Methyltransferase (MT)-catalyzed S-adenosyl-L-methionine (AdoMet)-dependent methylation is essential to all walks of life and alterations in methylation-dependent processes have direct relevance to microbial/fungal/viral pathogenesis and human disease. Yet, for many MTs, there is a lack of correlative fundamental knowledge regarding specific MT function and corresponding impact upon cellular fate and/or pathogenesis/disease. In addition, while simple chemical or MT-catalyzed methylation of a drug/drug lead can dramatically impact its corresponding ADMET (absorption, distribution, metabolism, excretion and/or toxicity), the structural complexity of many natural products often prohibits doing so in the context of natural products-based drugs/lead development. This proposal seeks to develop general chemoenzymatic alkylation strategies and reagents for that are expected to broadly facilitate the fundamental study, annotation and application of MTs. A centerpiece to the proposed universal platform development is the study, engineering and application of permissive methionine adenosyltransferases (MATs) and MTs, where the model MTs selected represent broad catalytic diversity (C-methylation, O-methylation and N-methylation) and directly act upon a selected set of complex natural product-based drugs, validated clinical candidates or marketed agricultural products. The proposed studies will integrate the chemical synthesis and application of unique methionine (Met) analogs, MAT/MT structure determination, high throughput MAT/MT assay development/application, structure-guided MAT/MT directed evolution, microbial strain engineering, complex natural product (NP) structure elucidation and bioactivity assessment for NP analogs generated. The anticipated outcomes of this study include highly permissive/proficient MATs/MTs engineered for medicinal chemistry applications, novel functional AdoMet orthologs designed as alternative alkyl donors and/or with improved stability, an expanded understanding of MAT/MT structure-activity relationships of potential relevance to MAT/MT inhibitor design, single vessel chemoenzymatic strategies to enable complex NP differential alkylation, engineered microbial strains to enable complex NP differential alkylation and functional MT annotation, and unprecedented differentially-alkylated NP analogs with potential therapeutic and/or agricultural applications. Within this context, the proposed studies will also provide the first bioorthogonal strategy to functionally annotate, interrogate or exploit a single MT within a cell containing a full complement of competing native MTs. While NP methylation has been selected as the model for platform development, it is important to note that reagents and concepts developed will likewise enable the similar study, annotation and application of other class I MTs relevant to cellular development, human disease or microbial/fungal/viral pathogenesis.
Enzyme-catalyzed methylation is essential to normal function and development of all species. However, disproportionate or aberrant methylation can also serve as a causative element in pathogenesis, human disease and directly impact a patient's response to treatment. This proposal seeks to develop a universal platform and innovative reagents to facilitate the fundamental study and practical application of enzyme-catalyzed methylation and will also provide novel natural product analogs with potential anticancer, immunosuppressant, anti-infective and/or insecticide activities.
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