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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 !!!