Development of novel compounds to inhibit PRMT5 enzyme in high grade astrocytomas

描述（由申请人提供）：高级别星形细胞瘤是侵袭性脑肿瘤，预后不佳，被认为是无法治愈的，尽管进行了强化的多模式治疗，平均生存期仍不到一年。当前治疗方式的局限性表明需要针对III级和IV级（多形性胶质母细胞瘤，GBM）的创新治疗方法。最近的研究表明，蛋白质的翻译后共价修饰和染色质的表观遗传调控在细胞生长、分化和增殖的控制中起着核心作用。染色质重塑酶如组蛋白去乙酰化酶（HDAC）、DNA甲基转移酶和蛋白精氨酸甲基转移酶5 （PRMT5）参与沉默促炎和肿瘤抑制基因（TSG）的表达，并促进细胞转化。PRMT5酶通过甲基化组蛋白上的精氨酸残基（在组蛋白4精氨酸残基3 （H4R3）和H3R8），有助于几个重要调控基因的转录沉默。我们的实验室已经证明，由PRMT5过表达驱动的表观遗传过程与高级别星形细胞瘤中可操作的关键致癌途径的调控相关。患者来源的GBM细胞系和原发性GBM肿瘤过度表达大量的PRMT5蛋白。正常脑组织和中低级别星形细胞瘤不过度表达PRMT5，提示过度表达可能选择性地发生在高级别、更具侵袭性的胶质瘤中。PRMT5的过表达程度与GBM患者的生存和GBM细胞系的增殖呈负相关。我们开发了小抑制RNA分子（SiRNA）来抑制PRMT5的表达，从而导致靶组蛋白尾部精氨酸残基的去甲基化以及肿瘤抑制蛋白和免疫调节蛋白的转录去抑制和快速翻译。抑制PRMT5过表达导致GBM细胞经历细胞周期阻滞、凋亡、完全抑制细胞迁移和使细胞对替马唑胺（一种被认为是GBM患者治疗标准的药物）的毒性作用敏感。我们相信我们的工作已经表明，PRMT5既是一个重要的预后因素，也是GBM的一个有吸引力的治疗靶点。我们利用同源PRMT酶的晶体结构开发了计算建模系统，允许构建PRMT5的分子模型并快速筛选超过10,000个小分子化合物。这种方法已经发现了几种抑制PRMT5活性的小分子。在这里，我们提出了一个多学科计划，利用新的策略来产生更有效和选择性的PRMT5活性小分子抑制剂。有希望的化合物将在体外和体内开发平台上进行严格评估，并将增强我们发现一类新的药物的能力，这些药物可以选择性地靶向GBM中有希望的治疗靶点。

DESCRIPTION (provided by applicant): High grade astrocytomas are aggressive brain tumors that are associated with a dismal prognosis and are considered incurable with a mean survival of less than one year despite intensive multimodality therapy. The limits of current treatment modalities indicate a need for innovative therapies targeting grade III and grade IV (glioblastoma multiforme, GBM). Recent studies have shown that post translational covalent modification of proteins and epigenetic regulation of chromatin play a central role in the control of cell growth, differentiation, and proliferation. Chromatin remodeling enzymes like histone deacetylase (HDAC), DNA methyltransferase and protein arginine methyltransferase 5 (PRMT5) are involved in silencing pro inflammatory and tumor suppressor gene (TSG) expression and contribute towards cellular transformation. The PRMT5 enzyme contributes towards transcriptional silencing of several important regulatory genes by methylating arginine residues on histone proteins (at histone 4 arginine residue 3 (H4R3) and H3R8). Our laboratory has demonstrated that epigenetic processes driven by PRMT5 over expression are relevant in regulation of key oncogenic pathways that are operable in high grade astrocytomas. Patient-derived GBM cell lines and primary GBM tumors over express abundant levels of PRMT5 protein. Normal brain tissue and low or intermediate grade astrocytomas do not over express PRMT5 suggesting that overexpression may selectively occur in high grade, more aggressive gliomas. The degree of PRMT5 over expression inversely correlated with survival of GBM patients and with proliferation of GBM cell lines. We developed small inhibitory RNA molecules (SiRNA) to inhibit PRMT5 expression leading to demethylation of target histone tail arginine residues and transcriptional de-repression and rapid translation of tumor suppressor and immune modulatory proteins. Inhibition of PRMT5 over expression led GBM cells to undergo cell cycle arrest, apoptosis, complete inhibition of cell migration and sensitized cells to the toxic effects of temazolomide, a drug considered standard of care in management of patients with GBM. We believe our work has shown that PRMT5 is both an important prognostic factor and an attractive therapeutic target for GBM. We have developed computational modeling systems utilizing crystallographic structure of homologous PRMT enzymes allowing for construction of a molecular model of PRMT5 and rapid screening of over 10,000 small molecule compounds. This method has led to the discovery of several small molecules that inhibit PRMT5 activity. Here we present a multidisciplinary plan utilizing novel strategies to generate more potent and selective small molecule inhibitors of PRMT5 activity. Promising compounds will be rigorously evaluated in both in vitro and in vivo development platforms and will enhance our ability to discover a new class of drugs that selectively target a promising therapeutic target in GBM. PUBLIC HEALTH RELEVANCE: High grade astrocytomas are the most common brain tumors afflicting both pediatric and adult populations and are associated with a dismal prognosis with most patients surviving less than 1 year despite aggressive multimodal therapy with surgery, radiation and chemotherapy. We have shown that the PRMT5 protein is selectively over expressed in high grade astrocytomas, enhances growth and survival, serves as an adverse prognostic factor in glioma patients and is an attractive therapeutic target. Here we propose methods to promote the rapid discovery of small molecules capable of selectively inhibiting PRMT5 activity and present a preclinical development plan to generate novel drugs inhibiting a new target that is selectively over expressed in high grade astrocytomas.