influenza a virus: emergence of drug resistance & quest
TRANSCRIPT
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Influenza A Virus: Emergence of Drug Resistance &
Quest for Solutions
Udayanga WanninayakeDepartment of ChemistryMichigan State University
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Influenza A Virus: A Global Threat
http://cagle.msnbc.com/news/BirdFlu05/1.asphttp://www.cagle.com/news/SwineFlu09/main.asp
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Statistics - WHO
Flu Type Infected Human Deaths1918 Spanish - > 50 Million1957 Asian - > 2 Million 1968 Hong Kong - > 1 Million2003 Avian (H5N1) - 2622009 Swine (H1N1) 1.5 Million >24,000Next?
http://www.flucount.org/http://www.who.int/csr/disease/swineflu/en/index.html
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Influenza Virus
A negative-sense single-stranded RNA virus of family Orthomyxoviridae
Three different genera: A, B and C
Matsubara T., Sumi M., Taki T., Okahata Y., Sato T., J. Med. Chem. 2009, 52, 4247-4256
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Influenza Virus
Matsubara T., Sumi M., Taki T., Okahata Y., Sato T., J. Med. Chem. 2009, 52, 4247-4256
Virus envelop proteins
Hemagglutinin16 subtypes
H1-H16
Neuraminidase9 subtypes
N1-N9
Group 1N1, N4, N5 & N8
H1N1, H5N1
Group 2N2, N3, N6, N7 & N9
H3N2
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Influenza Life Cycle
von Itzstein M., Nature Reviews Drug Discovery. 2007, 6, 967-974
M2 ion channel activity
Neuraminidase Activity
Hemagglutinin activity
RNA polymerase activity
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Strategies to Combat Influenza Virus
VaccinationNeutralized viral antigensInduces immunity against a virus
ChemotherapyChemical drugsInhibit the viral life cycle
Bhattacharya P. et al., J. Virology 2010, 84, 361-371
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Effective Vaccination?Vaccine effectiveness
Correct prediction of circulating virusesLimited by time constraint of manufacturing process
H1N1 2009Different from ordinary Influenza A virusesGenetic material; from different viral strainsRadically different phenotypes
Bhattacharya P. et al., J. Virology 2010, 84, 361-371Wang J. et al., Nature Rev. 2009, 459, 931-938
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Commercially Available Common Chemotherapeutics
Laohpongspaisan et al., J. Chem. Inf. Model. 2009, 49, 2323-2332.Erik De Clercq., Nature Rev. 2006, 5, 1015-1025.
Neuraminidase Inhibitors M2 Ion Channel Blockers
O CO2H
HN
OHN
NH
NH2
OH
HO
OH
O CO2Et
HN
ONH2
Zanamivir (Relenza®)
Oseltamivir (Tamiflu®)
NH2
NH2
Amantadine (Symetrel®)
Remantadine (Flumadine®)
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Emergence of Drug ResistanceM2 ion channel blockers
Amantadine and Rimantadine
In 2003-06 pandemic out break, avian influenza subtypes show resistance to Amantadine
Amantadine has not been recommended as a pandemic control drug since 2005 in USA
Amantadine type drug resistance of Influenza A viruses
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58
74
92
0102030405060708090
100
95-02 2003 2004 2005/06
Time / year%
resi
stan
ce
Laohpongspaisan et al., J. Chem. Inf. Model. 2009, 49, 2323-2332.
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Emergence of Drug Resistance
Neuraminidase Inhibitors (NAI) Zanamivir (Relenza®), Oseltamivir (Tamiflu®)
– Discovered in 1995 & 1997
Resistance in 16-18% of influenza patients
Hatakeyama S, Sugaya N, Ito M, Yamazaki M, Ichikawa M,. JAMA 2007, 297, 1435–42Gubareva L.V., Virus Res. 2004, 104, 199–203.
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Resistance!!! How?Antigenic Drift
Point mutations viral genomeResistant mutant strains
Antigenic ShiftReassortment of RNAs between different species (eg: H1N1 2009)Different antigenic determinants
Hong M. et al., JACS 2009, 131, 8066-8076
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How Should We Face the Problem?
