Final Report Summary - HIV ACE (Targeting assembly of infectious HIV particles)
AIDS is one of the major life threatening infectious diseases in the world today. There is an estimated 33.2 million people infected with HIV and no effective vaccine against HIV is currently available. To date, combinations of the currently available antiretroviral drug (ARVs) within highly active anti-retroviral therapy (HAART) have transformed the course of HIV infection into a chronic disease, while HAART is very effective at controlling HIV-1 replication, neither cure AIDS nor eradicate the virus. Therefore, there is a consensus that a constant supply of new antiretroviral molecules based on novel mechanisms of action is needed to respond to the major limitations of current drugs against HIV (viral resistance, viral persistence, drug toxicity). The therapeutic arsenal is expected to comprise antiviral molecules acting at almost every major step of the viral replication cycle, except for the crucial step of assembly, budding and release of HIV particles. Thus, drugs specifically targeting viral assembly and budding are urgently needed in order to complement the current pipeline of anti-HIV drugs with innovative molecules with a mechanism of action distinct from that of all drugs on the market or under late phase of development.
HIV-ACE's objective was to develop antiviral compounds acting during the assembly, budding and release of HIV particles and targeting interactions between host proteins and viral proteins instead of targeting the disruption of viral enzymes. The host proteins are much less prone to mutations than the viral enzymes, which would make it more difficult for the virus to develop resistance and escape treatment.
On the basis of novel and fully validated targets, the objectives of HIV-ACE were:
i) assay development, drug screening and pre-clinical development of small molecule inhibitors of Capsid assembly and Env incorporation, up to the stage of early drug candidates with acceptable toxicity profile determined by ADME / Tox studies;
ii) elucidation of 3D structures of these validated targets and rational drug design guided by molecular modeling and docking of inhibitors;
iii) validation and exploitation of the HIV-susceptible transgenic rat model to allow preclinical in vivo-evaluation of novel drug candidates from HIV-ACE;
iv) elucidation of the mechanisms responsible for activity of the validated inhibitors, and discovery/validation of novel targets in the budding pathway of HIV-1.
HIV-ACE consortium has been instrumental for major, recent breakthroughs concerning HIV capsid assembly, the characterisation of the mechanisms underlying BST2 antiviral activity on HIV release, the characterisation of HIV assembly in primary macrophages and the improvement of transgenic rat model. The effort of the consortium also demonstrated that it is possible to discover new HIV inhibitors based on completely different mechanism of action to currently available drugs, and on inhibition of protein-protein interactions instead of the more classical approach of inhibition of the catalytic activity of viral enzymes. A joint patent application has been filed for a compound inhibiting HIV assembly and having acceptable toxicity and a good selectivity index.
Project context and objectives:
AIDS is one of the major life-threatening infectious diseases in the world today, with over 33 million people living with the virus and 65 million people infected since the beginning of the epidemic. A constant supply of novel ARVs is needed to respond to the limitations of current drugs. Over thirty different treatments have been developed and approved since the first drug was made available in 1987. This currently approved therapeutic arsenal against HIV comprises ARVs blocking all major steps of the HIV replication, except for particle assembly and budding. Members of HIV-ACE were instrumental in recent breakthroughs concerning virus assembly and Envelope incorporation into virions. Hence the goal of the consortium is to translate progress in understanding the mechanisms involved in HIV-1 capsid assembly, budding and Env incorporation into innovative ARV.
