A dual-target herbicidal inhibitor of lysine biosynthesis

  1. Emily RR Mackie
  2. Andrew S Barrow
  3. Rebecca M Christoff
  4. Belinda M Abbott
  5. Anthony R Gendall
  6. Tatiana P Soares da Costa  Is a corresponding author
  1. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Australia
  2. School of Agriculture, Food and Wine, Waite Research Institute, The University of Adelaide, Waite Campus, Australia
  3. Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Australia
  4. Australian Research Council Industrial Transformation Research Hub for Medicinal Agriculture, AgriBio, La Trobe University, Australia
  5. Department of Animal, Plant and Soil Sciences, La Trobe University, Australia

Abstract

Herbicides with novel modes of action are urgently needed to safeguard global agricultural industries against the damaging effects of herbicide-resistant weeds. We recently developed the first herbicidal inhibitors of lysine biosynthesis, which provided proof-of-concept for a promising novel herbicide target. In this study, we expanded upon our understanding of the mode of action of herbicidal lysine biosynthesis inhibitors. We previously postulated that these inhibitors may act as proherbicides. Here, we show this is not the case. We report an additional mode of action of these inhibitors, through their inhibition of a second lysine biosynthesis enzyme, and investigate the molecular determinants of inhibition. Furthermore, we extend our herbicidal activity analyses to include a weed species of global significance.

Editor's evaluation

This paper presents the highly interesting and novel finding that the investigated compound inhibits two targets in lysine synthesis. Further, the first enzyme has allosteric inhibition, while the second enzyme is a competitive inhibitor. The authors do a good job explaining why this is high interest for herbicide resistance management in a new compound. The authors demonstrated that the compound is not a pro-herbicide, and propose that the higher in vivo activity relative to in vitro activity is due to the simultaneous in vivo inhibition of two separate steps in lysine synthesis.

https://doi.org/10.7554/eLife.78235.sa0

Introduction

Effective herbicides are critical for sustainable agriculture. However, our current options are dwindling as the prevalence of herbicide-resistant weeds continues to rise (Hall et al., 2020). Weeds have now evolved resistance to 21 out of the 31 herbicide modes of action, yet there has been a lack of herbicides with new modes of action brought to market over the last 30 years (Duke and Dayan, 2022; Heap, 2022).

Despite the success of targeting amino acid biosynthesis enzymes for the development of herbicides (e.g. glyphosate and chlorsulfuron), the inhibition of plant lysine biosynthesis has never been explored commercially. Our group was the first to discover inhibitors of lysine biosynthesis with herbicidal activity (Soares da Costa et al., 2021). We showed that the most potent of these inhibitors, (Z)-2-(5-(4-methoxybenzylidene)-2,4-dioxothiazolidin-3-yl)acetic acid (MBDTA-2) (Figure 1A) targets lysine production by inhibiting dihydrodipicolinate synthase (DHDPS), the enzyme that catalyses the first and rate-limiting step in the pathway (Soares da Costa et al., 2018). Interestingly, we found that the mode of DHDPS inhibition by MBDTA-2 was through binding at a novel allosteric site distinct from the allosteric lysine-binding site (Figure 1B), which enables regulation of the enzyme (Hall et al., 2021).

Structure and mode of binding of (Z)-2-(5-(4-methoxybenzylidene)-2,4-dioxothiazolidin-3-yl)acetic acid (MBDTA-2).

(A) Chemical structure of MBDTA-2. (B) The AtDHDPS1 quaternary structure with MBDTA-2 (green sticks) bound within a novel allosteric pocket (PDB ID: 7MDS) (Soares da Costa et al., 2021).

In the previous study, we revealed that the in vitro potency of MBDTA-2 against recombinant Arabidopsis thaliana (At) DHDPS enzymes was similar to the activity against agar-grown A. thaliana (Soares da Costa et al., 2021). Usually, herbicides inhibit their enzyme targets with greater potency than they inhibit in vivo plant growth. This is because the amount of herbicide reaching the target site is less than the amount applied. We proposed that this unusual similarity between in vitro and in vivo activity may be due to MBDTA-2 acting as a proherbicide, which is modified in vivo to a form that is more active at the target site. Proherbicides have been reported in the literature such as diclofop-methyl, which is demethylated via ester hydrolysis in vivo to produce the more active compound diclofop . Whilst MBDTA-2 could not undergo the same process as it does not contain an ester, we postulated that a similar proherbicidal effect may be observed through the in vivo demethylation of the aryl methyl ether.

The present study sought to extend our understanding of the mode of action of our previously developed DHDPS inhibitors (Christoff et al., 2021; Soares da Costa et al., 2021). Specifically, we used biochemical enzyme kinetic assays to demonstrate that MBDTA-2 does not act as a proherbicide, and that the apparent similarity between in vitro and in vivo potency may instead be explained by this series of compounds having a novel, dual-target mode of action through inhibition of the second lysine biosynthesis enzyme in the pathway, dihydrodipicolinate reductase (DHDPR). Static docking and biochemical assays revealed that in contrast to the allosteric binding of these inhibitors to DHDPS, active site binding is responsible for their inhibition of DHDPR. Additionally, we extended our previous in vivo activity studies on A. thaliana to include one of the most agriculturally problematic weeds in the world, rigid ryegrass (Lolium rigidum) (Bajwa et al., 2021; Busi and Beckie, 2020).

