Transcript Fighting the Flu: The Development of Influenza

Fighting the Flu: The
Development of Influenza
Neuraminidase Inhibitors
Matthew Dodge
Burke Group
December 1, 2005
1
Outline
I.
II.
III.
IV.
V.
Introduction and Background
Traditional Influenza Treatments
Development of Neuraminidase Inhibitors
Preparing for Future Pandemics
Summary and Conclusions
2
What is Influenza?
• Upper respiratory infection caused by the influenza virus
• Symptoms include fever, muscle ache, headache, and cough
• Healthy adults recover in one to two weeks
• “At-risk” patients are at risk of serious illness
• Affects 5-15% of the world population annually
• Globally, 3-5 million severe cases; 250,000-500,000 deaths
• In the US, 20-25 million physician visits; 10,000-40,000 deaths
• Economic burden of $12 billion annually in the United States
Oxford, J.S., Lambkin, R. Drug Discovery Today 1998, 3, 448-456.
World Health Organization. Influenza. http://www.who.int/mediacentre/factsheets/fs211/en/print.html
(accessed Nov 2005)
3
The Spread of Influenza
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Figure from http://www.vaccineinformation.org/photos/flu_iac001a.jpg (Accessed Nov 2005).
4
The Influenza Virus
• Orthomyxovirus
• Key Surface Proteins:
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• Hemagglutinin (HA)
• Neuraminidase (NA)
• M2 Protein
Figure from www.influenza.nl/ files/influenza.jpg (Accessed Nov 2005).
World Health Organization. Influenza.ww.who.int/mediacentre/factsheets/fs211/en/print.html (Accessed Nov 2005).
5
Virus Life Cycle
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Figure from http://www.chemsoc.org/exemplarchem/entries/2001/sanderson/images/lifecyc.gif
(Accessed Nov 2005).
6
Classification of Influenza Viruses
• Influenza exists in three types: A, B, and C
•Types A and B cause disease in humans
•Type A has a natural reservoir in birds and mammals
• Type A is further classified in subtypes
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Figure from http://www.brown.edu/Courses/Bio_160/Projects1990/av/influenza/html. Courtesy of International
Influenza Education Panel (accessed Nov 2005)
7
Influenza Pandemics
• 1918-1919: The Spanish Flu [A (H1N1)]
• 500,000 US deaths and 40 million deaths worldwide
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World Health Organization. Influenza. http://www.who.int/mediacentre/factsheets/fs211/en/print.html (accessed Nov 2005)
Center for Disease Control. Fact Sheet: Influenza (Flu): Information About Influenza Pandemics, October 17, 2005.
Picture from http://www.stanford.edu/group/virus/uda/ (Accessed Nov 2005)
8
Other Pandemics of the 20th Century
• 1957: Asian Influenza [A (H2N2)]
• 70,000 US Deaths
• 1968: Hong Kong Flu [A (H3N2)]
• 34,000 US Deaths
Pandemics are caused by new human viruses
World Health Organization. Influenza. http://www.who.int/mediacentre/factsheets/fs211/en/print.html (accessed Nov 2005)
Center for Disease Control. Fact Sheet: Influenza (Flu): Information About Influenza Pandemics, October 17, 2005.
9
Antigenic Drift and Shift
• Antigenic Drift
• Mutations arise from incremental changes in the amino
acid sequences of the surface proteins
• Flu vaccine is reformulated each year to account for
antigenic drift
• Antigenic Shift
• Mutations arise from the genetic reassortment of viruses
from different animal hosts
• New subtypes can result and cause pandemics
Varghese, J.N. Drug. Dev. Res. 1999, 46, 176-196
10
Mechanism of Antigenic Shift
Avian H3
Human H2
Human H3
Figure from http://virology-online.com/presentations/index.htm. Courtesy of Dr. Linda Stannard,
University of Cape Town, Virology Laboratory of the Yale-New Haven Hospital.
