Screening for Inhibitors for ERG in Prostate Cancer

描述（由申请人提供）：Ets家族成员ERG已被证明在前列腺癌中频繁过表达。也许更引人注目的是，ERG和Ets蛋白ETV 1最近已被证明是TMPRSS 2染色体易位的靶标，在80%的前列腺癌患者样品中观察到。事实上，TMPRSS 2的表达受雄激素调节，导致这些前列腺癌中ERG或ETV 1的过度表达。最近已经进行了关于前列腺细胞中ERG过度表达的影响的更广泛的研究。两项研究表明，ERG的过度表达导致侵袭增加。在前列腺癌细胞系VCaP中ERG的敲低已显示抑制侵袭。微阵列分析显示，VCaP细胞中的ERG敲低导致与分化的管腔前列腺上皮细胞相关的基因表达增加，这表明ERG过表达在前列腺癌中的作用之一可能是阻断分化。由Ets结构域介导的DNA结合对于Ets蛋白基因调控是必需的，并且当然对于由过度表达的ERG介导的失调是必需的。事实上，DNA结合已被证明是必不可少的EWS和Ets蛋白FLI-1，ERG的近亲之间的融合的情况下。EWS-FLI 1融合在高比例的尤文肉瘤患者中发现。EWS-FLI 1融合蛋白Ets结构域的点突变损害DNA结合，破坏EWS-FLI 1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来验证这一假设，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）来鉴定抑制这种蛋白质-DNA相互作用的初始先导化合物。我们将使用经过充分验证的荧光偏振测定来筛选化合物的MLPCN库。将使用HTRF测定和结合的NMR确认来验证最具活性的化合物。将通过针对不相关蛋白质-DNA相互作用以及针对其他Ets家族成员进行筛选来评估特异性。这些化合物随后将通过结构辅助药物设计方法与标准药物化学相结合来优化，以增加对Ets结构域的结合亲和力。最有效的化合物将在携带ERG易位的适当前列腺癌细胞系中进行测试，如果可行，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.