Results

Inhibitory activity of a demethylated MBDTA analogue

Given that proherbicides are metabolised in vivo to produce compounds with greater potency at the target site, it can be assumed that the metabolised form will have greater activity than the proherbicidal form against the target in vitro. As such, to assess whether MBDTA-2 is a proherbicide that is demethylated in vivo, we measured the inhibitory activity of the demethylated analogue, (Z)-2-(5-(4-hydroxybenzylidene)-2,4-dioxothiazolidin-3-yl)acetic acid (HBDTA), against both recombinant A. thaliana DHDPS enzymes (Figure 2; Christoff et al., 2021). The dose–response curves yielded IC50 values for AtDHDPS1 and AtDHDPS2 of 100 ± 0.95 and 105 ± 1.04 µM, respectively (Figure 2). These values are slightly greater than those we reported for MBDTA-2 (IC50 (AtDHDPS1) = 63.3 ± 1.80 µM, IC50 (AtDHDPS2) = 64.0 ± 1.00 µM) (Soares da Costa et al., 2021). These data suggest, conversely to our hypothesis, that the activity of MBDTA-2 at the target site is not influenced by the retention or loss of the methyl group in vivo.

Structure and in vitro potency of (Z)-2-(5-(4-hydroxybenzylidene)-2,4-dioxothiazolidin-3-yl)acetic acid (HBDTA).

(A) Chemical structure of HBDTA. (B) Dose–response curves of HBDTA against recombinant AtDHDPS1 (⚬) and AtDHDPS2 (+) enzymes. Initial enzyme rate was normalised against the vehicle control to determine % activity remaining. Data were fitted to a nonlinear regression model (solid line), resulting in R2 values of 0.99.

Dual-target activity of MBDTA-2

Given that the similarity in the in vitro and in vivo activity of MBDTA-2 could not be explained by enhanced target site activity of the demethylated compound, we sought to explore other mechanisms that may explain this observation.

We investigated whether additional modes of action beyond the inhibition of the DHDPS enzyme may account for the increased in vivo potency relative to the in vitro activity against the target enzyme. It is well established that proteins catalysing sequential reactions within a metabolic pathway often have conserved binding site features (Hsu et al., 2013; Jensen, 1976; Jenwitheesuk et al., 2008; Zhang et al., 2009). As such, we hypothesised that DHDPS inhibitors may also have activity against the enzyme following DHDPS in the plant lysine biosynthesis pathway, DHDPR. The activity of both recombinant A. thaliana DHDPR enzymes was measured whilst titrating MBDTA-2 to determine the IC50 values of 6.92 ± 0.92 µM against AtDHDPR1 and 8.58 ± 1.19 µM against AtDHDPR2 (Figure 3A). Given that these results revealed a new target site for MBDTA-2, we assessed whether the compound could be proherbicidal with respect to DHDPR. To do so, we assessed the inhibitory activity of HBDTA against both AtDHDPR isoforms to determine the IC50 values of 169 ± 0.92 and 155 ± 0.90 µM for AtDHDPR1 and AtDHDPR2, respectively (Figure 3B). HBDTA is therefore >18-fold less potent at the DHDPR target site relative to MBDTA-2, suggesting that the MBDTA-2 methyl group is beneficial for DHDPR inhibition. As such, MBDTA-2 is unlikely to be a proherbicide. Moreover, these results demonstrate that MBDTA-2 is a multi-targeted inhibitor of two consecutive enzymes in the lysine biosynthesis pathway, AtDHDPS and AtDHDPR. This compound represents the first example of a dual-target inhibitor of the lysine biosynthesis pathway.

In vitro potency of (Z)-2-(5-(4-methoxybenzylidene)-2,4-dioxothiazolidin-3-yl)acetic acid (MBDTA-2) and (Z)-2-(5-(4-hydroxybenzylidene)-2,4-dioxothiazolidin-3-yl)acetic acid (HBDTA) against AtDHDPR.

Dose–response curves of (A) MBDTA-2 and (B) HBDTA against recombinant AtDHDPR1 (⚬) and AtDHDPR2 (+) enzymes. Initial enzyme rate was normalised against the vehicle control to determine % activity remaining. Data were fitted to a nonlinear regression model (solid line), resulting in R2 values of (A) 0.99 and 0.95 and (B) 0.99 and 0.99 for AtDHDPR1 and AtDHDPR2, respectively.

