Kinetic, mechanistic and chemodynamic characterisation of non-nucleoside hepatitis C virus NS5B polymerase inhibitors using SPR biosensor technology

• Kinetic, mechanistic and chemodynamic aspects of the interaction between five non-nucleoside inhibitors and the HCV NS5B polymerase (genotype 2a) were assessed using SPR biosensor technology. The compounds were selected to represent different structural classes (benzothiadiazine, , α,γ-diketo acid, benzimidazole, thiophene carbocyclic acid and benzofuran), each known to interact with different binding sites. The viral polymerase interacted with the compounds with different kinetics and surprisingly also with different capacities. Cooperativity between the different allosteric inhibitor binding sites and the active site binding diketoacid was observed, but no cooperativity was seen between the allosteric sites. The interaction with diketoacid was stronger in phosphate buffer as compared to Tris buffer, indicating a phosphate ion-mediated interaction mechanism. The enzyme generally had reduced affinity for the inhibitors in the presence of RNA. Interaction parameters determined for human serum albumin revealed the propensity of the compounds to be distributed by HSA. This study provides important information for the design of optimized NS5B inhibitors and illustrates the complementarity of a biosensor-based analysis with inhibition studies, in particular for allosteric compounds with complex interaction mechanisms or when the target contains multiple ligand binding sites.

Nyckelord

Hepatitis C

NS5B

non-nucleoside inhibitors

biosensor

Publikations- och innehållstyp

vet (ämneskategori)

ovr (ämneskategori)