Development of small molecule RPN13 inhibitors for Treatment of Glioblastoma

抽象的。胶质母细胞瘤（GBM）是一种侵袭性和致命的脑癌，由于肿瘤的生长， 缺乏有效的针对性治疗。我们的目标是开发一种靶向RPN 13的小分子抑制剂， 19S蛋白酶体复合物中的蛋白作为GBM的新药。RPN 13是一种蛋白酶体泛素受体， 识别泛素结合的蛋白质，这些蛋白质因此被标记为通过20S蛋白酶体酶促降解 复杂. GBM对蛋白酶体抑制高度敏感，这是由于其侵略性生长和积累的蛋白酶体抑制剂。 错误折叠的多聚泛素化蛋白，尽管蛋白酶体水平增加，但仍不能充分管理， 蛋白酶体活性增强（即蛋白毒性应激）。Up Therapeutics明智地设计了类似药物的RPN 13 抑制剂，以克服在约翰霍普金斯大学确定的原型RPN 13抑制剂RA 190的局限性。 我们选择Up284作为先导化合物是基于我们的初步研究显示出药物样特征， 特异性（与RPN 13的Cys88结合）和蛋白酶体抑制，产生细胞毒性蓄积， polyUb蛋白、ER应激和活性氧。Up284处理选择性触发实体瘤细胞凋亡 肿瘤类型包括GBM细胞系和六钩蚴。通过测量来选择合适的Up284制剂。 水溶性、稳定性和在使用蛋白酶体依赖性体内报告物成像的小鼠中的靶活性。 Up284可以静脉内或口服给药，并且它穿透BBB。作为治疗概念的证明 在GBM中，Up284在颅内同基因GL261肿瘤模型中表现出靶向活性， 蛋白酶体在肿瘤组织中的功能。Up284还使同系、异种移植和自发移植中的肿瘤消退。 具有可接受的安全性特征的卵巢癌模型。中试毒理学数据表明，Up284相对较好 耐受，没有行为异常的证据，CBC和血液化学检查正常。鉴于 Up284作为一种新的抗癌药物和GBM作为其主要适应症的潜力，我们建议： 目标1a。确定脑中杀死GBM六钩蚴所需的Up284剂量（1 - 3个月）：可再生 培养物中六钩蚴的形成是某些脑肿瘤起始细胞的决定性特征。Up284活动 将测试在存在和不存在体外BBB层的情况下对GBM六钩蚴生长的抑制作用。 目标1b。雄性和雌性CD 1小鼠中IV与口服递送后的Up284血浆和组织分布研究 (3-6月）：Milestone：确定大脑中所需的GBM治疗水平以及如何将Up284输送到 实现它们。目标2。Up284对男性和女性中原位GBM六钩蚴生长的功效 雌性裸鼠（6 - 12个月）。我们将测试Up284对颅内原位移植的活性， 在携带颅内Br23 C的雌性裸鼠中使用表达萤火虫荧光蛋白酶的Br23 C六钩球的模型 六钩蚴或携带JHH-27六钩蚴的裸雄性小鼠，替莫唑胺耐药模型。Military： 证明Up284可提供RECIST定义的完全缓解（CR）或部分缓解（PR）， 肿瘤生长，在小鼠脑中使用瘤球衍生的GBM模型。

Abstract. Glioblastoma (GBM) is an aggressive and deadly brain cancer with a one year median survival due to the lack of effective targeted treatments. Our goal is to develop a small molecule inhibitor targeting RPN13 protein in the 19S proteasome complex as a new drug for GBM. RPN13 is a proteasome ubiquitin receptor that recognizes ubiquitin-conjugated proteins that are thus tagged for degradation via the 20S proteasome enzymatic complex. GBM is highly sensitive to proteasome inhibition due to its aggressive growth and accumulation of misfolded polyubiquitinated proteins that is insufficiently managed despite increased proteasome levels and enhanced proteasome activity (i.e. proteotoxic stress). Up Therapeutics judiciously designed drug-like RPN13 inhibitors to overcome limitations in the prototype RPN13 inhibitor RA190 identified at Johns Hopkins University. We selected Up284 as lead compound based upon our preliminary studies showing drug-like characteristics, specificity (binding to Cys88 of RPN13), and proteasome inhibition that produces a cytotoxic accumulation of polyUb proteins, ER stress, and reactive oxygen species. Up284 treatment selectively triggers apoptosis in solid tumor types including GBM cell lines and oncospheres. A suitable Up284 formulation was selected by measuring aqueous solubility, stability and on-target activity in mice imaged using a proteasome-dependent in vivo reporter. Up284 can be administered i.v. or orally, and it penetrates through the BBB. As a proof of concept for treatment of GBM, Up284 demonstrated on-target activity in intracranial syngeneic GL261 tumor model by inhibiting proteasome function in tumor tissues. Up284 also regressed tumors in syngeneic, xenograft and spontaneous models of ovarian cancer with acceptable safety profile. Pilot toxicological data indicated Up284 is relatively well tolerated with no evidence of behavioral abnormalities and normal CBC and blood chemistry panel. Given the potential of Up284 as a new cancer drug and GBM as its lead indication, we propose: Goal1a. Determine Up284 dose required in the brain to kill GBM oncospheres (1-3 months): Renewable oncosphere formation in culture is a defining characteristic of certain brain tumor initiating cells. Up284 activity against the growth of GBM oncospheres in the presence and absence of an in vitro BBB layer will be tested. Goal1b. Up284 plasma and tissue distribution studies after IV vs Oral delivery in male & female CD1 mice (3-6 months): Milestones: Identify GBM therapeutic levels needed in the brain and how to deliver Up284 to achieve them. Goal2. Efficacy of Up284 against the growth of orthotopic GBM oncospheres in male & female nude mice (6-12 months). We will test Up284 activity against an intracranial orthotopic transplantation model using firefly luciferase-expressing Br23C oncospheres in nude female mice bearing intracranial Br23C oncospheres or nude male mice bearing JHH-27 oncospheres, a temozolamide resistant model. Milestones: Demonstrate that Up284 provides RECIST-defined complete response (CR) or partial response (PR) against tumor growth using oncosphere-derived GBM models in mouse brain.