PRESENTATION TYPE: Oral or Poster
CURRENT CATEGORY: Viral Hepatitis C
CURRENT DESCRIPTORS: S02. HCV: Virology
TITLE: Discovery of a novel allosteric mechanism for the regulation of HCV NS3/4a protein function present across all genotypes
AUTHORS (FIRST NAME, LAST NAME): Susanne Saalau-Bethell1, Andrew Woodhead3, Gianni Chessari4, Maria G. Carr3, Joe Coyle5, Martyn Frederickson3, Brent Graham2, Chris Hamlett3, Steve Hiscock3, Finn P. Holding5, Harren Jhoti1, Rachel McMenamin2, Chris Murray4, Puja Pathuri1, Mike Reader3, David Rees3, Sharna J. Rich2, Caroline J. Richardson2, Neil Thompson2, Marcel L. Verdonk4, Mladen Vinkovic1, Pamela A. Williams1, Yon Jeff1
INSTITUTIONS (ALL): 1. Structural Biology, Astex Pharmaceuticals, Cambridge, United Kingdom.
2. Biology, Astex Pharmaceuticals, Cambridge, United Kingdom.
3. Chemistry, Astex Pharmaceuticals, Cambridge, United Kingdom.
4. Computational Chemistry, Astex Pharmaceuticals, Cambridge, United Kingdom.
5. Biophysics, Astex Pharmaceuticals, Cambridge, United Kingdom.
Background: The Hepatitis C virus NS3/4a 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 profoundly influence each other's enzymatic parameters. We have identified and validated a novel binding site on the full length NS3/4a protein present in all genotypes and using a chemical lead demonstrate its regulatory role on function.
Methods: Crystals of the full length HCV NS3/4a protein were soaked with cocktails of fragments and structures were solved to high resolution. The affinities of identified hits were measured by isothermal titration calorimetry (ITC) and in a fluorescence-based protease activity assay. Cell based antiviral activity was quantitated using the sub-genomic genotype 1b HCV-RNA replicon (pFKI3889luc-ubi-neo/NS3-3’/ET). Resistant mutations were elicited by long time exposure of cells to high concentrations of compound A. Identified mutations were incorporated into the wild-type enzyme and replicon by site directed mutagenesis for confirmation.
Results: Crystal structures of the protein in complex with various fragment hits unambiguously demonstrate the existence of a new binding site, distinct from the active site. The compounds inhibit the full length protein but not the isolated protease domain, consistent with the observed binding mode. Structure-based drug design was used to improve the affinity of the compounds to IC50 in the range of 10-100nM. They 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 and replicon resulted in weaker IC50/EC50 values, whilst not affecting the affinity of telaprevir. Sequence analysis show the site to be conserved across all genotypes and biophysical techniques demonstrate that compounds binding at this allosteric site inhibit the function of the NS3/4a protein by stabilizing an inactive conformation.
Conclusions: Ligands binding at this novel allosteric site inhibit enzyme function in vitro and in the cell based replicon. Mutants raised in the replicon system by selective pressure from compound A demonstrate that the site is relevant to viral RNA replication. We show that compounds binding at the novel site exert their inhibitory action by obstructing conformational changes essential for enzyme activity.
PRESENTATION TYPE: Poster Only
CURRENT CATEGORY: Viral Hepatitis C
CURRENT DESCRIPTORS: S06. HCV Therapy: Preclinical and Early Clinical Development
TITLE: Discovery of AT26893, a novel allosteric inhibitor of the regulation of HCV NS3/4a protein function
AUTHORS (FIRST NAME, LAST NAME): Nicola E. Wilsher1, Maria G. Carr1, Joe Coyle1, Lynsey Fazal1, Brent Graham1, Chris Hamlett1, Steve Hiscock1, Harren Jhoti1, Alessia Millemaggi1, Chris Murray1, David Norton1, Mike Reader1, David Rees1, Sharna J. Rich1, Susanne Saalau-Bethell1, Matt Sanders1, Neil Thompson1, Mladen Vinkovic1, Henriette M. Willems1, Andrew Woodhead1
INSTITUTIONS (ALL): 1. Astex Pharmaceutical, Cambridge, United Kingdom.
Background: The Hepatitis C viral NS3/4a 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 profoundly influence each other. Following the identification and validation of a novel binding site on the full length NS3/4a protein we have designed a preclinical candidate with drug-like properties which is currently undergoing IND enabling studies in preparation for clinical trials.
Methods: Astex Pharmaceuticals fragment screening platform (Pyramid™) was applied to the full length HCV NS3/4a protein to identify hits. These hits were used to start a structure guided optimization program. Compounds active against the full length NS3/4a protein were evaluated in Huh-7 cells persistently infected with a sub-genomic genotype 1b HCV-RNA construct to quantitate their antiviral activity. The ADMET properties of compounds with sub 100nM potency were evaluated using in vitro Clint, plasma protein binding, CYP inhibition, permeability assays and in vivo PK in rat and dog.
Results: Preclinical candidate AT26893 was identified with an EC50 of 4nM in the sub-genomic genotype 1b HCV-RNA replicon assay. In vitro ADMET assays showed AT26893 had a low drug-drug interaction potential with minimal inhibition of the major human CYPs, good pharmacokinetic properties with 30-60% bioavailability in preclinical species and was well tolerated in early toxicology studies.
Conclusions: Pharmacokinetic properties were optimized in preclinical species and in vitro in human based ADMET assays. The focused lead optimisation of cellular activity alongside ADMET properties resulted in the identification of a non-peptidic / non-macrocyclic preclinical candidate. The absence of CYP liabilities makes the candidate amenable to combination regimes required for effective treatment of HCV. The predicted human pharmacokinetics of the candidate are consistent with once daily dosing in man. The preclinical candidate is currently under evaluation in IND enabling studies.