- On their 3q2011 conference call, Astex (formerly SuperGen) revealed that data from their preclinical Hepatitis C Virus (HCV) program will be presented at the AASLD meeting in San Francisco (My apologies for not locating these abstracts when I posted the $GSK $LGND abstract for Promacta in HCV).
- Find more detailed company info on the ASTX research page.
- See more upcoming scientific conference and investor presentations for ASTX and other biotech companies I follow.
- Keep reading below for the complete abstracts about the new ASTX HCV NS3 allosteric inhibitors program - comments appreciated from those who follow the HCV space more closely than I do.
- Note that this program, being outside ASTX's main focus in oncology, will likely be partnered before entering the clinic. An IND is forecast for 2012.
Poster Session - November 5, 2011 #371
Cell based replicon validation of novel HCV NS3 allosteric inhibitors result in a new therapeutic approach
S. Saalau-Bethell1; B. Graham2; M. G. Carr4; G. Chessari5; J. Coyle3; C. Hamlett4; S. D. Hiscock4; P. Pathuri1; M. Reader4; S. J. Rich2; N. Thompson2; A. J. Woodhead4
1. Structural Biology, Astex Therapeutics, Cambridge, United Kingdom. 2. Biology, Astex Therapeutics, Cambridge, United Kingdom. 3. Biophysics, Astex Therapeutics, Cambridge, United Kingdom. 4. Chemistry, Astex Therapeutics, Cambridge, United Kingdom. 5. Computational Chemistry and Informatics, Astex Therapeutics, Cambridge, United Kingdom.
Background: A fragment-based drug discovery approach focused on the HCV full length NS3 protease-helicase enzyme resulted in the discovery of a novel binding site on the protein. Compounds binding at the new site were shown to inhibit the enzyme’s protease activity. Structure-based optimization efforts on the ligands aimed to improve the affinity of the molecules to enable testing in the subgenomic replicon system, as robust activity of the compounds in the replicon system was essential to test the relevance of the new site to viral RNA replication. Methods: Huh-7 cells persistently infected with a subgenomic genotype 1b HCV-RNA construct containing a firefly Luciferase reporter gene (pFKI3889luc-ubi-neo/NS3-3’/ET), were used to determine the cell based antiviral activity of compounds. HCV replication was quantified by luciferase activity as an indirect readout of HCV RNA load. Semilog dilutions of the compounds were added to the cells and incubated for 72 hours for EC50 and CC50 determinations. Cytotoxicity was measured with Alamar Blue. Luciferase readouts were confirmed by quantitative rtPCR using an NS5b primer. Resistant mutations against compound A were elicited by culturing cells in the presence of high concentrations of compound for an extended period of time. RNA from emerging colonies was isolated and sequenced. Identified mutations were incorporated into the wild-type enzyme by site directed mutagenesis for confirmation.
Results: Compounds binding at the new site inhibit the enzyme’s protease catalytic activity in vitro and show robust antiviral activity in the cell based genotype 1b replicon system with EC50 values in the 1-50nM range and >1000 fold cytotoxicity window. Long term selective pressure on the replicon at high concentrations of compound A resulted in mutations that map to the allosteric site. Incorporation of these mutations into the wild-type enzyme via site directed mutagenesis resulted in poorer IC50 values for the compounds, further validating the site and mode of action. Crystal structures of the identified mutations in the protein structure show how the size and shape of the pocket changes and explain the reduced affinity of the compounds. The identified mutations did not affect the affinity of protease active site inhibitors.
Conclusions: Mutants raised in the replicon system by selective pressure from compound A demonstrate that the site is relevant to viral RNA replication. Inhibitors that target this site display a new mode of action and represent a potential new therapeutic approach for the treatment of HCV infection.
Cell based replicon validation of novel HCV NS3 allosteric inhibitors result in a new therapeutic approach
S. Saalau-Bethell1; B. Graham2; M. G. Carr4; G. Chessari5; J. Coyle3; C. Hamlett4; S. D. Hiscock4; P. Pathuri1; M. Reader4; S. J. Rich2; N. Thompson2; A. J. Woodhead4
1. Structural Biology, Astex Therapeutics, Cambridge, United Kingdom. 2. Biology, Astex Therapeutics, Cambridge, United Kingdom. 3. Biophysics, Astex Therapeutics, Cambridge, United Kingdom. 4. Chemistry, Astex Therapeutics, Cambridge, United Kingdom. 5. Computational Chemistry and Informatics, Astex Therapeutics, Cambridge, United Kingdom.
