Final Report Summary - FLU-PHARM (New drugs targeting influenza virus polymerase)
Executive Summary:
In response to the perennial threat of pandemic influenza, the sporadic emergence of lethal avian strains that can infect humans and growing resistance to existing drugs, the Flupharm consortium brought together a leading academic researchers studying the structure and function of the polymerase, and SMEs specialized in drug development, to develop new anti-influenza drugs targeting the polymerase complex of the virus.
The influenza virus polymerase carries out two tasks vital for viral replication. It makes copies of the its genetic material – the viral RNA – to package into new viruses that can infect other cells; and it produces viral messenger RNA based on the virus’ genetic material, which is then used by the infected cell's machinery to make viral proteins.
Over the life of the project, around 2500 molecules were tested for their ability to block two essential functions of the influenza polymerase: its endonuclease and cap snatching activities. Of these 2500 molecules, half a dozen were good enough to be tested in mice, and 4 significantly improved the animals’ survival to influenza infection, thus giving the Flupharm project its first proof-of-concept.
Thanks to its success, the project’s drug development programme was subsequently taken over by pharmaceutical industry giant Roche.
Flupharm has not only fulfilled its initial objective of identifying potential molecules to block the influenza polymerase, it has progressed our fundamental understanding of how the polymerase works, and developed more effective, higher-throughput screening tests, thus paving the way for new, faster anti-polymerase drug discovery.
Scientific highlights from the basic research part of the programme include:
• Solving the atomic scale structure of the influenza ribonucleoprotein (RNP), a complex of polymerase and viral RNA coated in nucleoprotein, which will shed light on mechanisms of viral gene expression and RNP self-multiplication in infected cells (published in Science in 2012).
• The crystal structure at 2.7 Å resolution of the entire influenza A polymerase bound to the promoter, the first such structure of any polymerase from a negative strand RNA virus (published in Nature in 2014).
• Crystal structures of influenza A (FluA) and B (FluB) polymerases in complex with the viral RNA promoter, gave the first detailed mechanistic insights into their cap-snatching and RNA replication activities (published in Nature in 2014).
Project Context and Objectives:
In recent years, the serious threat posed by influenza virus to worldwide public health has been highlighted, firstly by the ongoing low level transmission of the highly pathogenic avian H5N1 strain to humans (59% mortality in reported cases of infected humans), and secondly by the unexpected emergence in 2009 of a novel pandemic strain A/H1N1, which rapidly spread around the world.
Whilst the 2009 pandemic strain was highly contagious, fortunately, it generally resulted in only mild illness. It has not become more virulent nor has acquired the single point mutation that confers Tamiflu resistance, in the same way as some seasonal H1N1 strains have recently done. Nevertheless it was a salutatory reminder of the unpredictability of influenza viruses.
Similarly, the highly-publicized demonstrations that H5N1 strains can acquire the ability to be transmitted between ferrets via aerosols, and the emergence of human cases of avian influenza A H7N9 associated with high mortality are both further warnings of the need for preparedness against future dangerous epidemics.
In such scenarios, the time needed to generate and deploy a vaccine (~6 months in the relatively favourable case of A/H1N1 and still not a completely solved problem for H5N1), could be catastrophically costly in terms of human lives and societal disruption. One could rightly say that mankind has been lucky so far.
It is widely acknowledged that a broader choice of anti-influenza drugs is needed to treat people before a new vaccine becomes available, to treat severe influenza cases generally, as well as to counter the problem of viral resistance. In a letter published in Science on 20/3/09 several eminent virologists stated “whatever strategies are adopted, it is clear that additional anti-influenza therapeutics are urgently needed”. So far, vaccines and antivirals have targeted three influenza envelope proteins: haemagglutinin, neuraminidase, and the M2 ion channel protein. New classes of antivirals that interfere with other necessary viral processes were needed.
In this context, Flupharm’s main objective was to develop novel inhibitors targeting the cap-snatching and endonuclease activities of the influenza virus polymerase complex. Its second objective was to advance our fundamental understanding of the structure and cellular function of the influenza polymerase.
