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.
|Etzkorn, Felicia A; Zhao, Song (2015) Stereospecific phosphorylation by the central mitotic kinase Cdk1-cyclin B. ACS Chem Biol 10:952-6|
|Mercedes-Camacho, Ana Y; Mullins, Ashley B; Mason, Matthew D et al. (2013) Kinetic isotope effects support the twisted amide mechanism of Pin1 peptidyl-prolyl isomerase. Biochemistry 52:7707-13|
|Nachman, Ronald J; Kim, Young-Joon; Wang, Xiaodong J et al. (2009) Potent activity of a PK/PBAN analog with an (E)-alkene, trans-Pro mimic identifies the Pro orientation and core conformation during interaction with HevPBANR-C receptor. Bioorg Med Chem 17:4216-20|
|Schroeder, Olivia E; Carper, Emily; Wind, Joshua J et al. (2006) Theoretical and experimental investigation of the energetics of cis-trans proline isomerization in peptide models. J Phys Chem A 110:6522-30|
|Wang, Xiaodong J; Xu, Bailing; Mullins, Ashley B et al. (2004) Conformationally locked isostere of phosphoSer-cis-Pro inhibits Pin1 23-fold better than phosphoSer-trans-Pro isostere. J Am Chem Soc 126:15533-42|
|Wang, Xiaodong J; Hart, Scott A; Xu, Bailing et al. (2003) Serine-cis-proline and serine-trans-proline isosteres: stereoselective synthesis of (Z)- and (E)-alkene mimics by Still-Wittig and Ireland-Claisen rearrangements. J Org Chem 68:2343-9|
|Hart, S A; Trindle, C O; Etzkorn, F A (2001) Solvent-dependent stereoselectivity in a Still-Wittig rearrangement: an experimental and ab initio study. Org Lett 3:1789-91|