Identifying novel selective PPO inhibitors through structure-based virtual screening and bio-evaluation

Abstract

Protoporphyrinogen oxidase (PPO) is a key enzyme in chlorophyll and heme biosynthesis, and the development of its inhibitors is of great importance both in the pharmaceutical and pesticide industries. However, the currently developed PPO inhibitors have insignificant bio-selectivity and have a serious impact on non-target organisms. In this study, a docking-based virtual screening approach combined with bio-activity testing was used to obtain novel selective inhibitors of PPO. The results of the bio-activity test showed that thirteen compounds showed 10-fold selectivity over human PPO. And the best selective compound, ZINC70338, has a K _i value of 2.21 μM for Nicotiana tabacum PPO and >113-fold selectivity for human PPO. The selectivity mechanism of ZINC70338 in different species of PPO was then analyzed by molecular dynamics simulations to provide a design basis and theoretical guidance for the design of novel selective inhibitors.

Fig. 1. Molecular structures. (A) The two channels of PPO; (B) the structure of the top 2 hits.

Fig. 2. Workflow of virtual screening and bio-activity assays for PPO inhibitors.

Fig. 3. (A) alignment of ntPPO (green) and hPPO (magenta) protein crystal structures; (B) sequence alignment of ntPPO and hPPO; (C) comparison of ntPPO (left) and hPPO (right) active cavities, the red area is the S2 and the cyan area is the S1 region.

Fig. 4. The binding modes of ZINC70338 with ntPPO (A) and hPPO (C) and 2D interaction diagram of ZINC70338 with ntPPO (B) and hPPO (D). ZINC70338 is shown as cyan sticks, and the key residues is shown as magenta sticks. The yellow dash is hydrogen bond, and the green is the π–π stacking interaction.

Fig. 5. (A) The RMSD of the complex formed by ntPPO with ZINC70338, the backbond atom of ntPPO and ZINC70338; (B) the RMSF in ntPPO system; (C) the RMSD of the complex formed by hPPO with ZINC70338, the backbond atom of hPPO and ZINC70338; (D) the RMSF in hPPO system; the SASA analysis of ZINC70338 with ntPPO (E) and hPPO (F); the radius of gyration of ZINC70338 with ntPPO (G) and hPPO (H).

Fig. 6. Per-residue decomposition studies and average structural analysis. (A) comparison of the key residues in ntPPO and hPPO; (B and C) a comparison of the average structure generated by extracting 1000 frames of conformation from the two complex traces.