Chen Yi, Ruben Eliza A, Rajadas Jayakumar, Teng Nelson N H
Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Stanford University, 300 Pasteur Drive, HH333, Stanford, CA 94305-5317, USA.
Biomaterials and Advanced Drug Delivery Laboratory, Stanford University, Stanford, CA, USA.
Bioorg Med Chem. 2015 Aug 1;23(15):4576-4582. doi: 10.1016/j.bmc.2015.06.002. Epub 2015 Jun 6.
Using TCGA database, we had demonstrated that aberrantly activated Forkhead box M1 (FOXM1) correlates to worse overall survival in a subgroup of platinum resistant patients. Application of thiostrepton, a natural thiazole antibiotics that inhibits FOXM1 transcription activity in the clinic is hampered by difficulties in synthesis, degradation potential, and solubility. In this study, we aim to identify potential FOXM1 small molecule inhibitors to develop a new class of therapeutic agents to address the challenges in treating chemotherapy resistant EOC.
We used in silico screening of compounds against a solved structure of FOXM1 and subsequently to derive a list of possible compounds that could inhibit FOXM1. Three compounds were tested for in vitro cytotoxicity and FOXM1 expression level was confirmed by RT-PCR and Western blot in EOC cell lines.
The FOXM1 structure obtained from 3G73 represented the DNA binding region of FOXM1 and possessed the winged helix fold representative of the Forkhead family of enzymes with two wings in direct contact with DNA. For ease of representation, we described both wings as a dimer and a single wing as a monomer. From this structure, we hypothesized two main models of how thiostrepton binding to FOXM1 could possibly curtail its transcriptional activity. In the first model thiostrepton could bind either of the wings or both wings and prevent association to DNA. In the second model thiostrepton bind the FOXM1/DNA complex and weaken association of FOXM1 to DNA. Subsequently, small molecular inhibitors could also use either of the models to inhibit transcription. To account for both models, the NCI diversity set was screened against the FOXM1 dimer:DNA complex (39 hits), dimer (11 hits) and monomer (14 hits). Those hits were further classified by chemical structure, biological function and chemical similarities to known molecules that target FOXM1. In cellular cytotoxicity assays, N-phenylphenanthren-9-amine (related to hit #225) successfully showed cytotoxicity to all three cell lines with IC50 around 1μM, and downregulate FOXM1 and transcription of its downstream molecules such as CCNB1.
By a combination of in silico screening coupled to cellular cytotoxicity studies, we have taken the first step towards identifying potential inhibitors of FOXM1 that can replace thiostrepton.
利用TCGA数据库，我们已经证明，在铂耐药患者亚组中，异常激活的叉头框M1（FOXM1）与较差的总生存期相关。硫链丝菌素是一种天然噻唑类抗生素，可抑制FOXM1转录活性，但由于合成困难、降解潜力和溶解性等问题，其在临床上的应用受到阻碍。在本研究中，我们旨在鉴定潜在的FOXM1小分子抑制剂，以开发一类新型治疗药物，应对化疗耐药性上皮性卵巢癌（EOC）治疗中的挑战。
我们利用计算机模拟筛选针对已解析的FOXM1结构的化合物，随后得出可能抑制FOXM1的化合物列表。测试了三种化合物的体外细胞毒性，并通过RT-PCR和蛋白质免疫印迹法在EOC细胞系中确认FOXM1表达水平。
从3G73获得的FOXM1结构代表了FOXM1的DNA结合区域，具有叉头酶家族典型的翼状螺旋折叠结构，两个翼直接与DNA接触。为便于表述，我们将两个翼描述为二聚体，单个翼描述为单体。基于该结构，我们推测了硫链丝菌素与FOXM1结合可能如何抑制其转录活性的两种主要模型。在第一个模型中，硫链丝菌素可以结合一个翼或两个翼，阻止与DNA结合。在第二个模型中，硫链丝菌素结合FOXM1/DNA复合物，削弱FOXM1与DNA的结合。随后，小分子抑制剂也可以使用这两种模型之一来抑制转录。为兼顾这两种模型，针对FOXM1二聚体：DNA复合物（39个命中物）、二聚体（11个命中物）和单体（14个命中物）筛选了美国国立癌症研究所（NCI）多样性集。这些命中物根据化学结构、生物学功能以及与已知靶向FOXM1分子的化学相似性进一步分类。在细胞毒性试验中，N-苯基菲-9-胺（与命中物#22相似）成功对所有三种细胞系显示出细胞毒性，IC50约为1μM，并下调FOXM1及其下游分子如细胞周期蛋白B1（CCNB1）的转录。
通过计算机模拟筛选与细胞毒性研究相结合，我们朝着鉴定可替代硫链丝菌素的潜在FOXM1抑制剂迈出了第一步。
Zotero 插件