| 2016 — 2019 |
Phillips, George Neal (co-PI) [⬀]
Thorson, Jon Scott [⬀] |
R01Activity Code Description:
To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Platform Technologies For Understanding and Exploiting Methylation
? DESCRIPTION (provided by applicant): 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.
|
0.915 |
| 2017 — 2021 |
Phillips, George Neal (co-PI) [⬀]
Thorson, Jon Scott [⬀]
Van Lanen, Steven Gary (co-PI) [⬀] |
R01Activity Code Description:
To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Studies to Understand and Exploit 10-Membered Enediyne Biosynthesis
ABSTRACT The 10-membered enediynes [exemplified by calicheamicin (CLM), esperamicin (ESP) and dynemicins (DYN)] are arguably among the most renowned natural products (NPs) discovered to date by virtue of their unprecedented complex molecular architectures, notable anticancer and anti-infective potencies and, in the case of CLM, demonstrated clinical utility. The current study builds on a longstanding collaborative effort of achievement and discovery relating to key aspects of 10-membered enediyne biosynthesis as well as parallel innovative efforts to co-opt key biosynthetic catalysts for synthetic applications. The studies put forth will take advantage of this strong foundation and a powerful combination of genetic, biochemical, chemical and protein structural tools to elucidate the remaining unusual biosynthetic transformations and to exploit select catalysts for enediyne non-native modification. Specifically, aims 1 and 2 will focus on extending our understanding of the fundamental steps of enediyne core biosynthesis common to CLM/DYN/ESP, DYN anthraquinone biosynthesis and a selected set of unique tailoring reactions (CLM/ESP thiosugar sulfur installation and aminopentose N- alkylation, ESP C6-hydroxylation and O-glycosylation). In parallel, aim 3 will focus on tactical structural studies to augment both aims 1 and 2 and the structural study of ?unknowns? to facilitate functional annotation. Additional studies in aim 2 with key catalysts and corresponding non-native substrates are designed to assess the potential for strategic installation of chemoselective handles to enable novel approaches for facile, mild bioconjugation of CLM to tumor-targeting mAbs (in collaboration with Pfizer).
|
0.915 |