A phosphorylated prodrug for the inhibition of Pin1

Abstract

Fmoc-pSer-Ψ[(Z)CH=C]-Pro-(2)-N-(3)-ethylaminoindole 1, showed moderate inhibition towards the mitotic regulator, Pin1 (IC 50 = 28.3μM). To improve the cell permeability, the charged phosphate was masked as the bis-pivaloyloxymethyl (POM) phosphate in Fmoc-(bisPOM)-pSer-Ψ[(Z)CH=C]-Pro-(2)-N-(3)-ethylaminoindole 2. Antiproliferative activity towards A2780 ovarian cancer cells of 1 (IC 50 = 46.2 μM) was improved significantly in 2 (IC 50 = 26.9 μM), comparable to the IC 50 of 1 towards Pin1 enzymatic activity.Cis-trans isomerization of proline-containing peptides has been implicated in a number of biologically important processes. 1, 2 Peptidyl-prolyl isomerase (PPIase) enzymes catalyze the cis-trans isomerization of Xaa-Pro amide bonds in proteins. 3 Pin1, a member of the PPIase family, 4 is unique because it isomerizes prolyl residues preceded by phosphorylated Ser or Thr with selectivity up to 1300-fold greater (k cat /K m ) than the unphosphorylated peptides. 5, 6The phosphorylation-dependent PPIase, Pin1, has been found to regulate mitosis through a simple conformational change, 6 the cis-trans isomerization of phospho-Ser/Thr-Pro amide bonds in a variety of key cell cycle regulatory phosphoproteins, including the Cdc25 phosphatase, the p53 oncogene, and the c-Myc oncogene. 6-8 Pin1 is essential for regulation of mitosis at G2 to M transition. 4 Cells depleted of Pin1 are characterized by premature entry into mitosis, followed by mitotic arrest, nuclear fragmentation, and apoptosis, while overexpression of Pin1 inhibits the G2 to M transition. 4, 7, 9 Therefore, Pin1 acts as a negative regulator for mitotic activity in G2, preventing lethal premature entry into mitosis. Pin1 is present at higher concentrations during mitosis, 10 making it a potential target in the continuously dividing cells of cancer. The central role Pin1 plays in the cell cycle makes it an interesting target for inhibition, both for potential anticancer activity and for elucidation of the mechanism of mitosis.Alkenes as cis-and trans-amide isosteres have been shown to be effective inhibitors of PPIases. 1, 11 We have shown previously that Pin1 binds a cis isotere more tightly than a trans isostere. 12 The cis alkene isostere was about 20-fold more potent than the trans alkene isostere in both the protease-coupled enzyme assay and the antiproliferative activity with A2780 cancer cells. 12 Based on these results, only the cis isostere was incorporated into inhibitors 1 and 2 ( Figure 1). The design of these two inhibitors was based on the selectivity of Pin1 for aromatic groups at the substrate termini. 6, 13, 14One common problem for phosphorylated compounds is that they are generally not effective at penetrating cell membranes because of the negative charge on the phosphate group. A general strategy for circumventing this problem involves masking the phosphate in a form that neutralizes its negative charge and enhances its cell permeability. 15-22 Upon cell entry, the Publisher's Disclaimer: This is a PDF file of an unedited manus...

A Reduced-Amide Inhibitor of Pin1 Binds in a Conformation Resembling a Twisted-Amide Transition State

“…This may be one reason that reduced amide 1 was only a 4.5-fold better inhibitor of Pin1 than the similarly substituted ground-state analogue, Fmoc-pSer–Ψ[( Z )CH=C]Pro–2-(indol-3-yl)-ethylamine (IC 50 = 28.3 μM). (39)…”

“…(35) No hydrogen bond was observed between any active-site residue and the tertiary amine nitrogen of the prolyl ring. (18) Inhibitors 1 and 2 represent a new application of reduced-amide peptide isosteres as Pin1 PPIase inhibitors with improved potency over similarly substituted alkene isosteres,(39) α-ketoamides,(40) or ketone substrate analogues. (35) These are among the best small molecule inhibitors we have synthesized to date, indicating that reduced-amide structures may be optimized to give potent inhibitors of Pin1.…”

The Ess1 prolyl isomerase: Traffic cop of the RNA polymerase II transcription cycle

“…These include peptidomimetics such as D-isomer and cyclic peptides, and conformationally-locked isosteres [84,90,102,108–111]. Selective WW-domain inhibitors have also been identified [112].…”

Cyclic cis-Locked Phospho-Dipeptides Reduce Entry of AβPP into Amyloidogenic Processing Pathway

“…All pCDPs used in this study (pCDP, pCDP-Bzl, pCDP-diBzl, pCDP-diPOM) were custom synthesized using green chemistry [35] and phosphorylation of Threonine completed [36–37] by Viva Biotech Ltd. (Shanghai, China) and purchased through Trillience (Toronto, Ontario). Deuterated-methanol NMR solvent (99.8%, CD3OD) was purchased from Cambridge Isotope Laboratories and used for each sample preparation.…”