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Virtual screening and MD simulation-guided discovery of CDK12-IN-3; a potent ALK inhibitor suppressing neuroblastoma growth in vitro and in vivo
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DOI：10.3389/fchem.2026.1870293.png)
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Abstract
En 中文
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.
Keywords:
molecular dynamics simulation
neuroblastoma
ALK
virtual screening
CDK12-IN-3
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