Proj 4: Chemical Chaperone Therapy of Batten Disease

项目总结(见说明)： 药理伴侣(如：AcGDap(Palm)VKIKK)可被细胞内化，并将错误折叠的蛋白质重新折叠为活性构型，但需要穿过血脑屏障，进入神经元和逃逸内小体。我们已经证明，棕榈酰化的多肽基序能够唯一地允许药物逃脱内体，并且这种伴侣可以重新激活错误折叠的蛋白质，如棕榈酰蛋白硫酯酶(PPT1)。我们现在建议设计序列，允许伴侣通过附着荧光团和目标基序(例如：狂犬病病毒糖蛋白外壳肽(RVG))或包覆在6-1onm量子点上来穿过血脑屏障。我们将测试Pro和组氨酸的短序列，使用 谷氨酰胺间隔物附着在635 nm红色量子点的表面，表面覆盖着4-硫醇聚乙二醇乙二醇配体。我们将在培养的出生后神经元和PPT1起源有明确点突变的患者的淋巴母细胞中进行测试，然后使用与项目I合作的E3胚胎鸡脊髓注射系统，或与项目II合作的大鼠海马片系统。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)、它们的有效性和最终的细胞分布，方法是在它们的实验系统中添加我们的伴侣。我们还将测试棕榈酰化肽基序通过特定定位于脂筏发挥作用的想法，脂筏是膜中极富含胆固醇和鞘磷脂的微域，似乎用于组装信号复合体。伴侣只能治疗20%-50%的突变，所以对于剩下的50%-80%的INCL患者，我们建议疏水性硫醇，如硫代胆固醇，可以在化学上促进储存材料本身的水解。最后，我们将把我们的方法扩展到最常见的巴顿病，这种病是由CLN2基因(三肽基肽酶-1)突变引起的。病情较轻的复合杂合子患者应该受益于基于抑制物硼酸盐复合体(AAFX)的伴侣疗法，该疗法通过我们独特的肽序列直接或包裹在量子点上传递到中枢神经系统。

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.