Project Results:
The Flupharm medicinal chemistry programme synthesized and tested 1348 candidate molecules for their ability to inhibit the polymerase’s endonuclease activity, while 1232 were tested against its cap-binding activity. A large number of these compounds have been characterised by physico-chemical analytic techniques. X-ray crystal structures were obtained of 30 compounds with the endonuclease domain and 48 with the cap-binding domain. In addition, 59 reference compounds and 64 dual cap/endo inhibitors were evaluated. Out of these, 6 candidate molecules were tested for their efficacy against lethal influenza infection in mice, including 4, both cap and endonuclease inhibitors, which gave the project its first proof-of-concept, by significantly improving the animals’ survival.
Various analysis tools were developed to aid the discovery of influenza polymerase inhibitors, including:
• An in vitro test suitable for medium to high-throughput screening, measuring the effects of candidate drugs on both RNA synthesis and endonuclease activities of the viral ribonucleoprotein
• A protocol to analyse the development of viral resistance against candidate drugs in mice.
• The crystal structure at 2.7 Å resolution of the entire influenza A polymerase bound to the promoter, the first such structure of any negative strand RNA virus, published in Nature in 2014. This breakthrough result shows the relative disposition of all the domains of the polymerase, gives detailed information on the polymerase active site and shows exactly how the viral RNA promoter is bound to the polymerase.
• Comparison of crystal structures of influenza A and B polymerases in complex with the viral RNA promoter, helped give the first detailed mechanistic insights into their cap-snatching and RNA replication activities, published in Nature in 2014.
• A high-resolution cryo-electron microscopy (cryo-EM) structure of the helical portion of the viral ribonucleoprotein (RNP), published in Science in 2012. This work led to a model based on the cryo-EM structure and the crystal structure of the viral nucleoprotein, explaining how the nucleoprotein fits into the RNP complex’s helical portion. This will shed light on mechanisms of viral gene expression, and RNP self-multiplication in infected cells
• The crystal structure of a monomeric form of the influenza nucleoprotein was determined giving new insight into the assembly process of RNPs.
• The first papers describing structures of complexes of designed inhibitors bound to the influenza endonuclease or cap-binding domain were published by the FluPharm consortium thus promoting structure-based optimisation of compounds.
• Using a novel replication competent influenza A virus encoding split-GFP tagged PB2 polymerase subunit that allows live cell imaging of the viral life cycle (developed by FluPharm partners), it was demonstrated that after being synthesized inside the cell nucleus, viral RNPs transiently accumulate around centrioles, before being transported across the cytoplasm. Viral RNPs move rapidly (about 1 μm/s), and directionally in the cytoplasm, and this movement is dependent on the presence of both actin and tubulin cytoskeleton networks
• Several host factors which interact with the influenza polymerase have been characterised. These include SFPQ, NXP2, RED-SMU1 and α-importins. The nuclear protein NXP2 was shown to be relevant for influenza virus infection and plays a role during virus transcription but not RNA replication. The cellular heterodimer RED-SMU1 was shown to regulate splicing of specific influenza virus mRNAs. Its deficit leads to a reduced nuclear export of virus progeny RNPs and reduced virus titres.
• A new screening system has been established based on recombinant viruses expressing the Gluc1 or Gluc2 complementation fragments of Gaussia princeps luciferase. Its usefulness has been verified with viral inhibitors and known cellular factors relevant for virus infection.
• Specific importin-α isoforms have been shown to be important for replication and pathogenicity of human or avian influenza virus in the murine model.
• Serial infection of importin-α7 knockout mice with non-pathogenic influenza virus strains leads to rapid adaptation and complete lethality. Adaptation takes place by mutations in the polymerase and NP genes and pathogenicity is increased by mutations in the surface virus genes.
• PB2 mutation E627K was shown to prevent destabilization of vRNP by the pathogen sensor RIG-I in mammalian cells and thus promotes host adaptation.
Potential Impact:
Potential impacts:
Overall, Flupharm has not only fulfilled its main objective of identifying lead compounds that block the influenza polymerase, it has opened new avenues for further anti-polymerase drug design programmes targeting multiple sites, using more effective, higher-throughput screening tests.
