Anti-influenza (M2 and NA) Drugs

Anti-influenza (M2 and NA) Drugs Alan J. Hay MRC National Institute for Medical Research, London Summer School on Influenza Siena 1-5 August 2011

Antiviral drugs licensed for use against influenza M2 (Flu A) inhibitors - amantadine (oral) - rimantadine (oral) NA inhibitors - zanamivir (inhaled; [i.v.]) - [laninamivir (inhaled) Japan)] - oseltamivir (oral) - [peramivir (i.v.) Japan)] HA fusion inhibitors - [arbidol (oral) Russia]

Control of Influenza Vaccines Vaccination most effective way of combatting seasonal flu (and pandemic flu - when available) Antivirals Early in pandemic situation (stockpile against H5N1 threat) Prophylaxis- 70-90% effective in prevention of infection (equivalent to vaccination) Treatment reduce duration and severity of disease. Short acute infection need to be administer early (within 48 hr of onset of symptoms) (special hotline in UK for 2009 pandemic). Prolonged replication (severe disease; immunocompromised patients) Resistance - selection during treatment - is a major consideration/limitation in their use - emergence during evolution of virus Co-circulation of drug-sensitive and -resistant viruses have complicated recommendations for antiviral use in recent years

Sites of action of antivirals Arbidol

Amantadine identified and developed in 1960s; target and mechanism of action not identified till 1980s Effective against influenza A (M2), but not influenza B (BM2) viruses Used as anti-parkinsons agent (concerns about side-effects) Resistance! Limited use of the drugs (amantadine used in 1968 pandemic) Rimantadine more effective (less side-effects) than amantadine drug of choice, but less widely licensed

Amantadine resistance mutations in the M2 proton channel FA T N E Emerge frequently in vitro; in epidemic H3N2 and H1N1 viruses Similar to wt in infectivity, virulence and transmissibility Well-defined mutations (residues 26, 27, 30, 31 or 34) - correlate with resistance in vitro and in vivo Screen for resistance by sequencing M gene (pyrosequencing)

M2 activities inhibited by amantadine Virus uncoating HP avian influenza H5 and H7 HAs are cleaved during transport through the trans Golgi Network (TGN) and become susceptible to low ph-induced conformation change transport of native HA requires active M2 to reduce the acidity of the TGN - increases sensitivity of virus production to amantadine in vitro

Locations of Val27Ala and Ser31Asn mutations in the M2 channel (tetramer) which causes resistance to amantadine

M2 forms a proton-selective channel specifically inhibited by amantadine N I MR Electrophysiology (patch-clamp) M2 forms a proton-selective channel Proton currents specifically inhibited by amantadine/rimantadine Amantadine-resistant mutations abrogate inhibition basis of clinical activity

Crystal structure of the transmembrane pore of the M2 proton channel and interaction of amantadine Stouffer et al, 2008

ph dependent changes in M2 structure in relation to proton transport and drug interaction His37 Trp41 High ph Channel closed Greater drug access Binds irreversibly Low ph Channel conducting More restricted drug access

Comparative structures of wt and mutant M2 channels (NMR) [Pielak and Chou, 2010] Reduced drug affinity Drug binds externally Low amantadine affinity Fast reversible inhibition Slow irreversible inhibition Slow reversible inhibition [S. Tokar] ph dependent High affinity for closed form ph independent

Preferable docking poses for aminoadamantane derivatives Amantadine (1) Rimantadine (20) Alkyl amantadines (0.08-0.04) Spiropiperidine (0.8) Spiropyrrolidine (0.3) Cyclic rimantadines (0.6-2.1) [Eleftheratos et al, 2010] (Binding affinities relative to amantadine)

Emergence of amantadine resistance in human and animal viruses Pre 1980 s - low incidence; approx. 1% Mid 1980 s - European swine viruses (sporadic human cases) 2000 - avian H5N1, H9N2 (SE Asia) 2003 - avian H5N1 clades 1; human cases H5N3 (SE Asia) H7N2 (N America) 2003 - - human H3N2 (China/Hong Kong - worldwide) 2006-09 - human H1N1 emergent variant (clade 2C) 2009 - pandemic H1N1 2009

M genes of H5N1 viruses amantadine resistance (Most sensitive) (Most sensitive) (Most resistant) (All resistant)

Emergence of amantadine-resistant H3N2 viruses, 1994-2005 Bright et al 2005

Changes in amantadine resistance of H3N2 viruses HA genes Brisbane/10/07 2006-7 resistant M genes Wisconsin/67/05 2005-6 resistant 2006-7 ~50% resistant California/7/04 2005-6 sensitive

