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

Maciej Janicki,Agata Cieśla,Małgorzata Marczak,Agnieszka Ludwików

doi:10.3389/fpls.2020.526460

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.

Highlights

Reversible protein phosphorylation is a key protein modification involved in the regulation of numerous plant cellular processes (Friso and Van Wijk, 2015; Gerotto et al, 2019)
We focused mainly on the structure of the abscisic acid insensitive1 (ABI1)-PYL-abscisic acid (ABA) ternary complex, which gives critical insights into ABI1-PYL binding
We screened for candidate inhibitors that bind via hydrogen bonds to the Gly180 and Glu142 residues, which are critical for ABI1 activity, are located at the active site, and are crucial for pyrabactin resistance-like1 abscisic acid receptor (PYL1) binding to this phosphatase (Figure 1A)

Summary

Introduction

Reversible protein phosphorylation is a key protein modification involved in the regulation of numerous plant cellular processes (Friso and Van Wijk, 2015; Gerotto et al, 2019). In Arabidopsis, 76 members of the protein phosphatase type 2C (PP2C) family have been identified, which can be divided into 10 groups according to sequence similarity and subdomain composition. Ongoing research has demonstrated that Arabidopsis group A PP2Cs Genetic analysis demonstrates that ABI1, HAB1, ABI2, and HAB2 are key regulators of drought tolerance (Saez et al, 2006; Bhaskara et al, 2012), and the respective signaling pathways are good candidates for genetic engineering to improve crop tolerance to drought

Methods

Results

Conclusion