• The fact that Flupharm’s drug development programme was taken over by pharmaceutical giant Roche is a testament of its success, and brings us one step closer to new, more effective treatments against influenza.
• Structural studies of the heterotrimeric influenza polymerase will give tremendous insights into how the polymerase works, but also provide information about the relative disposition of the cap-binding, endonuclease and polymerase active sites, which is invaluable for the design of future drugs targeting multiple sites simultaneously.
• Studies focusing on the function of the polymerase, and its interactions with host cells will potentially provide improved cellular assays for polymerase inhibitors, contribute to understanding how inhibitors work in the cellular context, and provide new targets for future drug-design.
Dissemination activities:
• The Flupharm consortium has published 29 peer-reviewed articles, reviews and book chapters, including 3 very high profile articles in Nature and Science on the structure of the influenza polymerase and ribonucleoprotein particle (RNP). Several additional articles are currently in preparation, covering different aspects of the drug development and fundamental research programmes, thus ensuring that the scientific community as a whole can benefit from the project’s advances. Numerous presentations have been made on Flupharm results by Flupharm scientists at international scientific meeting.
• In addition to getting wide recognition within the scientific community, Flupharm has consistently promoted dissemination activities towards a general European public. Overall, out of 55 dissemination activities, 15 were aimed at civil society and / or media.
As part of this, a Science Café about flu was organized in Grenoble, several Flupharm staff presented their work to the public at open days or science festivals. Targeting a public involved in education, we organised a Flupharm-themed training course about structural biology for science teachers. Over two days, 26 secondary school science teachers from all over Europe met with Flupharm scientists, enjoying a mixture of practical and formal training based on crystallography work in Flupharm. Towards the end of the project a ‘Simpleshow’ entitled “How can structural biology help to fight flu?” was commissioned and made. This short video clip explains in a comprehensive and engaging way how structural biology can be used to help fight influenza.
Exploitation of results:
Large efforts have been made by all partners to seize any opportunity to protect Intellectual Property resulting from the project. In total, 12 composition of matter patents for polymerase inhibitors have been filed by partners Savira and EMBL, together with Roche, since the project start (see reporting section on patents in the Participant Portal).
List of Websites:
http://flupharm.eu/
In response to the perennial threat of pandemic influenza, the sporadic emergence of lethal avian strains that can infect humans and growing resistance to existing drugs, the Flupharm consortium brought together a leading academic researchers studying the structure and function of the polymerase, and SMEs specialized in drug development, to develop new anti-influenza drugs targeting the polymerase complex of the virus.
The influenza virus polymerase carries out two tasks vital for viral replication. It makes copies of the its genetic material – the viral RNA – to package into new viruses that can infect other cells; and it produces viral messenger RNA based on the virus’ genetic material, which is then used by the infected cell's machinery to make viral proteins.
Over the life of the project, around 2500 molecules were tested for their ability to block two essential functions of the influenza polymerase: its endonuclease and cap snatching activities. Of these 2500 molecules, half a dozen were good enough to be tested in mice, and 4 significantly improved the animals’ survival to influenza infection, thus giving the Flupharm project its first proof-of-concept.
Thanks to its success, the project’s drug development programme was subsequently taken over by pharmaceutical industry giant Roche.
Flupharm has not only fulfilled its initial objective of identifying potential molecules to block the influenza polymerase, it has progressed our fundamental understanding of how the polymerase works, and developed more effective, higher-throughput screening tests, thus paving the way for new, faster anti-polymerase drug discovery.
Scientific highlights from the basic research part of the programme include:
• Solving the atomic scale structure of the influenza ribonucleoprotein (RNP), a complex of polymerase and viral RNA coated in nucleoprotein, which will shed light on mechanisms of viral gene expression and RNP self-multiplication in infected cells (published in Science in 2012).
• The crystal structure at 2.7 Å resolution of the entire influenza A polymerase bound to the promoter, the first such structure of any polymerase from a negative strand RNA virus (published in Nature in 2014).
• Crystal structures of influenza A (FluA) and B (FluB) polymerases in complex with the viral RNA promoter, gave the first detailed mechanistic insights into their cap-snatching and RNA replication activities (published in Nature in 2014).
