Virtual screening and MD simulation-guided discovery of CDK12-IN-3, a potent ALK inhibitor suppressing neuroblastoma growth in vitro and in vivo
TL;DR
CDK12-IN-3 is validated as a novel selective ALK inhibitor with potent anti-NB activity, providing a promising lead compound and an efficient dynamic simulation-guided drug repurposing paradigm for overcoming ALK inhibitor resistance in high-risk NB.
Abstract
Neuroblastoma (NB) is the most common extracranial malignant solid tumor in children, and aberrant activation of anaplastic lymphoma kinase (ALK)—driven primarily by hotspot mutations F1174L and R1275Q—represents a core oncogenic driver of high-risk and relapsed NB. Clinical ALK inhibitors are frequently hampered by acquired drug resistance and off-target toxicity, highlighting an unmet need for novel selective ALK inhibitors for NB treatment. Here, we employed a drug repurposing strategy integrating virtual screening, multi-dimensional molecular dynamics (MD) simulations, steered MD, and umbrella sampling to screen 7,322 compounds for novel ALK inhibitory activity, followed by systematic in vitro and in vivo validation. We identified CDK12-IN-3 (C18) as a high-affinity ligand for wild-type ALK and both NB-associated ALK mutants (F1174L, R1275Q), with favorable binding selectivity over 10 homologous tyrosine kinases. In vitro, CDK12-IN-3 exerted nanomolar-level antiproliferative, anti-clonogenic, and anti-migratory effects across three genetically distinct NB cell lines (SH-SY5Y, SK-N-BE (2), IMR-32), while showing no significant cytotoxicity in ALK-negative HEK293T normal cells even at 20 μM, demonstrating a wide in vitro safety window. Mechanistic studies confirmed that CDK12-IN-3 directly bound ALK (KD = 5.33 μM by surface plasmon resonance), downregulated ALK expression, suppressed the downstream PI3K/AKT/mTOR signaling pathway, and induced NB cell apoptosis. In vivo, oral administration of CDK12-IN-3 (50 mg/kg) significantly suppressed NB xenograft tumor growth in nude mice with no observable systemic toxicity. Collectively, our findings validate CDK12-IN-3 as a novel selective ALK inhibitor with potent anti-NB activity, providing a promising lead compound and an efficient dynamic simulation-guided drug repurposing paradigm for overcoming ALK inhibitor resistance in high-risk NB.