 and M2, and NS1 and NS2. Human viruses are characterized by their preferential binding of a-2,6 linked sialic acids in the airway.

However, there are inherent disadvantages associated with the preparation of conventional influenza vaccines such as the uncertainty of the actual circulating strain, the need for annual updating of the manufacturing process and preparation of reagents for vaccine lot release. Furthermore, mismatches between the strains selected for vaccine preparation and the circulating viruses were found to be responsible for much reduced efficacy of the seasonal influenza vaccines. Clearly, the drawbacks associated with traditional vaccine preparation would be dramatically exacerbated in the event of an outbreak of pandemic influenza, given a much shortened timeframe available for the production of prophylactic vaccines for global needs. All these problems concerning the influenza vaccines are largely due to one single biological property of the influenza virus itself: the constant mutations of the virus surface proteins hemagglutinin (HA) and neuraminidase (NA). HA is a lectin that mediates binding of the virus to target cells and entry of the viral genome into the target cell, while NA is involved in the release of progeny virus from infected cells.

SUMMARY OF THE INVENTION

According to an aspect of the invention, there is provided a method of treating an influenza virus infection comprising administering to an individual in need of such treatment an effective amount of a MASP1 (mannan-binding lectin serine protease 1) inhibitor or a PRSS33 (protease, serine, 33) inhibitor.

According to another aspect of the invention, there is provided use of a MASP1 (mannan-binding lectin serine protease 1) inhibitor, a PRSS33 (protease, serine, 33) inhibitor or a combination thereof to treat an influenza virus infection.

According to another aspect of the invention, there is provided use of a MASP1 (mannan-binding lectin serine protease 1) inhibitor or a PRSS33 (protease, serine, 33) inhibitor in the preparation of a medicament to treat an influenza virus infection.

According to an aspect of the invention, there is provided a method of preparing a medicament for treating an influenza virus infection comprising combining a MASP1 (mannan-binding lectin serine protease 1) inhibitor, a PRSS33 (protease, serine, 33) inhibitor or a combination thereof with a suitable excipient.

According to an aspect of the invention, there is provided a method of identifying an influenza virus antiviral compound comprising determining if a compound of interest is a MASP1 (mannan-binding lectin serine protease 1) inhibitor or a PRSS33 (protease, serine, 33) inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure . Bar graph showing the effect of siRNA-mediated protease knockdown on influenza A virus in Caco-2 cells as measured by plaque assay 48hpi. Proteases were knocked down by transfection with 20 nM of protease specific siRNA or a negative scramble siRNA as a control. Titers of each virus grown on wild-type cells are shown in column (WT). Cells were infected at a MOI of 0.001 in duplicate wells with either A/Canada/RV733/2007 (H1 N1) (RV733) or A/Hong Kong/1/68 (H3N2) (HK68) strains of influenza. Figure 2. Effect of MASP-1 knockdown on influenza virus replication. Stable MASP-1 deficient Caco2 cells (M2414) and Caco2 cells expressing GFP shRNA (GFP) as a negative control were generated. M24 4, GFP and wild-type Caco2 cells were infected with A/Can ad a/RV733/2007 (H1 N1) (RV733), A/Hong Kong/1/68 (H3N2) (HK68), A Singapore/1/57 (H2N2) (Sing/57), A/Anhui/1/20 3 (H7N9) (Anhui), and A/Canada/504/2004 (H7N3) at a MOI of 0.001. Virus titers were determined by plaque assays 48hpi. Results obtained from 3 independent experiments were subjected to unpaired t-test analysis, and significant p values are denoted by (*) on graph, where * = p < 0.05 and *** = p < 0.001.

Figure 3. Growth of a GFP expressing RV733 influenza virus in MASP-1 deficient cells. Wild-type Caco2 cells, Caco2 cells expressing GFP shRNA (GFP), and MASP-1 deficient Caco2 cells (M2414) were infected with RV733-GFP (H1 N1) at a MOI of 0.1 , in the presence or absence of 1 g/ml trypsin. Images were taken at 48hpi; all wild-type Caco2 and GFP cells infected with the addition of trypsin exhibited 100% cytopathic effect.