Project results:
The ordered assembly of infectious viral particles and the release of progeny virions from the host cell are crucial steps in virus replication. In the case of HIV, this involves the assembly of a protein capsid protecting the viral genome, the envelopment of the capsid by a lipid membrane which buds off from the membrane of the producing cell, and the incorporation of virus glycoprotein into this lipid envelope. The aim of WP1 was to identify small molecule inhibitors which interfere either with capsid assembly, viral budding or glycoprotein incorporation, respectively, and thereby inhibit HIV replication. In the first step, partners 1, 2 and 5 (partners' name at the end of the article) have established different in vitro assay systems suitable for medium to high throughput screening and have employed these systems to screen large libraries of small molecule compounds (Hermle et al., BMC Biotechnology, 2010). These efforts resulted in the identification of small molecules specifically interfering with HIV CA:CAI interaction, with HIV Env:TIP47 interaction, or with HIV particle release in tissue culture, respectively. These initial hits were subsequently characterised with respect to physicochemical properties, cytotoxicity and interaction with the viral protein of interest. Complementary cell biological and virological studies were performed in order to elucidate their mechanism of action. Antiviral activity of the compounds was confirmed by measuring their effect on HIV replication in tissue culture. Starting from initial hits with antiviral potential in vitro, iterative cycles of medicinal chemistry and inhibitor testing were performed by partners 1, 2, 5, and 7 in order to improve the inhibitory efficacy and the selectivity index of the respective compounds. While the antiviral potential of release inhibitors selected in tissue culture, as well as of inhibitors of Env:TIP47 interaction proved to be limited in these studies, two modified inhibitors of HIV capsid assembly with a significantly improved selectivity index as compared to the initial hits have entered ADME / Tox studies in mice, which will guide their further development. If successful, this would yield anti-HIV drugs acting through a mechanism different from that of all currently available antiretrovirals. Partner 2 and 7 are currently preparing a joint patent application for protection of intellectual property through the subsequent stages of development.
The aim of the rational drug design task force (WP2) was to elucidate the three-dimensional structures of potential targets validated in WP1, and to propose, through a rational drug design approach and the determination of target / ligand complex structures, new potential inhibitors molecules. This work package was delegated to partners 1, partners 2 and partners 6. Previous to this project, the crystal structure of the C-terminal domain of the HIV-1 capsid protein (CCA) was determined in complex with a capsid-assembly inhibitor (CAI) peptide. This peptide had been identified by partner 2, and the structure was done in collaboration with partner 6. Under WP2, in vitro and virological analyses carried out by partner 2 on mutated virus on capsid residues involved in CAI binding site allowed the characterisation of the residues forming a reactive groove crucial for the capsid assembly and maturation. By a crystallographic approach, partner 6 determined the structures of C-CA mutants alone or in complex with the CAI peptide (Bartonova et al., J. Biol. Chem., 2008 ). The structures of complexes between C-CA and CAI peptide showed that the peptide inhibited assembly not only by steric hindrance, but also by altering the CCA dimerisation interface. Because small peptides are in general not viable as drugs, a screening of chemical compounds was done by partner 7 to identify putative drugs that bind to this pocket. Six competitors of the inhibitory peptide, obtained from partner 7, were tested in crystallisation. In one case, crystals were obtained with the ligand positioned in the pocket. Additional work by WP2 partners is now needed to improve crystal quality and solve the structure of these complexes.
Under WP3, the suitability of the multi-transgenic rat model of HIV infection was demonstrated for testing late-phase drugs by means of a successful proof-of-principle validation of a prototypic protease inhibitor in vivo. Furthermore, remaining limitations to highly efficient HIV-1 replication in rat cells were identified and further characterized (Goffinet, Schmidt et al. J. Virol 2010a). In particular, the potent and Vpu-insensitive restriction factor rat CD317 imposes a relevant barrier to HIV-1 release in this species (Goffinet et al., Cell Host Microbe 2009). The mechanism by which Vpu counteracts CD317 in human cells was investigated in some detail and identified a subversion of intracellular trafficking of recycling and newly synthesized CD317/BST2 molecules as the key effector function of Vpu to downregulate the restriction factor from the plasma membrane (Goffinet, Homann, et al., J. Virol 2010b; Erikson et al., PNAS, 2011). Additional works complete the discoveries done by the consortium on CD317/BST2 restriction factor. Partner 3 in WP4 has shown that viruses related to HIV-1 that lack the vpu gene are able to overcome CD317/BST2 by a new activity encoded by SIV env (Gupta et al., PNAS, 2009 ). Moreover, two studies from partners 1 and 3 report the role of the ESCRT machinery and the endocytic pathway in the mechanism by which Vpu counteracts CD317/BST2 and promotes HIV-1 dissemination (Caillet, Janvier et al., PloS Pathogens, 2011 ). This knowledge now allows us to either introduce additional genetic changes in the host or evolve a rat CD317-targeting Vpu to overcome this limitation in rats.
Overall, this work demonstrates that novel antiviral compounds targeting steps in the HIV-1 replication cycle from entry, over reverse transcription and integration to virion egress can in principle be evaluated in this readily accessible, immunocompetent small animal model. Furthermore, strategies to further optimize replication in this animal model by overcoming the rat CD317 restriction have been designed.