Recent changes in amantadine resistance of H1N1 viruses HA genes 2C M genes 2B 2B 2C

Origin of amantadine resistance (M2 S31N) of pandemic H1N1 viruses

Resistance to amantadine/rimantadine Human viruses all resistant? - H3N2 - pandemic H1N1 - B Avian - H5N1 clades associated with human infection Swine - Eurasian H1N1, H3N2 - Pandemic H1N1 Amantadine/rimantadine currently of little use Attempts to develop alternative inhibitors with broader spectrum of activity against M2 of influenza B as well as amantadine-resistant A viruses continue

Structure-based Drug Design DANA low M Oseltamivir low nm

Inhibitors of influenza A and B neuraminidases Inhaled Oral (prodrug) Intravenous The guanidino group of zanamivir and the ethylpropoxy group of oseltamivir, both present in peramivir, are highlighted. Potential complementary resistance profiles of oseltamivir and zanamivir

Laninamivir long-lasting derivative of zanamivir (single administration) 3-O-octanoyl prodrug Laninamivir

Interaction of the active moieties of inhibitors with NA Guanidino group binds to a pocket formed by acidic residues Glu119, Asp151 and Glu227 Ethylpropoxy group binds a hydrophobic pocket exposed by reorientation of Glu276

Mutations affecting oseltamivir binding to the hydrophobic pocket I222R (N1) Deletion 244-247

Type and subtype specificity of resistance mutations and complementarity of drugs

Emergence of resistance to zanamivir and oseltamivir Clinical trials Zanamivir low (?) Oseltamivir adults: 0.3%(4/1228) children: 4%(17/421) Oseltamivir treatment studies Japanese children - H1N1: 16%(7/43) (Ward et al,2005) - H3N2: 18%(9/50) (Kiso et al, 2004) - B : 1.4%(1/74) (Hatakeyama et al, 2007) Japanese(untreated) B: 1.7%(7/422) (Hatakeyama et al) H5N1-infected patients: 25%(2/8) (de Jong et al, 2005) Seasonal H1N1 viruses (Late 2007 2009) Emergence and worldwide spread of oseltamivir resistance (H275Y) almost 100% by mid 2008 (in e.g. South Africa, Australia) Pandemic H1N1 Sporadic cases of resistance (H275Y) to oseltamivir (sensitive to zanamivir) 1% of >6000 isolates in Japan. Some from untreated patients transmission? Associated with typical illness.

Emergence of resistance to zanamivir and oseltamivir Clinical trials Zanamivir low (?) Oseltamivir adults: 0.3%(4/1228) children: 4%(17/421) Oseltamivir treatment Japanese children - H1N1: 16%(7/43) (Ward et al,2005) - H3N2: 18%(9/50) (Kiso et al, 2004) - B : 1.4%(1/74) (Hatakeyama et al, 2007) Japanese(untreated) B: 1.7%(7/422) (Hatakeyama et al) H5N1-infected patients: 25%(2/8) (de Jong et al, 2005) Seasonal H1N1 viruses (Late 2007 2009) Emergence and worldwide spread of oseltamivir resistance (H275Y) almost 100% by mid 2008 (in e.g. South Africa, Australia) Pandemic H1N1 Sporadic cases of resistance (H275Y) to oseltamivir (sensitive to zanamivir)

Effect of Oseltamivir Treatment on Virus Load in H5N1 Patients [arrows indicate resistant virus (H274Y)] Died Survived M. de Jong et al. N. Eng. J. Med. 2005

Genetic drift of H1 HA single substitutions cause major antigenic change

Emergence of oseltamivir-resistant seasonal H1N1 viruses during 2007-2008 (Amantadine-resistant)

Genetic differences which correlate with H275Y resistance mutation in clade 2B ( European ) NA D354G (reversion) PB2 P453S PB1-F2 L30R/E22G (truncated, 57 amino acids)

Emergence of oseltamivir-resistant seasonal H1N1 viruses during 2007-2008 (Amantadine resistant)

Proximity of recent amino acid changes in NA of H1N1 viruses to the catalytic site

