A fungal tolerance trait and selective inhibitors proffer HMG-CoA reductase as a herbicide mode-of-action

Herbicide resistance in weeds has escalated, diminishing the efficacy of existing chemistries and threatening global food security. Only one new herbicide mode of action has reached market in nearly four decades, underscoring the urgent need for new targets. Isoprenoid biosynthesis is essential across life and in plants proceeds via compartmentalized mevalonate (MVA, cytosol) and methylerythritol phosphate (MEP, plastid) pathways. No commercial herbicides currently target the plant MVA pathway. HMGR, the highly regulated, rate-limiting enzyme of the MVA pathway and the molecular target of statins in humans, presents a compelling candidate for herbicide development. Despite conservation of HMGR across kingdoms and concerns over off-target effects, differences may be exploitable for plant specificity. This study tests the hypothesis that plant HMGR possesses structural features enabling selective inhibition and that a tolerance trait can be engineered to support deployment of HMGR-targeting herbicides.

Prior work established that certain statins (e.g., lovastatin, mevastatin, pravastatin, atorvastatin) exhibit herbicidal activity in multiple species including Arabidopsis thaliana and Lemna gibba. The isoprenoid biosynthetic pathways (MVA vs MEP) differ by kingdom; plants uniquely possess both. HMGR enzymes are categorized into classes I and II, differing in catalytic core architecture, membrane domains, and cofactor preferences. Regulation of HMGR involves N-terminal ubiquitination and catalytic-domain phosphorylation in both plants and animals, though regulatory proteins vary and plants have multiple isoforms responsive to environmental cues. Structural studies of bacterial and human HMGRs elucidated statin binding and catalysis, with statins binding with far higher affinity than HMG-CoA. Recent selective HMGR inhibitors in insects demonstrate feasibility of species-selective inhibition within this enzyme family, motivating exploration of plant-specific inhibitors and crop tolerance mechanisms.

- Herbicidal activity assays: Arabidopsis thaliana (Col-0) and Eragrostis tef were grown on peat under controlled growth-room conditions (22 °C, 16:8 h light:dark, 60% RH). Pre-emergence (day 0) and post-emergence (days 4 and 7) treatments were applied on soil with eight statins (rosuvastatin, pravastatin, simvastatin, mevastatin, lovastatin, fluvastatin, atorvastatin, pitavastatin). Final spray solutions contained 2% DMSO and 0.02% wetting agent (Brushwet). Plants were imaged on day 16. Growth inhibition was quantified by ImageJ green-pixel analysis (Threshold Colour: hue 50–110, saturation 125–255, brightness 30–255), normalized to no-inhibitor controls. - Design and synthesis of atorvastatin analogues: Nine analogues (1–9) modifying the isopropyl group on the central pyrrole of atorvastatin were synthesized (Supplementary Method 1) to exploit a wider AtHMG1 pocket and introduce potential hydrogen-bond donors to compensate for altered Glu265 interactions. Compounds were tested for herbicidal activity on soil-grown A. thaliana (pre- and post-emergence) and for in vitro selectivity. - Protein expression and purification: The extracellular/catalytic domain of AtHMG1 (residues 121–592) and human HMGCR (HsHMGCR, residues 441–888) were cloned with N-terminal His-tags, expressed in E. coli (T7 SHuffle Express with pREP4), and purified by Ni-NTA affinity and size-exclusion chromatography in HEPES/NaCl/DTT buffers. Purity assessed by SDS-PAGE and concentration by spectrophotometry. - In vitro HMGR assays: Enzymatic activity monitored via NADPH oxidation at 340 nm with HMG-CoA as substrate in HEPES/NaCl/DTT buffer with 2% DMSO at 37 °C. Michaelis-Menten kinetics (Km, kcat) determined by nonlinear regression. Inhibitor potency assessed by preincubating enzyme with compounds (typically 500 nM) and generating IC50 curves using normalized response models. Selectivity tested for AtHMG1 versus HsHMGCR using NADPH-depletion assays. - Crystallography: The core domain of AtHMG1 (residues 121–576) was crystallized by sitting-drop vapor diffusion in ammonium sulfate/HEPES/poly(acrylic acid) conditions with additive screens. Apo and pitavastatin-bound complexes were obtained; crystals were cryoprotected with 25% glycerol and data collected at 100 K on the Australian Synchrotron MX2 beamline. Data processed with XDS and AIMLESS; structures solved by molecular replacement (PHASER) using a model based on HsHMGCR; manual building/refinement with Coot and REFMAC5; validation with MolProbity. PDB accession codes: 7ULI (apo) and 8ECG (pitavastatin complex). - Engineering and assessment of tolerance trait: A mutation (L558T) identified from fungal statin biosynthetic cluster HMGR sequences (e.g., Aspergillus terreus lurA) was introduced into AtHMG1 and tested in vitro for resistance to multiple statins and catalytic parameters. Full-length AtHMG1 and AtHMG1-L558T were overexpressed in A. thaliana under the CaMV 35S promoter via Agrobacterium floral dip. T2 lines were selected on hygromycin, then assayed on microplates for dose-response to hygromycin and rosuvastatin (0.16–5120 µM). Growth quantified via green-pixel analysis and IC50 values derived by nonlinear regression. - Statistics: For selected comparisons, two-tailed paired t-tests and one-way ANOVA with Dunnett’s multiple-comparison correction were applied as reported; data presented as mean ± s.d. or mean ± 95% CI.

