SBIR-STTR Award

Understanding the mechanisms by which chemical compounds and drugs interact with both the aryl hydrocarbon receptor (AhR) and estrogen receptor (ER) signaling pathways is important both for identifying potential environmental and endocrine disruptors and for screening compounds for therapeutic use. The existence of cross-talk between AhR and ER causes certain AhR ligands to exert estrogenic endocrine disrupting activity in the absence of binding to ER. Existing methods for studying these mechanisms are cumbersome and time consuming, and for cell-based methods, compound degradation by cellular metabolism significantly affects results. Recently, IA has developed biosensors for studying real-time association and dissociation kinetics between ER-alpha & -beta, and estrogenic nuclear response element (ERE) in the presence of agonists and antagonists. IA proposes to extend this technology to develop chemistries and methods which will allow the biosensors to be applied to study the complex AhR-mediated signaling pathways. During Phase I, biosensors for assessing the interaction of AhR with ligands, accessory proteins, and dioxin response element (DRE) will be developed. During Phase II, biosensors will be used to analyze cross-talk between ER and AhR, and to create screening methods for predicting effects of compounds based on hER & AhR mechanisms. PROPOSED COMMERCIAL APPLICATIONS: Biosensors which allow studying the real-time binding kinetics of compounds (agonists, antagonists, accessory proteins) to the AhR will provide researchers with powerful tools for screening environmental endocrine disruptors and for designing, developing, and testing new pharmaceuticals for treatment of cancer.

During Phase I work IA successfully applied evanescent sensing technology to the study of aryl hydrocarbon receptor (AhR)-mediated processes and provided discrimination between beneficial and harmful AhR ligands. When biosensors were used to assess the interaction of AhR with ligands, accessory proteins, and dioxin response element (DRE) distinctive sensorgrams emerged for each type of compound. Under Phase II work applications of this will be developed for the research and the environmental and food-testing markets. This will be achieved through the Specific Aims: (1) Optimize and standardize reagents and protocols; (2) Specify the detailed nature of the instrument; (3) Establish data interpretation methods that quantify and reliably distinguish between receptor agonists, antagonists and partial agonists; (4) Improve biosensor fluorometer instrument; (5) Improve design and manufacturing of biosensor cartridges; (6) Validate the system and methods on 30-50 compounds; and (7) Obtain regulatory approval for use of the AhR biosensor for dioxin screening. Existing methods for studying these mechanisms are cumbersome and time consuming, and for cell-based methods, compound degradation by cellular metabolism and instability of cell cultures significantly affects results. During Phase II, the biosensor instrument and reagents will be optimized and prototyped and will be used to assess the impact of ligands on the mechanisms affecting cross-talk between endocrine disrupting compounds at the level of receptor-DNA interactions and to predict the effects of compounds based on mechanism-based sensor evaluation. It is anticipated that parameterization of sensorgrams will provide criteria for discrimination between AhR agonists, antagonists, mixed agonist/antagonists and compounds which do not interact with AhR. The resulting biosensor, reagents and protocols will be validated by using them to categorize 30-50 blind coded compounds. This will initially be done at 1A, Inc. The proposal incorporates a time and resources extension which will support similar testing in 4 independent laboratories. Results will be submitted to the ICCVAM validation process. The biosensor system will also be tested for identification of dioxin-like activity in real-world samples obtained from the Great Lakes watershed. Once the system is validated it will have particular value in the food testing arena. Recent regulatory legislation by the EU requires that foods be certified free of dioxin contamination. This will cause the $6.5 billion industry which exports U.S. food to the EU to be in need of a cost effective, rapid, on-site screening test such as will be provided by this project.

Thesaurus Terms:
aromatic hydrocarbon receptor, biomedical equipment development, biosensor, chemical kinetics, dioxin, fiber optics, intermolecular interaction, toxicant screening endocrinology, estrogen receptor, flavonoid, hormone analog, hormone inhibitor, molecular chaperone bioengineering /biomedical engineering