Mode of AtDHDPR inhibition by MBDTA-2

To investigate the molecular determinants of inhibition of AtDHDPR, we sought to co-crystallise the enzyme with MBDTA-2. Given that our attempts were unsuccessful, we employed a static docking approach using the published AtDHDPR2 crystal structure (Watkin et al., 2018). The resulting data suggested that MBDTA-2 binds in the active site with a binding affinity of −6.2 kcal mol−1 (Figure 4). The hydrophobic pocket occupied by MBDTA-2 overlaps with the probable NADPH cofactor-binding site, based on the crystal structure of cofactor-bound Escherichia coli DHDPR (Reddy et al., 1996; Scapin et al., 1997). The predicted MBDTA-2 orientation suggests its stabilisation by polar interactions between the heterocyclic ring and Thr122 and Gly120. Additionally, the MBDTA-2 acid is within hydrogen bonding proximity to Asp185. To validate the mechanism of inhibition of MBDTA-2 against AtDHDPR, further enzyme kinetic experiments were performed. The previous dose–response experiments were conducted according to standard practice in that substrate and cofactor were kept at limiting concentrations to ensure that inhibition may be measured regardless of the kinetic mechanism of inhibition. Subsequently, the activity of AtDHDPR was measured whilst titrating MBDTA-2, this time in the presence of excess amounts of substrate and nucleotide cofactor, that is at concentrations 10-fold above the respective KM values (Figure 4B). The IC50 values determined were 72.7 ± 1.07 and 69.5 ± 1.06 µM for AtDHDPR1 and AtDHDPR2, respectively, which are 10- and 8-fold greater than those determined for AtDHDPR1 and AtDHDPR2 when substrate and cofactor were limiting. This apparent reduction in potency indicates that MBDTA-2 is a competitive inhibitor, and therefore likely binds at the AtDHDPR active site as suggested by the docking results. Interestingly, this contrasts with the allosteric mode of inhibition of MBDTA-2 against AtDHDPS.

Mode of AtDHDPR2 inhibition by (Z)-2-(5-(4-methoxybenzylidene)-2,4-dioxothiazolidin-3-yl)acetic acid (MBDTA-2).

(A) The predicted MBDTA-2 (green)-binding site resulting from static docking with AtDHDPR2 (PDB ID: 5UA0) overlaps with the probable NADPH cofactor-binding site (cyan, left panel). Hydrophobicity of the predicted binding pocket (right panel) is represented by white-red shading indicating hydrophilic–hydrophobic residues. (B) Dose–response curves of MBDTA-2 against AtDHDPR1 (⚬) and AtDHDPR2 (+) enzymes in the presence of saturating concentrations of substrate and cofactor. Data were fitted to a nonlinear regression model (solid line), resulting in R2 values of 0.97 and 0.98 for AtDHDPR1 and AtDHDPR2, respectively.

Herbicidal activity of MBDTA-2 against weeds

Previously, we showed that the MBDTA-2 compound has herbicidal activity against the model plant A. thaliana and is therefore the first example of a herbicidal lysine biosynthesis inhibitor. To further assess the potential of inhibiting plant lysine biosynthesis enzymes for the development of herbicides, the efficacy of MBDTA-2 against the economically significant invasive weed species rigid ryegrass L. rigidum was investigated. Treatment of L. rigidum with 1200 mg l−1 of MBDTA-2 resulted in inhibition of plant germination and growth, corresponding to a significant reduction in shoot fresh and dry weight and a significant reduction in root dry weight (Figure 5). Specifically, we observed ~4- and ~5-fold reductions in shoot fresh and dry weight, respectively, and a ~2-fold reduction in root dry weight (Figure 5). These results further exemplify the potential of lysine biosynthesis inhibitors for development as herbicide candidates.

Inhibition of Lolium rigidum germination and growth by (Z)-2-(5-(4-methoxybenzylidene)-2,4-dioxothiazolidin-3-yl)acetic acid (MBDTA-2).

(A) Day growth of L. rigidum treated with three pre-emergence treatments of vehicle control (2% (vol/vol) DMSO, 0.01% Agral), or 1200 mg l−1 of MBDTA-2, or 1200 mg l−1 of chlorsulfuron. Treatments were given by pipetting 2.0 ml per pot directly onto seeds. (B) Fresh weight of L. rigidum shoots and roots following treatment of plants with vehicle control (dots) or MBDTA-2 (lines). Shoots, p = 0.00002, roots, p = 0.05233, unpaired Student’s two-tailed t-test. (C) Dry weight of L. rigidum shoots and roots following treatment of plants with vehicle control (dots) or MBDTA-2 (lines). Shoots, p = 0.00088, roots, p = 0.00374, unpaired Student’s two-tailed t-test. Data were normalised against the vehicle control. Data represent mean ± standard error of the mean (SEM) (N = 3). **p < 0.01, ***p < 0.001, ****p < 0.0001.

Discussion

Herbicides with new modes of action are urgently needed to combat the rise in herbicide-resistant weed species, which pose a global threat to agriculture. In our previous study, we described the development of the first herbicidal lysine biosynthesis inhibitors, providing proof-of-concept for a novel herbicide mode of action. Whilst we previously explored the molecular mode of action of these inhibitors at their target site, namely the DHDPS enzyme, an explanation for their unusual similarity in in vitro and in vivo potency remained to be delineated. Specifically, we hypothesised that they may be acting as proherbicides, through demethylation of their aryl methyl ethers. While this mechanism of proherbicide conversion has not been reported to date, the demethylation of aryl methyl ethers on prodrugs, such as codeine, is well established (Dayer et al., 1988; Kirchheiner et al., 2007). Nevertheless, our finding that the demethylated analogue of the MBDTA-2 aryl methyl ether did not positively impact activity against the target enzyme DHDPS demonstrated that these compounds are not proherbicides.