Correlation between Km of NA activity (substrate affinity) and asparagine (N) or aspartic acid (D) at position 344 H1N1 Virus Km (µm) Ki (nm) Amino acid position Clade 2B Clade 2C Clade 2A Clade 1 Oseltamivir Zanamivir 222 249 274 344 354 A/Dnipro/117/08 * 24.9 ± 1.23 92 * 0.44 Q K Y N G A/England/26/08 * 31 ± 4 86 * 0.51 Q K Y N G A/Paris/341/07 * 21.2 ± 1.5 122 * 0.27 Q K Y N G A/Norway/1735/07 * 11.1 ± 0.6 36 * 0.18 Q K Y N G A/Norway/1758/07 6.4 ± 1.1 0.16 0.12 Q K H N D A/Kiev/245/08 25.1 ± 4.4 0.85 1.18 Q K H N D A/Yokohama/35/08 * 31.8 ± 3.2 123 * 0.66 Q K Y N D A/Yokohama/22/08 * 30.8 ± 2.8 167 * 0.59 Q K Y N D A/England/654/07 * 28 ± 2.5 22.3 * 0.31 Q K Y N D A/England/545/07 17 ± 2 0.25 0.18 Q K H N D A/Paris/194/07 14.6 ± 1.3 0.25 0.27 Q K H N D A/Brisbane/59/07 15.3 ± 2.7 0.21 0.17 Q K H N D A/Slovenia/246/08 10.5 ± 1.3 0.32 0.26 Q G H N G A/Hong Kong/2652/06 15 ± 2 0.45 0.31 Q G H N G A/St.Petersburg/10/07 28.6 ± 2.3 0.87 0.52 Q G H N G A/England/594/06 * 138 ± 12 270 * 1.36 Q R Y D D A/England/593/06 89 ± 4.6 0.59 0.65 Q R H D D A/St.Petersburg/96/07 62 ± 5 0.51 0.48 Q R H D D A/Fukushima/141/06 76 ± 8.5 0.97 0.68 R G H D G A/Solomon Islands/3/06 67.6 ± 10 1.02 0.74 R G H D G A/Egypt/39/05 214 ± 43 3.35 2.97 R G H D G A/England/493/06 176 ± 22 1.65 2.20 R G H D G A/England/494/06 * 133 ± 14 305 * 1.34 R G Y D G A/Thessaloniki/24/05 61.8 ± 8 0.54 0.49 R G H D G A/Morocco/69/01 17.6 ± 0.49 0.35 0.25 Q G H N G A/Iceland/1/01 33.1 ± 2.3 0.87 0.85 Q G H N G A/Lisbon/5/00 18 ± 0.5 0.34 0.25 Q G H N G A/New Caledonia/20/99 43.5 ± 5 0.64 0.42 R G H D G A/Ostrava/801/98 54.5 ± 5 0.93 0.68 Q G H D G A/Beijing/262/95 83 ± 6.4 1.15 1.02 R G H D G A/Durban/113/97 85 ± 7.2 1.05 1.11 Q G H D G A/Bayern/7/95 17.4 ± 1.2 0.32 0.25 Q G H N G A/Texas/36/91 61.2 ± 6.9 0.82 0.48 R G H D G A/Brazil/11/78 64.7 ± 5.8 0.71 0.21 Q G H D G * Oseltamivir Resistant Viruses Km < 35 µm (6 33µM) Km > 35µM (43 214µM) Increased activity of NA associated with D344N substitution - compensates for reduced activity due to H275Y resistance mutation?

Effects of the H275Y mutation on the location of Glu 276 of N1 of A/Vietnam/1203/04(H5N1) in complex with with oseltamivir or zanamivir Reduction in binding affinity: ~300 fold Reduction in binding affinity: 2 fold Wild type (yellow); H274Y mutant (green)

Locations of recent amino acid changes in the NAs of seasonal H1N1 viruses relative to the catalytic site and H275Y oseltamivir-resistant mutation Compensatory mutations: D344N - increase in NA activity R222Q + V234M increase in cell surface expression of NA (Bloom et al, 2010) H275Y adaptive change?; resistance to oseltamivir simply coincidental

Emergence of resistance to zanamivir and oseltamivir Clinical trials Zanamivir low (?) Oseltamivir adults: 0.3%(4/1228) children: 4%(17/421) Oseltamivir treatment studies Japanese children - H1N1: 16%(7/43) (Ward et al,2005) - H3N2: 18%(9/50) (Kiso et al, 2004) - B : 1.4%(1/74) (Hatakeyama et al, 2007) Japanese(untreated) B: 1.7%(7/422) (Hatakeyama et al) H5N1-infected patients: 25%(2/8) (de Jong et al, 2005) Seasonal H1N1 viruses (Late 2007 2009) Emergence and worldwide spread of oseltamivir resistance (H275Y) almost 100% by mid 2008 (in e.g. South Africa, Australia) Pandemic H1N1 Sporadic cases of resistance (H275Y;[ I223R]) to oseltamivir (sensitive to zanamivir) 1% of >6000 isolates in Japan. Some from untreated patients transmission? 4 of 8 patients infected by transmission of resistant virus in haematology unit in UK No transmission to the wider community.