Figure 4. Effect of trypsin on influenza replication in MASP-1 deficient cells. MASP-1 deficient Caco2 cells (M2414) were infected with either RV733 (H1 N1) or HK68 (H3N2) at a MOI of 0.001 in the presence or absence of 1 g/ml trypsin. Virus titers were determined by plaque assay 48hpi. Results are from 3 independent experiments. Unpaired t-test analysis determined significant p values that are denoted by (*) on graph, where ^* = p < 0.05 and ** = p < 0.01.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. A!l publications mentioned hereunder are incorporated herein by reference.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. . Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell Scientific Publications); T. E. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

Activation of influenza virus hemagglutinin (HA) by host cellular proteases is essential for influenza infection. Recently, members of the type II transmembrane serine proteases (TTSPs) have been shown to cleave influenza HA into HA1 and HA2 in vitro. Furthermore, HA cleavage has been described to occur in different cellular compartments and at various points during the influenza replication cycle, suggesting the involvement of multiple proteases during influenza infection.

As described herein, cellular proteases involved in the crucial HA activation step have been identified. As will be appreciated by one of skill in the art, identifying host proteases that are required for influenza replication provides new targets for therapeutic intervention.

The HA peptide in all human influenza viruses contains a monobasic recognition motif (R/K-X-X-X). However, avian influenza viruses can contain either a monobasic or multibasic recognition motif (R-X-R/K-R), which defines whether the virus has low pathogenicity or high pathogenicity, respectively. The monobasic recognition motif is typically recognized by a number of trypsin-like proteases, including but by no means limited to plasmin, tryptase clara, mini-plasmin, tryptase TC30, TMPRSS2 (transmembrane protease, serine 2), TMPRSS4 (transmembrane protease, serine 4) and HAT (human airway trypsin-like protease). Ali human influenza infections occur mainly in the lung tissues whereas low pathogenic avian influenza infections occur in the respiratory tract and Gl tract of birds. Most of the proteases that recognize monobasic cleavage sites are expressed in the respiratory and GI tracts of birds. Thus, viral replication is restricted to these compartments. In contrast, highly pathogenic avian influenza viruses (subtypes H5 and H7), contain a multibasic recognition motif (R-X-R/K-R) that is recognized by proteases that are found systemical!y in the avian host such as furin and PC5/6. This enables the infection to spread to other tissues, leading to severe disease symptoms. Occasionally, these highly pathogenic avian influenza viruses are able to infect humans and cause significant disease.

Most cell lines used to culture influenza viruses in the laboratory do not express proteases that can proteolytically activate the viral HA and the in vitro culture of most influenza viruses is dependent on the addition of exogenous trypsin for HA activation. To investigate proteases that are important in influenza virus growth, we utilized Caco-2 cells that promote trypsin-independent growth of seasonal (H1 N1 , H3N2) and pandemic (2009 H1N1 , 1918 H1 N1) influenza viruses. Furthermore, a seasonal H1 N1 GFP-expressing influenza virus was generated and used as a tool to screen a human siRNA protease library. Initial screens identified several proteases that play a role in influenza biology, including TMPRSS2 as previously reported by others. Validation of potential proteases by reduction in infectious viral titers and the generation of stably knocked down cell lines revealed novel proteases needed for efficient influenza replication

According to another aspect of the invention, there is provided use of a MASP1 inhibitor, a, a PRSS33 inhibitor or a combination thereof to treat an influenza virus infection.

In some embodiments of the invention, the inhibitor is a MASP-1 inhibitor.

According to another aspect of the invention, there is provided use of a MASP1 inhibitor or a PRSS33 inhibitor in the preparation of a medicament to treat an influenza virus infection. According to an aspect of the invention, there is provided a method of preparing a medicament for treating an influenza virus infection comprising combining a MASP1 inhibitor, a PRSS33 inhibitor or a combination thereof with a suitable excipient.

As will be appreciated by one of skill in the art, the ASP1 inhibitor or the

PRSS33 inhibitor may be formulated for systemic administration or for local administration, for example, administration to the lungs or Gl tract. In some embodiments, the MASP1 inhibitor or PRSS33 inhibitor may be administered orally.

As will be known to those of skill in the art, a number of MASP-1 inhibitors are known in the art, including antiproteases such as C1 -inhibitor, and anti-thrombin in the presence of heparin in addition to the siRNA inhibitors of MASP-1 and PRSS33 which are discussed herein. As will be apparent to one of skill in the art, the generation of other suitable siRNA inhibitors of MASP-1 and PRSS33 are well within the scope of knowledge of one of skill in the art. Methods of identifying other MASP-1 inhibitors and PRSS33 inhibitors are discussed herein and will also be apparent to one of skill in the art.

According to an aspect of the invention, there is provided a method of identifying an influenza virus antiviral compound comprising determining if a compound of interest is a MASP1 inhibitor, a or a PRSS33 inhibitor.

