Optimization of a cyclic peptide inhibitor of Ser/Thr phosphatase PPM1D (Wip1)

Abstract

PPM1D (PP2Cδ or Wip1) was identified as a wild-type p53-induced Ser/Thr phosphatase that accumulates after DNA damage and classified into the PP2C family. It dephosphorylates and inactivates several proteins critical for cellular stress responses, including p38 MAPK, p53, and ATM. Furthermore, PPM1D is amplified and/or overexpressed in a number of human cancers. Thus, inhibition of its activity could constitute an important new strategy for therapeutic intervention to halt the progression of several different cancers. Previously, we reported the development of a cyclic thioether peptide with low micromolar inhibitory activity toward PPM1D. Here, we describe important improvements in the inhibitory activity of this class of cyclic peptides and also present a binding model based upon the results. We found that specific interaction of an aromatic ring at the X1 position and negative charge at the X5 and X6 positions significantly increased the inhibitory activity of the cyclic peptide, with the optimized molecule having a K(i) of 110 nM. To the best of our knowledge, this represents the highest inhibitory activity reported for an inhibitor of PPM1D. We further developed an inhibitor selective for PPM1D over PPM1A with a K(i) of 2.9 μM. Optimization of the cyclic peptide and mutagenesis experiments suggest that a highly basic loop unique to PPM1D is related to substrate specificity. We propose a new model for the catalytic site of PPM1D and inhibition by the cyclic peptides that will be useful both for the subsequent design of PPM1D inhibitors and for identification of new substrates.

Figure 1

Schematic representation of the cyclic thioether peptide. The thioether bond as formed between the acylated N-terminal residue and the cysteine sidechain at the C terminus.

Figure 2

An aromatic residue at position X1 of the cyclic peptide is important for inhibitory activity. (A) Inhibitory activity of cyclic peptides (10 μM) substituted at position X1 in the presence of [S1981pS] human ATM(1976–1986) peptide (30 μM) as a substrate (*p<0.05 as compared with peptide 1). (B) Concentration dependence of inhibition by cyclic peptides: peptide 1, cyclo(M-pS-I-pY-VAC)-amide (closed circle); peptide 5, cyclo(F-pS-I-pY-VAC)-amide (open circle); peptide 6, cyclo(F-pS-pY-I-VAC)-OH (open triangle). (C) Inhibitory activity of cyclic peptides used at 10 μM and 1 μM, as indicated (*p<0.05 as compared with peptide 5).

Figure 3

The enantiomer of the Ile at position X3 is important for inhibition of PPM1D phosphatase activity. (A) Chemical structure of isoleucine analogs. (B) Concentration dependence of the inhibition of PPM1D by cyclic peptide [F-pS-(L-Ile)-pY-V-A] (peptide 5, open circle), [F-pS-(D-Ile)-pY-VAC] (peptide 22, cross), and [F-pS-(L-allo-Ile)-pY-VAC] (peptide 23, closed circle) in the presence of [S1981pS] human ATM(1976–1986) peptide (30 μM) as a substrate.

Figure 4

Acidic residues at positions X5 and X6 increase the inhibitory activity of the cyclic peptides. Inhibitory activity of cyclic peptide (F-pS-I-pY-X5-X6-C) in the presence of [S1981pS] human ATM(1976–1986) peptide (30 μM) as a substrate. Inhibitory activities of (A) cyclo(F-pS-I-pY-X5-AC)-amide and (B) cyclo(F-pS-I-pY-V-X6-C)-amide peptides were measured at 10 μM (closed bar) and 1 μM (open bar) (*p<0.05 as compared with peptide 5). (C) K_i values of cyclic thioether peptides substituted by acidic residues at both position X5 and X6. (D) Concentration dependence of cyclic thioether peptide (M-pS-I-pY-VAC)-amide (peptide 1, closed circle), (F-pS-I-pY-VAC)-amide (peptide 5, open circle), (F-pS-I-pY-DDC)-amide (peptide 40, closed triangle). (E) Inhibitory activity of cyclic peptide 37 in the presence of different Wip1 substrates (open circle, [T180pT, Y182pY]Human p38(175–185); closed circle, [S1981pS]Human ATM(1976–1986)).

Figure 5

The cyclic peptide is a competitive inhibitor of PPM1D. Double-reciprocal plot of PPM1D phosphatase activity against [S1981pS]human ATM(1976–1986) peptide concentration during the inhibition of PPM1D with different concentrations of cyclic thioether peptide (F-pS-I-pY-EEC)-amide (peptide 37) (cross, absent; open circle, 0.2 μM; closed circle, 0.5 μM).

Figure 6

Peptide 48 is a selective inhibitor of PPM1D. (A) Dephosphorylation of peptide 40 by PPM1A. (B) Inhibitory activity of cyclic peptides (25 μM) substituted at position X2 in the presence of [S1981pS] human ATM(1976–1986) peptide (30 μM) as a substrate (*p<0.05 as compared with a no-inhibitor control). †Retro-inverse peptide. (C) Concentration dependence of cyclic thioether peptides 48 (open circle), 49 (open triangle), and 50 (closed circle). (D) Phosphatase activity of PPM1A against cyclic peptides 1, 40 and 48 at 100 μM. Inset: PPM1A phosphatase activity against peptide 40 (50 μM) in the presence (open bar) or in the absence of peptide 48 (closed bar).

Figure 7

Model of cyclic peptide inhibitor 37 bound to the active site of PPM1D. Residues discussed in the text are labeled. Highlighted residues of the protein are colored cyan, blue, red and white, for carbon, nitrogen, oxygen and hydrogen. The metal ions are magenta. The unique colors of the cyclic peptide are green, yellow and orange, for carbon, sulfur and phosphorus.

Figure 8

Putative alignment of the B-loop region of PPM1D. (A) Comparison with PPM1A and structurally aligned PP2C homologues with similar flap sub-domain conformations, and with human PP2C proteins with unknown 3D structure. References for the structures are given in Supporting Table 1. The Swiss-Prot database (http://expasy.org/sprot/) accession numbers for PPM1D and the other human sequences are: PPM1D_HUMAN (O15297), PPM1A_HUMAN (P35813), PPM1B_HUMAN (O75688), PPM1E_HUMAN (Q8WY54), PPM1F_HUMAN (P49593), PPM1G_HUMAN (O15355), PPM1K_HUMAN (Q8N3J5), PPM1L_HUMAN (Q5SGD2). (B) Comparison with the structurally aligned sequences of PPM1A and PP2Ctg.

Figure 9

Characterization of the structure and activity of PPM1D mutants. (A) CD spectra of wild type and mutant PPM1D; wild type (black), [R243A]PPM1D (red), [K247A]PPM1D (blue). Protein concentration was 1.8–2.1 μM in 50 mM Tris-HCl, 30 mM MgCl₂, and 5% glycerol (pH 7.5). (B) K_m values and V_max values for wild type and mutant PPM1D. Phosphatase activities were measured using [S1981pS]human ATM(1976–1986) substrate peptide (closed circle, wild type; open circle, [R243A]PPM1D; open triangle, [K247A]PPM1D).