Proj 4: Chemical Chaperone Therapy of Batten Disease

项目总结（见说明）： 药理学伴侣（例如：AcGDap（Palm）VKIKK）可以被细胞内化并将错误折叠的蛋白质重新折叠成活性构型，但需要穿过血脑屏障，进入神经元并逃离内体。我们已经表明，棕榈酰化肽基序是唯一能够允许药物逃逸内体，并且这种分子伴侣可以重新激活错误折叠的蛋白质，如棕榈酰化蛋白硫酯酶（PPT 1）。我们现在建议设计序列，以允许分子伴侣通过连接荧光团和靶基序（例如：狂犬病病毒糖蛋白外壳肽（RVG））或6-1 Onm的量子点上的涂层穿过血脑屏障。我们将测试脯氨酸和组氨酸的短序列， 在一个实施方案中，该方法包括使用谷氨酰胺间隔物连接至用4-巯基PEG配体封端的635 nm红色QD的表面。我们将在培养的出生后神经元和来自在PPT 1起源中具有确定的点突变的患者的成淋巴细胞中测试这一点，然后与项目I合作使用E3鸡胚脊髓注射系统，或与项目II合作使用大鼠海马切片系统。这三个模型系统代表胚胎脑，新生儿脑和出生后脑，合作将使我们能够更好地评估这些药物的毒性（如果有的话），通过将我们的分子伴侣添加到他们的实验系统中，它们的功效和最终的细胞分布。我们还将测试棕榈酰化肽基序通过特异性定位于脂筏、膜中的微结构域而起作用的想法，所述膜中的微结构域极大地富含胆固醇和鞘脂，并且似乎用于组装信号复合物。伴侣只能治疗20-50%的突变，因此对于剩余的50-80%的INCL患者，我们提出疏水性硫醇如硫代胆固醇可以化学促进储存材料本身的水解。最后，我们将扩展我们的方法，以最常见的形式Batten病引起的突变CLN 2基因（三肽基肽酶-1）。患有较轻疾病的复合杂合子患者应受益于基于抑制剂（AAFX）的硼酸盐复合物的伴侣治疗，所述硼酸盐复合物与我们独特的肽序列直接或涂覆在量子点上递送至CNS。

PROJECT SUMMARY (See instructions): Pharmacological chaperones (eg: AcGDap(Palm)VKIKK)can be internalized by cells and re-fold misfolded proteins to an active configuration, but need to cross the blood brain barrier, enter neurons and escape endosomes. We have shown that the palmitoylated peptide motif is uniquely able to allow drugs to escape endosomes and that such chaperones can reactivate misfolded proteins such as palmitoyhprotein thioesterase (PPTl). We now propose to design sequences to permit the chaperones to cross the blood brain barrier by either attaching a fluorophore and target motif (eg: rabies virus glycoprotein coat peptide (RVG)) or coating on 6-1 Onm quantum dots. We will test short sequences of proline and histidine, with a glutamine spacer to attach to the surface of 635 nm red QDs capped with the 4-thiol PEG ligand. We will test this in cultured postnatal neurons, and lymphoblasts from patients with defined point mutations in PPTl origin, and then use either the E3 embryonic chick spinal cord injection system, in collaboration with Project I, or the rat hippocampal slice system through collaboration with Project II. The three model systems represent embryonic brain, neonatal brain and postnatal brain and the collaborations will allow us to better assess the toxicity (if any) of these drugs, their efficacy and their ultimate cellular distribution by adding our chaperones into their experimental systems. We will also test the idea that the palmitoylated peptide motif works by specifically localizing to lipid rafts, microdomains in membranes which are greatly enriched in cholesterol and sphingolipids and appear to be used to assemble signaling complexes. Chaperones can only treat 20-50% of the mutations so for the remaining 50-80% of INCL patients we propose that hydrophobic thiols such as thiocholesterol could chemically facilitate hydrolysis of the storage material itself. Finally we will extend our approach to the most common form of Batten disease caused by mutations in the CLN2 gene (tripepfidyl-peptidase-1). Compound heterozygote patients with milder disease should benefit from chaperone therapy based on borate complexes of the inhibitor (AAFX) delivered to the CNS with our unique peptide sequences either directly or coated on quantum dots.