Grantee Research Project Results
2002 Progress Report: Synthetic and Natural Small Molecule Zebra Mussel Anti-foulants
EPA Grant Number: R829421E03Title: Synthetic and Natural Small Molecule Zebra Mussel Anti-foulants
Investigators: Hamann, Mark T.
Institution: University of Mississippi
EPA Project Officer: Chung, Serena
Project Period: October 1, 2001 through September 30, 2003
Project Period Covered by this Report: October 1, 2001 through September 30, 2002
Project Amount: $109,732
RFA: EPSCoR (Experimental Program to Stimulate Competitive Research) (2001) RFA Text | Recipients Lists
Research Category: EPSCoR (The Experimental Program to Stimulate Competitive Research)
Objective:
The objectives of this research project are to: (1) measure the antifouling efficacy of natural and synthetic small molecules with tyrosine-based chemical structures; (2) determine the ability of these compounds to prevent the formation of 3,4-dihydroxyphenylalanine (DOPA)-containing glue proteins in zebra mussel adhesive plaques; and (3) correlate the antifouling and glue protein forming properties of these compounds with their tyrosine hydroxylase inhibiting activity. Our hypothesis is that small tyrosine-based molecules will be efficacious zebra mussel antifoulants. Our approach will be to test for zebra mussel antifouling activity using an in vivo zebra mussel reattachment bioassay and to correlate the antifouling efficacy of small tyrosine-derived molecules with their ability to both inhibit tyrosine hydroxylase activity and to prevent the formation of DOPA-containing glue proteins. The expected results are that small tyrosine-based molecules from synthetic and natural sources will be efficacious zebra mussel antifoulants. In addition, we anticipate a positive correlation between antifoulant properties and the ability to inhibit both the formation of DOPA and tyrosine hydroxylase activity. This research on the potential molecular mechanisms of zebra mussel antifouling agents will help to usher in a new generation of zebra mussel antifoulants that will result in billions of dollars of savings for both public and private coffers, and it may help to explain the general biology of antifoulant strategies utilized by a wide range of aquatic species. Understanding the principles of naturally occurring antifouling strategies promises to provide highly specific and environmentally friendly ways of combating zebra mussel infestations.
The colonization, and consequently the clogging of water intakes by zebra mussels (Dreissena polymorpha) is so severe that "The U.S. Fish and Wildlife Service forecasts 5 billion dollars in loses over the next decade to manufacturing, power, and municipal water intake facilities that use Great Lakes water." Zebra mussels and other freshwater and marine mussel species adhere to hard wet surfaces by secreting an adhesive polyphenolic glue protein. Remarkably, the glue proteins isolated from the adhesive plaques of different mussel species do not share much primary amino acid homology; however, they all contain an abundance of the exotic amino acid DOPA that is formed by the enzymatic addition of a second hydroxyl group to the tyrosine. Because the adhesive properties of mussel glue proteins require the presence of these DOPA residues, agents that interfere with the conversion of tyrosine to DOPA should prevent zebra mussels from adhering to aquatic substratum, and as a consequence, they should be effective zebra mussel antifoulants. Because tyrosine is a substrate for the enzyme, tyrosine hydroxylase, it follows that derivatives of tyrosine would compete with tyrosine for binding to the active site of the enzyme. For example, a-methyl-p-tyrosine methyl ester is an effective inhibitor of the enzyme tyrosine hydroxylase. Furthermore, capsaicin also has a chemical structure based upon tyrosine, and it has been shown to prevent zebra mussel reattachment. Moreover, a growing number of antifouling molecules isolated from marine organisms possess a tyrosine pharmacophore, including numerous examples isolated from marine sponges of the order Verongida. This is significant because Verongida sponges are classified chemotaxonomically by their production of tyrosine-derived brominated compounds, and classified ecologically by their clean (unfouled) exterior and the lack of foreign detritus within their structural fibers.
Progress Summary:
All relevant permits for the collection, handling, and transportation of live zebra mussels for their use in our research project, as well as full institutional approval, have been obtained and documented. An Associate Research and Development Biologist has been hired and trained to conduct live zebra mussel experiments. A live zebra mussel containment facility has been constructed and approved by state agencies and our institution.
Three different natural products relevant to this project have been purified to homogeneity and their structures have been fully characterized. A known natural product antifoulant, as well as a synthetic derivative of one of our lead antifouling compounds, recently have been synthesized and their structures have been confirmed. Single high dose pilot studies for several natural product and synthetic compounds have been performed using an in vivo zebra mussel reattachment assay as a measure of antifouling efficacy and a 48-hour post-experiment reattachment assay as a measure of zebra mussel toxicity. Within the last 2 months, two different compounds have been shown to have significant zebra mussel antifouling efficacy, but limited toxicity within the effective range. Full dose-response curves for one of our two lead compounds has been performed in three separate experiments and it has an antifouling potency (EC50) of approximately 15 µM and demonstrates full efficacy (100 percent effect) at approximately 25 µM. A biochemical assay for measuring the DOPA content in zebra mussel glue proteins has been initiated using dopamine as a catechol standard for the reference curve.
The identification of two zebra mussel antifouling lead compounds with no significant zebra mussel toxicity in the effective range is a major step toward our goal of developing environmentally friendly zebra mussel antifoulants and determining their mode of action. These findings are important, as all antifoulants currently in widespread use contain heavy metals (e.g., copper and tin), which pose a significant toxicity risk to many aquatic and terrestrial organisms, and to humans. Our findings have potential applications for the eventual development of environmentally friendly antifouling coatings that would prevent the attachment of zebra mussels to aquatic substratum.
Future Activities:
Future activities are to continue the single high-dose in vivo screening of synthetic and natural product compounds to identify additional compounds with the potential to be environmentally friendly zebra mussel antifoulants. In vivo dose-response curves for all compounds with demonstrable in vivo antifouling properties will be performed and the potency (EC50) and efficacy of these compounds will be established. Those compounds that do not display significant toxicity to zebra mussels at the lowest maximally effective antifouling dose will undergo additional testing designed to assess their toxic potential to other aquatic organisms using the very sensitive and standardized duck weed assay. Those compounds that are effective zebra mussel antifoulants and that display limited toxicity to zebra mussels and duck weed will be further investigated in an effort to establish their potential mode of antifouling action. Our initial efforts concerning the potential mode of antifouling efficacy will focus on testing our hypothesis that links zebra mussel antifouling efficacy to the ability of these compounds to prevent the post-translation formation of DOPA in zebra mussel glue protein and to inhibit tyrosine hydroxylase (an enzyme that converts tyrosine to DOPA in higher organisms). If we are unable to establish a link between zebra mussel antifouling efficacy, DOPA content in zebra mussel glue protein, and tyrosine hydroxylase inhibiting activity, we then will pursue to the extent possible alternative molecular mechanisms of action. Such studies will include the structure-activity relationships amongst derivatives with structural similarities to our lead antifouling compounds, as well as amongst compounds whose structures are dissimilar to our lead antifouling compounds, but that act on a common molecular target.
Journal Articles:
No journal articles submitted with this report: View all 2 publications for this projectSupplemental Keywords:
marine science, biology, central, zoology, industry., RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Ecology, Environmental Chemistry, Geochemistry, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Fate & Transport, Ecology and Ecosystems, fate and transport, chemical ecology, ecological effects, zebra mussel anti-foulants, biofoulfing, ecosystem assessment, aquatic habitat, chemical kinetics, ecosystem management, aquatic ecosystems, bioassay, aquatic ecologyProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.