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Merck

High-Throughput Screening and Triage Assays Identify Small Molecules Targeting c-MYC in Cancer Cells.

SLAS discovery : advancing life sciences R & D (2021-01-23)
Lorena A Kallal, Anna Waszkiewicz, Jon-Paul Jaworski, Anthony Della Pietra, Tom Berrodin, Pat Brady, Anthony J Jurewicz, Xin Zeng, Lisa Payne, Jesús R Medina, Carolyn Doepner-Buser, Biju Mangatt
ABSTRAKT

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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Sigma-Aldrich
Anti-c-myc Antibody, clone 3C7, clone 3C7, Chemicon®, from mouse
Sigma-Aldrich
Monoclonal Anti-α-Tubulin antibody produced in mouse, clone DM1A, ascites fluid