The overall objectives of WP4 (partners 1, 3 and 9) were to obtain fundamental information concerning the structure and function of host factors that are required for specific aspects of HIV-1 assembly, including the interaction between TIP47 and HIV-1 Env. Such information was obtained and will be critical to guide future development of drugs inhibiting this interaction that is critical to HIV-1 replication. Additionally, the role of TIP47 was confirmed and characterized in macrophages, one of the important cell types of the immune system that is targeted for infection by HIV-1 (Bauby et al., Traffic 2010). Other goals of the project included attempts to identify new host factors that regulate the production of infectious HIV-1. Several Rab proteins important generally for transport within cells were screened and Rab7a was found to play an important role in the formation of fully infectious virion particles and needed for one of the viral accessory proteins, Vpu, to counteract CD317/BST2 restriction factor and produce new virion particles (Caillet et al., Plos Pathogens, 2011 ). Rab7a, then, becomes a potential new target for inhibitors that block HIV-1 virion assembly. Additionally it was shown that the drug cyclosporine has a specific inhibitory effect on HIV-1 virion particle production and blocks the incorporation of the Env proteins essential for infectivity (Sokolskaja et al., J. Virol, 2010; Bernasconi et al., Plos One, 2010). This is the starting point for development of new HIV-1 inhibitors. Finally, number of additional fundamental discoveries made by the WP4 might be exploited in the future to inhibit HIV-1 assembly. These include the discovery of a discrete compartment within macrophages where HIV-1 assembles new virions (Pelchens-Mathews et al., Traffic, 2011) and the finding that the same transport pathway, the ESCRT machinery, that is required for HIV-1 virions to assemble also regulates a natural, cellular inhibitor of HIV-1 release known as CD317/BST2 (Janvier et al., PloS Pathogens, 2011).
Systems have also been developed to allow the direct observation of the assembly of individual HIV virions in live cells, which enables us to measure kinetics of intracellular HIV assembly and the interaction with cellular proteins involved in HIV release (Ivanchenko et al., PloS Pathogens, 2009; Eckhardt et al., PLos One, 2011; Baumgärtel et al., Nature cell biology, 2011 ). This system is being used for further characterisation of assembly inhibitory compounds.
In conclusion, HIV-ACE consortium has been instrumental for major, recent breakthroughs concerning HIV capsid assembly, BST-2 restriction, the characterisation of HIV assembly in primary macrophages and the improvement of transgenic rat model. The effort of the consortium also demonstrated that it is possible to discover new HIV inhibitors based on completely different mechanism of action to currently available drugs, and on inhibition of protein-protein interactions instead of the more classical approach of inhibition of the catalytic activity of viral enzymes.
Potential impact:
In conclusion, HIV-ACE consortium has been instrumental for major, recent breakthroughs concerning HIV capsid assembly, BST-2 restriction, the characterisation of HIV assembly in primary macrophages and the improvement of transgenic rat model. The effort of the consortium also demonstrated that it is possible to discover new HIV inhibitors based on completely different mechanism of action to currently available drugs, and on inhibition of protein-protein interactions instead of the more classical approach of inhibition of the catalytic activity of viral enzymes.
Furthermore a patent application has been filed for a compound able to disrupt the HIV-1 Capsid assembly.
Project website: http://hiv-ace.eu/
Contact:
Scientific coordinator: Clarisse Berlioz Torrent - clarisse.berlioz@inserm.fr
Project manager: Ibrahima Guillard - ibrahima.guillard@inserm-transfert.fr
Coordinator - partner 1: Institut National de la Santé et de la Recherche médicale
Clarisse Berlioz-Torrent - France - clarisse.berlioz@inserm.fr
Partner 2: Universitaetsklinikum Heidelberg
Hans-Georg Kraeusslich - Barbara Mueller - Oliver Keppler - Germany
Hans-Georg_Kraeusslich@med.uni-heidelberg.de; Barbara_Mueller@med.uni-heidelberg.de Oliver_Keppler@med.uni-heidelberg.de
Partner 3: Medical Research Council
Mark Marsh - United Kingdom - m.marsh@ucl.ac.uk
Partner 6: Institut Pasteur
Félix Rey - France - rey@pasteur.fr
Partner 7: Institute of organic chemistry and biochemistry of the ASCR
Jan Konvalinka - Czech Republic - konval@uochb.cas.cz
Partner 8: Inserm-Transfert SA
Ibrahima Guillard - France - ibrahima.guillard@inserm-transfert.fr
Partner 9: Université de Geneve
Jeremy Luban - Switzerland - jeremy.luban@unige.ch
Partner 10: Biodim
Richard Benarous - France - richard.benarous@mutabilis.fr
HIV-ACE's objective was to develop antiviral compounds acting during the assembly, budding and release of HIV particles and targeting interactions between host proteins and viral proteins instead of targeting the disruption of viral enzymes. The host proteins are much less prone to mutations than the viral enzymes, which would make it more difficult for the virus to develop resistance and escape treatment.
