Novel Probes for Studying Treatment of CNS-based Lysosomal Storage Diseases

描述（由申请人提供）：溶酶体贮积病（lsd）由42种不同溶酶体蛋白之一的基因突变引起，其中12种参与鞘脂和鞘脂糖（GSLs）的酶分解代谢或降解。虽然有三分之二的迷幻药与中枢神经系统有关，会导致认知和运动能力逐渐下降，但目前还没有有效的治疗方法。然而，有两种有效的方法来治疗周围的lsd。第一种是酶替代疗法（ERT），即用重组蛋白来补充有缺陷的酶，重组蛋白经过最终修饰后被溶酶体吸收。第二种方法需要用小分子抑制GSL合成（底物还原疗法或SRT），该策略已被临床证明对治疗戈歇1型病有效。迄今为止，SRT主要集中在抑制葡萄糖神经酰胺合成酶（GlcCer），该合成酶催化GSL合成的第一步。唯一被批准的药物是米卢司他，它是一种弱抑制剂，仅对戈谢氏1型有效，并且具有一些脱靶效应。一种更有效和选择性的抑制剂，酒石酸依格司他，目前正处于Gacher 1型的3期临床试验中，据报道其疗效优于ERT药物imiglucerase。不幸的是，酒石酸依利司他不能穿透中枢神经系统，因此对治疗基于中枢神经系统的lsd没有希望。我们最近证明了一种结构类似物（CCG-203586），设计成更具有中枢神经系统渗透性，能够显著降低小鼠大脑中glcer水平。然而，鉴于CCG-203586与依利司他在小鼠体内代谢迅速的密切结构关系，CCG-203586不太可能成为研究cns型lsd小鼠模型中GSL合成慢性抑制的最佳探针。这项工作的首要目标将是确定SRT是否是改善中枢神经系统lsd症状的有效方法。我们的方法将是：1)优化CCG-203586导联结构的代谢稳定性和CNS通透性，而不牺牲效能；2)评估最佳的新类似物进入小鼠CNS的外显性程度；3)选择最佳探针进行长期研究基于CNS的lsd小鼠模型：Sandhoff和tai - sachs。我们的建议在使用基于物理特性的设计来降低外排转运体（例如MDR1）在血脑屏障的识别方面具有创新性，并且通过使用双细胞检测GlcCer合成酶抑制，同时测量MDR1的活性和识别。这项工作将首次允许在中枢神经系统慢性抑制GlcCer合成酶对GSL动力学和中枢神经系统lsd动物模型中症状的发生和进展的影响进行研究。最后，我们工作的最终影响将是对未满足的医疗需求的第一种治疗方法的进展，即基于中枢神经系统的鞘糖脂病。

DESCRIPTION (provided by applicant): Lysosomal storage diseases (LSDs) result from genetic mutations in one of 42 different lysosomal proteins, 12 of which are involved in the enzymatic catabolism or degradation of sphingolipids and glycosphingolipids (GSLs). Although fully two-thirds of all LSDs have some CNS involvement that can result in progressive cognitive and motor decline, there are currently no effective therapies. There are, however, two effective approaches to treating LSDs in the periphery. The first is enzyme replacement therapy (ERT), in which the defective enzyme is supplemented with recombinant protein that has been terminally modified to be taken up into the lysosome. The second approach entails inhibition of GSL synthesis with small molecules (substrate reduction therapy or SRT), and this strategy has been clinically proven to be effective for the treatment of Gaucher type 1 disease. To date SRT has focused on inhibition of glucosylceramide (GlcCer) synthase, which catalyzes the first step in GSL synthesis. The only approved agent, miglustat, is a weak inhibitor which has limited efficacy only against Gaucher type 1 and possesses some off-target effects. A much more potent and selective inhibitor, eliglustat tartrate, is currently in phase 3 clinical trials for Gacher type 1 with reported efficacy superior to that of the ERT agent, imiglucerase. Unfortunately, eliglustat tartrate does not penetrate the CNS, so holds no promise for treating CNS-based LSDs. We recently demonstrated that a structural analog of eliglustat (CCG-203586), designed to be more CNS-permeable, was able to effect measurable reductions in GlcCer levels in the brains of mice. However, based on its close structural relationship to eliglustat, which is known to be rapidly metabolized in mice, it is unlikely that CCG-203586 will be an optimal probe for studying chronic inhibition of GSL synthesis in murine models of CNS-based LSDs. The overarching goal of this work will be to determine if SRT is an effective approach to ameliorating the symptoms of LSDs of the CNS. Our approach will be to: 1) optimize the CCG-203586 lead structure for metabolic stability and CNS-permeability without sacrificing potency, 2) evaluate the best new analogs for their degree of penetrance into the CNS of mice, and 3) select optimal probes for long-term studies in mouse models of the CNS-based LSDs: Sandhoff and Tay-Sachs. Our proposal is innovative in its use of physical property-based design to reduce recognition by efflux transporters (e.g. MDR1) at the blood brain barrier, and by the use of dual cell-based assays for GlcCer synthase inhibition that simultaneously measure both activity and recognition by MDR1. This work will be significant in allowing, for the first time, investigation o the effects of chronic inhibition of GlcCer synthase in the CNS on GSL dynamics and on development and progression of symptoms in animal models of CNS-based LSDs. Finally, the ultimate impact of our work will be progress toward the first therapy for an unmet medical need, viz. CNS-based glycosphingolipidoses.