Stop antigenic drift or antigenic shift? No
Modify existing drugs? May be
Discover novel drugs? Challenging
Look for novel drug targets? Challenging
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Drug Targets in Influenza Life Cycle
von Itzstein M. et al., Nature Reviews Drug Discovery. 2007, 6, 967-974
M2 ion channel BlockersEg: Amantadine, Rimantadine
Neuraminidase InhibitorsEg: Tamiflu®, Relenza®
Hemagglutinin Inhibitors
Eg: BMY27709
RNA polymerase InhibitorsEg: Ribavirin
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Outline
M2 ion channelM2 Ion Channel InhibitionEmergence of ResistanceNovel Ion Channel Inhibitors
Neuraminidase EnzymeEnzyme MechanismEnzyme InhibitionEmergence of ResistanceNovel Neuraminidase Inhibitors
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Mechanism of M2 Ion Channel
Schnell J.R., Chou J.J., Nature 2008, 451, 591-595Cady S.D., Luo W., Hu F., Hong M. Biochemistry 2009, 48, 7356-7364
•Homotetrameric transmembrane domain protein
•His37 – Proton sensor•Trp41 – Gate
PDB code 2RLF
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How Do Adamantane Based Drugs Inhibit M2 Ion Channel?
Two proposed mechanismsAllosteric inhibitionChannel pore blocking inhibition
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Allosteric Inhibition
Schnell J. R., Chou J. J., Nature 2008, 451, 591-595Pielak R.M., Schnell J. R., Chou J. J., PNAS 2009, 106, 7379-7384
NH2 NH2
Amantadine Rimantadine
PDB code 3CJ9
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What Happens in Resistant Mutants?S31N, in 99.9% of resistant viruses in 2005/06 flu season
Wild type- IC50= 16 µM
S31N- IC50= 199.9 µM
Other mutations; L26F, V27A, A30T, G34E and L38F
Schnell J.R., Chou J.J., Nature 2008, 451, 591-595Stouffer A.L. et al.,Nature 2008, 451, 596-597
PDB code 2RLF
NH2 NH2
Amantadine Rimantadine
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Channel Pore Blocking Mechanism
Stouffer, A.L. et al., Nature 2008, 451, 596-600
Trp41His37
Gly34
Ala30
Val27
V27
A30
PDB code 3CJ9
Val27, Ala30 and Ser31 make a pocket to accommodate Amantadine
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What Happens in Resistant Mutants?
S31N mutation gives rise to structural changes in the M2 transmembrane domain (M2TMD):
Extensive H-bonding between Asn carboxamides
High polarity
Stouffer, A.L. et al., Nature 2008, 451, 596-600
Ser31 Asn31
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M2TMD Structure Reveals Clues to Evolve Novel Drugs
Aqueous pore created, near highly conserved His37-Trp41 region
Drugs, binding to the N-terminal of the channel pore will not be successful due to frequent mutations
Drugs, which can bind to the altered allostericsite of mutants will be successful
Laohpongspaisan et al., J. Chem. Inf. Model. 2009, 49, 847-852
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Spiro[5,5]undecane CompoundsSpiranamine- potential drug against L26F & V27A mutantsL26- critical packing residue at the helix-helix interfaceV27- projects directly towards the pore
Ion Channel
Spiranamine
IC50 (µM)
Amantadine
IC50 (µM)
Spiro-piperidineIC50 (µM)
A/M2 12.6 15.8 0.92
A/M2 L26F 30.6 164.4 -
A/M2 V27A 84.9 1840.0 -
A/M2 S31N >10 mM 237.0 -
Wang J., Cady S.D., Balannik V., Pinto L.H., Degrado W.F., Hong M., JACS 2009, 131, 8066-8076.
N-terminal
C-terminal
NH2HN
NH2
Amantadine Spiranamine Spiro-piperidine
NH2HN
NH2
Amantadine Spiranamine Spiro-piperidine
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Neuraminidase (NA) Enzyme
Varghese, J.N., Colman, P.M., van Donkelaar, A., Blick, T.J., Sahasrabudhe, A., McKimm-Breschkin, J.L., Proc. Natl. Acad. Sci. USA 1997, 94, 11808–11812.
