Pin1 WW Domain Ligand Library Synthesized with an Easy Solid-Phase Phosphorylating Reagent.

Cell cycle regulatory enzyme Pin1 both catalyzes pSer/Thr--Pro isomerization and binds the same motif separately in its WW domain. To better understand the function of Pin1, a way to separate these activities is needed. An unnatural peptide library, RCO-pSer-Pro-NHR, was designed to identify ligands specific for the Pin1 WW domain.

Consensus design of a NOD receptor leucine rich repeat domain with binding affinity for a muramyl dipeptide, a bacterial cell wall fragment.

Repeat proteins have recently emerged as especially well-suited alternative binding scaffolds due to their modular architecture and biophysical properties. Here we present the design of a scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Consensus sequence design has emerged as a protein design tool to create de novo proteins that capture sequence-structure relationships and interactions present in nature.

Kinetic isotope effects support the twisted amide mechanism of Pin1 peptidyl-prolyl isomerase.

The Pin1 peptidyl-prolyl isomerase catalyzes isomerization of pSer/pThr-Pro motifs in regulating the cell cycle. Peptide substrates, Ac-Phe-Phe-phosphoSer-Pro-Arg-p-nitroaniline, were synthesized in unlabeled form, and with deuterium-labeled Ser-d3 and Pro-d7 amino acids. Kinetic data were collected as a function of Pin1 concentration to measure kinetic isotope effects (KIEs) on catalytic efficiency (kcat/Km).

A reduced-amide inhibitor of Pin1 binds in a conformation resembling a twisted-amide transition state.

The mechanism of the cell cycle regulatory peptidyl prolyl isomerase (PPIase), Pin1, was investigated using reduced-amide inhibitors designed to mimic the twisted-amide transition state. Inhibitors, R-pSer-Ψ[CH(2)N]-Pro-2-(indol-3-yl)ethylamine, 1 [R = fluorenylmethoxycarbonyl (Fmoc)] and 2 (R = Ac), of Pin1 were synthesized and bioassayed. Inhibitor 1 had an IC(50) value of 6.

Stereospecific gating of functional motions in Pin1.

Pin1 is a modular enzyme that accelerates the cis-trans isomerization of phosphorylated-Ser/Thr-Pro (pS/T-P) motifs found in numerous signaling proteins regulating cell growth and neuronal survival. We have used NMR to investigate the interaction of Pin1 with three related ligands that include a pS-P substrate peptide, and two pS-P substrate analogue inhibitors locked in the cis and trans conformations. Specifically, we compared the ligand binding modes and binding-induced changes in Pin1 side-chain flexibility.

Toward flexibility-activity relationships by NMR spectroscopy: dynamics of Pin1 ligands.

Drug design involves iterative ligand modifications. For flexible ligands, these modifications often entail restricting conformational flexibility. However, defining optimal restriction strategies can be challenging if the relationship between ligand flexibility and biological activity is unclear.

Enzyme-linked enzyme-binding assay for Pin1 WW domain ligands.

Peptidyl prolyl cis-trans isomerase (PPIase) interacting with NIMA-1 (Pin1) catalyzes the cis-trans isomerization of pSer/pThr-Pro amide bonds. Pin1 is a two-domain protein that represents a promising target for the treatment of cancer. Both domains of Pin1 bind the pSer/pThr-Pro motif; PPIase enzymatic activity occurs in the catalytic domain, and the WW domain acts as a recognition module for the pSer/pThr-Pro motif.