11
Outline
I. Introduction and Background
II. Traditional Influenza Treatments
A. Vaccines
B. Adamantane-Based Drugs
III. Development of Neuraminidase Inhibitors
IV. Preparing for Future Pandemics
V. Summary and Conclusions
12
Vaccines
• The prinicipal measure for preventing influenza
• In the elderly, reduces morbidity by 60% and mortality by
70-80%; In healthy adults, reduces morbidity by 70-90%
• Includes inactivated influenza A (H3N2), A (H1N1), and
influenza B viruses
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World Health Organization. Influenza. http://www.who.int/mediacentre/factsheets/fs211/en/print.html
(Accessed Nov 2005).
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More About Vaccines
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• Vaccine is grown in eggs; harvested from egg whites
• One dose per egg; 80 million eggs required annually for US
World Health Organization. Influenza. http://www.who.int/mediacentre/factsheets/fs211/en/print.html (accessed Nov 2005)
Center for Disease Control. Fact Sheet: Influenza (Flu): Information About Influenza Pandemics, October 17, 2005.
Picture from http://www.stanford.edu/group/virus/uda/ (Accessed Nov 2005)
14
Adamantane Derivatives
NH2
NH2
Amantadine
Symmetrel
Rimantadine
Flumadine
• Inhibit the activity of the M2 protein
• Effective only against influenza type A
• Inexpensive and readily available
• Severe side effects including delirium and seizures
Varghese, J.N. Drug. Dev. Res. 1999, 46, 176-196
15
Outline
I. Introduction and Background
II. Traditional Influenza Treatments
III. Development of Neuraminidase Inhibitors
A. Neuraminidase Enzyme
B. Zanamivir
C. Oseltamivir
D. Peramivir
IV. Preparing for Future Pandemics
V. Summary and Conclusions
16
Neuraminidase
Varghese, J.N., Laver, W.G., and Colman, P.M., Nature 1983, 303, 35-40; 41-44.
Laver, G., Garman, E., Science 2001, 293, 1776-1777.
17
Neuraminidase Crystal Structure
Varghese, J.N., McKimm-Breschkin, J.L., Caldwell, J.B., Kortt, A.A., and Colman, P.A. Proteins 1992, 14, 327-332.
18
Varghese, J.N. Drug. Dev. Res. 1999, 46, 176-196.
Sialic Acid Cleavage
HO
HO
H
COOO
OR
HO
AcHN
HO
HO
Neuraminidase
COO-
OH
R = H: Sialic Acid
(Neu5Ac)
R= sugars
HO
O
O
HO
AcHN
OH
HO
H
H OH
O
N
HO
COOOR
(Neu5Ac2en)
OH
HO H
O
N
Ac
HO OH
CO2H
19
Neu5Ac2en Binding
• S1, S2, S3, S4, and S5 binding subsites
• Neu5Ac2en is ~ 5M binder
Stoll, V. et al., Biochemistry, 2003, 42, 718-727.
20
Neuraminidase Inhibitors
OH
OH
H O
CO2H
HO
AcHN
HN
NH
COOEt
O
AcHN
NH2
NH2
Zanamivir
Oseltamivir
Relenza
Biota
GlaxoSmithKline
H2N
HN
Tamiflu
Gilead Sciences
Roche
NH
CO2H
OH
NHAc
Peramivir
H
AcHN
N
H
CH3
OCH3
COOiPr
ABT-675
BCX-1812
BioCryst Pharmaceuticals
Ortho-McNeil
Abbott Laboratories
21
Zanamivir
HO HO
H
O
CO2H
HO
AcHN
HN
NH
NH2
Zanamivir
Relenza
Biota
GlaxoSmithKline
22
Rational Design of Zanamivir
HO HO
H
HO HO
O
CO2H
HO
AcHN
H
HO HO
O
CO2H
Neu5Ac2en
CO2H
O
HO
AcHN
HO
AcHN
OH
H
NH2
4-amino-Neu5ac2en
HN
Zanamivir
NH
NH2
• S2 subsite designated a “hotspot”
• Amine should increase binding with the COO- of Glu 119
• Guanidinyl group should bind with even higher affinity
von Itzstein et al., Nature 1993, 363, 418-423.