On the basis of novel and fully validated targets, the objectives of HIV-ACE were:
i) assay development, drug screening and pre-clinical development of small molecule inhibitors of Capsid assembly and Env incorporation, up to the stage of early drug candidates with acceptable toxicity profile determined by ADME / Tox studies;
ii) elucidation of 3D structures of these validated targets and rational drug design guided by molecular modeling and docking of inhibitors;
iii) validation and exploitation of the HIV-susceptible transgenic rat model to allow preclinical in vivo-evaluation of novel drug candidates from HIV-ACE;
iv) elucidation of the mechanisms responsible for activity of the validated inhibitors, and discovery/validation of novel targets in the budding pathway of HIV-1.
HIV-ACE consortium has been instrumental for major, recent breakthroughs concerning HIV capsid assembly, the characterisation of the mechanisms underlying BST2 antiviral activity on HIV release, the characterisation of HIV assembly in primary macrophages and the improvement of transgenic rat model. The effort of the consortium also demonstrated that it is possible to discover new HIV inhibitors based on completely different mechanism of action to currently available drugs, and on inhibition of protein-protein interactions instead of the more classical approach of inhibition of the catalytic activity of viral enzymes. A joint patent application has been filed for a compound inhibiting HIV assembly and having acceptable toxicity and a good selectivity index.
Project context and objectives:
AIDS is one of the major life-threatening infectious diseases in the world today, with over 33 million people living with the virus and 65 million people infected since the beginning of the epidemic. A constant supply of novel ARVs is needed to respond to the limitations of current drugs. Over thirty different treatments have been developed and approved since the first drug was made available in 1987. This currently approved therapeutic arsenal against HIV comprises ARVs blocking all major steps of the HIV replication, except for particle assembly and budding. Members of HIV-ACE were instrumental in recent breakthroughs concerning virus assembly and Envelope incorporation into virions. Hence the goal of the consortium is to translate progress in understanding the mechanisms involved in HIV-1 capsid assembly, budding and Env incorporation into innovative ARV.
Project results:
The ordered assembly of infectious viral particles and the release of progeny virions from the host cell are crucial steps in virus replication. In the case of HIV, this involves the assembly of a protein capsid protecting the viral genome, the envelopment of the capsid by a lipid membrane which buds off from the membrane of the producing cell, and the incorporation of virus glycoprotein into this lipid envelope. The aim of WP1 was to identify small molecule inhibitors which interfere either with capsid assembly, viral budding or glycoprotein incorporation, respectively, and thereby inhibit HIV replication. In the first step, partners 1, 2 and 5 (partners' name at the end of the article) have established different in vitro assay systems suitable for medium to high throughput screening and have employed these systems to screen large libraries of small molecule compounds (Hermle et al., BMC Biotechnology, 2010). These efforts resulted in the identification of small molecules specifically interfering with HIV CA:CAI interaction, with HIV Env:TIP47 interaction, or with HIV particle release in tissue culture, respectively. These initial hits were subsequently characterised with respect to physicochemical properties, cytotoxicity and interaction with the viral protein of interest. Complementary cell biological and virological studies were performed in order to elucidate their mechanism of action. Antiviral activity of the compounds was confirmed by measuring their effect on HIV replication in tissue culture. Starting from initial hits with antiviral potential in vitro, iterative cycles of medicinal chemistry and inhibitor testing were performed by partners 1, 2, 5, and 7 in order to improve the inhibitory efficacy and the selectivity index of the respective compounds. While the antiviral potential of release inhibitors selected in tissue culture, as well as of inhibitors of Env:TIP47 interaction proved to be limited in these studies, two modified inhibitors of HIV capsid assembly with a significantly improved selectivity index as compared to the initial hits have entered ADME / Tox studies in mice, which will guide their further development. If successful, this would yield anti-HIV drugs acting through a mechanism different from that of all currently available antiretrovirals. Partner 2 and 7 are currently preparing a joint patent application for protection of intellectual property through the subsequent stages of development.
