Pin1 is an enzyme that catalyzes isomerization of phosphoserine/threonine-proline (pSer/Thr-Pro) amides. Pin1 is hypothesized to regulate signal transduction leading to mitosis by a conformational switch, isomerizing specific prolyl amides in cell cycle regulatory proteins. Pin1 has also been shown to regulate the activity of Alzheimer's associated tau protein. The unique peptidyl-prolyl isomerase (PPIase) enzymatic activity of Pin1 in the regulation of biological processes is interesting for fundamental reasons. Pin1 consists of two domains that both bind pSer/Thr-Pro motifs with distinct selectivity's. Specific ligands will be developed for each domain separately to be used as tools to study regulation by Pin1. In the first Specific Aim, we propose the design, parallel synthesis and assay of libraries of conformationally constrained trans- proline mimics targeted to the Pin1 WW domain. Parallel synthesis will use solid phase attachment through the invariant phosphate, and amide bond couplings to produce compounds of sufficient purity for screening. A high-throughput competitive enzyme-linked immunosorbent assay (ELISA) will be developed to assay WW domain binding. The WW domain ligands will also be tested for noncompetitive inhibition in the catalytic assay because we have shown inhibition of catalysis by a peptide binding to the WW domain. In the second Aim, libraries will be derived from compounds targeted to the catalytic domain that we have already synthesized: pSer-c/s-Pro isostere, reduced amide, ketone and alpha-ketoamide motifs. The catalytic domain inhibitors will be screened in a high-throughput enzymatic assay, and the mode of inhibition will be determined for the best inhibitors. In the third Aim, the two types of ligands for Pint will be combined into bivalent inhibitors to study the structural and dynamic interactions between the two domains of Pin1.
The fourth Aim concerns development of phosphate prodrugs, masked to improve membrane permeability. We will also develop diflurophosphonates stabilized towards phosphatases. These two modifications will allow these compounds to be used in whole cells and in vivo. The best ligands will be useful in collaborations designed to elucidate the structure, function and dynamic relationships within Pin1 and between Pin1 and its ligands.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA110940-08
Application #
7667517
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Lees, Robert G
Project Start
2000-09-01
Project End
2011-07-31
Budget Start
2009-08-01
Budget End
2011-07-31
Support Year
8
Fiscal Year
2009
Total Cost
$180,726
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
Mayfield, Joshua E; Fan, Shuang; Wei, Shuo et al. (2015) Chemical Tools To Decipher Regulation of Phosphatases by Proline Isomerization on Eukaryotic RNA Polymerase II. ACS Chem Biol 10:2405-14
Chen, Xingguo R; Fan, Shuang A; Ware, Rachel I et al. (2015) Stereochemical Control in the Still-Wittig Rearrangement Synthesis of Cyclohexyl (Z)-Alkene Inhibitors of Pin1. PLoS One 10:e0139543
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
Park, Joo Min; Hu, Jia-Hua; Milshteyn, Aleksandr et al. (2013) A prolyl-isomerase mediates dopamine-dependent plasticity and cocaine motor sensitization. Cell 154:637-50
Zhang, Mengmeng; Wang, Xiaodong J; Chen, Xi et al. (2012) Structural and kinetic analysis of prolyl-isomerization/phosphorylation cross-talk in the CTD code. ACS Chem Biol 7:1462-70
Xu, Guoyan G; Slebodnick, Carla; Etzkorn, Felicia A (2012) Cyclohexyl ketone inhibitors of Pin1 dock in a trans-diaxial cyclohexane conformation. PLoS One 7:e44226
Namanja, Andrew T; Wang, Xiaodong J; Xu, Bailing et al. (2011) Stereospecific gating of functional motions in Pin1. Proc Natl Acad Sci U S A 108:12289-94
Xu, Guoyan G; Zhang, Yan; Mercedes-Camacho, Ana Y et al. (2011) A reduced-amide inhibitor of Pin1 binds in a conformation resembling a twisted-amide transition state. Biochemistry 50:9545-50
Mercedes-Camacho, Ana Y; Etzkorn, Felicia A (2010) Enzyme-linked enzyme-binding assay for Pin1 WW domain ligands. Anal Biochem 402:77-82

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