The peptidyl prolyl cis/trans isomerase, PPIase, has been the focus of numerous studies in the field of cell cycle regulation since proline-directed phosphorylation is an essential signaling mechanism that might arrest cancer proliferation. Pin1 is the first phosphorylation-dependent PPIase enzyme to be discovered. The Pin1 regulatory mechanism, acting on other mitotic proteins in vivo and in vitro, remains largely unknown. For the study of Pin1 function, two types of assays were used to identity ligands for Pin1: (1) The Enzyme-Linked Enzyme Binding Assay (ELEBA) for the identification of WW domain ligands, (2) a catalytic assay to identified inhibitors of Pin1 catalytic activity. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain from chemical libraries. By using the ELEBA, a pSer-Pro peptidomimetic library of 315 ligands was screened, identifying three promising ligands cis-D2, O2, and M18. Competitive Kd values for cis-D2, O2, and M18 were determined to be 263 ± 6.4, 206 ± 3.4, and 130 ± 3.0μM, respectively. Furthermore, we screened the pSer-Pro peptidomimetic library using a Pin1 discontinuous-catalytic assay to identify inhibitors of Pin1. Ligands D20 and K7 were identified to decrease more than 90% of the Pin1 catalytic activity. To investigate the nature of the Pin1 interaction with c-Myc, we synthesized and characterized four peptides corresponding to the c-Myc sequence. These peptides were used in NMR isomerization studies of Pin1 by our collaborator Dr. Jeffry Peng (University of Notre Dame). Preliminary work shows that Pin1 binds and isomerizes the Ac–LLPpTPPLSPS–NH₂ peptide at the cMyc pThr58 position. Finally, we measured a secondary kinetic isotope effect (2º KIE) to study the Pin1 catalytic mechanism of proline isomerization. The ratio of kH/kD for unlabeled and [d₃]Ser-labeled substrate gave a SKIE value of 1.34 ± 0.01. The normal 2º KIE value indicates that carbonyl-serine hybridization is not changing from sp² to sp³. This result supports substrate analogue inhibitor studies, and previous solvent and SKIE results on Pin1, that suggest a twisted amide mechanism assisted by a transient hydrogen bond in the transition state. / Ph. D.

Pin1 is a peptidyl prolyl isomerase (PPIase) enzyme with two domains, the catalytic domain and the WW domain. Both domains specifically bind pSer/pThr–Pro motifs. Pin1 plays an important role in regulating the cell cycle, and it is involved in many diseases, such as cancer, HIV-1, Alzheimer's disease, asthma, hepatitis B, and rheumatoid arthritis. Pin1 is a very promising target for new drug development. Three stereoisomers: (R,S)-, (S,R)- and (S,S)-Ac–pSer–Ψ[(Z)CH=C]–Pip–2-(2-naphthyl)ethylamine were synthesized as inhibitors binding to the Pin1 catalytic domain. The (R,S)- and (S,R)-isomers were synthesized via a 13-step route, with overall yields of 2.0% and 1.4%, respectively. The newly formed stereogenic center in the piperidyl ring was introduced by a Luche reduction, followed by a stereoselective [2,3]-Still-Wittig rearrangement. The configuration of the stereocenter was determined by NOESY of a bicyclic derivative. The (Z)- to (E)-alkene ratio in the rearrangement was (5.5:1). The (S,S)-isomer was obtained as the epimerized by-product resulting from the (S,R)-isomer in the Na/NH3 deprotection step. The IC50 values for Pin1 inhibition were: 52, 85, and 141 μM, respectively. We concluded that in this Z-alkene isostere, the R-configuration would be preferred at both stereogenic centers, as mimics of L-Ser and L-Pip, to improve the affinity. Combinatorial chemistry is a powerful method to discover biologically active compounds, and solid-phase synthesis is most commonly used to synthesize combinatorial libraries. To identify ligands for the Pin1 WW domain, a library, R1CO–pSer–Pro–NHR2, was designed. A new solid-phase phosphorylating reagent (SPPR) containing a phosphoramidite function was synthesized in one step from commercially available Wang resin. The SPPR was applied in the preparation of a designed library through parallel synthesis. The library contained 357 members (17 Ã 21), and was screened by an enzyme-linked enzyme binding assay (ELEBA). The best hits were resynthesized, and the competitive dissociation constants, Kd-rel, were measured by ELEBA, with a Kd-rel value of 130 μM for the best ligand. The absolute dissociation constants will be measured by our collaborator, Prof. Jefferey Peng, University of Notre Dame, using NMR methods. Besides the identification of the Pin1 WW domain ligands, I created a practical method for solid-phase synthesis of phosphopeptides. / Ph. D.