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.
There are several thousand children in the US who are dying slowly from the effects of mutations in lysosomal hydrolases and at present there is no treatment for those which involve mental retardation. We propose novel approaches to develop chaperones based on palmitoylated peptides which can cross the blood-brain barrier, stabilize misfolded proteins and restore enzyme activity and/or degrade storage material.
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