Given that we could not attribute the similarity between the in vitro and in vivo potency of our compounds to their modification to a more active form in vivo, we postulated that we may have previously failed to capture the totality of their target site effects. Our discovery that MBDTA-2 is an inhibitor of not only DHDPS, but also of the subsequent enzyme in the lysine biosynthesis pathway DHDPR, supported this hypothesis. Moreover, the ~8-fold greater potency of MBDTA-2 against DHDPR than DHDPS reveals that the in vitro potency is actually ~6-fold greater than the in vivo potency. Furthermore, the low-micromolar potency of MBDTA-2 at the DHDPR target site is comparable to the potency of glyphosate, the most successful commercial herbicide active ingredient, at its enzyme target 5-enolpyruvylshikimate-3-phosphate synthase (Mao et al., 2016; Sammons et al., 2018). The phenomenon of inhibitors having dual-target activity against consecutive enzymes in metabolic pathways has sometimes been attributed to conserved active site features (Hsu et al., 2013; Toulouse et al., 2020). However, there are also many examples of inhibitors of multiple targets from distinct pathways, which have been identified regardless of binding site similarities (Allen et al., 2015; Hashmi et al., 2021; Wang et al., 2019). Nevertheless, to our knowledge, this is the first time a dual-target inhibitor has been shown to have allosteric and orthosteric inhibitory activity at two different enzymes. The advantages of multi-target inhibitors over single-target inhibitors have been well reported for the development of novel drugs and fungicides (Allen et al., 2015; Gullino et al., 2010; Oldfield and Feng, 2014). Such advantages include a reduced susceptibility to the generation of resistance, which is also a highly desirable property in herbicide development (Gressel, 2020; Hall et al., 2020). Indeed, there has been a focus on the use of herbicide mixtures inhibiting multiple molecular targets in attempts to reduce the generation of resistance to existing herbicides (Gressel, 2020). Despite the recognition of the potential of inhibiting multiple targets for the reduction of target site resistance generation, little work has been done on the development of new herbicides that do so (Fu et al., 2019; Giberti et al., 2017). Dual-target compounds such as MBDTA-2 are therefore promising candidates for progressing the herbicide development field beyond the ‘one target-one herbicide’ approach.

For the future development of dual-target herbicides, the previously published DHDPS co-crystal structure and the DHDPR-binding model presented here could be used for the rational design of new MBDTA-2 analogues with increased target site activity (Soares da Costa et al., 2021). Such rational design efforts could also be guided by additional kinetic assays in the presence of sub-saturating amounts of MBDTA-2. Examining any changes in the KM values for the DHDPR substrate and cofactor under these conditions may provide further insights into MDBTA-2 interactions at the binding site. Additionally, exploring the structure–activity relationship of MBDTA-2 through the screening of analogues against whole plants may also provide insights into modifications that are advantageous to in vivo potency.

Although these methods offer opportunities to optimise inhibitor potency, formulation also has a substantial impact on the performance of herbicide active ingredients and is therefore an important avenue to pursue for potency maximisation. Whilst the in vivo assays conducted here provide proof-of-concept for the potential of dual-target lysine biosynthesis inhibitors as herbicide active ingredients, controlled-dosage spraying experiments with the formulated compound will be pertinent to assess the application rate required for herbicidal efficacy in comparison to commercial herbicides. Furthermore, metabolomics experiments to quantify changes in lysine and other aspartate-derived amino acid levels in response to treatment with dual-target lysine biosynthesis inhibitors would be of interest to validate the mode of action at the target site, as well as elucidate whether pathway deregulation contributes to the in vivo activity.

The development of new herbicides that are effective against L. rigidum, particularly those with a reduced propensity to generate resistance, is of the highest priority given the economic impact of this species. An overreliance on a small number of herbicide modes of action has culminated in the widespread evolution of multiple resistance mechanisms in L. rigidum (Bajwa et al., 2021; Owen et al., 2014). In Australia alone, herbicide-resistant L. rigidum invades 8 million hectares of cropping land, resulting in revenue losses of AUD$93 million annually (Llewellyn et al., 2016). Our finding that MBDTA-2 can significantly reduce L. rigidum germination and growth further illustrates the potential utility of lysine biosynthesis inhibitors in combatting the global herbicide resistance crisis.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Arabidopsis thaliana)DHDPS1TAIRAtG60880
Gene (Arabidopsis thaliana)DHDPS2TAIRAtG45440
Gene (Arabidopsis thaliana)DHDPR1TAIRAt2G44040
Gene (Arabidopsis thaliana)DHDPR2TAIRAt3G59890
Software, algorithmPyRXSource ForgeVersion 0.8

Chemical synthesis

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Compounds were synthesised as previously described (Christoff et al., 2021; Perugini et al., 2018).

Protein expression and purification

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Recombinant AtDHDPS1, AtDHDPS2, AtDHDPR1, and AtDHDPR2 proteins were produced as previously described (Mackie et al., 2022; Soares da Costa et al., 2021).

Enzyme inhibition assays

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DHDPS enzyme activity was measured using methods previously described (Soares da Costa et al., 2021). DHDPR enzyme activity was measured using methods previously described (Mackie et al., 2022). Briefly, reaction mixtures were incubated at 30℃ for 12 min before a second 60-s incubation following the addition of excess E. coli DHDPS (51 µg ml−1) for generation of the DHDP substrate. The relevant DHDPR isoform (2.6 µg ml−1) was added to initiate the reaction, and substrate turnover measured spectrophotometrically at 340 nm via the associated oxidation of the cofactor NADPH. Experiments were performed in technical triplicates.