Project Context and Objectives:
In recent years, the serious threat posed by influenza virus to worldwide public health has been highlighted, firstly by the ongoing low level transmission of the highly pathogenic avian H5N1 strain to humans (59% mortality in reported cases of infected humans), and secondly by the unexpected emergence in 2009 of a novel pandemic strain A/H1N1, which rapidly spread around the world.
Whilst the 2009 pandemic strain was highly contagious, fortunately, it generally resulted in only mild illness. It has not become more virulent nor has acquired the single point mutation that confers Tamiflu resistance, in the same way as some seasonal H1N1 strains have recently done. Nevertheless it was a salutatory reminder of the unpredictability of influenza viruses.
Similarly, the highly-publicized demonstrations that H5N1 strains can acquire the ability to be transmitted between ferrets via aerosols, and the emergence of human cases of avian influenza A H7N9 associated with high mortality are both further warnings of the need for preparedness against future dangerous epidemics.
In such scenarios, the time needed to generate and deploy a vaccine (~6 months in the relatively favourable case of A/H1N1 and still not a completely solved problem for H5N1), could be catastrophically costly in terms of human lives and societal disruption. One could rightly say that mankind has been lucky so far.
It is widely acknowledged that a broader choice of anti-influenza drugs is needed to treat people before a new vaccine becomes available, to treat severe influenza cases generally, as well as to counter the problem of viral resistance. In a letter published in Science on 20/3/09 several eminent virologists stated “whatever strategies are adopted, it is clear that additional anti-influenza therapeutics are urgently needed”. So far, vaccines and antivirals have targeted three influenza envelope proteins: haemagglutinin, neuraminidase, and the M2 ion channel protein. New classes of antivirals that interfere with other necessary viral processes were needed.
In this context, Flupharm’s main objective was to develop novel inhibitors targeting the cap-snatching and endonuclease activities of the influenza virus polymerase complex. Its second objective was to advance our fundamental understanding of the structure and cellular function of the influenza polymerase.
Project Results:
The Flupharm medicinal chemistry programme synthesized and tested 1348 candidate molecules for their ability to inhibit the polymerase’s endonuclease activity, while 1232 were tested against its cap-binding activity. A large number of these compounds have been characterised by physico-chemical analytic techniques. X-ray crystal structures were obtained of 30 compounds with the endonuclease domain and 48 with the cap-binding domain. In addition, 59 reference compounds and 64 dual cap/endo inhibitors were evaluated. Out of these, 6 candidate molecules were tested for their efficacy against lethal influenza infection in mice, including 4, both cap and endonuclease inhibitors, which gave the project its first proof-of-concept, by significantly improving the animals’ survival.
Various analysis tools were developed to aid the discovery of influenza polymerase inhibitors, including:
• An in vitro test suitable for medium to high-throughput screening, measuring the effects of candidate drugs on both RNA synthesis and endonuclease activities of the viral ribonucleoprotein
• A protocol to analyse the development of viral resistance against candidate drugs in mice.
• The crystal structure at 2.7 Å resolution of the entire influenza A polymerase bound to the promoter, the first such structure of any negative strand RNA virus, published in Nature in 2014. This breakthrough result shows the relative disposition of all the domains of the polymerase, gives detailed information on the polymerase active site and shows exactly how the viral RNA promoter is bound to the polymerase.
• Comparison of crystal structures of influenza A and B polymerases in complex with the viral RNA promoter, helped give the first detailed mechanistic insights into their cap-snatching and RNA replication activities, published in Nature in 2014.
• A high-resolution cryo-electron microscopy (cryo-EM) structure of the helical portion of the viral ribonucleoprotein (RNP), published in Science in 2012. This work led to a model based on the cryo-EM structure and the crystal structure of the viral nucleoprotein, explaining how the nucleoprotein fits into the RNP complex’s helical portion. This will shed light on mechanisms of viral gene expression, and RNP self-multiplication in infected cells
• The crystal structure of a monomeric form of the influenza nucleoprotein was determined giving new insight into the assembly process of RNPs.
• The first papers describing structures of complexes of designed inhibitors bound to the influenza endonuclease or cap-binding domain were published by the FluPharm consortium thus promoting structure-based optimisation of compounds.
