Developing Approaches to Treat Canavan Disease
Başlık:
Developing Approaches to Treat Canavan Disease
Yazar:
Wang, Qinzhe, author.
ISBN:
9780438097308
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (143 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Committee members: Robert Blumenthal; Dragan Isailovic; Donald Ronning; Ronald Viola.
Özet:
Canavan disease is a progressive, fatal neurological disorder that is caused by defects in the human ASPA gene, which leads to an interruption in the metabolism of N-acetylaspartic acid (NAA). NAA is the second most abundant amino acid in the human brain. It is synthesized by aspartate N-acetyltransferase in neuronal mitochondria and is hydrolyzed by aspartoacylase in oligodendrocytes. Multiple therapies are currently under investigation to identify a treatment for Canavan disease.
Aspartoacylase has previously been successfully expressed, kinetically characterized, and structurally characterized. To provide further insights about this enzyme, a series of mass spectrometry-based protein analysis studies have been performed. The amino acid sequence of aspartoacylase was confirmed by high-performance liquid chromatography/matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry. No post-translational modifications, in particular asparagine-linked glycosylation that were previously implicated, were identified. Aspartate N-acetyltransferase (ANAT) has been shown to be a membrane associated enzyme with a putative membrane region of about 30 amino acid residues. While detergent extraction and several protein engineering approaches in the putative membrane domain failed to produce a soluble and active form of the enzyme, a tandem affinity purification approach using maltose-binding protein as fusion tag was able to produce an active and soluble enzyme form suitable for inhibitor development.
Aspartate N-acetyltransferase was found to have high substrate specificity. Only three compounds, beta-methylaspartate, 2,3-diaminosuccinate, and L-glutamate, were identified as alternative substrates from 160 different amino acid analogs that were examined. Several factors that could affect ANAT activity were also tested. Triton X-100 and Tween 20 were identified to have the least impact on ANAT activity after a screening of a detergent library. The effect of variations in pH identified a pK value of 6.8, which could reflect an active site histidine residue functioning as a general base.
As an initial effort to develop selective inhibitors for ANAT as a novel substrate reduction therapy for Canavan disease, several weak to moderate ANAT inhibitors were identified through focused library screening of amino acid analogs and small molecule metabolites. A surprisingly potent hit with carboxybenzyl-aspartic acid led to the exploration of a series of di-carboxylic acids. Optimizing the chain lengths, and the nature of the side chain linkage resulted in inhibitors with low micromolar inhibition constants. This was followed by targeted inhibitor optimizations starting with a benzylaminoethylphthalate core structure, which has led to several sub-micromolar inhibitors against this important human enzyme. These sets of newly synthesized ANAT inhibitors are now being modified to improve their cellular uptake properties in order to start efficacy studies in an animal model of Canavan disease.
Notlar:
School code: 0232
Tüzel Kişi Ek Girişi:
Mevcut:*
Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(687193.1) | 687193-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
On Order
Liste seç
Bunu varsayılan liste yap.
Öğeler başarıyla eklendi
Öğeler eklenirken hata oldu. Lütfen tekrar deneyiniz.
:
Select An Item
Data usage warning: You will receive one text message for each title you selected.
Standard text messaging rates apply.