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

描述（由申请方提供）：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种参与鞘脂和鞘糖脂（GSL）的酶促催化或降解。尽管所有LSD中有三分之二的患者都有一些CNS受累，可能导致进行性认知和运动能力下降，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边缘地区的LSD。第一种是酶替代疗法（ERT），其中有缺陷的酶补充有重组蛋白，该重组蛋白已被末端修饰以被溶酶体吸收。第二种方法需要用小分子抑制GSL合成（底物减少疗法或SRT），并且该策略已被临床证明对治疗1型戈谢病有效。到目前为止，SRT的重点是抑制葡萄糖神经酰胺（GlcCer）合酶，催化GSL合成的第一步。唯一获批的药物麦格司他是一种弱抑制剂，仅对1型戈谢病疗效有限，并具有一些脱靶效应。一种更有效和选择性更强的抑制剂，酒石酸依格列他，目前正处于针对Gacher 1型的III期临床试验中，报告的疗效上级于ERT药物伊米苷酶。不幸的是，酒石酸依格列他不能渗透CNS，因此不能用于治疗CNS型LSD。我们最近证明，eliglustat（CCG-203586）的结构类似物（设计为更具CNS渗透性）能够实现小鼠脑中GlcCer水平的可测量降低。然而，基于其与eliglustat的密切结构关系（已知eliglustat在小鼠中快速代谢），CCG-203586不太可能成为研究基于CNS的LSD小鼠模型中GSL合成慢性抑制的最佳探针。这项工作的总体目标是确定SRT是否是改善CNS LSD症状的有效方法。我们的方针是：1）优化CCG-203586先导结构的代谢稳定性和CNS渗透性而不牺牲效力，2）评估最佳新类似物进入小鼠CNS的渗透程度，以及3）选择最佳探针用于基于CNS的LSD的小鼠模型中的长期研究：Sandhoff和Tay-Sachs。我们的提案在以下方面具有创新性：使用基于物理性质的设计来减少血脑屏障上外排转运蛋白（例如MDR 1）的识别，以及使用基于细胞的双重GlcCer合酶抑制测定，同时测量活性和识别通过MDR 1。这项工作将是有意义的，允许，第一次，研究慢性抑制的GSL动力学和CNS为基础的LSD动物模型中的症状的发展和进展的CNS中的GlcCer合酶的影响。最后，我们工作的最终影响将是为未满足的医疗需求提供第一种治疗方法，即基于CNS的鞘糖脂增多症。

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.