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Design and Synthesis of Amino Acid-based Inhibitors Against Key Enzymes

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2017, Doctor of Philosophy, University of Toledo, Chemistry.
Canavan disease (CD), a fatal neurological disorder observed in newborns, is caused by an inherited genetic defect. CD is a leukodystrophy, causing disruption in the growth and maintenance of myelin sheath which is responsible for efficient transmission of nerve impulses. Therefore, infants affected with CD do not develop motor skills and speech, and they usually also develop conditions such as hypotonia and macrocephaly. This neurological disorder is caused by multiple mutations in the aspA gene that codes for aspartoacylase, an enzyme responsible for catalyzing the conversion of N-acetyl-L-aspartate (NAA) to L-aspartic acid and acetate (an important chemical in the biosynthesis of myelin sheaths). For several decades researchers primarily focused on the acetate deficiency as the major cause for the brain disorders that are associated with Canavan patients. Recently, a study has shown that a knockout of the Nat8l gene that codes for ANAT, the enzyme responsible for NAA synthesis, seems to overcome these adverse effects through normal myelination. In addition, overexpression of ANAT and the subsequent rise in NAA levels have been linked with different life-threatening cancers. High NAA levels were detected in a large percentage of adenocarcinoma and squamous cell carcinoma cells isolated from patients with late-stage lung cancer, and these elevated levels of NAA and high expression of ANAT was also identified in high-grade ovarian cancer tissue samples. In each case these elevated levels have been correlated with worse overall patient survival in these and in several other forms of cancers. This work is primarily focused on the synthesis of potent inhibitors against ANAT. In addition to the screening amino acids, metabolites, and constrained analog libraries, a library of 105 dioic acid and phthalate analogs were synthesized, out of which a 50 % hit rate of modest enzyme inhibitors was identified. An expansion of this synthetic inhibitor library is in progress by systematically optimizing the structures of these dioic acid and phthalate derivative hits to obtain more potent inhibitors, with the best compounds having inhibitor Ki values in the low micromolar range. As an alternative approach, several bisubstrate analogs were synthesized which exhibits extremely high affinity towards ANAT, with inhibitor values in the nanomolar range. An in-depth study of the electronic interactions between these bisubstrate analogs and their truncated derivatives with the enzyme has been performed. This study will allow us to understand and design new potent inhibitors which can be further developed into selective drug candidates to treat Canavan disease. As a second project, the design and synthesis of inhibitors against a key enzyme in the aspartate pathway was also conducted. The aspartate pathway is the pathway through which most of the microorganisms like prokaryotes, fungi and some higher plants synthesize many essential amino acids for their survival. This pathway is also responsible for the biosynthesis of diaminopimelate, which plays a crucial role in bacterial cell wall formation. Pathogenic bacterial species such as Staphylococcus, Enterococcus, Salmonella, Gonococcus, Streptococcus and Mycobacterium which were previously susceptible to antibiotics are now showing multidrug resistance, which poses a serious threat to human health. The enzyme aspartate-ß-semialdehyde dehydrogenase (ASADH) catalyzes a critical step in this aspartate pathway at a branch point leading in the production of four essential amino acids, L-lysine, L-methionine, L-leucine, L-isoleucine. The inhibition of ASADH prevents the production of these amino acids, which has been shown to be fatal to these microorganisms. The absence of this entire pathway in mammals makes the enzymes in the aspartate pathway more selective targets, giving rise to the possibility of developing new antibiotics with novel mechanisms. An initial set of glutamic and adipic acid derivatives was synthesized as bisubstrate inhibitors against ASADH, out of which (3,5-bis(trifluoromethyl)benzoyl)glutamic acid exhibits fairly weak inhibition, but was found to be specific for the ASADH form found in Vibrio cholerae. Currently, derivatizations on compound G6 are being performed to develop additional species-selective potent inhibitors. Simultaneously, ebselen derivatives were synthesized as potential covalent modifiers of ASADH. The preliminary docking results on newly synthesized ebselen derivatives show promising in-silico results, with high predicted binding affinity to the enzyme ASADH. The synthesis of these newly designed inhibitors would lead to identifying more potent inhibitors which can be further developed into species-selective drug candidates. In the final project, small thiol containing compounds were synthesized as potent protein reducing agents. Two compounds, dithiopropylamine (DTPA) and 2-methyldithopropylamine (MDTPA) were synthesized and their qualitative reduction potentials were compared to dithiothreitol (DTT) and to a newly developed commercial compound dithiobutylamine (DTBA) by performing kinetic screening experiments against the enzyme papain. More in-depth analysis is necessary to directly measure the reduction potentials of the synthesized reagents, and this study would provide an understanding of the relationship between dithiane ring formation and the reduction ability of this series of compounds.
Viola Ronald (Committee Chair)
Andreana Peter (Committee Member)
Zhu Jianglong (Committee Member)
Viranga Tillekeratne L.M (Committee Member)
144 p.

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Citations

  • Mutthamsetty, V. (2017). Design and Synthesis of Amino Acid-based Inhibitors Against Key Enzymes [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1513014525316672

    APA Style (7th edition)

  • Mutthamsetty, Vinay. Design and Synthesis of Amino Acid-based Inhibitors Against Key Enzymes. 2017. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1513014525316672.

    MLA Style (8th edition)

  • Mutthamsetty, Vinay. "Design and Synthesis of Amino Acid-based Inhibitors Against Key Enzymes." Doctoral dissertation, University of Toledo, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1513014525316672

    Chicago Manual of Style (17th edition)