| المؤلفون: |
Kallal LA; Screening, Profiling, and Mechanistic Biology, GlaxoSmithKline, Collegeville, PA, USA., Waszkiewicz A; Screening, Profiling, and Mechanistic Biology, GlaxoSmithKline, Collegeville, PA, USA., Jaworski JP; Early Pipeline Project Management, GlaxoSmithKline, Collegeville, PA, USA., Della Pietra A; Oncology, GlaxoSmithKline, Collegeville, PA, USA., Berrodin T; Oncology, GlaxoSmithKline, Collegeville, PA, USA., Brady P; Computational Sciences, GlaxoSmithKline, Collegeville, PA, USA., Jurewicz AJ; Screening, Profiling, and Mechanistic Biology, GlaxoSmithKline, Collegeville, PA, USA., Zeng X; Screening, Profiling, and Mechanistic Biology, GlaxoSmithKline, Collegeville, PA, USA., Payne L; Protein and Cell Sciences, GlaxoSmithKline, Collegeville, PA, USA., Medina JR; Chemistry, GlaxoSmithKline, Collegeville, PA, USA., Doepner-Buser C; Arvinas, New Haven, CT, USA., Mangatt B; Oncology, GlaxoSmithKline, Collegeville, PA, USA. |
| مستخلص: |
While c-MYC is well established as a proto-oncogene, its structure and function as a transcription factor have made c-MYC a difficult therapeutic target. To identify small-molecule inhibitors targeting c-MYC for anticancer therapy, we designed a high-throughput screening (HTS) strategy utilizing cellular assays. The novel approach for the HTS was based on the detection of cellular c-MYC protein, with active molecules defined as those that specifically decreased c-MYC protein levels in cancer cells. The assay was based on a dual antibody detection system using Förster/fluorescence resonance energy transfer (FRET) and was utilized to detect endogenous c-MYC protein in the MYC amplified cancer cell lines DMS273 and Colo320 HSR. The assays were miniaturized to 1536-well plate format and utilized to screen the GlaxoSmithKline small-molecule collection of approximately 2 million compounds. In addition to the HTS assay, follow-up assays were developed and used to triage and qualify compounds. Two cellular assays used to eliminate false-positive compounds from the initially selected HTS hits were (1) a cellular toxicity assay and (2) an unstable protein reporter assay. Three positive selection assays were subsequently used to qualify compounds: (1) 384-well cell cycle flow cytometry, (2) 384-well cell growth, and (3) c-MYC gene signature reverse transcription quantitative PCR (RT-qPCR). The HTS and follow-up assays successfully identified three compounds that specifically decreased c-MYC protein levels in cancer cells and phenocopied c-MYC siRNA in terms of cell growth inhibition and gene signatures. The HTS, triage, and three compounds identified are described. |
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