OBJECTIVE  Neuroblastoma(NB) is a common extracranial solid tumor in children, accounting for about 7%−10% of childhood malignant tumors and about 15% of all childhood cancer deaths. Approximately 50% of cases are high-risk with a relatively low overall survival rate. Even with intensive myeloablative chemotherapy, recurrence is still common and fatal. Therefore, new treatment methods must be explored by targeting molecular and genetic abnormalities during tumorigenesis and disease progression. Anaplastic lymphoma kinase(ALK) is a cell surface neural receptor tyrosine kinase that is significantly expressed only in the developing embryo and neonatal brain and belongs to the insulin receptor tyrosine kinase superfamily. Studies have shown that mutations or gene amplifications of ALK are associated with NB susceptibility in 15% of patients. Studies have shown that activated ALK can promote cell growth and survival and inhibit the proliferation of NB cells. In a significant number of cases, high ALK expression is associated with a poor prognosis of NB. ALK is rarely expressed in the tissues of normal healthy people but is highly expressed in some cancer tissues. This characteristic makes ALK an ideal therapeutic target for neuroblastoma. This study is dedicated to using computer-aided drug design methods to screen for inhibitors with significant selectivity for ALK from natural products of traditional Chinese medicine, thus hopefully solving the resistance problem of existing ALK inhibitors such as crizotinib and opening up a new way for the treatment of neuroblastoma.

METHODS  Obtained the structures of ALK(4FOB, 4FOC, and 4FOD) from the PDB database. Used the traditional Chinese medicine natural product database YaTCM as the ligand library and screen with 4FOB, 4FOC and 4FOD as receptors respectively. Compounds with scores in the top 15% in the previous step entered the next round of screening. Finally, taken the intersection of the screened compounds obtained from the three virtual screenings, and the final intersection compounds were the hit compounds. And further verified through MD simulation; deeply studied the characteristics of the ALK-hit compound system by calculating MM/PBSA, RMSD, and RMSF. Conducted CCK-8 experiments to verify the killing effect of compounds on NB cells, and performed Western blotting experiments to detect the inhibitory effect on targets.

RESULTS Through virtual screening and molecular dynamics simulation, six hit compounds were finally screened out. The MM/PBSA calculation results showed that all six hit compounds can bind tightly to the target protein ALK. The RMSD calculation results for the hit compounds and the target protein ALK during the 100 ns simulation showed that morindone and quercetagetin had the best robustness in binding to the target protein. The RMSF calculation results showed that compared with morindone, quercetagetin could significantly reduce the RMSF value of amino acid residues in the DFG region. This indicated that quercetagetin can weaken the fluctuation of amino acid residues in the above region. Therefore, quercetagetin could better interact with the DFG region of ALK and was expected to become an inhibitor of ALK. The CCK-8 experiment results showed that quercetagetin could have a strong killing effect on SK-N-BE(2) cells at a relatively low concentration(IC₅₀=7.096 μmol·L⁻¹). The Western blotting experiment results showed that quercetagetin could inhibit the expression of ALK and induce apoptosis of NB cell SK-N-BE(2).

CONCLUSION Quercetagetin may be an effective candidate drug for the treatment of NB.