转移,癌细胞迁移到其他组织并形成新的肿瘤,是癌症死亡和治疗失败的主要原因。在之前的研究中,苯丙哌林(Benp)被鉴定为癌细胞迁移抑制剂和肌动蛋白相关蛋白2/3复合物亚基2(ARPC2)的抑制剂。然而,Benp是外消旋混合物,和哪个立体异构体是活性异构体仍不清楚。在这项研究中,我们发现S-Benp是一种活性异构体,比R-Benp更强烈地抑制癌细胞的迁移和侵袭,对正常细胞没有影响。S-Benp的转移抑制作用也在动物模型中得到证实。验证抑制剂在细胞和组织中与它们的靶标结合一直是药物发现中非常具有挑战性的任务。通过表面等离子体共振分析(SPR)验证了ARPC2和S-Benp之间的直接相互作用,细胞热转移测定(CETSA),和药物亲和力响应靶标稳定性(DARTS)。在ARPC2F225A细胞的突变研究中,根据CETSA和DARTS,S-Benp不与ARPC2F225A结合。此外,我们根据CETSA使用Cy3偶联的S-Benp验证了S-Benp在癌细胞中与ARPC2共定位,并在肿瘤组织中直接与ARPC2结合.最后,肌动蛋白聚合测定和免疫细胞化学表明,S-Benp抑制了肌动蛋白的重塑,例如lamellipodium的形成。一起来看,我们的数据表明S-Benp是Benp的活性立体异构体,并且是通过ARPC2结合的潜在转移抑制剂.
Metastasis, in which cancer cells migrate to other tissues and form new tumors, is a major cause of both cancer death and treatment failure. In a previous study, benproperine (Benp) was identified as a cancer cell migration inhibitor and an inhibitor of actin-related protein 2/3 complex subunit 2 (ARPC2). However, Benp is a racemic mixture, and which stereoisomer is the active isomer remains unclear. In this study, we found that S-Benp is an active isomer and inhibits the migration and invasion of cancer cells much more strongly than R-Benp, with no effect on normal cells. The metastasis inhibitory effect of S-Benp was also verified in an animal model. Validating that inhibitors bind to their targets in cells and tissues has been a very challenging task in drug discovery. The direct interactions between ARPC2 and S-Benp were verified by surface plasmon resonance analysis (SPR), a cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS). In the mutant study with ARPC2F225A cells, S-Benp did not bind to ARPC2F225A according to CETSA and DARTS. Furthermore, we validated that S-Benp colocalized with ARPC2 in cancer cells and directly bound to ARPC2 in tumor tissues using Cy3-conjugated S-Benp according to CETSA. Finally, actin polymerization assays and immunocytochemistry showed that S-Benp suppressed actin remodeling such as lamellipodium formation. Taken together, our data suggest that S-Benp is an active stereoisomer of Benp and a potential metastasis inhibitor via ARPC2 binding.