23
Binding of 4-amino-Neu5Ac2en
Binding Predicted by GRID
von Itzstein et al., Nature 1993, 363, 418-423.
X-ray structure
24
Binding of Zanamivir
Binding Predicted by GRID
von Itzstein et al., Nature 1993, 363, 418-423.
X-ray structure
25
Synthesis of 4-amino-Neu5Ac2en
OH
HO
O
HO
OH
CO2H
H
AcHN
OH
HO
OH
O
MeOH, HCl
HO
94%
a) Ac2O, pyridine, DMAP
CO2Me
H
b) TMSOTf, EtOAc
AcHN
OH
OH
62% over 2 steps
Neu5Ac
OAc
AcO
O
AcO
CO2Me TMSN3,
tBuOH,
OAc
AcO
80°C
AcO
H
76%
N
CO2Me
O
H
NaOMe, MeOH
71%
AcHN
O
N3
Me
OH
HO
O
HO
H
CO2Me
1) Et3N, H2O
2) Lindlar catalyst, H2, H2O
3) Dowex 2 x 8 resin
AcHN
OH
HO
N3
56% over 3 steps
O
HO
CO2H
H
AcHN
NH2
4-amino-Neu5Ac2en
26
Chandler, M. et al, J. Chem. Soc., Perkin Trans. 1 1995, 1173-1180.
Synthesis of Zanamivir
NH
H2N
OH OH
H O
CO2H
HO
AcHN
S
O
OH
NaOH, K2CO3
48%
OH OH
H O
CO2H
HO
AcHN
HN
NH2
4-amino-Neu5Ac2en
NH
NH2
Zanamivir
Ki
50 nM
0.2 nM
• Potent inhibition of influenza neuraminidase activity
• Zanamivir is currently available as Relenza®
• Reduces the duration of symptoms by 1-1.5 days
Chandler, M. et al, J. Chem. Soc., Perkin Trans. 1 1995, 1173-1180.
GlaxoSmithKline.
Relenza® Prescribing
Information. April 2003.
27
Disadvantages of Zanamivir
OH OH
H O
CO2H
HO
AcHN
HN
Zanamivir
NH
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NH2
• Low oral bioavailability
• Relenza® contains lactose as the delivery vehicle
• Relenza® has been shown to cause bronchospasm and
decline in lung function
GlaxoSmithKline. Relenza® Prescribing Information. April 2003.
28
Oseltamivir
COOEt
O
AcHN
NH2
Oseltamivir
Tamiflu
Gilead Sciences
Roche
29
Design of Oseltamivir
HO
COOH
H OH
HO
OR
O
N
HO
O
Neu5Ac
CO2H
RO
AcHN
NH2
HO
or
HO
HO
O
HO
CO2H
H
O
CO2H
HO
AcHN
HO
AcHN
CO2H
RO
H
OH
OH
AcHN
NH2
Zanamivir
Kim, C.U., et al, J. Am. Chem. Soc. 1997, 119, 681-690.
30
Importance of Double Bond Position
CO2H
HO
AcHN
AcHN
NH2
IC50
CO2H
HO
6.9 M
NH2
>200 M
• Oxonium ion mimic provides significantly better activity
• Cause of difference in unknown
Kim, C.U., et al, J. Am. Chem. Soc. 1997, 119, 681-690.
