3.7.2 From Primary Hit to Confirmed Hit

Compounds showing activity in a single-concentration primary HTS campaign ('primary hits') are not immediately considered validated actives; they are...

Compounds showing activity in a single-concentration primary HTS campaign ('primary hits') are not immediately considered validated actives; they are retested in the original assay format to eliminate false positives arising from pipetting error or transient assay artefacts, then subjected to full dose-response ('hit confirmation') testing to establish a genuine IC50 or EC50. Confirmed hits are further triaged using orthogonal assays employing a different detection technology, to rule out compounds that are artefacts of the primary assay's specific readout (for example, fluorescent or coloured compounds that interfere with an optical detection method), before progressing to the medicinal chemistry and SAR workflow described in Phase 2.