1. Institute of Molecular Virology Westfälische-Wilhelms-University Münster Pursuing New Avenues in Anti-Influenza Therapy Stephan Ludwig Options for the Control of Influenza VII, Hongkong, Sep, 7, 2010

2. Outline Introduction Viral targets Cellular/host targets - Immunemodulators - Factors directly regulating viral functions Traditional Medicine

3. Urgent need for novel antiviral agents against influenza viruses - Wide availability - Broad activity - Low cost - No resistance Increasing incidence of resistance to clinically approved classes of drugs

4. Novel viral targets Viral Polymerase Favipiravir, T-705 (Toyama Chemical Co., Ltd.) Clincial Trail Phase 2->3 FLU-PHARM - New drugs targeting influenza virus polymerase Coordinator: Stephen Cusack, EMBL (France) 14 Partners from 7 European Countries FLUCURE - Development of novel antiviral drugs against Influenza Coordinator: Heather Marshall-Heyman, VIRONOVA AB (Sweden) 9 Partners from 7 European Countries Viral Nonstructural Protein 1 JJ3297 and other blockers of interferon antagonistic NS1 function, Basu et al. (P-460), (Basu et al. (2009) J Virol. 83:1881-91) Viral Nucleoprotein Nucleozin triggers aggregation of NP and inhibits nuclear accumulation (Kao et al. (2010) Nat Biotech 28: 600-5)

5. Cellular/Host targets A) Modulators or inducers of the immune response B) Inhibitors of cellular factors or pathways that regulate the virus life cycle

6. Cellular targets A)Modulators or inducers of the immune response - IFN inducing agents, e.g. ASN2 (Ortigoza et al., O-869) - Low dose interferon treatment for prophylaxis (Bennet et al., O-822) - Protease-activated receptor (PAR) agonists act antiviral through induction of IFN gamma (Khoufache et al., P-446) - anti-influenza activity of PS-341 (Velcade) through induction of an antiviral state (Dudek et al. (2010) J Virol. 84:9439-51) - COX-2 inhibitors down modulate hyperinduction of immune responses (Lee et al., O-868) (Zheng et al. PNAS (2008) 105: 8091-6) - Use of existing immunmodulatory drugs (Statins, Glycyrrhizin, Glitazones) (Fedson, P-447, Korossy-Horwood et al. P-458, Allevea et al. P-459)

7. Taken from: Watanabe et al. (2010) Cell Host Microbe, 7, 427-439 Cellular targets B) Inhibitors of cellular factors that regulate the virus life cycle Cell 139, 1243–1254 Nature 454, 890–893 Nature 463, 813–817 Nature 463, 818–822 Cell 139, 1255–1267 Virology 387, 473–481

8. Adsorption Endocytosis Fusion and Release vRNA (-) cRNA (+) mRNA Import Translation Posttranslational Processing RNP- Export Budding Packaging Entry Cellular targets B) Inhibitors of cellular factors that regulate the virus life cycle DAS181 Importins NUPs Rabs, V-type ATPases PKC CRM1 Hsc70 NUP153 Actin Rab 11 Hillaire et al. (P-452) Collectin pSP-D Nicol et al. (P-449) FLUPEP

9. Adsorption Endocytosis Fusion and Release vRNA (-) cRNA (+) mRNA Import Translation Posttranslational Processing RNP- Export Budding Packaging Cellular targets Viral penetration of cellular barriers is controlled by cellular signaling cascades Entry PI3K RTKs (e.g. EGFR) Eierhoff et al. (2010) PLoS Pathog (in press) Raf/MEK/ERK Pleschka et al. (2001) Nat Cell Biol. IKK/NF-kB Wurzer et al. (2004) Cell Microbiol. Wurzer et al. (2003) EMBO J.

