Confirm Target Engagement in Live Cells

Hit confirmation requires quantitative evidence that initial screening hits engage the intended RNA-binding protein in cells. At this stage, prioritized compounds are re-evaluated using RBP-TE™ assays established in selected cellular matrices to confirm cellular target engagement under physiologically relevant conditions and to define objective criteria for compound progression.

Assay design—including cell model selection and assay format—is chosen to maximize biological relevance and measurement fidelity. In parallel, control schemes, plate layouts, and predefined quality-control metrics are implemented to ensure robust, reproducible engagement measurements. Retesting strategies typically begin with single- or limited multi-concentration assessments, followed by dose–response experiments to derive cellular target engagement IC₅₀ values.

By anchoring hit confirmation to direct cellular target engagement, this step establishes whether observed functional or phenotypic activity is supported by engagement of the nominated target rather than nonspecific, indirect, or assay-dependent effects. Counter-screens, interference assessments, and target-specific control conditions are incorporated as needed to ensure that only compounds with validated on-target engagement advance into follow-up biology and SAR-focused optimization.