Background: A fragment-based drug discovery approach focused on the HCV full length NS3 protease-helicase enzyme resulted in the discovery of a novel binding site on the protein. Compounds binding at the new site were shown to inhibit the enzyme’s protease activity. Structure-based optimization efforts on the ligands aimed to improve the affinity of the molecules to enable testing in the subgenomic replicon system, as robust activity of the compounds in the replicon system was essential to test the relevance of the new site to viral RNA replication. Methods: Huh-7 cells persistently infected with a subgenomic genotype 1b HCV-RNA construct containing a firefly Luciferase reporter gene (pFKI3889luc-ubi-neo/NS3-3’/ET), were used to determine the cell based antiviral activity of compounds. HCV replication was quantified by luciferase activity as an indirect readout of HCV RNA load. Semilog dilutions of the compounds were added to the cells and incubated for 72 hours for EC50 and CC50 determinations. Cytotoxicity was measured with Alamar Blue. Luciferase readouts were confirmed by quantitative rtPCR using an NS5b primer. Resistant mutations against compound A were elicited by culturing cells in the presence of high concentrations of compound for an extended period of time. RNA from emerging colonies was isolated and sequenced. Identified mutations were incorporated into the wild-type enzyme by site directed mutagenesis for confirmation.
Results: Compounds binding at the new site inhibit the enzyme’s protease catalytic activity in vitro and show robust antiviral activity in the cell based genotype 1b replicon system with EC50 values in the 1-50nM range and >1000 fold cytotoxicity window. Long term selective pressure on the replicon at high concentrations of compound A resulted in mutations that map to the allosteric site. Incorporation of these mutations into the wild-type enzyme via site directed mutagenesis resulted in poorer IC50 values for the compounds, further validating the site and mode of action. Crystal structures of the identified mutations in the protein structure show how the size and shape of the pocket changes and explain the reduced affinity of the compounds. The identified mutations did not affect the affinity of protease active site inhibitors.
Conclusions: Mutants raised in the replicon system by selective pressure from compound A demonstrate that the site is relevant to viral RNA replication. Inhibitors that target this site display a new mode of action and represent a potential new therapeutic approach for the treatment of HCV infection.
Poster Session #352: November 5, 2011
Discovery of a novel allosteric binding site on the full length HCV NS3/4a enzyme by fragment-based X-ray screening
S. Saalau-Bethell1; J. Coyle3; P. A. Williams1; C. J. Richardson2; A. J. Woodhead4; G. Chessari5; M. G. Carr4; M. Frederickson4; S. D. Hiscock4; F. P. Holding3; H. Jhoti1; R. McMenamin2; C. W. Murray5; D. C. Rees4; S. J. Rich2; M. L. Verdonk5; M. Vinkovic1; J. Yon1
1. Structural Biology, Astex Therapeutics, Cambridge, United Kingdom.
2. Biology, Astex Therapeutics, Cambridge, United Kingdom.
3. Biophysics, Astex Therapeutics, Cambridge, United Kingdom.
4. Chemistry, Astex Therapeutics, Cambridge, United Kingdom. 5. Computational Chemistry and Informatics, Astex Therapeutics, Cambridge, United Kingdom.
Background: The Hepatitis C virus NS3 protein is a bi-functional enzyme with protease and helicase activities, both of which are essential for viral replication and consequently attractive targets for therapeutic intervention. The functional domains of the protein remain attached in vivo and published biochemical studies have demonstrated that the domains profoundly influence each other's enzymatic parameters. We have identified a novel binding site on the full length NS3 protein through the application of our proprietary fragment-based screening platform, PyramidTM
Methods: We established a robust system to produce high quality and reproducible crystals of the full length HCV NS3/4a protein. These crystals were soaked with cocktails of fragments (low molecular weight compounds) at high concentrations (50mM) and the protein-ligand crystal structures were solved to high resolution in order to identify hits. The affinities of the resulting hits for the full length protein were measured directly by isothermal titration calorimetry (ITC) and in a fluorescence-based protease activity assay utilising a substrate peptide derived from the NS4A/B cleavage site (Anaspec).
