Identification of G-Quadruplex-Binding Inhibitors of Myc Expression through Affinity Selection–Mass Spectrometry

Abstract

The Myc oncogene is overexpressed in many cancers, yet targeting it for cancer therapy has remained elusive. One strategy for inhibition of Myc expression is through stabilization of the G-quadruplex (G4), a G-rich DNA secondary structure found within the Myc promoter; stabilization of G4s has been shown to halt transcription of downstream gene products. Here we used the Automated Ligand Identification System (ALIS), an affinity selection-mass spectrometry method, to identify compounds that bind to the Myc G4 out of a pool of compounds that had previously been shown to inhibit Myc expression in a reporter screen. Using an ALIS-based screen, we identified hits that bound to the Myc G4, a small subset of which bound preferentially relative to G4s from the promoters of five other genes. To determine functionality and specificity of the Myc G4-binding compounds in cell-based assays, we compared inhibition of Myc expression in cells with and without Myc G4 regulation. Several compounds inhibited Myc expression only in the Myc G4-containing line, and one compound was verified to function through Myc G4 binding. Our study demonstrates that ALIS can be used to identify selective nucleic acid-binding compounds from phenotypic screen hits, increasing the pool of drug targets beyond proteins.

Targeting RNA with Small Molecules: Identification of Selective, RNA-Binding Small Molecules Occupying Drug-Like Chemical Space

“…In fact, DNA G-quadruplexes previously screened in ALIS also resulted in a high number of binding compounds. 26…”

“…In fact, DNA G-quadruplexes previously screened in ALIS also resulted in a high number of binding compounds. 26 Though the ALIS platform was able to identify small molecule binders for diverse RNA targets, we wondered whether the observed low hit rate across targets was due to inherent bias in the interrogated screening libraries that are traditionally used for protein-targeted drug discovery. The Functionally Annotated Library, assembled using pharmacogenomic data, and the Diversity Library, assembled from synthetic and commercial acquisition efforts, have historically both been applied toward the discovery of proteinbinding ligands.…”

Label‐free Bio‐affinity Mass Spectrometry for Screening and Locating Bioactive Molecules

“…One attractive route to pharmacological inhibition of MYC has been the prevention of its expression through small‐molecule‐mediated stabilization of the G‐quadruplex present in its promoter (Felsenstein et al, 2016). Using the ALIS‐based screen system, Flusberg et al (2019) identified 10 hits that selectively bound to the Myc G‐quadruplex from a set of ~50,000 compounds. Binding affinities were determined using the ALIS ligand titration method.…”

Target-Directed Approaches for Screening Small Molecules against RNA Targets

“…126,[129][130][131] Recently, Merck (Kenilworth, NJ) applied their AS-MS method, referred to as the Automated Ligand Detection System (ALIS), to screen compounds against numerous RNA targets, an approach validated with ncRNA riboswitches, their ligands, and a large unbiased smallmolecule library. [132][133][134] Here, RNA is incubated with mass-encoded small-molecule mixtures, small molecule-RNA complexes are purified by size exclusion chromatography (SEC), and the complexes dissociate through reverse-phase chromatography. Bound ligands are detected by high-resolution MS (Fig.…”