Docking

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The AtDHDPR2 crystal structure was retrieved from the Protein Data Bank and hydrogens added using AutoDock Tools. Three-dimensional MBDTA-2 was docked with AtDHDPR2 using an unlimited search space in the PyRX interface using AutoDock Vina with default parameters. The resulting ligand poses were visualised in PyMol.

Herbicidal activity analyses

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The herbicidal efficacy of MBDTA-2 against L. rigidum was assessed using methods similar to those reported previously (Mackie et al., 2022). Pre-wet seed-raising soil (pH 5.5) (Biogro, Dandenong South, VIC, Australia) supplemented with 0.22% (wt/wt) Nutricote N12 Micro 140 day-controlled release fertiliser (Yates, Sydney, NSW, Australia) was used. Ten seeds were sown at a depth of 0.5 cm into pots of pre-wet soil, following stratification at 4℃ for 21 days in the dark. Compounds dissolved in DMSO were diluted to working concentrations in H2O containing 0.01% (vol/vol) Agral (Syngenta, North Ryde, NSW, Australia) to a final DMSO concentration of 2% (vol/vol). Treatments were given by pipetting 2.0 ml of MBDTA-2, vehicle control or positive control (chlorosulfuron PESTANAL [Sigma-Aldrich, North Ryde, NSW, Australia]) directly onto seeds upon sowing and on each of the subsequent 2 days. Plants were grown in a chamber at 22℃ under a 16 hr light (100 µmol m−2 s−1)/8 hr dark schedule for 14 days before photos were taken. Roots and shoots were separated prior to drying at 70℃ for 72 hr. Experiments were performed in biological triplicates.

Data availability

Figure 2 - Source Data 1, Figure 3 - Source Data 1, Figure 4 - Source Data 1 and Figure 5 - Source Data 1 contain the numerical data used to generate the figures.

References

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    1. Llewellyn R
    2. Ronning D
    3. Ouzman J
    4. Walker S
    5. Mayfield A
    6. Clarke M
    (2016)
    Impact of Weeds on Australian Grain Production: the cost of weeds to Australian grain growers and the adoption of weed management and tillage practices
    Report for GRDC, Grains Research and Development Corporation and the Commonwealth Scientific and Industrial Research Organisation.
  2. Book
    1. Perugini MA
    2. Abbott B
    3. Costa T
    (2018)
    Heterocyclic Inhibitors of Lysine Biosynthesis via the Diaminopimelate Pathway
    DHDPS1.

Decision letter

  1. Todd Gaines
    Reviewing Editor; Colorado State University, United States
  2. Detlef Weigel
    Senior Editor; Max Planck Institute for Biology Tübingen, Germany
  3. Todd Gaines
    Reviewer; Colorado State University, United States
  4. Paul Schmitzer
    Reviewer; Corteva Agriscience, United States

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "A dual-target herbicidal inhibitor of lysine biosynthesis" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Todd Gaines as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Detlef Weigel as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Paul Schmitzer (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Please provide the chlorsulfuron positive control data for the Lolium rigidum bioassay figure. Alter the claims for biological activity on Lolium rigidum and remove the extrapolated application rate, or conduct a new bioassay with methods suggested in the reviews.

2) The question arose in the reviews as to whether the compound is a pro-herbicide with respect to DHPDR. Please alter the discussion to raise this question, or consider conducting the experiment to add the demethylated compound to the DHPDR inhibition assay to test its activity.

Reviewer #1 (Recommendations for the authors):

Excellent paper. The authors could include a reference to work in fungicides to provide further evidence that multi-site inhibitors are lower risk for resistance evolution than single-site inhibitors. The bioassay method used for Lolium rigidum is unusual. The authors pipetted 2 mL of a 1200 mg/L solution directly onto seeds when sowing. Alternatives that could be used would include soaking seeds for 24 hour in herbicide solution; using an agarose based assay; spraying post-emergence on emerged seedlings; or spraying directly onto seeds or seedlings in soil. The authors report a calculated application equivalent of 48 kg/ha. It is not clear how this extrapolated number is obtained, nor is it realistic. No pesticide could be applied at such a high rate (the compound would be coated like salt onto everything in the field), and the sheer volume of water application required to achieve that would also represent drenching a field. I suggest that the authors remove this extrapolated calculation, and perhaps instead discuss the relative activity/toxicity of their compound in comparison to other known herbicides based on in vitro inhibition of the two enzymes. I don't think that more experiments are needed for this paper, but future work could conduct bioassays using methods that would provide more accurate ways to extrapolate to potential field use rates. Future work could also conduct biochemical measurements to ask whether lysine is reduced in Lolium rigidum following treatment with this herbicide, supporting the prediction that the mode of action is two-site inhibition of lysine synthesis. I also suggest that the authors provide more methods in the figure caption for Figure 5 so that it stand alone. Currently the caption only states the application concentration of 1200 mg/L, but it does not provide the application volume of 2 mL pipetted onto each seed, so in the current presentation the reader must examine the methods to understand the total dose applied to the plants. Suggest adding the 2 mL pipetting onto each seed to the caption.