Oseltamivir-resistant pandemic H1N1 viruses: sporadic (~340 cases to January 2011) (Effect of H275Y on transmission in animals contradictory data)

Effects of anti-na drugs on infection Inhibits receptor destroying activity Inhibits receptor binding by D151G mutants - selected in cell culture (clinical significance?) Reduces synergistic bacterial infection prevents exposure of cryptic receptors

Location of residue 151 (D151G/N) at the edge of the catalytic site of NA Sialic acid Substitution of aspartic acid(d) 151 by glycine(g) (or asparagine(n)) causes: No change in enzyme activity of NA NA to bind additional receptors, different from HA, and refractory to enzymatic cleavage Residue 151 important in restricting interaction with sialic acid receptors, to match cleavage activity and HA binding Natural significance (low level in clinical specimens)? [Lin et al, 2010]

Effects of anti-na drugs on infection Inhibits receptor destroying activity Inhibits receptor binding by D151G mutants (clinical significance?) Reduces synergistic bacterial infection (mice, ferrets) prevents exposure of cryptic receptors

Oseltamivir reduces secondary infection by S. pneumoniae McCullers and Bartmess. JID 187:1000; 2003

Anti-NA antivirals Only antivirals widely used Resistance major concern in relation to use (especially oseltamivir) Resistance to zanamivir less frequent Complementary resistance profiles Zanamivir (laninamivir) effective against oseltamivir-resistant viruses

Activation of HA by proteolytic cleavage and membrane fusion at low ph ph ~5 Site of cleavage Low ph triggers: - conformational transition - promotes membrane fusion Proteolytic cleavage activates infectivity

Inhibitors of HA Fusion (tend to be subtype-specific) Tert butyl hydroquinone (TBHQ) (H3) [Bodian et al, 1993] Qinolizidines (BMY 27709) (H1 + H2) [Luo et al 1996] Substituted piperidines (H1 + H2) [Plotch et al, 1999] Arbidol (effective against influenza A and B) [Glushkov et al, 1992] Licensed in Russia in 1990 mechanism of action?

Arbidol causes increase in acid stability of HA of wt (A) - abrogated by Arbidol-resistant mutation (HA2 K51N) (B) Wild type HA2 K51N mutant Control Arbidol Conformational change occurs at 0.2 ph units lower in presence of Arbidol Leneva et al 2009

Arbidol-resistant mutations (in vitro) cause increase in ph of the conformational change in HA (A) and haemolysis (B) by the H7 virus (i.e. reduce acid stability to counteract the stabilising effect of Arbidol A. Conformational change determined by reactivity with native- and low phspecific antibodies B. Haemolysis Mutant Wild type Leneva et al 2009

Locations of Arbidol-resistance mutations - putative binding site Leneva et al 2009

Binding of TBHQ in the crystal structure of X-31 H3 HA Russell et al 2008 Structural differences explain the subtype-specificity of TBHQ inhibition of H3 but not H1 TBHQ binds to each subunit of the HA trimer Arbidol binds in a similar location in H3 HA what is the basis for its broader specificity against viruses with group 1 (H1) and group 2 (H3) HAs? No in vivo Arbidol-resistant mutants identified Is stabilisation of the HA the basis of its in vivo antiviral action?

Inhibition of influenza replication X XArbidol

Antiviral drugs licensed for use against influenza A M2 inhibitors - amantadine - rimantadine NA inhibitors - zanamivir (inhaled) - laninamivir (inhaled) - oseltamivir (oral) - peramivir (i.v.) HA fusion inhibitors arbidol (Russia)

NIMR (Mill Hill) Acknowledgements WHO Flu Centre John McCauley Rod Daniels Yi Pu Lin Victoria Gregory Lynne Whittaker Xiang Zheng Nicholas Castle Johannes Kloess Patrick Collins John Skehel Steve Gamblin Steve Wharton WHO Influenza Network Collaborating Centres National Influenza Centres CNRL Network (EISS/VirGil) HPA, Colindale Maria Zambon Angie Lackenby Sergiy Tokar Irina Leneva