The aim of the rational drug design task force (WP2) was to elucidate the three-dimensional structures of potential targets validated in WP1, and to propose, through a rational drug design approach and the determination of target / ligand complex structures, new potential inhibitors molecules. This work package was delegated to partners 1, partners 2 and partners 6. Previous to this project, the crystal structure of the C-terminal domain of the HIV-1 capsid protein (CCA) was determined in complex with a capsid-assembly inhibitor (CAI) peptide. This peptide had been identified by partner 2, and the structure was done in collaboration with partner 6. Under WP2, in vitro and virological analyses carried out by partner 2 on mutated virus on capsid residues involved in CAI binding site allowed the characterisation of the residues forming a reactive groove crucial for the capsid assembly and maturation. By a crystallographic approach, partner 6 determined the structures of C-CA mutants alone or in complex with the CAI peptide (Bartonova et al., J. Biol. Chem., 2008 ). The structures of complexes between C-CA and CAI peptide showed that the peptide inhibited assembly not only by steric hindrance, but also by altering the CCA dimerisation interface. Because small peptides are in general not viable as drugs, a screening of chemical compounds was done by partner 7 to identify putative drugs that bind to this pocket. Six competitors of the inhibitory peptide, obtained from partner 7, were tested in crystallisation. In one case, crystals were obtained with the ligand positioned in the pocket. Additional work by WP2 partners is now needed to improve crystal quality and solve the structure of these complexes.
Under WP3, the suitability of the multi-transgenic rat model of HIV infection was demonstrated for testing late-phase drugs by means of a successful proof-of-principle validation of a prototypic protease inhibitor in vivo. Furthermore, remaining limitations to highly efficient HIV-1 replication in rat cells were identified and further characterized (Goffinet, Schmidt et al. J. Virol 2010a). In particular, the potent and Vpu-insensitive restriction factor rat CD317 imposes a relevant barrier to HIV-1 release in this species (Goffinet et al., Cell Host Microbe 2009). The mechanism by which Vpu counteracts CD317 in human cells was investigated in some detail and identified a subversion of intracellular trafficking of recycling and newly synthesized CD317/BST2 molecules as the key effector function of Vpu to downregulate the restriction factor from the plasma membrane (Goffinet, Homann, et al., J. Virol 2010b; Erikson et al., PNAS, 2011). Additional works complete the discoveries done by the consortium on CD317/BST2 restriction factor. Partner 3 in WP4 has shown that viruses related to HIV-1 that lack the vpu gene are able to overcome CD317/BST2 by a new activity encoded by SIV env (Gupta et al., PNAS, 2009 ). Moreover, two studies from partners 1 and 3 report the role of the ESCRT machinery and the endocytic pathway in the mechanism by which Vpu counteracts CD317/BST2 and promotes HIV-1 dissemination (Caillet, Janvier et al., PloS Pathogens, 2011 ). This knowledge now allows us to either introduce additional genetic changes in the host or evolve a rat CD317-targeting Vpu to overcome this limitation in rats.
Overall, this work demonstrates that novel antiviral compounds targeting steps in the HIV-1 replication cycle from entry, over reverse transcription and integration to virion egress can in principle be evaluated in this readily accessible, immunocompetent small animal model. Furthermore, strategies to further optimize replication in this animal model by overcoming the rat CD317 restriction have been designed.
