Probing Functional States and Inhibition of Flaviviral Proteases Using Nanopore Tweezers

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.

Grant Number: 5R01AI156187-05
NIH Institute/Center: NIH
Principal Investigator: Min Chen