• Using a novel replication competent influenza A virus encoding split-GFP tagged PB2 polymerase subunit that allows live cell imaging of the viral life cycle (developed by FluPharm partners), it was demonstrated that after being synthesized inside the cell nucleus, viral RNPs transiently accumulate around centrioles, before being transported across the cytoplasm. Viral RNPs move rapidly (about 1 μm/s), and directionally in the cytoplasm, and this movement is dependent on the presence of both actin and tubulin cytoskeleton networks
• Several host factors which interact with the influenza polymerase have been characterised. These include SFPQ, NXP2, RED-SMU1 and α-importins. The nuclear protein NXP2 was shown to be relevant for influenza virus infection and plays a role during virus transcription but not RNA replication. The cellular heterodimer RED-SMU1 was shown to regulate splicing of specific influenza virus mRNAs. Its deficit leads to a reduced nuclear export of virus progeny RNPs and reduced virus titres.
• A new screening system has been established based on recombinant viruses expressing the Gluc1 or Gluc2 complementation fragments of Gaussia princeps luciferase. Its usefulness has been verified with viral inhibitors and known cellular factors relevant for virus infection.
• Specific importin-α isoforms have been shown to be important for replication and pathogenicity of human or avian influenza virus in the murine model.
• Serial infection of importin-α7 knockout mice with non-pathogenic influenza virus strains leads to rapid adaptation and complete lethality. Adaptation takes place by mutations in the polymerase and NP genes and pathogenicity is increased by mutations in the surface virus genes.
• PB2 mutation E627K was shown to prevent destabilization of vRNP by the pathogen sensor RIG-I in mammalian cells and thus promotes host adaptation.
Potential Impact:
Potential impacts:
Overall, Flupharm has not only fulfilled its main objective of identifying lead compounds that block the influenza polymerase, it has opened new avenues for further anti-polymerase drug design programmes targeting multiple sites, using more effective, higher-throughput screening tests.
• The fact that Flupharm’s drug development programme was taken over by pharmaceutical giant Roche is a testament of its success, and brings us one step closer to new, more effective treatments against influenza.
• Structural studies of the heterotrimeric influenza polymerase will give tremendous insights into how the polymerase works, but also provide information about the relative disposition of the cap-binding, endonuclease and polymerase active sites, which is invaluable for the design of future drugs targeting multiple sites simultaneously.
• Studies focusing on the function of the polymerase, and its interactions with host cells will potentially provide improved cellular assays for polymerase inhibitors, contribute to understanding how inhibitors work in the cellular context, and provide new targets for future drug-design.
Dissemination activities:
• The Flupharm consortium has published 29 peer-reviewed articles, reviews and book chapters, including 3 very high profile articles in Nature and Science on the structure of the influenza polymerase and ribonucleoprotein particle (RNP). Several additional articles are currently in preparation, covering different aspects of the drug development and fundamental research programmes, thus ensuring that the scientific community as a whole can benefit from the project’s advances. Numerous presentations have been made on Flupharm results by Flupharm scientists at international scientific meeting.
• In addition to getting wide recognition within the scientific community, Flupharm has consistently promoted dissemination activities towards a general European public. Overall, out of 55 dissemination activities, 15 were aimed at civil society and / or media.
As part of this, a Science Café about flu was organized in Grenoble, several Flupharm staff presented their work to the public at open days or science festivals. Targeting a public involved in education, we organised a Flupharm-themed training course about structural biology for science teachers. Over two days, 26 secondary school science teachers from all over Europe met with Flupharm scientists, enjoying a mixture of practical and formal training based on crystallography work in Flupharm. Towards the end of the project a ‘Simpleshow’ entitled “How can structural biology help to fight flu?” was commissioned and made. This short video clip explains in a comprehensive and engaging way how structural biology can be used to help fight influenza.
Exploitation of results:
Large efforts have been made by all partners to seize any opportunity to protect Intellectual Property resulting from the project. In total, 12 composition of matter patents for polymerase inhibitors have been filed by partners Savira and EMBL, together with Roche, since the project start (see reporting section on patents in the Participant Portal).
List of Websites:
http://flupharm.eu/