Homotetrameric glycoprotein anchored in viral membrane
Hydrolysis of glycosidic linkage between sialic acid and glycoprotein
PDB code 1MWER= glycosidic chain
OHO2C
NH
OOH
RO
OH
OH
HO
Sialyl glycoside
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Neuraminidase Mechanism
Taylor N.R., von Itzstein M., J Med Chem. 1994, 37, 616-624
PDB 1MWE
Sialyl glycoside
Sialic acid α-anomer
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Neuraminidase InhibitorsRelenza®
Inhaled / intranasal treatmentLow bioavailability
Tamiflu®
Oral drugHigh bioavailability
von Itzstein M., Wu W.Y., Kok G.B., Pegg M.S., Dyason J.C., Nature 1993, 363, 418–423 Kim C.U., Lew W., Williams M.A., Liu H., Zhang L., JACS. 1997, 119, 681–690
Relenza® Tamiflu®Sialic Acid
2
2
2
2
O CO2H
HN
OOH
OH
HO
HO
OH
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Binding of Relenza® to the NA Active Site
Relenza®
Glu119 –Guanidium gpKi = 5 x 10-8M
(PDB code 2QWK)
OH
Varghese J.N., Epa V.C., Colman P.M., Protein Sci. 1995, 4, 1081–87
HO
OHO2C
NH
ONH
OH
NH
H2N
PDB 1NNC
OHO2C
NH
OOH
HO
OH
OH
HO
Sialic Acid
PDB 1MWE
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Binding of Tamiflu® to the NA Active SiteTamiflu®
Glu276 - Arg224 ionic interactionsKi = 2 x 10-10 M
(PDB code 2QWK)
Kim C.U., Lew W., Williams M.A., Liu H., Zhang L., et al. J. Am. Chem. Soc. 1997, 119, 681–690
OHO2C
NH
O
NH2
PDB 2QWK
OHO2C
NH
OOH
HO
OH
OH
HO
Sialic Acid
PDB 1MWE
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Mutations in NA Active Site
H274Y in NA, 99.4% of all isolated Tamiflu® resistant H1N1 viruses in USA in 2009
Tamiflu® activity loss by 900-2500 fold
Q136K in NA; Relenza® resistant Influenza strain – H1N1
Relenza® activity loss by 300 fold
Hurt A.C., Holien J.K, Barr G.I., AAC 2009, 53, 4433-4440Nguyen J.T. et al., AAC 2009, 53, 4115-4126Hurt A.C., Holien J.K., Parker M., Kelso A., Barr I.G., J Virology 2009, 83, 10366-10373
PDB 2QWK
PDB 1NNC
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Mechanisms of Drug Resistance
Collins P.J. et al., Nature Letters 2008, 453, 1258-1261Hurt A.C., Holien J.K., Parker M., Kelso A., Barr I.G., J Virology 2009, 83, 10366-10373
1. Glu276 is pushed into the active site: Hydrophobic cavity is lost
2. H-bonding network (Gln136-Asp151-Arg156) is lost: Guanidinium group is not stable
Tamiflu®
Relenza®
Relenza®
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Novel NA Inhibitors
1. Structural Modifications2. Natural Products3. Computer Simulations/ Virtual Screening4. Novel NA Target
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Structural Modifications
Influenza Virus H1N1
A-315675 IC50 (nM) & fold increase with WT
Tamiflu® IC50 (nM) & fold increase with WT
WT 0.3 (+/-0.06) 1.74 (+/-0.01)
H274Y 5.5 >1000
R292K 7.0 >3000
Baz M., Abed Y., Nehme B., Boivin G., Antiviral Research 2007, 53, 791-793Maring et al. J. Med. Chem., 2005, 48, 3980–3990
πsta
cking
A-315675
HN
CO2H
O
NHO
H
A-315675
O
CO2H
HN
O
NH2
PDB 2QWKPDB 2HTU
Tamiflu®
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Natural Products - Diarylheptanoids
Alpinia katsumadai is a Medicinal plant used in traditional Chinese medicineAn antiviral herbal remedy
Compound IC50 value (μM)
1 4.67 +/- 0.36
2 6.10 +- 1.52
3 29.75 +/- 8.15
4 1.05 +/- 0.42
5 4.13 +/- 1.50
In vitro inhibition of NA of Influenza virus (H1N1) A/PR/8/34
Grienke U., Schmidtke M., Kirchmair J., Pfarr K, Wutzler P., Dorrwald R., Wolber G., Liedl K.R., Stuppner H., Rollinger J. M., J Med. Chemistry 2010, 53, 778-786
O
O
O
O
O OHOHOH
O
OH
1 2 3
4
5O
O
O
O
O OHOHOH
O
OH
1 2 3
4
5
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Natural Products - Diarylheptanoids