- Statin herbicidal activity: All tested statins inhibited plant growth, with stronger effects post-emergence and greater activity against the dicot A. thaliana than the monocot E. tef. Rosuvastatin was most potent, lethal to A. thaliana at ~15 µM without formulation beyond a wetting agent; formulated glyphosate (Roundup) was lethal at ~35 µM under the same conditions. - Plant HMGR structural features: Crystal structures of A. thaliana HMGR (AtHMG1) apo (1.9–2.1 Å) and pitavastatin-bound revealed a conserved fold but a uniquely arranged Lβ2–Lα1 loop and increased flexibility in the neighboring Nα4–Lβ1 loop (plant-conserved Pro at position 236 versus Val in human). This reorientation widens the active site pocket (solvent-accessible volume ~357 Å3 in AtHMG1 vs ~314 Å3 in HsHMGCR) and shifts Glu265 such that it cannot hydrogen bond to the O5-hydroxyl of the statin HMG moiety, altering the binding environment. - Species-selective inhibitor design: Atorvastatin analogues (1–9) modifying the isopropyl substituent were synthesized. Compound 7 exhibited >20-fold selectivity for plant over human HMGR in vitro: AtHMG1 IC50 = 32 nM ± 12 nM; HsHMGCR IC50 = 890 nM ± 143 nM, while retaining herbicidal activity. Compounds 4 and 7 showed preferential inhibition of AtHMG1 at 500 nM in initial screens; longer side chains generally reduced activity. - Tolerance trait from fungal biosynthetic clusters: A Leu558Thr mutation (L558T) in AtHMG1, analogous to a substitution in Aspergillus terreus statin-biosynthetic HMGR (lurA), reduced statin sensitivity while retaining catalytic activity. In vitro, L558T conferred >20-fold resistance to rosuvastatin (WT IC50 53 nM ± 20 nM; L558T IC50 > 1000 nM). Catalytic parameters without inhibitors: WT Km 69 µM ± 19 µM, kcat 10.7 ± 1.0 s−1; L558T Km 24 µM ± 16 µM, kcat 2.4 ± 0.3 s−1. - In planta validation of tolerance: A. thaliana overexpressing 35S::AtHMG1-L558T were over sixfold more resistant to rosuvastatin than lines overexpressing WT AtHMG1 and >100-fold more resistant than WT plants. IC50 values: 35S::AtHMG1-L558T 300 µM ± 18 µM; 35S::AtHMG1 46 µM ± 5 µM; WT 3 µM ± 1 µM. 35S::AtHMG1-L558T lines were up to 16-fold less sensitive to rosuvastatin than 35S::AtHMG1 lines in susceptibility assays. Both transgenics showed similar hygromycin resistance, indicating comparable transgene expression.

The study validates HMGR as a viable herbicide mode-of-action by demonstrating that statins can act as potent herbicides and that plant HMGR possesses structural features enabling selective inhibition. The widened, uniquely configured active site in AtHMG1 disrupts a key hydrogen bond observed in human HMGR, creating an opportunity for species-selective inhibitor design. Rational modifications to the atorvastatin scaffold yielded analogues, notably compound 7, with strong preference for plant HMGR while retaining herbicidal activity, addressing concerns over potential mammalian off-target effects. Furthermore, leveraging insights from fungal biosynthetic self-resistance, introduction of the L558T mutation into plant HMGR conferred substantial statin resistance both in vitro and in planta, establishing a plausible crop tolerance trait to pair with HMGR-targeting herbicides. Collectively, these findings support HMGR as a tractable herbicide target and provide structural, biochemical, and genetic tools for selective herbicide development and trait deployment.

This work establishes HMGR as a credible herbicide target by integrating structural biology, chemical design, and plant genetics. The AtHMG1 crystal structures reveal a plant-conserved, wider active site that can be exploited for species-selective inhibition. Rationally designed atorvastatin analogues achieved >20-fold selectivity for plant over human HMGR while maintaining herbicidal activity. A single amino acid substitution (L558T), inspired by fungal biosynthetic self-resistance, confers significant statin tolerance in vitro and in transgenic Arabidopsis, demonstrating a potential crop tolerance trait. Future research should optimize plant-selective inhibitor potency and pharmacology, explore additional selectivity strategies (e.g., targeting the divergent N-terminal regulatory domain), assess the tolerance trait across crop species, refine trait expression and regulation, evaluate impacts on sterol metabolism and seed set, and characterize environmental and residue profiles of HMGR inhibitors.

- Species scope: Most biological validation was conducted in Arabidopsis thaliana, with limited testing in a monocot (Eragrostis tef) for herbicidal sensitivity; broader crop and weed species evaluations are needed. - Off-target and safety: While in vitro selectivity over human HMGR was demonstrated for certain analogues, comprehensive mammalian in vivo selectivity, toxicity, and pharmacokinetics were not assessed. - Trait performance: The L558T tolerance trait was validated in Arabidopsis under controlled conditions; effectiveness across diverse crops, agronomic environments, and against various HMGR inhibitors remains to be tested. - Plant physiology: Potential effects of HMGR overexpression/mutation on sterol homeostasis, lipid metabolism, development, and seed set were not fully characterized. - Environmental fate: Residue persistence, soil interactions, and non-target organism impacts of HMGR inhibitors were not addressed. - Structural generalizability: Structural insights are from AtHMG1; conservation of the widened pocket and loop conformations across crop HMGR isoforms requires further structural and functional validation.