Identification of Novel Inhibitors of a Plant Group A Protein Phosphatase Type 2C Using a Combined In Silico and Biochemical Approach

Abstract

Type 2C protein phosphatases (PP2Cs) of group A play a significant role in the regulation of various processes in plants including growth, development, ion transport, and stress acclimation. In this study, we selected potential PP2C group A inhibitors using a structure-based virtual screening method followed by biochemical and in vitro validation. Over twenty million chemical compounds from the ZINC database were used for docking studies. The precision of the calculations was increased by an induced-fit docking protocol and the molecular mechanics/generalized Born surface area (MM/GBSA) method, which yielded approximate values for the binding energy of the protein-ligand complex. After clustering and ranking their activity, the top-ranking compounds were tested against PP2C group A members in vitro and their in vivo activity was also explored. Phosphatase activity assays identified two compounds with significant inhibitory activity against ABI1 protein ranging from around 57 to 91% at a concentration of 100 μM. Importantly, this in vitro activity correlated well with in vivo inhibition of seed germination, as expected for PP2C inhibitors. The results should promote the design of novel inhibitors with improved potency against ABI1-like and other PP2Cs that might be used in agriculture for the protection of crops against stress.

Keywords: PP2C inhibitor; protein phosphatase 2C group A; protein-ligand complexes; structure-based virtual screening.

Figure 1

Initial docking study. (A) Protein target ABI1 from crystallographic structure PDB code 3KDJ (complex of PYL1-ABI1) showing proposed binding site (cyan-red-blue sticks) with residues crucial for the interaction labeled; (B) Fifteen chemical compounds from our docking studies were chosen for validation of ABI1 phosphatase activity; (C) In the ABI1 active site, the docking pose of ZINC33009768 is shown as a magenta backbone, and the docking pose of ZINC09438755 is shown as a yellow backbone. PYL1 is shown in gray (the gate-loop of PYL1 is visible adjacent to each chemical compound ZINC33009768 and ZINC09438755); (D) Chemical structure of ZINC09438755 and ZINC33009768 compounds generated in Maestro (Schrödinger Suite); (E) Effect of ZINC33009768 and ZINC09438755 on ABI1 protein phosphatase activity. Normalized results of phosphatase activity are shown in the presence of ZINC09438755 and ZINC33009768, respectively. The enzyme reactions were performed in a 50 µl final volume containing 3 µg of His–ABI1 with or without 100 μM ZINC33009768 or 100 μM ZINC09438755. The results presented are the means from three independent biological replicates.

Figure 2

Strategy for identification of novel inhibitors. (A) Structure of PYL1-ABI1 complex (PDB code: 3KDJ). PYL1 is shown in color scale, while ABI1 PP2C is shown in grayscale. The manganese ion is shown as a pink sphere; (B) Target (gray surface) and a putative binding site (shown as blue red mesh): a metal ion is included at the binding site; (C) Comparison between proposed binding site (blue spheres) and interaction surface of PYL1 (shown as red cartoons) and SnRK2.6 (shown as light blue cartoons) and ABI1 (shown in gray surface representation).

Figure 3

Testing of docking model. (A) Superposition of human PP2Ca structure (shown as magenta cartoons, PDB: 4RA2) and plant group A PP2C (shown as green cartoons, PDB: 3KDJ); (B) Monophosphate ion binding pose. Ions shown as spheres; the position of the monophosphate ion was taken from the superposition of those two phosphatase structures; (C) Redocking pose of the monophosphate ion using our proposed binding model and comparison with position of the monophosphate ion present in mammalian phosphatase PP2Ca; (D) Docking pose of pSER, pTHR, and pNPP. Docked molecules are shown as stick models.

Figure 4

Virtual screening procedure. (A) The 325 top-ranked compounds resulting from the virtual screening procedure on a target ABI1 protein surface are shown. Selected molecules are shown as stick models on the protein surface; (B) The virtual screening procedure is shown as a set of filtering procedures, which gradually remove chemical compounds in each step. Initially, 22 million chemical compounds from the ZINC database were considered, and these were ultimately reduced to 325 candidate molecules. A three-step docking algorithm was used with different precision modes (HTVS, SP, XP). Small compounds were removed on the basis of number of atoms and ABA-like molecules were also filtered out computationally.

Figure 5

Effect of Candidate Inhibitors on ABI1 Protein Phosphatase Activity. (A) Normalized results of phosphatase activity in the presence of each potential PP2C inhibitor are shown. Phosphatase activity was obtained from three independent biological replicates. IC50 was determined for six different concentrations of ZINC05273880 (B) and ZINC59151964 (C), respectively, in the range 0 to 200 µM. Results are an average value of two independent experiments (n = 12). IC50 was determined by setting the inhibition rate of phosphatase activity versus inhibitor concentration. Error bars represents standard deviation. (D) K_m value for ABI1 in a substrate range from 0 to 200 µM; (E, F) Effect of ZINC05273880 and ZINC59151964 on ABI1, ABI2 and non-clade A PP2Cs. Recombinant ABI1, ABI2, PP1, PP2A, and PPH1 proteins were assayed with (100 µM) and without (control) the indicated ZINC compounds. Values are expressed as a percentage of control and as the mean of two independent experiments (n = 6–12). The asterisks marks a significant difference by Student’s t-test (p < 0.001).

Figure 6

Interaction sites of best experimentally validated compounds. (A) ZINC05273880 interaction diagram and proposed binding pose with the crucial interactions between amino acid residues and the docked molecule highlighted; (B) ZINC59151964 interaction diagram and proposed binding pose with the crucial interactions between amino acid residues and the docked molecule highlighted. Amino acids within a 5 Å radius of the docking pose of the chemical compound are labeled.

Figure 7

PYL1 and SnRK2.6 interaction loops, and docking pose structural correlation. (A) SnRK2.6 is shown as a blue cartoon, docked molecules are shown as sticks, and PP2C is shown as a surface. (B) The PYL1 receptor is shown as a red cartoon, docked molecules are shown as sticks, and PP2C is shown as a surface.

Figure 8

Effect of ZINC59151964, ZINC05273880 and ABA on seed germination. Seeds of WT (A, C), and snrk2.2/2.3 (B, D) mutant lines were grown on MS medium supplemented with ABA, the indicated concentration of ZINC compound or both. Right panel (A, B) presents germination results for ZINC59151964. Left panel (C, D) presents germination results for ZINC05273880. Seeds are considered germinated when green cotyledons have expanded. Values are the mean germination frequency from three to four separate plates (22–32 seeds per plate) for each genotype. Error bars indicate SD; “a” indicates a significant change (P < 0.05, Student’s t-test) compared with the mock control; “b” indicate a significant change (P < 0.05, Student’s t-test) between ABA and ABA/inhibitor treatment.

Figure 9

Cell-free degradation of recombinant GST-ACS7 protein. (A) Recombinant ACS7 protein was incubated with WT Col-0 protein extracts treated with or without MG132, ZINC95151964, and ZINC05273880 for the indicated times. The GST-ACS7 level was visualised by immunoblotting using anti-GST antibodies. Equal protein loading was shown by Ponceau S staining; (B) Half-life plot for cell-free degradation of ACS7 after MG132, ZINC95151964, and ZINC05273880 treatment. The GST-ACS7 bands were quantified using ImageJ software. Error bars indicate the SD (n = 4–6 replicates per time point) and the asterisks indicate a significant difference between mock and inhibitor treatment (based on Student’s t-test **p < 0.0001; *p < 0.03).