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Principal Investigator: Robert E London
Organization: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
Fiscal Year: 2020
Award: $1,046,440
Funding agency: National Institute of Environmental Health Sciences
Independent Project: Folate-dependent enzymes play a central role in single carbon metabolism and in nucleotide biosynthesis; its perturbation can be mutagenic and ultimately lethal. More generally, dysregulation of folate metabolism appears to be broadly linked to deficiencies in genome methylation, stability and repair. Alternatively, anti-folate drugs such as methotrexate are widely used in the treatment of neoplastic disease, but low doses have found increasing application in the treatment of rheumatoid diseases and other illnesses that are associated with chronic inflammation. Our previous studies of folate metabolism indicated that the NSAID fenbufen was able to interact with the enzyme dihydrofolate reductase (DHFR) by binding to the region of the folate binding site that interacts with the p-aminobenzoyl-L-glutamate (pABG). We are currently performing a more general assessment of the interaction of other NSAIDs with this subsite, their ability to bind to this site, and their inhibition of DHFR activity. These studies have quantified the DHFR inhibitory strength of various NSAIDs, and demonstrated that some possess even greater inhibitory potency than fenbufen. The potential anti-folate activity of NSAIDs suggests the possibility of additional applications in the treatment of chronic inflammatory diseases without some of the toxicity of more potent antifolate compounds such as methotrexate.
NMR Core Activities:
There are number of fluorinated anesthetics that are used in neurobiology, which can be difficult to solubilize. The NMR group utilized 19F NMR spectroscopy to assess the solution concentrations of several anesthetics for the Ion Channel Physiology Group in the Neurobiology Laboratory.
The Stem Cell Biology Group in the Epigenetics and Stem Cell Biology Laboratory was working on new methods to create random mutagenesis by altering the chemistry of DNA bases. The NMR group utilized 1D NMR to assess the chemical structure and effectiveness of catalytic reactions to better optimize the procedures.
The Macromolecular Structure Group in the Epigenetics and Stem Cell Biology Laboratory is looking at RNA structures to better understand how structural changes affect RNA regulatory pathways. The NMR group has been acquiring NMR data to determine the structure of the RNA targets.
The Nuceolar Integrity Group in the Signal Transduction Laboratory is trying to understand the molecules involved in the pathway of ribosome biogenesis. The NMR group acquired data with the goal of providing information on how las1/grc3 interacts with RNA targets during biogenesis.
The Pediatric Neuroendocrinology Group has been interested in how the protein SMCHD1, another chromatin remodeler, modulates ACE2 expression and to utilize our structural biology program to determine if SMCHD1 may be a druggable target. Downregulation of ACE2 expression is considered a potential treatment for SARS-CoV-2 infection. The NMR group has acquired data on conformations of SMCHD1 and are helping preparing samples for this project.
The Matrix Biology group has been interested in the interactions of hyaluronic-acid (HA) with the RBD of the SARS-CoV-2 spike protein. The NMR group has run assays to assess the binding of different size HA with RBD and spike protein constructs. If HA blocks RBD binding of the spike protein of SARS-CoV-2 to ACE2, it may be a useful therapeutic.
The Mutagenesis and DNA Repair Regulation Group is interested in how oxidative damage leads to mutagenesis. The group is studying now various yeast strains, and mutated yeast strain respond to oxidative chemical agents. In order to better understand the metabolic implications and how chemical pathways are affected by the oxidative chemical agents, the NMR group has been assessing the yeast metabolome from the strains and treatment groups. Metabolic pathways were assessed for similarities and differences for comparison with mutagenic profiles of the oxidative agents to better understand the chemical mechanisms of mutagenesis.
The Mitochondrial DNA Replication Group tries to understand factors influence mutagenesis in the only organelle to maintain its own genome. One factor may be the formation of TT dimers due to UV irradiation near proposed G quadraplexes in the mitochondrial genome. The NMR group collected chemical data to assess the extent of TT dimer formation in different DNA samples based on sequence context and UV dose. The data was useful in optimizing further experiments to measure rates of mutagenesis.
The Structural Cell Biology group is interested in how DNA damage is recognized and repaired. One important protein in sensing DNA damage is APTX. The NMR group has measured reaction rates for APTX under various conditions, and assessed the conformation of the enzyme in response to various substrates by examining the histidine residues. These studies contribute to a better understand of DNA repair pathways that help cells avoid mutagenesis and maintain genomic integrity.
The General Toxicology and Cancer Group of the National Toxicology program has been assessing the toxicological properties of complex mixtures, like those found in dietary botanical supplements. Toward this end, the NMR group measured the concentrations of compounds in Ginkgo biloba extracts from various manufacturers in order to assess the similarity. The data was useful in comparing various technical methods, which evaluate the similarity, for the best procedure. The data also demonstrated that many products reported to contain Ginkgo biloba did not contain a significant amount of Ginkgo biloba extract.
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