Reviewer #2 (Recommendations for the authors):

The paper was a joy to read as it represents many processes in the design of new herbicides. I don't like creating additional work but through my review of the paper I kept wondering if the analogs presented in your previous publications (FBDTA and MBDTA-1), as well as any other available analogs, could build the knowledge of the inhibition of DHDPR and show a limited SAR. This may be a topic for a future publication so please disregard it if not appropriate for this paper.

Reviewer #3 (Recommendations for the authors):

This is a well-written description of experiments to further determine the mode of action of novel lysine synthesis inhibitor. The expansion of the mode of action to two consecutive enzymes of the lysine synthesis pathway is a surprising result, especially with different binding modes for the two enzymes. Again, the in vitro activity MBDTA-2 is rather poor for an effective herbicide. Perhaps inhibition of two enzymes makes up for the poor activity on each of the enzymes. Was the demethylated form of the compound tested in an in vitro assay of AtDHDPR1 and AtDHDP2? This must be done. If HBDTA is much more active than MBDTA-2 on DHDPR, the results would make more sense to me.

The bioassay method is very weak, as it is impossible to determine whether MBDTA-2 is an effective herbicide from these results. Herbicides are not pipetted onto weed seeds in the field. MBDTA-2 was applied three times, on three consecutive days. In the field, a herbicide is applied once. I suggest that the compounds be applied once to the soil containing weeds seeds and watered into soil, as soil applied herbicide are used, or applied to foliage as a foliar spray with an adjuvant. I would try both methods, as some herbicides that are excellent soil-applied herbicides are poor foliar herbicides and vice versa. This way, you can compare the efficacy in g/ha with the positive control. Finally, herbicides are expected to kill weeds. Figure 5 shows only growth stunting. Where are the chlorsulfuron results?

https://doi.org/10.7554/eLife.78235.sa1

Author response

Essential revisions:

1) Please provide the chlorsulfuron positive control data for the Lolium rigidum bioassay figure. Alter the claims for biological activity on Lolium rigidum and remove the extrapolated application rate, or conduct a new bioassay with methods suggested in the reviews.

The chlorsulfuron positive control data has been added to Figure 5, panel A. The Figure 5 legend has been updated accordingly with the addition of ‘or 1200 mg·L-1 of chlorsulfuron.’ The claims for biological activity on Lolium rigidum have been altered as follows: Line 216: ‘as we have demonstrated that MBDTA-2 possesses herbicidal activity against one of the most problematic weed species to global agriculture’ deleted; Line 81:

“we successfully extended our previous herbicidal activity studies’ changed to ‘we extended our previous in vivo activity studies’; Line 271: ‘herbicidal’ deleted. Line 211: the extrapolated application rate has been removed by deleting ‘(equivalent to 48 kg·ha-1)”.

2) The question arose in the reviews as to whether the compound is a pro-herbicide with respect to DHPDR. Please alter the discussion to raise this question, or consider conducting the experiment to add the demethylated compound to the DHPDR inhibition assay to test its activity.

The inhibition of DHDPR by the demethylated compound has now been measured and added to Figure 3B and described in the Results section starting at Line 134: Given that these results revealed a new target site for MBDTA-2, we assessed whether the compound could be proherbicidal with respect to DHDPR. To do so, we assessed the inhibitory activity of HBDTA against both AtDHDPR isoforms to determine the IC50 values of 169 ± 0.92 µM and 155.3 ± 0.90 µM for AtDHDPR1 and AtDHDPR2, respectively (Figure 3B). HBDTA is therefore >18-fold less potent at the DHDPR target site relative to MBDTA-2, suggesting that the MBDTA-2 methyl group is beneficial for DHDPR inhibition. As such, MBDTA-2 is unlikely to be a proherbicide.

Reviewer #1 (Recommendations for the authors):

Excellent paper. The authors could include a reference to work in fungicides to provide further evidence that multi-site inhibitors are lower risk for resistance evolution than single-site inhibitors.

We thank the reviewer for the feedback. Line 263: ‘and fungicides’ and Gullino et al., 2010 reference added to provide further evidence that multi-site inhibitors are lower risk for resistance evolution than single-site inhibitors. Line 262: ‘dual-target’ changed to ‘multi-target’ to encompass the use of multi-site fungicides.

The bioassay method used for Lolium rigidum is unusual. The authors pipetted 2 mL of a 1200 mg/L solution directly onto seeds when sowing. Alternatives that could be used would include soaking seeds for 24 hour in herbicide solution; using an agarose based assay; spraying post-emergence on emerged seedlings; or spraying directly onto seeds or seedlings in soil.

We thank the reviewer for the insightful comment. Direct pipetting onto seeds on soil, as employed in this study, is similar to soaking the seeds prior to planting, but is more relevant to herbicide use in the field. Pipetting directly onto seeds also functions similarly to spraying directly onto the seeds, however it provides a more consistent and controlled dosage as over-spray can occur with spray application. Experiments with Arabidopsis indicated that these inhibitors are more effective when applied pregermination. The value of further bioassays, as highlighted by the reviewer, has been acknowledged with the addition of the following at Line 287:

“Whilst the in vivo assays conducted here provide proof-of concept for the potential of dual-target lysine biosynthesis inhibitors as herbicide active ingredients, controlled-dosage spraying experiments with the formulated compound will be pertinent to assess the application rate required for herbicidal efficacy in comparison to commercial herbicides.”

