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Molecular Mechanisms of Estrogen Biosynthesis

We have been investigating the structure-function relationships and mechanisms of action of the enzymes responsible for estrogen biosynthesis and metabolism.  In these studies, we use biophysical and biochemical tools such as X-ray crystallography, structural/chemical biology, enzymology, calorimetry, mass spectroscopy, molecular biology/recombinant DNA technology and cell biology.

The major objectives include elucidation of the molecular mechanism of estrogen biosynthesis by the three-enzyme system in human (Cytochrome P450 aromatase, 17b-hydroxysteroid dehydrogenase type 1 and steroid sulfatase), as well as rational design, synthesis and evaluation of inhibitors against the enzyme targets for the treatment and prevention of hormone-dependent breast cancer. We are pursuing structure-function and inhibitor design studies on all three enzymes with the hypothesis that selective inhibition of these enzymes may be the most effective control of estrogen biosynthesis in breast tumors.  A journey that began in early 1990’s with the goal to crystallize all three human enzymes reached an important landmark in 2009 with the successful elucidation of the crystal structures of all three enzymes purified in their pristine forms from human placenta

Aromatase is a membrane-bound hemeprotein of the endoplasmic reticulum.  It is the only known enzyme to catalyze the biosynthesis of estrogens from androgens. Crystallization of aromatase (CYP19A1), arguably the most unique cytochrome P450 in vertebrates, and a major breast cancer drug target, remained elusive for decades.  Our unusual approach of crystallizing the human placental enzyme, as in the case of steroid sulfatase, has finally been vindicated by the growth of the first crystal of the full-length microsomal P450.  The structure provides an unprecedented glimpse into the active site of a substrate-specific estrogen-synthesizing catalytic machine, unlike those of the drug- and xenobiotic-metabolizing P450s or numerous published homology models, and has opened a floodgate of opportunities for the rational design of next generation aromatase inhibitors. 

Currently, structure-based design, synthesis and evaluation of the next generation aromatase inhibitors constitute a major objective of the aromatase project.  Several compounds synthesized by structure-guided design have shown great promise in our laboratory evaluation.  We have conducted anti-proliferative assays on these promising candidates in a MCF-7 breast cancer cell line.  Some show inhibitory and anti-proliferative properties better than those of the well-known breast cancer drug exemestane.  For decades many laboratories have made attempts to crystallize various recombinant forms of human aromatase by deleting the N-terminal trans-membrane domain, but no crystal was reported.  Using a bacterial construct of the amino terminus truncated aromatase we have now been able to address this problem.  We have crystallized the recombinant aromatase and determined the crystal structure.  In addition, some of the mutagenesis studies that we have already conducted raise interesting possibilities with regard to its oligomerization and functional issues.  By incorporating the recombinant aromatase into our research plan, we have taken the aromatase and novel inhibitor discovery research to the next level.  We have also initiated an investigation into the effects of environmental chemicals and endocrine-disrupting agents such as fungicides on aromatase and estrogen biosynthesis, and their mechanisms of action.  Additionally, collaborative efforts are underway to extend our structure-function research into elucidating the possible roles of aromatase in the CNS and neuroendocrine effects of estrogen, as well as into rational design of antibodies as diagnostic tools for the detection of a super-active form of aromatase in estrogen-dependent breast tumors.

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