Pin 1, a newly discovered regulator of mitosis signal transduction, is an enzyme that acts on phosphoserine-proline amides. The unique phosphorylation dependent peptidyl-prolyl isomerase activity of Pin1 in regulating mitosis makes it a very attractive novel target for potential cancer chemotherapeutics. Pin 1 is hypothesized to regulate mitosis by a conformational switch, isomerizing key proline amides in a set of proteins that are upregulated during mitosis. Cdc25 specifically is known to interact with Pin1 through two phosphoserine-proline motifs. Cdc25 phosphatase regulates the activity of the mitosis-specific kinase, Cdc2 complexed with cyclin B. Many prolines require isomerization of the amide bond preceding proline between the trans and cis conformations in order to fold into the biologically active conformation. the folding process is accelerated by the peptidyl-prolyl isomerase (PPIase enzymes). A better mechanistic understanding of the native PPIase activity of the Pin 1 enzyme will give insight into PPIase-dependent processes. The first Specific Aim concerns Pin1 inhibition by conformationally constrained cis- and trans- proline substrate mimics. The mimics will be synthesized by stereoselective organic synthesis techniques developed in the PI's laboratory. The (Z) and (E)-alkene proline dipeptide mimics cannot be isomerized, so they will be assayed as competitive inhibitors of Pin1. The relative levels of Pin1 inhibition by cis and trans proline mimics of the Cdc25 substrate will help elucidate the mechanism by which Pin regulates mitosis. In the second specific aim, the conformational selectivity of kinases upstream of Pin1 that phosphorylate Cdc25 will be investigated. The role of Pin 1 in mitosis will be investigated: 1) as an enzyme that isomerases phosphoSer-Pro bonds in the substrate, Cdc25 phosphatase; 2) as a cofactor that binds to Cdc25 to regulate its activity in mitosis and 3) in Cdc25 protein folding and chaperone studies. In the third specific aim, the mechanism of Pin1 peptidyl-proline isomerase activity will be investigated using isotope effects and active site thiol titration. Using this information, a series of mechanism-based inhibitors will be synthesized and assayed for Pin1 inhibition. The mechanism-based inhibitors are considered rational design leads for anti-cancer drugs. The successful completion of this proposal will lead to an understanding of the mechanism of Pin1 PPIase activity to help elucidate its essential role in signal transduction leading to mitosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063271-03
Application #
6526115
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Schwab, John M
Project Start
2000-09-01
Project End
2005-08-31
Budget Start
2002-09-01
Budget End
2005-08-31
Support Year
3
Fiscal Year
2002
Total Cost
$178,850
Indirect Cost
Name
Virginia Polytechnic Institute and State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
003137015
City
Blacksburg
State
VA
Country
United States
Zip Code
24061
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