Grantee Research Project Results
2004 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, 2003 through September 30, 2004
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 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; they all contain, however, an abundance of the exotic amino acid 3,4-dihydroxyphenylalanine (DOPA) that is formed by the enzymatic addition of a second hydroxyl group to the tyrosine. Since 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. Since 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, α-methyl-p-tyrosine methyl ester is an effective inhibitor of the enzyme tyrosine hydroxylase. Capsaicin also has a chemical structure based on tyrosine, and has been shown to prevent zebra mussel reattachment. 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 ecologically by their clean (unfouled) exterior and lack of foreign detritus within their structural fibers.
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 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 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 usher in a new generation of zebra mussel antifoulants that will result in billions of public and private dollars saved 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.
Progress Summary:
All relevant permits for the collection, handling, and transportation of live zebra mussels for use in the research project as well as full institutional approval have been obtained, documented, and updated for the present work. The associate research and development biologist hired for this project has continued to maintain the live zebra mussels and conduct experiments in the specially designed containment facility that has been constructed and approved by state agencies and our institution.
To date 36 synthetic, semi-synthetic, and natural products have been assayed with full dose-response curves in vivo in our zebra mussel antifouling assay. Of these classes of compounds 11 were inactive, whereas 16 were determined to be effective antifoulant compounds. Of the 16 active compounds, 1 structural class of natural products containing 7 active compounds appears to be a very promising lead. The remaining nine compounds were initially determined as too toxic or having insignificant activity. Select compounds determined to be active, partially active and toxic in the zebra mussel assay will be further assayed in field exposure studies in paint matrices. An additional 10 compounds not previously tested in the zebra mussel assay will be added to the paint exposure studies due to the lack of adequate sample material to complete the full dose-response zebra mussel assay. If the most active compounds in the zebra mussel assay are determined to have decreased activities in the paint matrix, modifications may be made to the compounds to increase activity or hydrophobicity.
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 but has yet to be utilized because of limitations in design. DOPA content and glue protein studies will commence when a modified procedure and positive lead compounds have officially been identified through zebra mussel and field tests and have passed toxicity screening with duckweed and medaka.
The identification of several zebra mussel antifouling compounds with no significant zebra mussel toxicity in the effective range is a major step toward our goal of developing environment-friendly zebra mussel antifoulants and determining their mode of action(s). The use of these natural products as environmentally benign antifouling alternatives to metal-based paints and preservatives is significant, not only as a potential control of fouling organisms, but also to highlight the ecological importance of these and similar biochemical defenses. These findings are important as currently all antifoulants in widespread use contain heavy metals (e.g., copper and tin) that pose a significant toxicity risk to many aquatic and terrestrial organisms and to humans. Our findings have potential applications for the development of environment-friendly antifouling coatings that would prevent the attachment of zebra mussel and similar fouling organisms to aquatic substratum.
Future Activities:
We have three manuscripts in preparation for the zebra mussel antifouling work (patent applications pending). We also should have four publications in print and at least three others in various stages of preparation (patent applications pending) over the next year or so. During the period from 10/20/04 to 05/07/05, we plan to continue the single high-dose in vivo screening of synthetic and natural product compounds to identify additional compounds with the potential to be environment-friendly antifoulants. In vivo dose-response curves for all compounds with demonstrable in vivo antifouling properties will be performed and the potency (EC50) and efficacy (percent effect) of these compounds will be established. Select compounds determined to be active, partially active and toxic in the zebra mussel assay will be further assayed in field exposure studies (6–12 months) in paint matrices. Many of the more toxic compounds in the zebra mussel assay may have marked decrease of toxicity in a paint matrix. Additionally, the highly active compounds may be rendered totally inactive in the same paint matrix studies. If the most active compounds from the zebra mussel assays are determined to have decreased activities in the paint matrix, modifications may be made to the compounds to increase activity or hydrophobicity. Compounds that show significant antifouling activity will be analyzed to assess their toxic potential to other aquatic organisms using the very sensitive and standardized phytotoxicity assay with duckweed (Leman pausicostata) and fish toxicity assay with Japanese medaka (Oryzias latipes). Those compounds that are effective zebra mussel antifoulants and that display limited toxicity to zebra mussels, fish, and duckweed 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 have met with limited success on testing the 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). The mode of action studies to prevent the formation of DOPA will be studied at a later date after a modified procedure is developed. We now are pursuing alternative molecular mechanisms of action, such as the structure-activity relationships among derivatives with structural similarities to our lead antifouling compounds as well as compounds whose structures are dissimilar to our lead antifouling compounds, but that act on a common molecular target.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 2 publications | 1 publications in selected types | All 1 journal articles |
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Diers JA, Pennaka HK, Peng J, Bowling JJ, Duke SO, Hamann MT. Structural activity relationship studies of zebra mussel antifouling and antimicrobial agents from Verongid sponges.. Journal Of Natural Products. 2004;67(12):2117-2120. |
R829421E03 (2004) |
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Supplemental Keywords:
marine science, biology, natural product, structure-activity relationships (SAR), 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.