Primary and Confirmatory Screening for Flavivirus Genomic Capping Enzyme Inhibition
Mosquito-borne flaviviruses (family Flaviviridae, genus flavivirus), including dengue, yellow fever and West Nile viruses can cause significant morbidity and mortality worldwide. The Aedes aegypti mosquito, which is found on almost every continent of the world, is the primary vector for both dengue and yellow fever viruses. Flavivirus infection can cause a wide range of disease symptoms ranging more ..
BioActive Compounds: 1013
Assay Provider: Brian Geiss, Colorado State University
Mosquito-borne flaviviruses (family Flaviviridae, genus flavivirus), including dengue, yellow fever and West Nile viruses can cause significant morbidity and mortality worldwide. The Aedes aegypti mosquito, which is found on almost every continent of the world, is the primary vector for both dengue and yellow fever viruses. Flavivirus infection can cause a wide range of disease symptoms ranging from mild febrile illness to hemorrhagic disease in dengue infection and liver and kidney failure in yellow fever infection. 50-100 million cases of dengue fever and 200,000 cases of yellow fever are reported each year resulting in respectively ~20,000 and ~30,000 deaths annually throughout the world. Despite the morbidity and mortality caused by flavivirus infection there is currently no effective chemotherapeutic treatment for infection by any member of the flavivirus family. As such, the identification and characterization of novel drug target sites is critical to developing new classes of antiviral drugs. The flavivirus NS5 N-terminal capping enzyme (CE) is critical to the formation of the viral RNA cap structure, which directs viral polyprotein translation and stabilizes the 5' end of the viral genome. The structure of the flavivirus CE has been solved and a detailed understanding of the CE:guanosine triphosphate (GTP) and CE:RNA cap interactions is available. Because of the essential nature of the CE:GTP interaction for viral replication, disrupting CE:GTP binding is an attractive approach for structure-based drug development. A robust solution-based assay for monitoring CE:GTP binding in real-time using fluorescence polarization (FP) has been developed. We adapted this assay for high-throughput screening, and performed a screen of 340K compounds in our MLPCN library in single dose. Compounds determined to be active in the single dose screen were then run in a ten point dose response confirmatory assay.
A fluorescence polarization assay was adapted for high-throughput compound screening in 1536-well plates. The assay evaluated the ability of a small molecule inhibitor to compete with GTP-BODIPY for the GTP-binding site of the CE enzyme and was performed in 1536-well format using low-binding opaque black microtiter plates (Corning 3728).
Compound Dosing/Plating: For the single dose assays 10 nL of compound was added to each well to give a concentration of 25 uM. For the dose response assay 10 concentrations of each compound ranging from 100-0.2 uM were dispensed into 1536-well black non-binding surface plates.
Assay Setup: 2 uL of reagent mix, which included the GTP-Bodipy substrate in assay buffer, was added to each well of the previously compound dosed 1536-well plates. The reaction was initiated with the addition of 2 uL of the CE enzyme diluted in assay buffer. The final concentrations in the reaction were 10 nM GTP-Bodipy and 2 microM CE enzyme diluted in assay buffer (50 mM Tris (pH 7.5), 0.1% NP-40, and 2% DMSO). The test plate was incubated at room temperature for 60 minutes, and then transferred to a Perkin Elmer Envision microplate reader and fluorescence polarization (mP) was measured at an excitation wavelength of 480 nm and a polarized emission wavelength of 535 nm. Each plate had 256 control wells in the eight outside columns with 128 wells containing the complete reaction mixture with carrier control (Full Rxn) and 128 wells in which the CE enzyme had been left out (Bkg).
Data Analysis: 128 background control wells containing the GTP-Bodipy substrate only and 128 full reaction control wells containing GTP-Bodipy substrate and 2 uM CE enzyme were included on each assay plate and used to calculate a Z' value for each plate and to normalize the data on a per plate basis. Data were analyzed using the IDBS Activity Base software. Results for each concentration were expressed as percent inhibition (% Inhibition) and was calculated as: 100*((Med Full Rxn mP- Med Bkg mP) - (Cmpd mP - Med Bkg mP))/ ((Med Full Rxn mP - Med Bkg mP)). IC50 values were determined using a four parameter logistic fit to the data (Excel Fit equation 205) with the maximum and minimum locked at 100 and 0.
Possible artifacts in this assay include, but are not limited to, compounds that fluoresce at 480/535 nm, that absorb at either 480 or 535 nm, or that precipitate.
Outcome: From the primary screen, the criteria to define active compounds is % inhibition > 28.49 (the mean inhibition of the compound population plus three standard deviations). Subsequently, compounds exhibiting autofluorescent characteristics were excluded. Available compounds from the remaining list were screened in the confirmatory assay. Compounds that showed activity in the primary screen, but were not available for further study are labeled as inconclusive. In the confirmatory assay, compound activity is defined as 30% or greater inhibition at any tested concentration. IC50 values were calculated for these compounds and used to determine the relative score.
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score: Compounds in the primary screen are scored on a scale of 0-40 based on % activity; a score of 40 corresponds to 100% activity. In the confirmatory dose response screen of primary screen hits, active compounds are scored on a scale of 41-80 based on IC50 result while compounds where activity was not confirmed are given the score 0. Confirmatory dose response and secondary screens of purified and/or resynthesized compounds, indicating the highest degree of confidence) are scored on a scale of 81-100 based on IC50 result. Inactive compounds are given the score 0.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)