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Development of Selective Inhibitors against Enzymes Involved in the Aspartate Biosynthetic Pathway for Antifungal Drug Development

Dahal, Gopal Prasad
http://orcid.org/0000-0002-8510-5615
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1532889045486984

Abstract Details

2018, Doctor of Philosophy, University of Toledo, Chemistry.
Aspartate semialdehyde dehydrogenase (ASADH) functions at a critical junction in the aspartate biosynthetic pathway and represents a validated target for antimicrobial drug design. This enzyme catalyzes the NADPH-dependent reductive dephosphorylation of ß-aspartyl phosphate to produce the key intermediate aspartate semialdehyde. Production of this intermediate represents the first committed step for the biosynthesis of several essential amino acids in fungi and in bacteria. The absence of this enzyme in humans and other mammals will allow the selective targeting of any ASADH inhibitors against pathogenic microorganisms. We have accumulated significant structural and mechanistic information about the bacterial ASADHs, but have only limited knowledge of their fungal counterparts. To bridge this gap we have determined the high-resolution structures of three pathogenic fungal forms of ASADH, from C. neoformans, A. fumigatus, and from B. dermatitidis in both apo- and ligand-bound forms. While the overall structures of the fungal ASADHs are similar to the bacterial orthologs, some critical differences both in biological assembly and in secondary structural features can potentially be exploited for the development of species-selective drugs with selective toxicity against infectious fungal organisms. As an initial study ~1,000 compounds from our customized fragment libraries were screened against ASADH, followed by determination of the inhibition constant (Ki) values of the most potent hits. Encouragingly, these results showed that most of the hits obtained already have Ki values in the lower micromolar range. Several of these most potent and structurally diverse initial hits were selected for structural-activity relationship (SAR) evaluation, leading to optimized inhibitors against fungal ASADH with inhibition constants in the low micromolar/high nanomolar range, while still retaining high ligand efficiency values. The efficacy of these hits was further validated using a sensitive cell-based assay against the growth of Candida cells. These ASADH inhibitors suppress fungal growth, with most potent hits in this cell-based assay have low micromolar IC50 values. Further screening of ~1000 drug-like compounds from an NIH clinical collection library and a Prestwick drug library revealed that many of the hits from these libraries have core scaffolds that are representative of those obtained from the fragment library hits. These results confirm that fragments can serve as hit predictors and as a guide for optimized inhibitor selection. Native PAGE studies and small angle x-ray scattering (SAXS) analysis showed that the fungal ASADHs exist as a functional tetramer, in contrast to the bacterial ASADHs that exist as dimers. Some of these newly identified inhibitors show a non-competitive mode of inhibition, functioning to bind and cause oligomer dissociation into inactive dimers. These compounds are being used as starting points for designing protein-protein interaction inhibitors with a unique mode of action against our fungal enzyme target. High throughput docking against a sub-set of the ZINC compound library (70,000 compounds) also yielded hits with similar sub-structures from fragment library screening, with measured Ki values as low as 4 µM. These docking results are being used to guide the design and development of more potent inhibitors. The second project focused on purification, characterization and crystallization of membrane bound aspartate N-acetyltransferase (ANAT) enzyme, and performing inhibitor screening against mouse and human forms of this enzyme for the treatment of Canavan Disease (CD). ANAT catalyzes the biosynthesis of N-acetyl aspartate (NAA) in neurons, a major source of acetyl groups for lipid synthesis during brain development. CD is a fatal autosomal-recessive neurodegenerative disease caused by mutations in the acy2 gene that leads to the deficiency of the enzyme aspartoacylase, the enzyme responsible for the deacetylation of NAA in the oligodendrocytes of the brain. An approach in which selective ANAT inhibitors are used to lower brain NAA levels back into the physiological range has the potential to treat the symptoms of CD without introducing higher risks. We have now screened several bisubstrate analog inhibitors and their truncated series against ANAT, and the most potent hits have been found to inhibit ANAT in the lower nanomolar range. These bisubstrate inhibitors are also used for co-crystallization of ANAT in order to determine the crystal structure of the enzyme. This study will allow us to understand the mechanism of the enzyme and develop selective inhibitors which, upon further modifications, will produce lead compounds for the treatment of Canavan Disease.
Ronald Viola, PhD (Committee Chair)
Donald Ronning, PhD (Committee Member)
Jianglong Zhu, PhD (Committee Member)
Viranga Tillekeratne, PhD (Committee Member)
170 p.
Biochemistry; Biophysics; Chemistry
Antifungal drugs, amino acid biosynthesis, crystallography, drug design

Recommended Citations

Citations

  • Dahal, G. P. (2018). Development of Selective Inhibitors against Enzymes Involved in the Aspartate Biosynthetic Pathway for Antifungal Drug Development [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1532889045486984

    APA Style (7th edition)

  • Dahal, Gopal. Development of Selective Inhibitors against Enzymes Involved in the Aspartate Biosynthetic Pathway for Antifungal Drug Development. 2018. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1532889045486984.

    MLA Style (8th edition)

  • Dahal, Gopal. "Development of Selective Inhibitors against Enzymes Involved in the Aspartate Biosynthetic Pathway for Antifungal Drug Development." Doctoral dissertation, University of Toledo, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1532889045486984

    Chicago Manual of Style (17th edition)

toledo1532889045486984
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