A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

Abstract

In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC (50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions. b s t r a c tIn drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC 50 values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC 50 values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other proteinligand interactions. Ó 2009 Elsevier Inc. All rights reserved.High-throughput screening (HTS) 2 is a major method in drug discovery for new small lead molecules. In high-throughput enzyme inhibition assays, more than a million compounds are usua...

Ligand-Bound Structures of the Dengue Virus Protease Reveal the Active Conformation

“…2A). This agrees with results of previous nuclear magnetic resonance (NMR) spectroscopy experiments, which showed that the ␤ hairpin of NS2B is largely disordered in solution for DENV and that substrate binding or small molecules are capable of inducing the closed conformation of the enzyme (5,34).…”

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“…The construction and preparation of luciferase DENV-2 were described elsewhere (43). At 0, 2,4,6,8,10,13,16,20, and 24 h p.i., 2 M NITD-2636 was added to the infected cells. Dimethyl sulfoxide (DMSO) (0.9%) was used as a negative control.…”

A Novel Dengue Virus Inhibitor, BP13944, Discovered by High-Throughput Screening with Dengue Virus Replicon Cells Selects for Resistance in the Viral NS2B/NS3 Protease

“…By analogy with the success of the HIV and HCV protease inhibitors, a number of approaches have been employed to search for DENV NS3 protease inhibitors (reviewed in references 8 and 34-37). One strategy used for this purpose involves a specific target-based approach, e.g., an enzyme-based biochemical assay or structure-based virtual screen (38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49). An alternative strategy is to use a replication-based approach, e.g., screening through virus infection assays or replicon cell lines (50)(51)(52).…”