31
Hydrophobic Interaction
HO HO
9
8
7
HO
AcHN
H
1
O
2
6
5
COOH
3
4
OH
CO2H
RO
AcHN
NH2
Neu5Ac2en
• C7 hydroxyl of Neu5Ac2en has little contribution to binding
• C8 makes hydrophobic contact with Arg224’s carbon chain
• Hydrophobic optimization could provide additional binding
and increase lipophilicity
Kim, C.U., et al, J. Am. Chem. Soc. 1997, 119, 681-690.
32
C6 Aliphatic Group Optimization
CO2H
RO
AcHN
NH2
R
NA IC50 (nM)
Plaque IC50 (nM)
H
6300
-------
Ethyl
2000
-------
n-butyl
300
-------
Iso-butyl
200
-------
3-pentyl
1
16
4-amino-Neu5Ac2en
150
2500
Zanamivir
1
15
Kim, C.U., et al, J. Am. Chem. Soc. 1997, 119, 681-690.
* GS-4104
33
Crystal Structure of GS-4104
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Kim, C.U., et al, J. Am. Chem. Soc. 1997, 119, 681-690.
34
Industrial Synthesis of Oseltamivir
COOH
HO
HO
EtOH, SOCl2 (0.5 eq)
reflux, 3 h
97%
HO
OH
OH
(-)-shikimic acid
MsCl (1.3 eq)
COOEt Et3N (2.0 eq)
EtOAc, 0-5°C
O
O
89%
O
98%
COOEt NaHCO3 (1.6 eq)
EtOH/H2O, 60°C, 1.5 h
O
HO
OMs
95%
OMs
Et3SiH (1.3 eq)
COOEt TiCl4 (1.1 eq)
CH2Cl2, -34°C, 2-6 h
O
Me2C(OMe)2 (2.0 eq)
TsOH, (0.01 eq)
EtOAc, 35°C
3-pentanone (15 eq)
COOEt CF3SO3H (0.045 eq)
40°C, 100 mbar; Et3N
O
OH
O
COOEt
HO
OMs
Federspiel, M., et al, Org. Process. Res. Dev., 1999, 3, 266-274.
80% over 2 steps
>98% pure by HPLC
COOEt
O
O
35
Completion of the Synthesis
COOEt
O
aq. EtOH
HN
COOEt
2) Ac2O, NaHCO3
AcHN
44%
CH3CN
OH
COOEt
O
1) NaN3, NH4Cl, DMF
PMe3
N3
N3
86%
COOEt
O
HO
O
O
COOEt
O
NaN3, NH4Cl
N3
1) Raney-Ni, H2, EtOH
2) 85% H3PO4
97%
O
COOEt
AcHN
NH2•H3PO4
71%
• ~ 17% yield over 12 steps from (-)-shikimic acid
• No chromatography
• Concerns: (-)-shikimic acid and use of azide chemistry
Rohloff, J.C., et al. J. Org. Chem., 1998, 63, 4545-4550.
36
Harrington, P.J., et al, Org. Process. Res. Dev., 2004, 8, 86-91
Oseltamivir Phosphate as a Drug
O
COOEt
O
Hepatic esterases
COO-
AcHN
AcHN
NH3+
NH2•H3PO4
• Reduces duration of
symptoms by 1.5 days
• Reduces incidence of
disease from 12% to 1%
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• Must be used within 48
hours of symptom onset
Roche Pharmaceuticals. Tamiflu® Complete Product Information. June 2004.
37
Peramivir
H 2N
HN
NH
CO2H
OH
NHAc
Peramivir
BCX-1812
BioCryst Pharmaceuticals
Ortho-McNeil
38
Crystal Structure Analysis
HO HO
H
O
CO2H
HO
AcHN
OH
Neu5Ac2en
HO
OH
HO
O
AcHN
OH
CO2H
HO
Can cyclopentane be used as a scaffold?
39
Babu, Y. S. et al, J. Med. Chem. 2000, 43, 3482-3486.