Suitable compounds of interest will be readily apparent to one of skill in the art.

For example, libraries of small molecules, aptamers, siRNAs, antibodies, intrabodies (synthetic cloned antibodies modified for intracellular targeting) and the like may be screened for MASP1 inhibition or PRSS33 inhibition and then subsequently confirmed to have influenza inhibitory activity as discussed herein.

A variety of suitable means for determining inhibition of MASP1 and/or

PRSS33 will be readily apparent to the skilled artisan.

For example, MASP-1 could be incubated with the compound of interest under suitable conditions for MASP-1 protease activity and a fluorogenic substrate of MASP- 1 added (which are available commercially) to the reaction mix. The activity of MASP- 1 in the presence and absence of the compound of interest could then be determined by measuring levels of fluorescence generated by the cleavage of the fluorogenic substrate. As will be apparent to one of skill in the art, the level of fluorescence in the absence of a compound of interest does not necessarily need to be repeated every time and instead a suitable threshold level could be determined which would distinguish compounds of interest as inhibiting or non-inhibiting.

MASP-1 is a thrombin-like peptide that cleaves fibrinogen and Factor VIII. It plays a role in coagulation and in the complement pathway. MASP-1 inhibitors include but are by no means necessarily limited to C1 -inhibitor, α-2-macroglobuiin, anti- thrombin and heparin as well as by suitable siRNAs, as discussed herein.

PRSS33 (protease, serine, 33) is a serine protease that has amidolytic activity and cleaves before arginine residues in its substrates. It is expressed predominately in macrophages, and has been found to be present in many organs including the spleen, small and large intestine, lung and brain. This serine protease may have a role in macrophage related biological and pathological functions (Chen et al. 2003).

As will be appreciated by one of skill in the art, as used herein, "an individual in need of such treatment" is an individual who has been or is suspected of having been infected by an influenza virus. This individual may be exhibiting or suffering from one or more symptoms associated with an influenza virus infection, for example, fever, chills, cough, sore throat, runny nose, congestion, muscle aches, body aches, headache, fatigue, vomiting and/or diarrhea. Accordingly, an effective amount of the compound will result in amelioration and/or reduction in severity of one or more of these symptoms.

As will be appreciated by one of skill in the art, "an effective amount" may depend on several factors, including but by no means limited to age, weight and general condition of the patient or individual as well as the severity of the disease and/or symptoms associated with the disease. As such, a suitable "effective amount" can be readily determined through routine experimentation.

The invention will now be further described by way of examples. However, the examples are intended for illustrative purposes and the invention is not necessarily limited by the examples. A siRNA protease library comprising siRNA from 389 known proteases was screened for its ability to inhibit influenza virus replication and/or viral protein expression.

While most cell lines used to culture influenza viruses lack proteases that can activate HA and therefore require exogenous trypsin to be added, Caco-2 cells are capable of supporting trypsin-independent growth of seasonal and pandemic influenza viruses.

In this example, Caco2 cells were transfected with 20 nM of siRNA from the protease library and 0.25 μΙ of Lipofectamine RNAiMAX™. A portion of the cells were tested for viability 3 days post transfection to confirm that the siRNA was not having non-specific toxicity. 48 hours post transfection, the cells were infected with a modified RV733 (H1 N1) influenza virus engineered to express green fluorescent protein (RV733-GFP) at a multiplicity of infection of 0.01. GFP expression was subsequently measured.

From this initial protease screen, PRSS33, MASP1 and TMPRSS2 were identified as proteases that could significantly reduce influenza A virus replication compared to the control. As TMPRSS2 is well known from published literature to play a role in influenza HA cleavage, this helped confirm the validity of the screening approach used.

As can be seen from Figures 1 and 2, viral replication is reduced for a broad range of viruses tested: RV733 (H1 N1), HK68 (H3N2), Sing/57 (H2N2), H7N3 and Anhui (H7N9).

Based on the preliminary screen with the siRNA, a knockout cell line was generated to further analyze the importance and role of the proteases.

As shown in Figure 2, stably knocked down Caco-2 cells targeting MASP-1

(M2414) and GFP as a negative control were infected at a MOl of 0.001 with RV733, HK68, Sing/57, H7N3 and Anhui (H7N9), and titers determined 48hpi by plaque assay. Results obtained from 3 independent experiments were subjected to unpaired t-test analysis, and significant p values are shown on the graph. As can be seen, plaque forming units were greatly reduced for all viruses in the MASP-1 deficient cells (M2414), with reductions in replication by at least 2-logs.