H1N1 Influenza Virus Compnd 4 (µM)
0.73
0.59
1.11
1.64
Tamiflu (nM)
A/Postdam/15/81 0.16
A/Belzig/2/01 0.20
A/Horneburg/IDT7489/08 0.06
A/Brest/IDT7490/08 0.19
NA Inhibitory Activity of Oseltamivir and 4 against Several Porcine H1N1 Isolates
Grienke U., Schmidtke M., Kirchmair J., Pfarr K, Wutzler P., Dorrwald R., Wolber G., Liedl K.R., Stuppner H., Rollinger J. M., J Med. Chemistry 2010, 53, 778-786
Tamiflu®
Relenza®
O
O
O
O
4
O
O
O
O
4
O
HOHO
OHNH
ONH
NHH3N
O
O
HN
O
O
ONH3
O
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Structural Insights
Grienke U., Schmidtke M., Kirchmair J., Pfarr K, Wutzler P., Dorrwald R., Wolber G., Liedl K.R., Stuppner H., RollingerJ. M., J Med. Chemistry 2010, 53, 778-786
O
O
O
O
4
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Structural InsightsCation−π interactions; Arg152, Arg224, and Arg371
H-bonding; cyclic ether and Arg292 and between the lactoneand Glu119
His274Tyr mutation; shift of Glu276 toward the binding pocket
Grienke U., Schmidtke M., Kirchmair J., Pfarr K, Wutzler P., Dorrwald R., Wolber G., Liedl K.R., Stuppner H., RollingerJ. M., J Med. Chemistry 2010, 53, 778-786
O
O
O
O
4
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Discovery of a Second Binding Site?N1 NA subtype - An additional site adjacent to the active site150-loop region (147-152) (10 Å x 5 Å x 5 Å)Open conformation; preferred in group 1 NAs, Closed; preferred in group 2 NAs
Russell R.J. et al Nature 2006, 443, 45-49, Amaro R.E. et al., JACS, 2007, 7764-7765
H274Y mutation is specific to N1 subtype (H5N1, H1N1)
Key: design drugs with bulkier moieties to fit the adjacent cavity
Loop 150
Loop 430
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A Novel Drug Binds to Cavity 150Synergistic effect with Tamiflu®
Binds simultaneously with Tamiflu®
OCl
NH
N
N
OH
H2N
An J. et al., Journal of Medicinal Chemistry 2009, 52, 2667-2672
NSC89853
Val116, Arg156 & Thr439
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Possible Drug Candidates
O
CO2H
NH
O
H2N
OCl
NH
N
N
OH
H2N
1
NH
CO2H
OHN
O
H
OCl
NH
N
N
OH
H2N
2
OH
OHO
CO2H
NH
O
NH OH
HN
NH2
OCl
NH
N
N
OH
H2N
3
An J. et al., Journal of Medicinal Chemistry 2009, 52, 2667-2672
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Future Insights
RNA polymerase Inhibitors
Hemagglutinin Inhibitors
von Itzstein M., Nature Reviews Drug Discovery. 2007, 6, 967-974
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Future Insights
RNA polymerase Inhibitors
Promoter RNA binding site of viral RNA polymerase- A new
target for drugs
Kuzuhara T. et al., JBC 2009, 284, 6855-6860
Hemagglutinin Inhibitors
Small molecule inhibitors saponins with 3-O-β-chacotriosyl
Song G. et al., J. Med. Chem. 2009, 52, 7368-7371
von Itzstein M., Nature Reviews Drug Discovery. 2007, 6, 967-974
OOOO
OH
HOOHHO
HO
OH
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ConclusionPandemics of Influenza virus are global threatsEmergence of resistance to common drugs has become critical health and economical problemFinding a novel effective drug against Influenza viruses has become an immediate global needNovel M2 ion channel inhibitors have not improved up to a satisfactory levelResearch on novel neuraminidase inhibitors have improved to a promising point after the discovery of novel drug binding site
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AcknowledgementsDr. WalkerDr. BabakDr. ChryssoulaMy Group Members
Danielle, Dennis, Dilini, Getrude, Irosha, Koyeli, Mark, Noelle, Ruth, Sean, Washington
Kahsay, Salinda, and all my friends
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Thank You All !!!