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
Outline
Introduction
Viral targets
Cellular/host targets
- Immunemodulators
- Factors directly regulating viral functions
Traditional Medicine
Increasing incidence of resistance to
clinically approved classes of drugs
Urgent need for novel antiviral agents against
influenza viruses
- Wide availability
- Broad activity
- Low cost
- No resistance
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 Nucleoprotein
Nucleozin triggers aggregation of NP and inhibits nuclear accumulation
(Kao et al. (2010) Nat Biotech 28: 600-5)
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)
Cellular/Host targets
A) Modulators or inducers of the immune response
B) Inhibitors of cellular factors or pathways
that regulate the virus life cycle
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)
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
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
Hillaire et al. (P-452) Collectin pSP-D
Nicol et al. (P-449) FLUPEP
DAS181
Adsorption
Budding
Packaging
Entry
Posttranslational
Processing
Endocytosis
Actin
Rab 11
Translation
Rabs, V-type ATPases
PKC
Fusion and
Release
mRNA
vRNA (-)
Import
Importins
NUPs
cRNA (+)
RNPExport
CRM1
Hsc70
NUP153
Cellular targets
Viral penetration of cellular barriers is controlled
by cellular signaling cascades
Budding
Adsorption
RTKs (e.g. EGFR)
Eierhoff et al. (2010)
PLoS Pathog (in press)
Packaging
PI3K
Posttranslational
Processing
Entry
Translation
Endocytosis
Fusion and
Release
mRNA
vRNA (-)
Import
cRNA (+)
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.
RNPExport
NF-kB inhibition efficiently blocks viral RNP export
0h
DAPI Inh.
NF-kB
-M
- INH
-JNK1
-NS1
-ERK2
-NP
+ INH
-ERK2
-PB1
-ERK2
-
2h
-
4h
8h
10h
- + - + - + merge
+ anti-NP
The NF-kB inhibitor SC75741 blocks replication of influenza viruses
A/FPV/Bratislava/79 (H7N7)
Ehrhardt et al, P- 457
Reiling et al, P-453
Hours post infection
A/Thailand/KAN-1/2004 (H5N1)
Virustiter
% der unbehandelten Kontrolle
1000
100
10
1
0,1
0,01
0,001
DMSO
0,1%
KH1
1µM
DMSO
0,25%
KH1
2,5µM
DMSO KH1 5µM
0,5%
SC75741 shows no tendency to induce resistant virus variants
Control
SC75741
Oseltamivir
Progeny virus titer supon repeated passaging in the presence and absence of the drugs
Therapeutic treatment of H5N1 infected mice with SC75741
A/Mallard/Bavaria/2005 (H5N1), induces severe disease in mice without adaptation (LD50: 8x101)
once daily 15 mg/Kg i.p.
1.2
1.2
1001.0
1001
SC75741
Wahrscheinlichkeit
SC75741
0.8
% Survival
Wahrscheinlichkeit
% Survival
twice daily 7,5mg/Kg i.p.
0.6
50
Placebo
0.4
0.6
50
Placebo
0.4
0.2
0.2
00.0
0.8
00
0
5
10
Tage/Dauer
Days after infection
15
0
5
10
DaysTage/Dauer
after infection
SC75741 shows a significant (p = 0.05) anti H5N1 activity, when
treatment starting 4 days after infection
15
SC75741 results in reduced
cytokine/chemokine expression in vivo
120
100
80
60
40
20
0
Control
 Cytokine/Chemokine specific
real time RT-PCR
IP-10
Comparative delta CT value relative to control
(100%)
Comparative delta CT value relative to control
(100%)
IL6
SC75741
120
 Primer: Qiagen RT-PCR
100
 RNA isolated from the lung 48h
p.i.
80
 SC75741 15mg/kg
60
 Mallard/Bavaria/2005 (H5N1)
40
20
0
Control
SC75741
SC75741 leads to a reduced transcription of IL-6 and IP-10 in H5N1
infected mice
Targeting signal transduction pathways as an antiviral approach
-broad activity
-no emergence of resistent variants detectable
-can be done by using existing drugs
-NF-kB or MAPK blockers have indirect beneficial effects
due to their immunemodulatory function
Traditional Medicine
4.2 Improve Clinical Management of Patients
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.
.....
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 theaflavindigallate from green tea:
Unspecific binding of the HA and agglutination of virus particles
(Nakayama et al.; Antiviral Res. 21,289–299)
CYSTUS052 possess anti-influenza activity
Viral titers
100000
CYSTUS052 is very rich in highly
polymeric polyphenols
x102PFU/0.5ml
10000
1000
Acts antiviral by inhibiting binding of virus
particles to cells
100
10
1
0,1
8h
24h
50µg/ml CYSTUS052
25µg/ml CYSTUS052
untreated
36h
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
Ehrhardt et al. 2007, Antiviral Res.
Droebner et al. 2007, Antiviral Res.
Kalus et al. 2009, Antiviral Res.
CYSTUS052 protects mice against
H7N7 influenza virus infection
Bodyweight
CYSTUS052
weight in %
110
100
Control
90
80
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
days p.i.
Survival Control: 4/10
CYSTUS052: 10/10
(O-871)
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