Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


Qinzhe Wang-Dissertation_final.pdf (10.33 MB)

ETD Abstract Container

Abstract Header

Developing Approaches to Treat Canavan Disease

Wang, Qinzhe
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1493301078219765

Abstract Details

2017, Doctor of Philosophy, University of Toledo, Chemistry.
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, ß-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.
Ronald Viola (Committee Chair)
Donald Ronning (Committee Member)
Dragan Isailovic (Committee Member)
Robert Blumenthal (Committee Member)
142 p.
Analytical Chemistry; Biochemistry; Chemistry
Canavan Disease; N-acetylaspartic acid; NAA; Aspartoacylase; ASPA; Aspartate N-acetyltransferase; ANAT; Asp-NAT; NAT8L; Glycosylation; MALDI; metabolism; therapy; detergent; Protein engineering; Dual Affinity Purification; inhibitor; membrane; SPEG; DTNB

Recommended Citations

Citations

  • Wang, Q. (2017). Developing Approaches to Treat Canavan Disease [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1493301078219765

    APA Style (7th edition)

  • Wang, Qinzhe. Developing Approaches to Treat Canavan Disease. 2017. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1493301078219765.

    MLA Style (8th edition)

  • Wang, Qinzhe. "Developing Approaches to Treat Canavan Disease." Doctoral dissertation, University of Toledo, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1493301078219765

    Chicago Manual of Style (17th edition)

toledo1493301078219765
383
© 2017, all rights reserved.
This open access ETD is published by University of Toledo and OhioLINK.