Project Summary Flavivirus are major mosquito-borne pathogens infecting millions of people worldwide each year. Currently there is no antiviral therapy available for treating West Nile, Dengue and Zika viral infections. The first vaccine CYD- TDV (Dengvaxia) against DENV was approved last year but shows only 56% overall efficacy against the four dengue serotypes. The flaviviral two-component NS2B/NS3 protease is required for viral replication and thus an attractive antiviral target. However, extensive screening and rational design efforts have failed to identify any clinically viable inhibitors at this point. Two key factors have likely contributed to the challenge. First, traditional screening efforts rely primarily on binding affinity to predict the drug efficacy. Yet, increasing evidence has emerged to show that the residence time of drug-target interaction is a more reliable predictor of in vivo pharmacological activity. These kinetic rate parameters are generally not available during early stages of drug discovery. Second, NS2B/NS3 proteases display complex conformational dynamics during function and inhibition, which is still poorly understood. This project aims to develop a new label-free single molecular approach to resolve the conformational states of NS2B/NS3 proteases. Key to the approach is the use of an innovative nanopore tweezers where the protease is confined with the pore lumen, allowing dynamic structural changes during substrate or inhibitor binding to be continuously monitored by current fluctuation signals. Analysis of the current traces will provide a complete profile of binding affinity and kinetic rates as well as the distribution of conformational states. Specifically, we will first build a nanopore tweezers tool set that is readily tunable for trapping various flaviviral proteases. Secondly, we will track and analyze the functional states of the NS2B/NS3 protease in the presence of various substrates. Influence of critical residues, substrate, construct design on the dynamic equilibrium between the “open” and “closed” states will be assessed to provide insight into the mechanism of protease activity. Finally, the nanopore tweezers will be deployed to determine the structural dynamics and binding thermodynamics and kinetics profiles of NS2B/NS3 interacting with various inhibitors. Once the inhibition profiles are established, the nanopore tweezers confined NS2B/NS3 system will be tested for screening a diverse compound library to identity novel allosteric inhibitors with improved drug-like properties compared to active-site inhibitors. This work will provide unprecedented kinetic information on the function- structural dynamics relationship of NS2B/NS3 complex and mechanisms of substrate binding and inhibition, as well as establish a new paradigm for high-throughput drug screening that is independent of enzymatic activity.

Project Narrative Flavivirus lead to recurrent epidemic outbreaks and are a global threat to human health without any effective antiviral treatment. This project will develop and deploy a novel nanopore tweezers approach that can provide a complete conformational dynamics, binding thermodynamics and kinetics profile of the flaviviral protease to guide the discovery and design of new inhibitors. The nanopore tweezers platform, operating independent of the enzyme activity, will also provide a new paradigm for label-free, single molecule and high-throughput drug screening.