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 structurebased 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 mM. 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.

Perspective Chapter: Plant Abiotic Stress Factors – Current Challenges of Last Decades and Future Threats

“…Today, the ABA signaling pathway from a signaling cascade consisting of the PYL ABA receptors, to type 2C protein phosphatases (PP2Cs) and Snf1-related protein kinases 2 (SnRK2s) has been enlightened by multiple structural studies which provided insight on regulating each level of the ABA signal transduction pathway [29]. In this core ABA signaling pathway model, PYLs [14 members; Pyrabactin Resistance 1 (PYR1) and PYR1-like 1-13 (PYL1-PYL13) or regulatory component of ABA Receptor (RCAR1-RCAR14)] bind to ABA to inhibit PP2Cs, which represent a major phosphatase family in plants and consisted of single subunit Mg 2+ /Mn 2+ -dependent Ser/Thr phosphatases [30]. ABA response is kept silent at the basal level by negative regulators of ABA as PP2Cs, including ABI1, ABI2, and HAB1.…”

A dynamic model of the ABA Signaling pathway with its core components: translation rate of PP2C determines the kinetics of ABA-induced gene expression

“…To examine the hypothesis, we conducted an experiment with the RD29A::LUC transgenic plants and pyridostatin hydrochloride, a recently identified chemical inhibitor that is specific for the PP2C phosphatase activity against SnRK2 (Janicki et al, 2020). On addition of 100 µM but not 25 M pyridostation hydrochloride, an increase in luminescence as well as a delay of the peak time was observed, indicating inhibitor-concentration dependent changes (Fig.…”

Genome-wide identification and characterization of protein phosphatase 2C (PP2C) gene family in sunflower (Helianthus annuus L.) and their expression profiles in response to multiple abiotic stresses

“…Subgroup A includes nine members, 6 of them (ABI1, ABI2, AHG1, HAB1, HAB2, and AHG3/ATPP2CA) negatively modulate ABA signaling and others three (HAI1, HAI2/AIP1, and HAI3) responded individually to stress [ 16 ]. Two candidate inhibitors of ABI1 protein phosphatase were identified, and they have the potential to play a role in modulating ABA responses [ 17 ]. The PP2C gene family plays crucial roles in the modulation of stress signaling and plant development.…”