Lawrence J. Marnett
Organic, Bioorganic and Biochemistry
Our research group has interests in the areas of protein structure and function, nucleic acid chemistry, drug design and synthesis, and chemical genomics. Much of our work focuses on the biochemistry and molecular biology of oxidation of natural and synthetic chemicals. Areas of interest to us include: mechanisms of oxidation of arachidonic acid and endocannabinoids by cyclooxygenase and lipoxygenase enzymes, design, synthesis, and biochemical evaluation of lipoxygenase and selective cyclooxygenase-2 (COX-2) inhibitors; chemistry and biology of DNA damage by lipid oxidation products; and endogenous pathways of DNA damage in the genesis of human cancer.
The following studies are underway in our labs:
Structure and Function of Fatty Acid Oxygenases
Our laboratory has a long-standing interest in enzymes of arachidonic acid oxygenation. This includes lipoxygenases, which incorporate one molecule of O2 into the carbon framework and cyclooxygenases, which incorporate two molecules of O2. The products of both pathways of oxygenation are substrates for metabolizing enzymes that generate a panoply of lipid mediators. Leukotrienes and prostaglandins are involved in multiple physiological and pathophysiological events, and inhibition of their action is the molecular basis for the pharmacological activities of several important drugs. Foremost among these are non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 (COX-2) inhibitors. We have conducted extensive functional studies with lipoxygenases and cyclooxygenases based on available crystal structures and employing exhaustive site-directed mutagenesis.
Design, Synthesis, and Evaluation of Novel COX-2 Inhibitors
Cyclooxygenase-2 (COX-2) is the molecular target of non-steroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors. Our laboratory has combined structural analysis with functional studies to define the molecular determinants of the interaction of ligands (substrates and inhibitors) with COX-2. For example, we recently reported the identification of a critical H-bonding interaction that leads to the selectivity of aspirin for acetylation of Ser-530 in COX-2. Many NSAIDs are aralkyl carboxylic acids. Comparative analysis of the effect of site-directed mutation of active site residues on the binding of substrates and inhibitors to COX-1 and COX-2 led us to hypothesize that neutral derivatives of esters and amides would bind selectively to COX-2. We tested this hypothesis by synthesizing a series of neutral derivatives of NSAIDs and demonstrating increases in selectivity for COX-2 of several orders of magnitude. We are exploiting this discovery to prepare novel COX-2 inhibitors as anti-inflammatory drugs and cancer preventive agents.
Chemistry and Biology of Endogenous DNA Damage by Lipid Peroxidation Products
Our laboratory has focused on DNA damage by aldehydes produced endogenously in mammalian cells as a result of lipid peroxidation. Malondialdehyde is the major mutagenic product of lipid peroxidation and is produced ubiquitously in animal and human tissues. It reacts with DNA to form a series of adducts that we and others have identified. The major adduct is a pyrimidopurinone that we have abbreviated M1G. This adduct possesses a blocked Watson-Crick base-pairing region so it is expected to be mutagenic. We have evaluated its mutagenicity by synthesizing viral genomes containing M1G at defined positions. Following transfection into bacterial or mammalian hosts, the replicated genome is interrogated to determine the outcome of replication at the site of the adduct. These experiments indicate that M1G is indeed mutagenic. We have used a variation of this approach to establish that M1G is repaired by nucleotide excision repair. To support and extend these in vivo studies, we conduct experiments utilizing adduct-containing duplex DNA molecules or template-primers as substrates for purified DNA polymerases or repair enzymes. These investigations provide more detailed information about the structural and functional basis for induction of mutation. Our laboratory has had a long-standing collaboration with the Stone laboratory in the Chemistry Department at Vanderbilt, which has provided precise information about the structural perturbations introduced into DNA by adducts such as M1G.
Signal Transduction by Lipid Mediators and Lipid Peroxidation Products
A relatively new area of research in the laboratory is the definition of signal transduction pathways stimulated or interrupted by lipid mediators or lipid peroxidation products. The work on lipid mediators is focused on endocannabinoid oxygenation products of COX-2 and 15-lipoxygenase whereas the work on lipid peroxidation products is focused on malondialdehyde, 4-hydroxynonenal, and structurally related molecules. We are particularly interested in events important in controlling the growth and metastasis of cancer cells such as proliferation, migration, apoptosis and angiogenesis.
Each of the projects utilizes a range of the outstanding core facilities available at Vanderbilt (microarray, proteomics, molecular recognition, and high throughput screening) and involves stimulating collaborations with colleagues at Vanderbilt and elsewhere.