Design, synthesis, and structure-activity relationship exploration of 1-substituted 4-aroyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-one analogues as inhibitors of the annexin A2-S100A10 protein interaction

Abstract

S100 proteins are small adaptors that regulate the activity of partner proteins by virtue of direct protein interactions. Here, we describe the first small molecule blockers of the interaction between S100A10 and annexin A2. Molecular docking yielded candidate blockers that were screened for competition of the binding of an annexin A2 peptide to S100A10. Several inhibitory clusters were identified with some containing compounds with potency in the lower micromolar range. We chose 3-hydroxy-1-(2-hydroxypropyl)-5-(4-isopropylphenyl)-4-(4-methylbenzoyl)-1H-pyrrol-2(5H)-one (1a) as a starting point for structure-activity studies. These confirmed the hypothetical binding mode from the virtual screen for this series of molecules. Selected compounds disrupted the physiological complex of annexin A2 and S100A10, both in a broken cell preparation and inside MDA-MB-231 breast cancer cells. Thus, this class of compounds has promising properties as inhibitors of the interaction between annexin A2 and S100A10 and may help to elucidate the cellular function of this protein interaction.

Chart 1. Clusters of Blockers of the Annexin A2−S100A10 Protein Interaction Identified by GOLD Docking and Biochemical Assay

Figure 1

Structure-based design of annexin A2−S100A10 inhibitors. (a) The interaction between the helical N-terminal annexin A2 peptide (gray CPK coloring) and a binding pocket of S100A10 (green CPK surface); the side chains, including the N-terminal acetyl group of the annexin peptide involved in intimate contact with the receptor through either hydrophobic or hydrogen-bonding (dashed lines) interactions, are shown as solid sticks and are labeled. (b) The hydrophobic (H1 and H2) and hydrophilic (H3) subsites of the S100A10 binding pocket are colored in cyan, yellow, and magenta, respectively. Predicted binding poses of verified virtual screening hits (gray CPK stick models): (c) 1a, (d) 2a, (e) 3, (f) 4, and (g) 5 (refer Chart 1).

Scheme 1. Synthesis of 1-Substituted 4-Aroyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-ones

Reagents and conditions: (a) 1,4-Dioxane, 15 min. (b) 1,4-Dioxane, overnight reaction. (c) (i) Microwave irradiation, 250 W, 30 °C, 5 min, 2 M NaOMe/MeOH; (ii) H⁺ (pH 3−4).

Figure 2

Alternative binding poses for N1-methoxyalkyl and allyl 1H-pyrrol-2(5H)-ones. Highly scoring S100A10-docked poses of compounds 35 (a), 36, (b), and 47 (c) are shown.

Figure 3

Biological activity of peptide and small-molecule annexin A2−S100A10 inhibitors. (A) Dose−response curves for the 14-residue N-terminal annexin A2 peptide (⧫) and compounds 1a (▲), 47 (◼), and 50 (▼) in the FRET-based competitive annexin A2−S100A10 binding assay are shown. Curve fit was performed in Graphpad Prism as described in Table 1. Error bars represent the standard error of the mean of four (compounds) or eight (peptide) observations. (B) As under A except that compound 27 (●; n = 28 ± standard error of the mean) or 22 (○; n = 4 ± standard error of the mean) was analyzed.

Figure 4

Analysis of the native complex of S100A10 and annexin A2. (A) MDA-MB-231 cells were lysed and incubated with 50 μM compound as indicated or with the annexin A2 N terminus peptide (P). S100A10 was immunoprecipitated, and the amount of annexin A2 in the immunoprecipitates was analyzed by SDS-PAGE followed by Western blotting using an annexin A2 antibody. Annexin A2 was identified based upon comigration with an annexin A2 reference on the blot. The section of the blot with the annexin A2 signal is shown. (B) MDA-MB-231 cells were lysed and incubated with compound at concentrations as indicated or with the annexin A2 N terminus peptide (P). Western blots were obtained and scanned, and scans were quantified using Scion Image software. Quantified data were expressed as % of nontreated control. Error bars represent the standard error of the mean (n = 3 from three separate lysates). (C) As in B, except that MDA-MB-231 cells were treated with compound for 6 h after which the cells were lysed and washed, and immunoprecipitations were performed as above. Error bars represent the standard error of the mean (n = 3 from three separate lysates). *p < 0.05 (nonpaired t test).