Here is the press release issued this morning.
Visit the ASTX research page for info on all of Astex's clinical programs (most of the pipeline is cancer focused, and thus they will seek to out-license this HCV candidate early in development).
See below for complete text of the two abstracts being presented on 4/20 poster session.
Pre-clinical characterisation of a novel direct acting antiviral agent with a new mode of action
Author: N. Wilsher*, S. Saalau-Bethell, A. Woodhead, A. Millemaggi, B. Graham, C. Hamlett, C. Murray, D. Norton, D. Rees, E. Chiarparin, H. Willems, J. Lewis, L. Fazal, M. Carr, M. Hodder, M. Reader, N. Thompson, P. Pathuri, S. Rich, S. Hiscock, V. Martins, M. Sanders
Affiliation: Astex Pharmaceuticals, Cambridge, UK. *email@example.com
Background: The HCV NS3 protein is a bi-functional enzyme with protease and helicase activities, both of which are essential for viral replication. X-ray crystallographic fragment-based screening of the full length NS3/4a protein identified a novel allosteric pocket at the interface of the functional domains. Subsequent structure-based drug design led to the discovery of a series of potent allosteric inhibitors with promising drug-like properties.
Methods: Huh-7 cells persistently infected with a subgenomic genotype 1b HCV-RNA construct containing a firefly Luciferase reporter gene, were used to determine the cell based antiviral activity of compounds. Resistant mutations were elicited by culturing cells in the presence of high concentrations of compound 1 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. Pharmacokinetics were characterised in rat and dog, administered with compound at 1 (IV) and 5 (PO) mg/kg. Plasma samples were analysed by positive ion LC / MSMS using reverse-phase chromatography following protein precipitation.
Results: Compounds binding at the novel site 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 compound concentrations resulted in mutations that map to the allosteric site, confirming the physiological relevance of the allosteric site. Unlike peptide derived active site inhibitors, this new class of compounds are low molecular weight (< 400), low lipophilicity and highly water soluble, with in vivo DMPK properties predictive of once daily oral dosing in humans.
Conclusions: Using fragment screening we have identified a novel binding site on the full length NS3/4a protein. Structure based drug design and profiling compounds in the replicon assay allowed us to demonstrate the drugability of this novel site. Optimisation of cellular activity alongside DMPK properties and off target activities resulted in the identification of the non-peptidic / non-macrocyclic pre-clinical candidate.
Fragment based discovery and genotypic profiling of a series of direct acting antiviral agents with a novel mode of action
Author: S. Saalau-Bethell*, A. Woodhead, B. Graham, C. Richardson, C. Murray, F. Holding, G. Chessari, H. Jhotti, H. Willems, J. Coyle, M. Verdonk, M. Vinkovic, N. Thompson, P. Williams, S. Rich, S. Hiscock
Affiliation: Astex Pharmaceuticals, Cambridge, UK. *firstname.lastname@example.org
Background: The HCV NS3 protein is a bi-functional enzyme with protease and helicase activities, both of which are essential for viral replication. 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 and validated a novel binding site on the full length NS3/4a protein through the application of our proprietary fragment-based screening platform, Pyramid™.
Methods: We established a robust system for solving protein-ligand crystal structures of the full length HCV NS3/4a protein. The affinities of identified hits were measured directly by isothermal titration calorimetry (ITC) and in a fluorescence-based protease activity assay using full length NS3/4a from a range of genotypes. Cellular activity was measured in Huh-7 cells persistently infected with a sub-genomic genotype 1b HCV-RNA construct containing a firefly Luciferase reporter gene.
Results: Protein-ligand crystal structures unambiguously demonstrate the existence of a new binding site, distinct from the protease active site. Compounds binding at the novel site, located at the interface of the 2 functional domains were shown to inhibit the enzymes' catalytic activites, by stabilizing an auto-inhibited conformation. Cross genotypic sequence analysis of this site revealed a high level of conservation, suggesting that this pocket may have a functional role. Data will be presented on the cross genotypic sequence analysis and the activity profile of a range of allosteric inhibitors in both the isolated enzyme and replicon systems.
Conclusions: Compounds binding at this novel allosteric site represent a new class of direct acting antivirals. These compounds show in vitro activity against a range of genotypes and resistance variants. The results warrant further exploration of these compounds as therapeutic agents for the treatment of HCV.