The authors report a calculated application equivalent of 48 kg/ha. It is not clear how this extrapolated number is obtained, nor is it realistic. No pesticide could be applied at such a high rate (the compound would be coated like salt onto everything in the field), and the sheer volume of water application required to achieve that would also represent drenching a field. I suggest that the authors remove this extrapolated calculation, and perhaps instead discuss the relative activity/toxicity of their compound in comparison to other known herbicides based on in vitro inhibition of the two enzymes.

We thank the reviewer for the comment. Line 211: The extrapolated application rate has been removed by deleting ‘(equivalent to 48 kg·ha-1)’. The relative in vitro activity of our compound in comparison to known herbicides is now discussed with the following addition at Line 252:

“Furthermore, the low-μM potency of MBDTA-2 at the DHDPR target site is comparable to the potency of glyphosate, a commercial herbicide active ingredient, at its enzyme target 5-enolpyruvylshikimate-3phosphate synthase (Mao et al., 2016; Sammons et al., 2018)”.

I don't think that more experiments are needed for this paper, but future work could conduct bioassays using methods that would provide more accurate ways to extrapolate to potential field use rates. Future work could also conduct biochemical measurements to ask whether lysine is reduced in Lolium rigidum following treatment with this herbicide, supporting the prediction that the mode of action is two-site inhibition of lysine synthesis.

We thank the reviewer for their feedback. The raised points about future work have now been added to the Discussion as follows: Line 287: the value of further bioassays has been acknowledged with the addition of ‘Whilst the in vivo assays conducted here provide proof-of-concept for the potential of dual-target lysine biosynthesis inhibitors as herbicide active ingredients, controlled-dosage spraying experiments with the formulated compound will be pertinent to assess the application rate required for herbicidal efficacy in comparison to commercial herbicides.’ Line 291:

“quantification of lysine levels following treatment with these compounds has been added as a future direction by adding ‘Furthermore, metabolomics experiments to quantify changes in lysine and other aspartate-derived amino acid levels in response to treatment with dual-target lysine biosynthesis inhibitors would be of interest to validate the mode of action at the target site, as well as elucidate whether pathway deregulation contributes to the in vivo activity.”

I also suggest that the authors provide more methods in the figure caption for Figure 5 so that it stand alone. Currently the caption only states the application concentration of 1200 mg/L, but it does not provide the application volume of 2 mL pipetted onto each seed, so in the current presentation the reader must examine the methods to understand the total dose applied to the plants. Suggest adding the 2 mL pipetting onto each seed to the caption.

We thank the reviewer for the comment. Line 221: ‘Treatments were given by pipetting 2.0 mL per pot directly onto seeds’ added to Figure 5 legend.

Reviewer #2 (Recommendations for the authors):

The paper was a joy to read as it represents many processes in the design of new herbicides. I don't like creating additional work but through my review of the paper I kept wondering if the analogs presented in your previous publications (FBDTA and MBDTA-1), as well as any other available analogs, could build the knowledge of the inhibition of DHDPR and show a limited SAR. This may be a topic for a future publication so please disregard it if not appropriate for this paper.

Indeed, future work will involve the synthesis of a comprehensive series of analogues to understand the structure-activity relationship of dual-target DHDPS/DHDPR inhibitors. This has been added to the Discussion at Line 281 as follows:

“Additionally, exploring the structure-activity relationship of MBDTA-2 through the screening of analogues against whole plants may also provide insights into modifications that are advantageous to in vivo potency.”

Reviewer #3 (Recommendations for the authors):

This is a well-written description of experiments to further determine the mode of action of novel lysine synthesis inhibitor. The expansion of the mode of action to two consecutive enzymes of the lysine synthesis pathway is a surprising result, especially with different binding modes for the two enzymes. Again, the in vitro activity MBDTA-2 is rather poor for an effective herbicide. Perhaps inhibition of two enzymes makes up for the poor activity on each of the enzymes. Was the demethylated form of the compound tested in an in vitro assay of AtDHDPR1 and AtDHDP2? This must be done. If HBDTA is much more active than MBDTA-2 on DHDPR, the results would make more sense to me.

We thank the reviewer for raising an interesting point. The IC50 values of MBDTA-2 against AtDHDPR1 and AtDHDPR2 are 6.9 and 8.6 µM, respectively. These values are similar to the IC50 of the herbicide active ingredient glyphosate against its enzyme target. The potency of MBDTA-2 relative to current herbicides is now discussed with the addition of Line 252: ‘Furthermore, the low-μM potency of MBDTA-2 at the DHDPR target site is comparable to the potency of glyphosate, the most successful commercial herbicide active ingredient, at its enzyme target 5-enolpyruvylshikimate-3-phosphate synthase.’ We have now tested the demethylated compound against AtDHDPR1 and AtDHDPR2 and included the results in the revised manuscript as described in Response 2 above.

The bioassay method is very weak, as it is impossible to determine whether MBDTA-2 is an effective herbicide from these results. Herbicides are not pipetted onto weed seeds in the field. MBDTA-2 was applied three times, on three consecutive days. In the field, a herbicide is applied once. I suggest that the compounds be applied once to the soil containing weeds seeds and watered into soil, as soil applied herbicide are used, or applied to foliage as a foliar spray with an adjuvant. I would try both methods, as some herbicides that are excellent soil-applied herbicides are poor foliar herbicides and vice versa. This way, you can compare the efficacy in g/ha with the positive control. Finally, herbicides are expected to kill weeds. Figure 5 shows only growth stunting.

