Coxsackievirus A16 2A protease binding assay for compound screening using grating-coupled interferometry v1

Résumé

This protocol aims to measure kinetic parameters (ka, kd, and KD) for compounds against coxsackievirus A16 (CVA16) 2A protease using the Creoptix Wave system. CVA16 2A protease is used as a surrogate for the related enterovirus A71 (EV-A71) 2A protease in the pan-enteroviral drug discovery efforts by our collaborator, the AI-Driven Structure-Enabled Antiviral Platform (ASAP) Discovery Center, and was chosen as the target for OpenBind's first data release package. Grating-coupled interferometry (GCI) is a label-free technique to measure kinetics and affinity for different targets (proteins, small molecules, fragments, etc.) with enhanced sensitivity. Biotin-tagged CVA16 2A protease was captured on the streptavidin-coated chip surface via biotinylation. This immobilisation technique provides oriented and active protein immobilisation for binding assays and can be applied to different protein targets. Binding analysis was performed with the Repeated Analyte Pulses of Increasing Duration (RAPID) method, which involves the injection of samples at a single concentration with varied association times. Kinetic parameters were obtained from the Creoptix Wave software (v 4.5.18) and the in-house developed, open-source, Python-based tool 'SensoFit' (xchem/sensofit).

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