Chalcone-based Selective Inhibitors of a C4 Plant Key Enzyme as Novel Potential Herbicides

Abstract

Weeds are a challenge for global food production due to their rapidly evolving resistance against herbicides. We have identified chalcones as selective inhibitors of phosphoenolpyruvate carboxylase (PEPC), a key enzyme for carbon fixation and biomass increase in the C4 photosynthetic pathway of many of the world's most damaging weeds. In contrast, many of the most important crop plants use C3 photosynthesis. Here, we show that 2',3',4',3,4-Pentahydroxychalcone (IC50 = 600 nM) and 2',3',4'-Trihydroxychalcone (IC50 = 4.2 μM) are potent inhibitors of C4 PEPC but do not affect C3 PEPC at a same concentration range (selectivity factor: 15-45). Binding and modeling studies indicate that the active compounds bind at the same site as malate/aspartate, the natural feedback inhibitors of the C4 pathway. At the whole plant level, both substances showed pronounced growth-inhibitory effects on the C4 weed Amaranthus retroflexus, while there were no measurable effects on oilseed rape, a C3 plant. Growth of selected soil bacteria was not affected by these substances. Our chalcone compounds are the most potent and selective C4 PEPC inhibitors known to date. They offer a novel approach to combat C4 weeds based on a hitherto unexplored mode of allosteric inhibition of a C4 plant key enzyme.

Figure 1. Binding affinities of butein (8), robtein (9), and okanin (12) to C₄ PEPC from F. trinervia.

(a) ITC binding curves of the compounds binding to the wild-type (wtFt). (b) ITC binding curves of the compounds binding to the mutant G884R at lower affinities.

Figure 2. Competition between okanin (12) and the feedback inhibitor aspartate in binding to the wild-type F. trinervia PEPC.

ITC binding curves of okanin (12) binding to C₄ PEC from F. trinervia in the presence of 0.5 mM or 17 mM aspartate.

Figure 3. Prediction of the binding mode of chalcones.

(a) Pose of 12 (magenta) within C₄ PEPC after MAB minimization of the configuration docked with GLIDE with the lowest energy in the largest cluster. Very similar binding poses are also found for all other chalcones tested here (see Fig. S3). Ring A (red circle) is located in between the two arginines (R641 and R888) that are important for Asp binding. Ring B (blue circle) is deeply buried in a subpocket unused by Asp. (b) Starting configuration (magenta) of 12 and configuration after 200 ns of MD simulations (gold) in C₃ PEPC. During the MD simulations, 12 shifts by ~3 Å RMSD. (c,d) 2D schemes of the binding poses of 12 in C₄ PEPC (c) and 11 in C₃ PEPC (d). The red circles mark G884 and R884, which is the selectivity-determining residue. (e,f) Representative RMSD with respect to the starting structure over the course of three independent MD simulations each of 12 in C₄ PEPC (e) and 12 in C₃ PEPC (f) for the backbone atoms of PEPC (orange) and side-chains atoms of PEPC within 5 Å distance of the starting configuration of the ligand (yellow). The blue, green, and violet lines depict RMSD values of the ligands with respect to the starting configuration for the three independent MD simulations each of a PEPC-ligand complex. For better visibility smoothing was applied for all plots. (a,b) Ring B is buried inside a subpocket, which is formed by A132, E135, Q673, H679, L680, C681, R683, and R687. For clarity, residues A132, E135, Q673, H679, and C681 are not represented; none of the ligands investigated in this study interact with these amino acids.

Figure 4. Effects of chalcones on A. retroflexus and B. napus.

(a) Leaf area of A. retroflexus and B. napus six days after treatment. Results of experiment 1 (top) and experiment 2 (bottom) are displayed. 2′,3′,4′-Trihydroxychalcone (10) and 2′,3′,4′,3,4-Pentahydroxychalcone (12) significantly reduced A. retroflexus leaf area in both experiments according to Tukey’s HSD at α = 0.05. None of the compounds had an effect on leaf area of B. napus. Bars display standard deviations (n = 6; B. napus experiment 2: n = 5). Dashed lines represent the average of the corresponding control treatments. (b) Effect of 2′,3′,4′-Trihydroxychalcone (right) on growth of A. retroflexus (top) and B. napus (bottom) in comparison to the control treatment (left). (c) Effect of 2′,3′,4′,3,4-Pentahydroxychalcone (right) on growth of A. retroflexus (top) and B. napus (bottom) in comparison to the control treatment (left). (d) false-color image of anthocyanin reflectance index (ARI) of A. retroflexus two days after treatment with 2′,3′,4′,3,4-Pentahydroxychalcone (bottom) and the corresponding control (top). ARI values given inside the image are the average values of the 3^rd leaf for six replicates. Standard errors are given in brackets. (e) false-color image of photochemical reflectance index (PRI) of A. retroflexus two days after treatment with 2′,3′,4′,3,4-Pentahydroxychalcone (bottom) and the corresponding control (top). PRI values given inside the image are the average values of the 3^rd leaf for six replicates. Standard errors are given in brackets.