Monday, October 22, 2007 : 2:40 p.m.

Assessment of EDCs Exhibiting Estrogen Mimicry using a Multi-tiered Approach for Predicting Ligand Structural Requirements

James L. Wittliff, Sarah A. Andres, D. Alan Kerr II, Irina A. Smolenkova, Stefanie B. Bumpus and Huy T. Le, University of Louisville

Estrogens and their mimics form one of the most abundant classes of hormone-like molecules in the world.  Diverse chemical features of estrogenic ligands are illustrated by the promiscuous nature of their interactions with the ligand binding domain (LBD) of human estrogen receptor alpha (hERα).  To predict structural characteristics of ligand and LBD recognition, a three-tiered approach consisting of (1) a ligand competition array using Lundon OneSite®, Compete® and GraphPad Prism® software, (2) an electrophoretic mobility shift assay with [α32P]Estrogen Response Element sequences and (3) a yeast cell-based bioassay containing an hERa expression plasmid (YEpE12) and a reporter plasmid (YRG2) with an ERE sequence upstream of β-galactosidase was employed. This allowed determination of the influence of estrogen mimics on hERα activity for modeling structural requirements of the ligand. Biological activities of ligands were modeled using the pharmacophoric alignment software GALAHAD™ and DiscoTECH™ operating on a Silicon Graphics workstation.  Pharmacophores generated were also subjected to a Comparative Molecular Field Analysis and Partial Least Square Analysis to determine quantitatively the relationship between three-dimensional ligand structures and corresponding activities.  Using published results of LBD structure (Leduc et al. 2003, Ruff et al. unpublished), SurFLEX-Dock™ software was employed to challenge fitting of three-dimensional characteristics of ligands into the LBD. Collectively these results are revealing features of estrogen mimics that promote endocrine disruption to improve exposure risk/benefit assessment.

Supported in part by NIEHS/SBIR #1R43 ES10076-01, #2R44-ES10076-02 & Phi Beta Psi Charity Trust grants. SAA & DAK are recipients of IPIBS graduate fellowships & HTL is a recipient of a Cancer Education Grant Fellowship (NCI 5R25CA44789).

 

James L. Wittliff, University of Louisville Professor of Biochemistry & Molecular Biology Research Professor of Surgery Acting Director, Institute for Molecular Diversity & Drug Design University of Louisville

Sarah A. Andres, University of Louisville Sarah A. Andres, B.S., University of Louisville, is a Senior Graduate Research Fellow who has a broad background in the biochemistry of estrogens and their mimics as well as in the genomics of human breast carcinoma. She and Dr. Wittliff have identified a clinically relevant subset of genes that predicts the behavior of certain types of breast cancer.

D. Alan Kerr II, University of Louisville D. Alan Kerr II, B.A., University of Louisville, is a Graduate Research Fellow who is pursuing a combined degree program culminating in an M.D. and a Ph.D. He has extensive experience in the biochemistry of human cancer focusing on genomics and clinical correlations.

Irina A. Smolenkova, University of Louisville Irina A. Smolenkova, M.D., University of Louisville, is a Research Scholar with an emphasis in molecular endocrinology and clinical chemistry. Her research interests include use of radioligand-based assays for detecting estrogen mimics in natural products and synthetically prepared medicinals.

Stefanie B. Bumpus, University of Louisville Stefanie B. Bumpus, B.S., University of Louisville, graduated with Honors from the Department of Chemistry and accepted a position as Graduate Research Fellow in the Department of Chemistry at the University of Illinois at Urbana-Champaign.

Huy T. Le, University of Louisville Huy T. Le, University of Louisville, is undergraduate scholar in the Department of Chemistry where he is pursuing an Honors Thesis as part of his Bachelor of Science degree which combines the principles and technologies of biochemistry and molecular biology.


6th International Conference on Pharmaceuticals and Enocrine Disrupting Chemicals in Water