神经母细胞瘤是婴儿期最常见和最致命的肿瘤。WDR5(WD重复域5),通过其WBM位点支持N-myc转录复合物并通过其WIN位点与染色体相互作用的关键因素,促进神经母细胞瘤的进展,从而使其成为潜在的抗神经母细胞瘤药物靶点。到目前为止,已经报道了一些WIN位点抑制剂,WBM网站的破坏者很少见。在这项研究中,我们进行了虚拟筛选以鉴定靶向WDR5的WBM位点的候选命中化合物。因此,选择60个化合物作为候选WBM位点抑制剂。细胞增殖试验证明了6种结构不同的WBM位点抑制剂,编号为化合物4、7、11、13、19和22,可有效抑制3种神经母细胞瘤细胞系(MYCN扩增的IMR32和LAN5细胞系,和MYCN-未扩增的SK-N-AS细胞系)。其中,化合物19抑制IMR32和LAN5细胞的增殖,EC50值为12.34和14.89μM,分别,对SK-N-AS细胞有一定程度的抑制作用,在20μM下不影响HEK293T细胞。化合物19对WDR5的高分辨率晶体复合物结构的分析表明,它竞争性地占据了V264所在的疏水口袋,这可能会破坏MYC与WDR5的相互作用,并进一步破坏MYC药物的基因转录。通过进行RNA-seq分析,我们证明了化合物19和WIN位点抑制剂OICR-9429的分子作用机制的差异。最有趣的是,我们通过联合使用WBM位点抑制剂19和WIN位点抑制剂OICR-9429确立了特别高的协同率,为神经母细胞瘤提供了新的治疗途径.
Neuroblastoma is the most common and deadliest tumor in infancy.
WDR5 (WD Repeat Domain 5), a critical factor supporting an N-myc transcriptional complex via its WBM site and interacting with chromosome via its WIN site, promotes the progression of neuroblastoma, thus making it a potential anti-neuroblastoma drug target. So far, a few WIN site inhibitors have been reported, and the WBM site disruptors are rare to see. In this
study we conducted virtual screening to identify candidate hit compounds targeting the WBM site of
WDR5. As a result, 60 compounds were selected as candidate WBM site inhibitors. Cell proliferation assay demonstrated 6 structurally distinct WBM site inhibitors, numbering as compounds 4, 7, 11, 13, 19 and 22, which potently suppressed 3 neuroblastoma cell lines (MYCN-amplified IMR32 and LAN5 cell lines, and MYCN-unamplified SK-N-AS cell line). Among them, compound 19 suppressed the proliferation of IMR32 and LAN5 cells with EC50 values of 12.34 and 14.89 μM, respectively, and exerted a moderate inhibition on SK-N-AS cells, without affecting HEK293T cells at 20 μM. Analysis of high-resolution crystal complex structure of compound 19 against
WDR5 revealed that it competitively occupied the hydrophobic pocket where V264 was located, which might disrupt the interaction of MYC with
WDR5 and further MYC-medicated gene transcription. By performing RNA-seq analysis we demonstrated the differences in molecular action mechanisms of the compound 19 and a WIN site inhibitor OICR-9429. Most interestingly, we established the particularly high synergy rate by combining WBM site inhibitor 19 and the WIN site inhibitor OICR-9429, providing a novel therapeutic avenue for neuroblastoma.