As shown in Figure 3, replication of a modified RV733 virus to express GFP (RV733-GFP) in ASP-1 deficient cells (M2414) was greatly reduced compared to wild-type Caco2 cells or Caco2 cells expressing GFP shRNA (GFP) as a negative control. In the absence of trypsin, single cell infections for M24 4 and limited spread of the virus was seen compared to Caco-2 and GFP-Caco2 infections where GFP expression was detected in multiple cells. When 1 μg ml of trypsin was added to the infection, all wild-type Caco2 and GFP cells exhibited significant GFP expression and 100% cytopathic effect compared to M2414 cells, where the addition of trypsin did not help restore infectivity of the virus or spread of the virus.

As shown in Figure 4, the ability of trypsin to restore infectivity in the MASP-1 stable knock-down cell line (M2414) was further tested. In this experiment, M2414 cells were infected in the presence or absence of trypsin with either RV733 or HK68 at a MOI of 0.001. For RV733, the addition of trypsin had little effect on restoring infectivity by increasing virus titers by less than 1-log. These results correspond well to the limited effect trypsin had on RV733-GFP replication (Figure 3). However, for HK68, the addition of trypsin was able to restore virus replication, by increasing virus titers by 2-3 logs to levels similar to wild-type Caco-2 cells.

While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fail within the spirit and scope of the invention.

Claims

1. A method of treating an influenza virus infection comprising administering to an individual in need of such treatment an effective amount of a MASP1 (mannan-binding lectin serine protease 1) inhibitor or a PRSS33 (protease, serine, 33) inhibitor.

2. The method according to claim 1 wherein the MASP1 inhibitor or PRSS33 inhibitor is an siRNA.

3. The method according to claim 1 wherein a MASP1 inhibitor is administered.

4. The method according to claim 3 wherein the MASP-1 inhibitor is selected from the group consisting of: C1-inhibitor; α-2-macroglobulin; anti-thrombin and heparin; and a suitable siRNAs.

5. Use of a MASP1 (mannan-binding lectin serine protease 1) inhibitor, a PRSS33 (protease, serine, 33) inhibitor or a combination thereof to treat an influenza virus infection.

6. The use according to claim 5 wherein the MASP1 inhibitor or PRSS33 inhibitor is an siRNA.

7. The use according to claim 5 wherein a MASP1 inhibitor is used.

8. The use according to claim 7 wherein the MASP-1 inhibitor is selected from the group consisting of: C1-inhibitor; α-2-macroglobulin; anti-thrombin and heparin; and a suitable siRNAs.

9. Use of a MASP1 (mannan-binding lectin serine protease 1) inhibitor or a PRSS33 (protease, serine, 33) inhibitor in the preparation of a medicament to treat an influenza virus infection.

10. The use according to claim 9 wherein the MASP1 inhibitor or PRSS33 inhibitor is an siRNA.

11. The use according to claim 9 wherein a MASP1 inhibitor is used.

12. The use according to claim 1 wherein the MASP-1 inhibitor is selected from the group consisting of: C1-inhibitor; α-2-macroglobulin; anti-thrombin and heparin; and a suitable siRNAs.

13. A method of preparing a medicament for treating an influenza virus infection comprising combining a ASP1 (mannan-binding lectin serine protease 1) inhibitor, a PRSS33 (protease, serine, 33) inhibitor or a combination thereof with a suitable excipient.

14. The method according to claim 13 wherein the MASP1 inhibitor or PRSS33 inhibitor is an siRNA.

15. The method according to claim 13 wherein a ASP1 inhibitor is administered.

16. The method according to claim 15 wherein the MASP-1 inhibitor is selected from the group consisting of: C -inhibitor; α-2-macroglobulin; anti-thrombin and heparin; and a suitable siRNAs.

17. A method of identifying an influenza virus antiviral compound comprising determining if a compound of interest is a MASP1 (mannan-binding lectin serine protease 1) inhibitor or a PRSS33 (protease, serine, 33) inhibitor.

18. The method according to claim 17 wherein the compound of interest is added to a mixture of MASP-1 protease and a fluorogenic substrate of MASP-1 and levels of fluorescence generated by the cleavage of the fluorogenic substrate in the absence and presence of the compound of interest are measured.

19. The method according to claim 17 wherein the compound of interest is selected from the group consisting of: small molecule;, aptamers; siRNAs; antibodies; and intrabodies.