Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


Bharani Thangavelu_Dissertation.pdf (9.26 MB)

ETD Abstract Container

Abstract Header

Development of Selective Inhibitors against Metabolic Enzymes Involved In Aspartate Pathway for Antibiotic Development

Thangavelu, Bharani
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1466587873

Abstract Details

2016, Doctor of Philosophy, University of Toledo, Chemistry.
Aspartate-ß-semialdehyde dehydrogenase (ASADH) is located at the first branch point in the aspartate metabolic pathway, which leads to the biosynthesis of several essential amino acids and important metabolites. This pathway is present only in plants and microbes but absent in mammals. The Aspartate pathway is crucial for bacteria for various metabolic processes and the microbial enzymes involved in this pathway are attractive targets for new antibiotic and antifungal compounds. The structures of ASADHs have been determined from Gram-positive bacteria, Gram-negative bacteria and fungal species. These enzymes share the same substrate binding and active site catalytic groups; however they show different inhibition patterns when screened against low molecular weight fragment libraries. Methionine is a sulfur-containing amino acid that is synthesized via a branch point in the aspartate metabolic pathway. S-adenosyl methionine (AdoMet) is subsequently synthesized from methionine, and plays a critical role in the transfer of methyl groups to various biomolecules, including DNA, proteins and small-molecule secondary metabolites. The branch point that leads to the synthesis of methionine and S-adenosyl methionine starts with the activation of the hydroxyl group of homoserine. The mode of activation of homoserine differs from plants to microorganisms, as well as within different microbial systems. At this point, depending on the species, at least three different activation routes have been identified. In addition, the route of sulfur assimilation in these systems can also vary from species to species. Homoserine acyl transferases catalyze the primary routes to homoserine activation in microbes, and these enzymes are members of the a/ß hydrolase superfamily. While the two different families of homoserine acyl transferases use the same kinetic and chemical mechanisms to catalyze this related reaction, they do so by using significantly different overall structures, as well as subtle differences in their closely related active site structures. In all fungi, and in many Gram-positive bacterial species such as Bacillus, Brevibacterium and Corynebacterium, an acetyl group is transferred to homoserine from acetyl-CoA to form O-acetyl homoserine (OAH) catalyzed by homoserine transacetylase (HTA) and coded by the met2 gene. In most enteric bacteria, including E. coli and other Gram-negative facultative and anaerobic bacteria, a succinyl group is transferred to homoserine from succinyl-CoA to form O-succinylhomoserine (OSH) catalyzed by homoserine transsuccinylase (HTS). The catalytic activity of both HTS and HTA has been shown to be regulated by coordinated feedback inhibition by both L-methionine and AdoMet. No organism has been identified to date that contains the genes for both acyltransferases. In addition to their variant in species distribution, these two enzymes display virtually no primary sequence similarity and also show significant structural differences. Since this pathway produces metabolites that play a number of critical biochemical roles in microorganisms, and because of the complete absence of related enzymes in mammals, the enzymes in this pathway represent novel targets for future antibiotic drug development. As a second project within this dissertation, development of enzyme inhibitors for the treatment of a rare neurological disorder is studied. Aspartate N-acetyl transferase (ANAT), an enzyme which catalyzes the N-acetyl aspartate (NAA). NAA is a major source of acetyl groups for lipid synthesis during brain development. Canavan disease (CD) is a rare, but fatal autosomal-recessive neurodegenerative disease caused by mutations in the acy2 gene that leads to the deficiency in the encoded aspartoacylase (ASPA), the enzyme responsible for the deacetylation of NAA in the brain. Recent work has shown that a knockout of the Nat8l gene that codes for ANAT leads to a reversal of the CNS demyelination in an animal model of CD. An approach in which selective ANAT inhibitors are used to adjust brain NAA levels back into the physiological range has the potential to treat the symptoms of CD without introducing increased risks. In the present study we describe the crystal structure of Staphylococcus aureus HTA and several approaches, including fragment based drug discovery, docking studies, kinetic and crystal structure studies which were combined to identify and characterize selective inhibitors of ASADH. The initial phthalate based inhibitors were modified by employing coupling reactions around the secondary amine, and systematically produced a series of inhibitors against our target enzyme. Structure-guided development of these lead compounds has now produced potent inhibitors of our target enzyme, with superior selectivity observed between the Gram-negative and Gram-positive orthologs of ASADH. This combined approach of inhibitor design, along with kinetic and structural characterization, illustrates the potential for potent drug development against these essential enzymes.
Ronald Viola, Ph.D. (Committee Chair)
Max Funk, Ph.D. (Committee Member)
Donald Ronning, Ph.D. (Committee Member)
Amanda Bryant-Friedrich, Ph.D. (Committee Member)
229 p.
Biochemistry; Chemistry

Recommended Citations

Citations

  • Thangavelu, B. (2016). Development of Selective Inhibitors against Metabolic Enzymes Involved In Aspartate Pathway for Antibiotic Development [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1466587873

    APA Style (7th edition)

  • Thangavelu, Bharani. Development of Selective Inhibitors against Metabolic Enzymes Involved In Aspartate Pathway for Antibiotic Development. 2016. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1466587873.

    MLA Style (8th edition)

  • Thangavelu, Bharani. "Development of Selective Inhibitors against Metabolic Enzymes Involved In Aspartate Pathway for Antibiotic Development." Doctoral dissertation, University of Toledo, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1466587873

    Chicago Manual of Style (17th edition)

toledo1466587873
324
© , some rights reserved.Creative Commons License Development of Selective Inhibitors against Metabolic Enzymes Involved In Aspartate Pathway for Antibiotic Development by Bharani Thangavelu is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by University of Toledo and OhioLINK.