The bioassays conducted in this study provide proof-of-concept work for the potential utility of dual-target lysine biosynthesis inhibitors for development as herbicides. Future work will focus on optimisation of the inhibitors presented in this study in order to develop them as herbicides. This point is now included in the Discussion at line 281 as follows: ‘Additionally, exploring the structure activity relationship of MBDTA-2 through the screening of analogues against whole plants may also provide insights into modifications that are advantageous to in vivo potency.’ Future work will also optimise the formulation of these compounds to improve efficacy and this is included in the Discussion at Line 285 as follows: ‘Although these methods offer opportunities to optimise inhibitor potency, formulation also has a substantial impact on the performance of herbicide active ingredients and is therefore an important avenue to pursue for potency maximisation (Pacanoski, 2015). Whilst the in vivo assays conducted here provide proof-of-concept for the potential of dual-target lysine biosynthesis inhibitors as herbicide active ingredients, controlled-dosage spraying experiments with the formulated compound will be pertinent to assess the application rate required for herbicidal efficacy in comparison to commercial herbicides.’ The compounds presented demonstrate that they inhibit the germination and growth of a weed species and therefore could potentially be developed into herbicides with future optimisation. Accordingly, the claims for biological activity on Lolium rigidum have been altered as discussed in Response 1 above.

Where are the chlorsulfuron results?

The chlorsulfuron positive control data has been added to Figure 5, panel A. Line 220:

“The Figure 5 legend has been updated accordingly with the addition of ‘or 1200 mg·L-1 of chlorsulfuron.”

https://doi.org/10.7554/eLife.78235.sa2

Article and author information

Author details

  1. Emily RR Mackie

    1. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia
    2. School of Agriculture, Food and Wine, Waite Research Institute, The University of Adelaide, Waite Campus, Glen Osmond, Australia
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0851-5741
  2. Andrew S Barrow

    Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – review and editing
    Contributed equally with
    Rebecca M Christoff
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3661-5173
  3. Rebecca M Christoff

    Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – review and editing
    Contributed equally with
    Andrew S Barrow
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2111-3218
  4. Belinda M Abbott

    Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia
    Contribution
    Methodology, Resources, Supervision, Writing – review and editing
    Competing interests
    B.M.A. are listed as inventors on a patent pertaining to inhibitors described in the manuscript. Patent Title: Heterocyclic inhibitors of lysine biosynthesis via the diaminopimelate pathway; International patent (PCT) No.: WO2018187845A1; Granted: 18/10/2018
  5. Anthony R Gendall

    1. Australian Research Council Industrial Transformation Research Hub for Medicinal Agriculture, AgriBio, La Trobe University, Bundoora, Australia
    2. Department of Animal, Plant and Soil Sciences, La Trobe University, Bundoora, Australia
    Contribution
    Methodology, Resources, Supervision, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2255-3939
  6. Tatiana P Soares da Costa

    1. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia
    2. School of Agriculture, Food and Wine, Waite Research Institute, The University of Adelaide, Waite Campus, Glen Osmond, Australia
    Contribution
    Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing
    For correspondence
    tatiana.soaresdacosta@adelaide.edu.au
    Competing interests
    T.P.S.C. is listed as inventors on a patent pertaining to inhibitors described in the manuscript. Patent Title: Heterocyclic inhibitors of lysine biosynthesis via the diaminopimelate pathway; International patent (PCT) No.: WO2018187845A1; Granted: 18/10/2018
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6275-7485

Funding

Australian Research Council (DE190100806)

  • Tatiana P Soares da Costa

Australian Research Council (IH180100006)

  • Anthony R Gendall

Grains Research and Development Corporation (9176977)

  • Emily RR Mackie

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

T.P.S.C. acknowledges the Australian Research Council for funding support through a DECRA (DE190100806) and The University of Adelaide for a Future Making Fellowship. Work in A.R.G.’s laboratory is supported by the Australian Research Council Research Hub for Medicinal Agriculture (IH180100006). E.R.R.M. acknowledges the Grains Research and Development Corporation (9176977) for support through a PhD scholarship and operational funding. E.R.R.M. and R.M.C. are recipients of Australian Government Research Training Program Scholarships. R.M.C. is also the recipient of a LIMS Write-Up Award.

Senior Editor

  1. Detlef Weigel, Max Planck Institute for Biology Tübingen, Germany

Reviewing Editor

  1. Todd Gaines, Colorado State University, United States

Reviewers

  1. Todd Gaines, Colorado State University, United States
  2. Paul Schmitzer, Corteva Agriscience, United States

Publication history

  1. Preprint posted: March 4, 2022 (view preprint)
  2. Received: March 4, 2022
  3. Accepted: June 10, 2022
  4. Version of Record published: June 20, 2022 (version 1)

Copyright

© 2022, Mackie et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Emily RR Mackie
  2. Andrew S Barrow
  3. Rebecca M Christoff
  4. Belinda M Abbott
  5. Anthony R Gendall
  6. Tatiana P Soares da Costa
(2022)
A dual-target herbicidal inhibitor of lysine biosynthesis
eLife 11:e78235.
https://doi.org/10.7554/eLife.78235
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