Results: Crystal structures of the protein in complex with a range of fragment hits unambiguously demonstrate the existence of a new binding site, which is distinct from the active site currently targeted by telaprevir, boceprevir and other protease inhibitors in clinical development. The compounds inhibited the full length protein but showed no inhibition of the isolated protease domain, consistent with the observed binding mode. Sequence analysis revealed that the residues lining this novel site are conserved across all genotypes, suggesting that this pocket may have a functional role. Structure-based drug design was used to improve the affinity of the compounds against the full length enzyme and resulted in concentration dependent inhibition of protease activity with IC50 values in the range of 10-100nM [see above abstract]. The functional inhibition is consistent with the low nanomolar Kd’s determined by ITC and supports a model in which compounds binding at the novel site exert their inhibitory action by obstructing conformational changes necessary for enzyme activity.
Conclusions: Ligands binding at this novel allosteric site inhibit enzyme function via a new mode of action with nanomolar potency. These compounds show in vitro activity against a number of resistance variants elicited by protease active site inhibitors reported in the literature and in clinical trials. The results warrant further exploration of these molecules as a potential new class of therapeutic agents.
Discovery of a novel allosteric binding site on the full length HCV NS3/4a enzyme by fragment-based X-ray screening
S. Saalau-Bethell1; J. Coyle3; P. A. Williams1; C. J. Richardson2; A. J. Woodhead4; G. Chessari5; M. G. Carr4; M. Frederickson4; S. D. Hiscock4; F. P. Holding3; H. Jhoti1; R. McMenamin2; C. W. Murray5; D. C. Rees4; S. J. Rich2; M. L. Verdonk5; M. Vinkovic1; J. Yon1
1. Structural Biology, Astex Therapeutics, Cambridge, United Kingdom.
2. Biology, Astex Therapeutics, Cambridge, United Kingdom.
3. Biophysics, Astex Therapeutics, Cambridge, United Kingdom.
4. Chemistry, Astex Therapeutics, Cambridge, United Kingdom. 5. Computational Chemistry and Informatics, Astex Therapeutics, Cambridge, United Kingdom.
Background: The Hepatitis C virus NS3 protein is a bi-functional enzyme with protease and helicase activities, both of which are essential for viral replication and consequently attractive targets for therapeutic intervention. The functional domains of the protein remain attached in vivo and published biochemical studies have demonstrated that the domains profoundly influence each other's enzymatic parameters. We have identified a novel binding site on the full length NS3 protein through the application of our proprietary fragment-based screening platform, PyramidTM
Methods: We established a robust system to produce high quality and reproducible crystals of the full length HCV NS3/4a protein. These crystals were soaked with cocktails of fragments (low molecular weight compounds) at high concentrations (50mM) and the protein-ligand crystal structures were solved to high resolution in order to identify hits. The affinities of the resulting hits for the full length protein were measured directly by isothermal titration calorimetry (ITC) and in a fluorescence-based protease activity assay utilising a substrate peptide derived from the NS4A/B cleavage site (Anaspec).
Results: Crystal structures of the protein in complex with a range of fragment hits unambiguously demonstrate the existence of a new binding site, which is distinct from the active site currently targeted by telaprevir, boceprevir and other protease inhibitors in clinical development. The compounds inhibited the full length protein but showed no inhibition of the isolated protease domain, consistent with the observed binding mode. Sequence analysis revealed that the residues lining this novel site are conserved across all genotypes, suggesting that this pocket may have a functional role. Structure-based drug design was used to improve the affinity of the compounds against the full length enzyme and resulted in concentration dependent inhibition of protease activity with IC50 values in the range of 10-100nM [see above abstract]. The functional inhibition is consistent with the low nanomolar Kd’s determined by ITC and supports a model in which compounds binding at the novel site exert their inhibitory action by obstructing conformational changes necessary for enzyme activity.
Conclusions: Ligands binding at this novel allosteric site inhibit enzyme function via a new mode of action with nanomolar potency. These compounds show in vitro activity against a number of resistance variants elicited by protease active site inhibitors reported in the literature and in clinical trials. The results warrant further exploration of these molecules as a potential new class of therapeutic agents.
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