10. NF- B Inh. - M - PB1 - NP - ERK2 -- + --- +++ - JNK1 - NS1 - ERK2 0h 2h 4h 8h 10h NF- B inhibition efficiently blocks viral RNP export DAPI anti-NP merge - INH + INH

11. The NF- B inhibitor SC75741 blocks replication of influenza viruses A/FPV/Bratislava/79 (H7N7) A/Thailand/KAN-1/2004 (H5N1) Hours post infection Ehrhardt et al, P- 457 Reiling et al, P-453

12. SC75741 shows no tendency to induce resistant virus variants Progeny virus titer supon repeated passaging in the presence and absence of the drugs Control SC75741 Oseltamivir

13. Therapeutic treatment of H5N1 infected mice with SC75741 SC75741 shows a significant (p = 0.05) anti H5N1 activity, when treatment starting 4 days after infection twice daily 7,5mg/Kg i.p. SC75741 Placebo Days after infection 100 50 0 % Survival once daily 15 mg/Kg i.p. SC75741 Placebo Days after infection 100 50 0 % Survival A/Mallard/Bavaria/2005 (H5N1), induces severe disease in mice without adaptation (LD 50 : 8x10 1 )

14. SC75741 results in reduced cytokine/chemokine expression in vivo SC75741 leads to a reduced transcription of IL-6 and IP-10 in H5N1 infected mice Cytokine/Chemokine specific real time RT-PCR Primer: Qiagen RT-PCR RNA isolated from the lung 48h p.i. SC75741 15mg/kg Mallard/Bavaria/2005 (H5N1)

15. Targeting signal transduction pathways as an antiviral approach -broad activity -no emergence of resistent variants detectable -can be done by using existing drugs -NF- B or MAPK blockers have indirect beneficial effects due to their immunemodulatory function

16. Expansion and optimization of the current repertoire of antiviral drugs and development of clinical research to assess efficacy of putative adjuvant treatment modalities such as immunomodulators, passive immunotherapy and traditional medicine that are suitable for use in under-resourced areas would be most beneficial. Research Recommendations:..... 4.2.4 Develop novel and effective treatment strategies including adjunctive treatments (e.g. immunomodulators, immunoglobulin, natural products) that are applicable in low resource settings and easy to administer...... 4.2 Improve Clinical Management of Patients Traditional Medicine

17. Traditional Herbal Medicine Numerous reports of the anti-influenza activity of medicinal plant extracts and plant products Korrossy-Horwood et al. (P-458) Glycyrrhizin from licorice roots Tsai et al. (P-450) Platform to screen Chinese herbal medicines Ehrhardt et al. (O-871) Cystus052, a polypenol-rich extract from pink rockrose Plant polyphenols possess ant-influenza activity: Anti-influenza activity of resveratrol from red grapes: Improved survival and reduced lung titers in infected mice (Palamara et al. J.Infect. Dis.191,1719–1729) Epigallocatechin-3-gallate and theaavindigallate from green tea: Unspecific binding of the HA and agglutination of virus particles (Nakayama et al.; Antiviral Res. 21,289–299)

18. CYSTUS052 possess anti-influenza activity 0,1 1 10 100 1000 10000 100000 8h24h36h x10 2 PFU/0.5ml untreated 25µg/ml CYSTUS052 50µg/ml CYSTUS052 CYSTUS052 is very rich in highly polymeric polyphenols Acts antiviral by inhibiting binding of virus particles to cells Does not interfere with cell viablity, metabolism, intracellular signaling or binding of cytokines to cellular receptors No pharmacological effects Does not induce resistant virus variants Viral titers Ehrhardt et al. 2007, Antiviral Res. Droebner et al. 2007, Antiviral Res. Kalus et al. 2009, Antiviral Res.

19. Bodyweight Control CYSTUS052 CYSTUS052 protects mice against H7N7 influenza virus infection Survival Control: 4/10 CYSTUS052: 10/10 (O-871)

20. Novel Antiviral Approaches - Perspectives -Numerous promising approaches under investigation, but still in an early stage -Inhibitors of cellular targets may be best suited to cover a broad range of viruses (also newly emerging strains) and to prevent emergence of resistant variants - Inhibitors that possess dual action (immunemodulation and direct antiviral activity) might be of major advantage - Use of existing drugs against cellular targets - Drug combinations should be considered - Medicinal products from traditional medicine should be given more attention to meet WHO recommendations for low-resource settings and to provide safe options for prophylactics