Membrane Permeable Cyclic Peptidyl Inhibitors against Human Peptidylprolyl Isomerase Pin1

Abstract

Peptidyl-prolyl isomerase Pin1 regulates the function and/or stability of phosphoproteins by altering the conformation of specific pSer/pThr-Pro peptide bonds. In this work, a cyclic peptide library was synthesized and screened against the catalytic domain of human Pin1. The selected inhibitors contained a consensus motif of D-pThr-Pip-Nal (where Pip is L-piperidine-2-carboxylic acid and Nal is L-2-naphthylalanine). Representative compounds were tested for binding to Pin1 by isothermal titration calorimetry and inhibition of Pin1 activity and the most potent inhibitors had KD (and KI) values in the low nanomolar range. Treatment of breast cancer cells with the inhibitors, which were rendered membrane permeable by attachment of an octaarginine sequence, inhibited cell proliferation and increased the protein levels of two previously established Pin1 substrates, PML and SMRT. Finally, a second generation of cell permeable Pin1 inhibitors was designed by replacing the noncritical residues within the cyclic peptide ring with arginine residues and shown to have anti-proliferative activity against the cancer cells.

The Peptidyl-prolyl Isomerase Pin1 Up-regulation and Proapoptotic Function in Dopaminergic Neurons

“…Neurobasal medium, RPMI 1640 medium, hygromycin B, B27 supplement, fetal bovine serum, L-glutamine, penicillin, and streptomycin were purchased from Invitrogen. The Pin1-cyclic peptide inhibitor (peptide inhibitor F, sequence cyclo(D-Arg-D-Arg-DThr(P)-Pip-Nal-Arg-Gln), where Pip is L-piperidine-2-carboxylic acid and Nal is L-2-naphthylalanine) was kindly provided by Dr. Pei Dehua (Ohio State University) and generated as described previously (26).…”

“…PiB, a small chemically synthesized compound, was originally recognized as a potent Pin1 inhibitor through a library screen (39). The cyclic peptide inhibitor F (sequence, cyclo(D-Arg-D-Arg-D-Thr(P)-Pip-Nal-Arg-Gln)) belongs to a novel family of cyclic peptidyl Pin1 inhibitors showing levels of affinity and specificity higher than small molecular Pin1 inhibitors (26). The cyclic peptide inhibitor F inhibits Pin1 both in vitro and in intact cells at low micromolar concentrations (26).…”

“…The cyclic peptide inhibitor F (sequence, cyclo(D-Arg-D-Arg-D-Thr(P)-Pip-Nal-Arg-Gln)) belongs to a novel family of cyclic peptidyl Pin1 inhibitors showing levels of affinity and specificity higher than small molecular Pin1 inhibitors (26). The cyclic peptide inhibitor F inhibits Pin1 both in vitro and in intact cells at low micromolar concentrations (26). MN9D cells were treated with 300 M MPP ϩ for 24 h in the presence or absence of PiB (5 M), juglone (1 M), or peptide inhibitor (5, 10, and 15 M), and Pin1 expression was determined by Western blot.…”

Cell-Permeable Bicyclic Peptide Inhibitors against Intracellular Proteins

“…19 Thus, we fused cFΦR 4 with the previously reported monocyclic peptide 5 , which is a potent inhibitor against Pin1 in vitro ( K D = 258 ± 66 nM) but membrane-impermeable 20 (Scheme 3). In addition, we replaced the l -Tyr at the pThr + 3 position with Arg to improve the aqueous solubility.…”

Peptide‐Based Inhibitors of Enzymes

“…All of the isolated peptides contained phosphoThr-Pip-Nal (where Pip is l-piperidine-2-carboxylic acid and Nal is l-2-napthylalanine) and were rendered membrane-permeable by incorporating an Arg 8 sequence onto a side chain or into the peptide backbone. These cyclic peptides successfully entered cells and slowed down cell proliferation, displaying the first example of macrocyclic Pin1 inhibitors active in vivo [136].…”