Identification of novel HCV NS3 helicase inhibitors through the application of optimized unwinding and ATPase assays in high throughput screens (HTS)
K. Kossen1; J. Kraemer2; D. Winkler2; T. Hesterkamp2; S. Misialek1; R. Rajagopalan1; B. O. Buckman1; L. M. Blatt1; S. Seiwert1; L. Beigelman1
1. Intermune, Inc, Brisbane, CA, USA.
2. Evotec AG, Hamburg, Germany.
Background: The RNA helicase activity of HCV NS3 is essential for viral replication and thus represents an attractive target for new antiviral compounds. As the ATPase and unwinding activities of the NS3 motor protein are coupled, we performed HTS assays against both enzymatic activities to maximize the chances of identifying tractable chemical matter.
Methods: The ATPase activity of NS3 is robust whereas duplex unwinding requires more specialized conditions. Therefore, assay optimization focused on improving the unwinding activity by varying the duplex substrate and assay buffer. The optimized conditions were incorporated into fluorescence polarization (FP)-based assays for both ATPase and unwinding, miniaturized to an assay volume of ~ 1μL, and employed in parallel HTS assays of ~300,000 compounds each. The ATPase assay uses a format originally developed for the characterization of kinase inhibitors. The unwinding assay uses a novel design in which opposite strands of a duplex DNA substrate incorporate either a fluorophore or biotin, respectively. NS3 helicase promotes the dissociation of these two strands. Detection is accomplished by addition of streptavidin which forms a high FP complex with substrate but not product.
Results: The performance of the unwinding assay is significantly influenced by both the choice of duplex substrate and the buffer conditions. Optimization of duplex length and fluorescent labels identified an optimized substrate that afforded an approximately 2-fold increase in dynamic range. Optimization of reaction buffer resulted in further enhancement of unwinding activity. Interestingly, under the optimized assay conditions NS3 shows significant unwinding activity when duplex substrate is in excess over enzyme. The final unwinding assay is linear over ~1 hr with a dynamic range of 120 mP. The conditions of the unwinding assay required minimal modifications when transferred to the ATPase assay which yielded a dynamic range of 80 mP. Application of these conditions to HTS provided excellent assay performance with average Z’ values of 0.76 and 0.74 for the unwinding and ATPase end-points, respectively. Following resynthesis and hit verification, a total of 12 structural families of active leads were identified.
Conclusions: NS3 helicase is considered a difficult target for small molecule drug discovery and to date no inhibitors have entered clinical trials. Here, the application of optimized assay conditions for NS3 catalyzed unwinding and ATPase activities in parallel HTS screens resulted in identification of 12 chemically tractable lead families. Optimization of these leads is currently ongoing.