The overall objectives of WP4 (partners 1, 3 and 9) were to obtain fundamental information concerning the structure and function of host factors that are required for specific aspects of HIV-1 assembly, including the interaction between TIP47 and HIV-1 Env. Such information was obtained and will be critical to guide future development of drugs inhibiting this interaction that is critical to HIV-1 replication. Additionally, the role of TIP47 was confirmed and characterized in macrophages, one of the important cell types of the immune system that is targeted for infection by HIV-1 (Bauby et al., Traffic 2010). Other goals of the project included attempts to identify new host factors that regulate the production of infectious HIV-1. Several Rab proteins important generally for transport within cells were screened and Rab7a was found to play an important role in the formation of fully infectious virion particles and needed for one of the viral accessory proteins, Vpu, to counteract CD317/BST2 restriction factor and produce new virion particles (Caillet et al., Plos Pathogens, 2011 ). Rab7a, then, becomes a potential new target for inhibitors that block HIV-1 virion assembly. Additionally it was shown that the drug cyclosporine has a specific inhibitory effect on HIV-1 virion particle production and blocks the incorporation of the Env proteins essential for infectivity (Sokolskaja et al., J. Virol, 2010; Bernasconi et al., Plos One, 2010). This is the starting point for development of new HIV-1 inhibitors. Finally, number of additional fundamental discoveries made by the WP4 might be exploited in the future to inhibit HIV-1 assembly. These include the discovery of a discrete compartment within macrophages where HIV-1 assembles new virions (Pelchens-Mathews et al., Traffic, 2011) and the finding that the same transport pathway, the ESCRT machinery, that is required for HIV-1 virions to assemble also regulates a natural, cellular inhibitor of HIV-1 release known as CD317/BST2 (Janvier et al., PloS Pathogens, 2011).
Systems have also been developed to allow the direct observation of the assembly of individual HIV virions in live cells, which enables us to measure kinetics of intracellular HIV assembly and the interaction with cellular proteins involved in HIV release (Ivanchenko et al., PloS Pathogens, 2009; Eckhardt et al., PLos One, 2011; Baumgärtel et al., Nature cell biology, 2011 ). This system is being used for further characterisation of assembly inhibitory compounds.
In conclusion, HIV-ACE consortium has been instrumental for major, recent breakthroughs concerning HIV capsid assembly, BST-2 restriction, the characterisation of HIV assembly in primary macrophages and the improvement of transgenic rat model. The effort of the consortium also demonstrated that it is possible to discover new HIV inhibitors based on completely different mechanism of action to currently available drugs, and on inhibition of protein-protein interactions instead of the more classical approach of inhibition of the catalytic activity of viral enzymes.
Potential impact:
In conclusion, HIV-ACE consortium has been instrumental for major, recent breakthroughs concerning HIV capsid assembly, BST-2 restriction, the characterisation of HIV assembly in primary macrophages and the improvement of transgenic rat model. The effort of the consortium also demonstrated that it is possible to discover new HIV inhibitors based on completely different mechanism of action to currently available drugs, and on inhibition of protein-protein interactions instead of the more classical approach of inhibition of the catalytic activity of viral enzymes.
Furthermore a patent application has been filed for a compound able to disrupt the HIV-1 Capsid assembly.
Project website: http://hiv-ace.eu/
Contact:
Scientific coordinator: Clarisse Berlioz Torrent - clarisse.berlioz@inserm.fr
Project manager: Ibrahima Guillard - ibrahima.guillard@inserm-transfert.fr
Coordinator - partner 1: Institut National de la Santé et de la Recherche médicale
Clarisse Berlioz-Torrent - France - clarisse.berlioz@inserm.fr
Partner 2: Universitaetsklinikum Heidelberg
Hans-Georg Kraeusslich - Barbara Mueller - Oliver Keppler - Germany
Hans-Georg_Kraeusslich@med.uni-heidelberg.de; Barbara_Mueller@med.uni-heidelberg.de Oliver_Keppler@med.uni-heidelberg.de
Partner 3: Medical Research Council
Mark Marsh - United Kingdom - m.marsh@ucl.ac.uk
Partner 6: Institut Pasteur
Félix Rey - France - rey@pasteur.fr
Partner 7: Institute of organic chemistry and biochemistry of the ASCR
Jan Konvalinka - Czech Republic - konval@uochb.cas.cz
Partner 8: Inserm-Transfert SA
Ibrahima Guillard - France - ibrahima.guillard@inserm-transfert.fr
Partner 9: Université de Geneve
Jeremy Luban - Switzerland - jeremy.luban@unige.ch
Partner 10: Biodim
Richard Benarous - France - richard.benarous@mutabilis.fr
