(1220-A) Applying RT-qPCR for HTS of Transcription Factors

Transcription factors have traditionally been deemed “undruggable” due to a lack of ligand binding sites and structural complexity [1]. The challenges associated with targeting a transcription factor drove an internal project team to focus on a phenotypic screening funnel, starting with a proximal readout to measure regulation of transcripts. Reverse transcription quantitative polymerase chain reaction assay (RT-qPCR) was previously associated with high cost and logistical challenges that prevented teams from utilizing this technology for primary screens. This assay format was miniaturized and protocol streamlined to be amenable to 384 well screening with human M1 primary macrophages. Over 200,000 library compounds were screened in this format. The hits identified in the RT-qPCR assay were confirmed through a protein expression (TR-FRET) assay with a 44% confirmation rate. Here we describe the development of a HTS compatible one-step RT-qPCR assay and employment of RT-qPCR in screening funnels targeting transcription factors.