Machine learning-driven discovery of potent isocitrate dehydrogenase 1 mutant inhibitors from ultralarge ligand libraries for targeting malignant glioma.

Glioblastoma (GBM) represents one of the most lethal and therapy-resistant forms of brain tumors, characterized by high heterogeneity, metabolic reprogramming, and recurrence. In the current study, we aimed to identify novel small molecule inhibitors targeting mutant isocitrate dehydrogenase 1 (IDH1), a crucial enzyme involved in GBM tumor metabolism. For this aim, machine learning-based quantitative structure-activity relationships modeling was combined with structure-based e-Pharmacophore screening to virtually screen ultralarge chemical libraries containing around 157 million compounds. The best hits were selected based on docking score, predicted pIC50 value, and molecular mechanics/generalized Born surface area binding energy calculations. Furthermore, molecular dynamics (MD) simulations were conducted to validate the selected hit compounds. In total, 36 compounds were subjected to short MD simulations (10 ns), and 16 molecules showing low binding free energies (below -90 kcal/mol) were further analyzed through long MD simulations (100 ns). Among these, 11 synthetically available hits were ordered and experimentally tested on human glioblastoma U87 and U251 cell lines. Our experimental results showed that 5 of the tested compounds (hits 1, 4, 5, 6, and 7) reduced spheroid formation by nearly 80%-90% and inhibited cell proliferation. Moreover, these hits decreased the oxygen consumption rate and extracellular acidification rate (ECAR), by up to 62% and 55%, respectively, indicating inhibition of both mitochondrial respiration and glycolysis. Furthermore, Western blot and quantitative real-time polymerase chain reaction analyses revealed downregulation of glycolytic enzymes and stemness markers. Moreover, steered MD and free energy perturbation analyses confirmed the stable interactions of these compounds at the IDH1 mutant active site. This multistage in silico-in vitro approach allowed the identification of metabolically disruptive novel mutant IDH1 inhibitors that suppress glycolysis, mitochondrial respiration, and cancer stemness in glioblastoma cells. These compounds represent promising scaffolds for the development of next-generation GBM therapeutics. SIGNIFICANCE STATEMENT: This study integrate machine learning-guided quantitative structure-activity relationships modeling with structure-based pharmacophore screening to discover small molecule inhibitors of mutant IDH1, a central mediator of metabolic reprogramming in glioblastoma. Lead compounds identified through this pipeline inhibit mutant IDH1 activity, disrupt metabolic pathways required for glioblastoma cell viability, and concomitantly reduce stem-like phenotypes in vitro, consistent with a dual mechanism of action that targets both bulk tumor cells and cancer stem-like populations.