Design of BCX-1812
NH
N
H
CO2H
CO2H
CO2H
HO
NH2
NH
NH
O
• Guanidinyl group binds
with Asp151, Glu119, and
Glu227 in the same
manner as zanamivir
N
H
NH2
NH
O
NH
N
H
NH2
NH
O
• Guanidinyl group binds
differently than zanamivir
• Guanidinyl group binds
differently than zanamivir
• n-butyl chain interacts
with Ala246, Ile222, and
Arg224
• 3-pentyl chain interacts with
two hydrophobic pockets
• Active isomer was chosen
by the NA enzyme
• Retains activity against
zanamivir-resistant mutants
40
Babu, Y. S. et al, J. Med. Chem. 2000, 43, 3482-3486.
Synthesis of BCX-1812
HN
CO
a) HCl, MeOH
BocHN
CO2Me PhCNO, Et3N,
2-ethyl-1-nitrobutane
BocHN
70%
b) (Boc)2O, Et3N
N
92%
CO2Me
HCl, ether
O
b) Ac2O, Et3N
67%
N
BocHN
CO2Me a) H , PtO , MeOH,
2
2
HCl (100 psi)
H2N
CO2Me
a) (iPr)2EtN H2N
N
NH
•HCl
NH
H2N
NH
CO2H
b) NaOH
OH
NHAc
OH
NHAc
50% over 3 steps
OH
NHAc
• 21.6% over 8 steps from the commercially available lactam
41
Babu, Y. S. et al, J. Med. Chem. 2000, 43, 3482-3486.
Binding of BCX-1812
42
Babu, Y. S. et al, J. Med. Chem. 2000, 43, 3482-3486.
Peramivir as a Drug Candidate
NH
H2N
NH
CO2H
• Reduced duration of
symptoms by 0.53-0.64 days
OH
NHAc
IC50 Values Against N9 NA Enzyme
Drug
Influenza A
Influenza B
Relenza®
0.3 – 2.3 nM
1.5 – 17 nM
Tamiflu®
0.01 – 2.2 nM
6.4 – 24 nM
Peramivir
0.1 – 1.4 nM
0.6 – 11 nM
• Ortho-McNeil terminated their
agreement with BioCryst
• Intramuscular injection
Peramivir in pre-clinical stages
Babu, Y. S. et al, J. Med. Chem. 2000, 43, 3482-3486.
43
BioCryst Pharmaceuticals Press Release
Outline
I.
II.
III.
V.
VI.
Introduction and Background
Traditional Influenza Treatments
Development of Neuraminidase Inhibitors
Preparing for Future Pandemics
Summary and Conclusions
44
H5N1: The Next Pandemic?
Feb 2003
2004
1996
1997
Dec 2003
2 Human deaths
in Hong Kong
2005
Found in Vietnam (H),
Japan, Thailand (H),
Cambodia, Indonesia,
Laos, China, and
Malaysia
18 Human cases in
Hong Kong; kills 6
H5N1 detected in
geese in China
Korea confirms
poultry cases
Found in Russia,
Kazakhstan,
Mongolia, Turkey,
Romania, and Croatia
World Health Organization, H5N1 Avian Influenza: Timeline, October 28, 2005.
45
H5N1: Human Deaths
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Plans to Combat H5N1
Three Main Pillars of US National Strategy
• Preparedness and Communication
• WHO recommends antiviral stockpiles for 20% of the population
• US plans to stockpile enough for 25% of the population
• Survelliance and Detection
• Response and Containment
• Containing outbreaks by limiting travel in and out of the country
World Health Organization; Antiviral drugs: Their role during a pandemic; November 2005
National Strategy for Pandemic Influenza; Homeland Security Council, November 2005
HHS Pandemic Influenza Plan; US Department of Health and Human Services, November 2005
47
Significance of Antivirals Against H5N1
• M2 Inhibitors could provide a frontline defense
• These have been in use in Asian poultry since 1990
• Most H5N1 strains are resistant
• NIs provide the only viable option
• H5N1 should be susceptible to Relenza® and Tamiflu®
• WHO warns against prophylactic use
World Health Organization; Antiviral drugs: Their role during a pandemic; November 2005
48
Challenges to Meet NI Demand
• Limited Production Capacity
• Cost and availability of (-)-shikimic acid
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• Time required for manufacturing process
• Cost
• WHO will have 3 million doses for immediate response
• Tamiflu: $78-$91 per regimen
• Relenza: $67-$78 per diskhaler
World Health Organization; Antiviral drugs: Their role during a pandemic; November 2005
49
Outline
I.
