Date Available
10-17-2020
Year of Publication
2019
Degree Name
Doctor of Philosophy (PhD)
Document Type
Doctoral Dissertation
College
Pharmacy
Department/School/Program
Pharmaceutical Sciences
First Advisor
Dr. Jon S. Thorson
Abstract
Methyl group transfer from S-adenosyl-l-methionine (AdoMet) to various substrates including DNA, proteins, and natural products (NPs), is accomplished by methyltransferases (MTs). Analogs of AdoMet, bearing an alternative S-alkyl group can be exploited, in the context of an array of wild-type MT-catalyzed reactions, to differentially alkylate DNA, proteins, and NPs. This technology provides a means to elucidate MT targets by the MT-mediated installation of chemoselective handles from AdoMet analogs to biologically relevant molecules and affords researchers a fresh route to diversify NP scaffolds by permitting the differential alkylation of chemical sites vulnerable to NP MTs that are unreactive to traditional, synthetic organic chemistry alkylation protocols.
The full potential of this technology is stifled by several impediments largely deriving from the AdoMet-based reagents, including the instability, membrane impermeability, poor synthetic yield and resulting diastereomeric mixtures. To circumvent these main liabilities, novel chemoenzymatic strategies that employ methionine adenosyltransferases (MATs) and methionine (Met) analogs to synthesize AdoMet analogs in vitro were advanced. Unstable AdoMet analogs are simultaneously utilized in a one-pot reaction by MTs for the alkylrandomization of NP scaffolds. As cell membranes are permeable to Met analogs, this also sets the stage for cell-based and, potentially, in vivo applications.
In order to further address the instability of AdoMet and analogs thereof, MAT-catalyzed reactions utilizing Met and ATP isosteres generated highly stable AdoMet isosteres that were capable of downstream utilization by MTs. Finally, the development, use, and results of a high-throughput screen identified mutant-MAT/Met-analog pairs suitable for postliminary bioorthogonal applications.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2019.398
Funding Information
NIH Protein Structure Initiative (U01 GM098248, GNP and JST), NIH R37 AI52188 (JST), and NIH GM109456 (GNP)
National Center for Advancing Translational Sciences (UL1TR000117)
University of Kentucky College of Pharmacy and Markey Cancer Center
Recommended Citation
Huber, Tyler D., "TOWARD AN ENZYME-COUPLED, BIOORTHOGONAL PLATFORM FOR METHYLTRANSFERASES: PROBING THE SPECIFICITY OF METHIONINE ADENOSYLTRANSFERASES" (2019). Theses and Dissertations--Pharmacy. 106.
https://uknowledge.uky.edu/pharmacy_etds/106
Included in
Amino Acids, Peptides, and Proteins Commons, Biochemical Phenomena, Metabolism, and Nutrition Commons, Biochemistry Commons, Biotechnology Commons, Chemical and Pharmacologic Phenomena Commons, Enzymes and Coenzymes Commons, Medicinal and Pharmaceutical Chemistry Commons, Medicinal Chemistry and Pharmaceutics Commons, Medicinal-Pharmaceutical Chemistry Commons, Molecular Biology Commons, Natural Products Chemistry and Pharmacognosy Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons, Organic Chemistry Commons, Structural Biology Commons
