Screening for Inhibitors for ERG in Prostate Cancer

描述(申请人提供)：ETS家族成员ERG已被证明在前列腺癌中经常过度表达。也许更引人注目的是，ERG和ETS蛋白ETV1最近被证明是TMPRSS2染色体易位的目标，在80%的前列腺癌患者样本中观察到了这种易位。事实上，TMPRSS2的表达是受雄激素调节的，导致这些前列腺癌中ERG或ETV1的过度表达。最近，对ERG在前列腺细胞中过度表达的影响进行了更广泛的研究。两项研究表明，ERG的过度表达会导致侵袭性增加。前列腺癌细胞系VCaP中ERG基因的敲除已被证明可以抑制侵袭。基因芯片分析显示，ERG在VCaP细胞中的敲除导致与分化的管腔上皮细胞相关的基因表达增加，提示ERG在前列腺癌中的过度表达可能是阻止分化的作用之一。Ets结构域介导的DNA结合是Ets蛋白基因调控所必需的，也是过度表达ERG所介导的失调所必需的。事实上，DNA结合在EWS和ETS蛋白Fli-1之间的融合中是必不可少的，Fli-1是ERG的近亲。EWS-FLI1融合在尤文肉瘤患者中发现的比例很高。EWS-FLI1融合蛋白ETS结构域的点突变会破坏DNA结合，从而破坏EWS-FLI1的转化能力。DNA结合对这些蛋白质功能的重要性强烈表明，抑制ERG的DNA结合活性可能是治疗前列腺癌以及ERG活性起关键作用的其他癌症的机制。我们的目标是开发这种相互作用的小分子抑制剂作为探针，以检验这一假说，并为开发针对ERG的靶向治疗奠定基础。为此，我们建议使用高通量筛选(HTS)来识别抑制这种蛋白质-DNA相互作用的初始先导化合物。我们将使用一种经过验证的荧光偏振分析来筛选化合物的MLPCN文库。最活跃的化合物将使用HTRF分析和结合的核磁共振确认来验证。特异性将通过筛查无关的蛋白质-DNA相互作用以及筛查其他ETS家族成员来评估。随后，这些化合物将通过结合标准药物化学的结构辅助药物设计方法进行优化，以增加Ets结构域的结合亲和力。最有效的化合物将在含有ERG易位的适当的前列腺癌细胞系中进行测试，如果可行的话，还将在适当的前列腺癌小鼠模型中进行测试。

DESCRIPTION (provided by applicant): The Ets family member ERG has been shown to be frequently over-expressed in prostate cancer. Perhaps more strikingly, ERG and the Ets protein ETV1 have recently been shown to be the targets of chromosomal translocations with TMPRSS2 which are observed in 80% of prostate cancer patient samples. Indeed, the expression of TMPRSS2 is androgen regulated, resulting in over-expression of ERG or ETV1 in these prostate cancers. More extensive studies of the effects of over-expression of ERG in prostate cells have recently been carried out. Two studies have shown that over-expression of ERG results in increased invasion. Knockdown of ERG in the prostate cancer cell line VCaP has been shown to inhibit invasion. Microarray analysis shows that ERG knockdown in VCaP cells results in increased expression of genes associated with differentiated luminal prostate epithelial cells, suggesting that one of the roles of ERG overexpression in prostate cancer may be to block differentiation. DNA binding mediated by the Ets domain is essential for Ets protein gene regulation and certainly essential for the dysregulation mediated by over-expressed ERG. Indeed, DNA-binding has been shown to be essential in the context of the fusion between EWS and the Ets protein FLI-1, a close relative of ERG. The EWS-FLI1 fusion is found in a high percentage of Ewing's sarcoma patients. Point mutations in the Ets domain of the EWS-FLI1 fusion protein which impair DNA binding disrupt the transforming ability of EWS-FLI1. The importance of DNA-binding to the function of these proteins strongly suggests that inhibiting the DNA binding activity of ERG may be a mechanism to treat prostate cancer as well as other cancers where the activity of ERG plays a key role. Our goal is to develop small molecule inhibitors of this interaction as probes to test this hypothesis and lay the groundwork for the development of targeted therapies against ERG. To that end, we are proposing to use high throughput screening (HTS) to identify initial lead compounds which inhibit this protein-DNA interaction. We will use a well-validated fluorescence polarization assay to screen the MLPCN library of compounds. The most active compounds will be verified using an HTRF assay and NMR confirmation of binding. Specificity will be assessed by screening against an unrelated protein-DNA interaction as well as screening against other Ets family members. The compounds will subsequently be optimized to increase binding affinity for the Ets domain by structure-aided drug design approaches combined with standard medicinal chemistry. The most potent compounds will be tested in appropriate prostate cancer cell lines harboring ERG translocations and, if feasible, in appropriate mouse models of prostate cancer.