Machine-Learning-Guided Identification of CCT245737 as a Functional Pan-G-Quadruplex Ligand for the Treatment of Multiple Myeloma

Abstract

Multiple myeloma (MM) still lacks curative therapies, creating an urgent demand for innovative agents targeting unique molecular vulnerabilities. G-quadruplex (G4) stabilization modulates oncogenic regulatory regions and displays potent preclinical antitumor effects. Nevertheless, most existing G4 ligands act on single G4 motifs. Pan-G4 agents capable of perturbing multiple oncogenic pathways are scarce, while conventional high-throughput screening is costly and labor-intensive. Herein, we constructed a repurposing-driven screening pipeline combining curated fluorescence resonance energy transfer (FRET)-derived melting-temperature shift (ΔTm) data sets, 1024-bit Chemistry Development Kit (CDK) molecular fingerprints, three machine-learning classifiers, and multi-G4 molecular docking. In nested cross-validation, our Random Forest (RF) classifier yielded a receiver operating characteristic area under the curve (ROC AUC) of 0.840 and a Matthews correlation coefficient (MCC) of 0.708, superior to label-randomized controls. Consensus screening of 4270 compounds from the TargetMol Anticancer Library yielded 812 high-confidence hits; the top-ranked 487 candidates underwent docking against four structurally diverse oncogenic G4s (c-Myc, c-KIT, Bcl-2, k-RAS). CCT245737 ranked in the top ten across all four independent docking analyses. Functional assays revealed that CCT245737 suppressed the viability of U266 and RPMI-8226 MM cells (IC50 = 0.58 ± 0.06 and 0.68 ± 0.04 μM) and enhanced nuclear BG4 signals. FRET-melting and NMM-displacement assays verified its concentration-dependent stabilization of the four G4s with minimal duplex-DNA interference. qPCR and immunoblotting confirmed downregulation of c-Myc, c-KIT, Bcl-2, and k-RAS at both mRNA and protein levels. In summary, CCT245737 is validated as a promising pan-G4 ligand, and our FRET-guided workflow offers a feasible repurposing strategy for discovering anti-MM G4-targeted therapeutics.