II.
III.
IV.
V.
Introduction and Background
Traditional Influenza Treatments
Development of Neuraminidase Inhibitors
Preparing for Future Pandemics
Summary and Conclusions
50
Summary and Conclusions
• Computer-aided design has allowed the
development of efficient neuraminidase inhibitors (NIs)
• NIs are effective against all strains of influenza
• Relenza® and Tamiflu® should be effective against H5N1
• Challenges in the use of Tamiflu® include prohibitive
costs, the availability of (-)-shikimic acid, and the current
manufacturing capabilities
51
Acknowledgements
Professor Burke
Burke Group Members
Practice Talk Attendees
Andrew Dilger
Brian Lucas
Andy Hawk
Vicki Wilde
Sarah Jewell
Laura Wysocki
Grant Geske
Misty Dodge
52
ABT-675
H
AcHN
N
H
CH3
OCH3
COOiPr
ABT-675
Abbott Laboratories
53
Design of ABT-675
+H
C OO-
3N
N
O
O
tBu
Racemate
Ki = 58 M
Stoll, V. et al., Biochemistry, 2003, 42, 718-727.
54
High-Throughput X-Ray Crystallograhy
+H
C OO-
3N
N
O
N
Me
Me
• Modeling failed to be predictive
Me
• An iterative structure-based design
program was initiated using X-ray
feedback on a large number of compounds
Ki = 0.5 M
O
H3CO
AcHN
H
N
H
• Further potency gains were troublesome
due to inconsistent structure-activity
relationships
COO-
• >120 compounds were soaked into NA
crystals and X-ray structures were obtained
• This permitted the rapid discovery of several
subnanomolar inhibitors
Ki = 0.8 nM
Stoll, V. et al., Biochemistry, 2003, 42, 718-727.
55
Novel Binding Mode
O
H3CO
O
NH3+
COO-
OCH3
Stoll, V. et al., Biochemistry, 2003, 42, 718-727.
56
Final Structure Optimization
O
H3CO
AcHN
H
H
N
H
COO-
AcHN
N
H
CH3
OCH3
COOiPr
Ki = 0.8 nM
DeGoey, D.A.., et al, J. Org. Chem., 2002, 67, 5445-5453.
57
Barnes, D.M., et al, Org. Lett. 2002, 4, 1427-1430.
Synthesis of ABT-675
TrSN
OTBS
N
Boc
TfOH (0.8 eq)
Me
OMe
TrSHN
THF/heptane
-40°C,
H
MeO Me
O
N
Boc
BrMg
TrSHN
H
MeO Me
96%, 18:1
a) DIBAL-H
TrSHN
H
MeO Me
N
Boc
a) PPTS, MeOH, reflux
H
b) PPTS, MeOH
c) TMSCN, BF3•Et2O
Cu(I)Br•DMS,
TMSCl
89%
TrSHN
O
O
N
Boc
MeO Me
N
Boc
CN
b) Ac2O, Et3N
69%
67% over 3 steps
AcHN
CN 6 N HCl, 60°C
N
H
99%
Boc
Me
MeO
AcHN
H
MeO Me
N
H
DeGoey, D.A.., et al, J. Org. Chem., 2002, 67, 5445-5453.
Barnes, D.M., et al, Org. Lett. 2002, 4, 1427-1430.
CO2H
58