A dual-target herbicidal inhibitor of lysine biosynthesis
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Curated by eLife
Evaluation Summary:
This excellent paper presents the highly interesting finding of a compound that inhibits two targets in lysine synthesis. Further, the mechanisms for the first enzyme is allosteric inhibition, while for the second enzyme the compound is a competitive inhibitor. The authors nicely explain why this is of significant interest for herbicide resistance management with a new compound. The authors convincingly demonstrate that the compound is not a pro-herbicide, and instead that the higher in vivo relative to in vitro activity is due to the additional inhibition of the second step in lysine synthesis. The work is of interest to those studying enzymology, herbicide action and evolution of herbicide resistance.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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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.
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Author Response
Reviewer #2 (Public Review):
Use of binding models is common in rational drug design so it is understandable for the authors to pursue a binding model for MBDTA-2. It is difficult to assess the utility of the docking model for SAR development without a better understanding of how many docking conformation predictions the software provided and/or a measure of the docking score.
We thank the reviewer for the comment. The predicted binding affinity is now described as follows: Line 158: ‘with a binding affinity of -6.2 kcal·mol-1’.
The measure of the apparent Km values for the substrate and co-factor with MBDTA-2 at the sub-saturated IC50 values (6.92 and 8.58 micromolar) would help better understand the potential interaction between MBDTA-2 and the substrate and co-factor at the binding site.
The point raised by the …
Author Response
Reviewer #2 (Public Review):
Use of binding models is common in rational drug design so it is understandable for the authors to pursue a binding model for MBDTA-2. It is difficult to assess the utility of the docking model for SAR development without a better understanding of how many docking conformation predictions the software provided and/or a measure of the docking score.
We thank the reviewer for the comment. The predicted binding affinity is now described as follows: Line 158: ‘with a binding affinity of -6.2 kcal·mol-1’.
The measure of the apparent Km values for the substrate and co-factor with MBDTA-2 at the sub-saturated IC50 values (6.92 and 8.58 micromolar) would help better understand the potential interaction between MBDTA-2 and the substrate and co-factor at the binding site.
The point raised by the reviewer has been incorporated as a discussion point at Line 278: ‘Such rational design efforts could also be guided by additional kinetic assays in the presence of subsaturating 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 MBDTA-2 interactions at the binding site.’
Interpretation of the whole plant data for such an application would be clearer with the inclusion of the application rate and whole plant data for the positive control, chlorosulfuron PESTANAL.
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)’.
It would be interesting to get the authors' perspectives on opportunities to utilize the binding data for MBDTA-2 on DHDPS and the docking model data for MBDTA-2 on DHDPR to identify new analogs that could have increased affinity for both enzymes with the goal to increase the whole plant activity.
We thank the reviewer for the comment. The possibility of utilising the crystallography and docking data for the rational design of more potent analogues has been noted in the Discussion with the following addition to Line 275: ‘For the future development of dual-target herbicides, the previously published DHDPS co-crystal structure (Soares da Costa et al., 2021) and the DHDPR binding model presented here could be used for the rational design of new MBDTA-2 analogues with increased target site activity.’
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Evaluation Summary:
This excellent paper presents the highly interesting finding of a compound that inhibits two targets in lysine synthesis. Further, the mechanisms for the first enzyme is allosteric inhibition, while for the second enzyme the compound is a competitive inhibitor. The authors nicely explain why this is of significant interest for herbicide resistance management with a new compound. The authors convincingly demonstrate that the compound is not a pro-herbicide, and instead that the higher in vivo relative to in vitro activity is due to the additional inhibition of the second step in lysine synthesis. The work is of interest to those studying enzymology, herbicide action and evolution of herbicide resistance.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also …
Evaluation Summary:
This excellent paper presents the highly interesting finding of a compound that inhibits two targets in lysine synthesis. Further, the mechanisms for the first enzyme is allosteric inhibition, while for the second enzyme the compound is a competitive inhibitor. The authors nicely explain why this is of significant interest for herbicide resistance management with a new compound. The authors convincingly demonstrate that the compound is not a pro-herbicide, and instead that the higher in vivo relative to in vitro activity is due to the additional inhibition of the second step in lysine synthesis. The work is of interest to those studying enzymology, herbicide action and evolution of herbicide resistance.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Overall a very interesting paper. There have been calls for discovery of herbicides that are multi-site inhibitors as a predicted way to delay resistance evolution to those herbicides. Fungicides are known that are multi-site inhibitors and these are known to have lower risk for resistance evolution. The authors provide evidence that their novel inhibitors of lysine synthesis inhibit both the first enzyme (previously shown) and the second enzyme in the lysine synthesis pathway. Inhibition of the first enzyme was shown to be due to inhibition at an allosteric site, while the same compound is shown in this paper to be a competitive inhibitor of the second enzyme. This two-site inhibition explains the relatively higher in vivo activity of the compound compared to its in vitro activity on the first enzyme alone. …
Reviewer #1 (Public Review):
Overall a very interesting paper. There have been calls for discovery of herbicides that are multi-site inhibitors as a predicted way to delay resistance evolution to those herbicides. Fungicides are known that are multi-site inhibitors and these are known to have lower risk for resistance evolution. The authors provide evidence that their novel inhibitors of lysine synthesis inhibit both the first enzyme (previously shown) and the second enzyme in the lysine synthesis pathway. Inhibition of the first enzyme was shown to be due to inhibition at an allosteric site, while the same compound is shown in this paper to be a competitive inhibitor of the second enzyme. This two-site inhibition explains the relatively higher in vivo activity of the compound compared to its in vitro activity on the first enzyme alone. The authors show that this two-site inhibitor of lysine synthesis has biological activity to reduce growth of the global weed Lolium rigidum. The modeling work to show the specific amino acids in the target binding site that are predicted to interact with the compound is really interesting and gives insights into how target site resistance could eventually evolve to this herbicide.
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Reviewer #2 (Public Review):
The paper contains a continued investigation of previously described dihydrodipicolinate synthase (DHDPS) inhibitor (MBDTA-2) to determine if the MBDTA-2 could be activated in planta to a more potent inhibitor. The hypothesis of potential demethylation of the methoxy was solid and the authors clearly showed that the hydroxy analog has lower affinity for inhibition of both forms of Arabidopsis DHDPS. The authors show diligence looking at MBDTA-2 inhibition of other plant enzymes to explain the previously described in vivo data. Dose response curves for MBDTA-2 inhibition of the second enzyme in the lysine biosynthesis pathway, dihydrodipicolinate reductase (DHDPR), show that MBDTA-2 provides about 10-fold greater inhibition for both forms of Arabidopsis DHDPR than it did for DHDPS with IC50 values for …
Reviewer #2 (Public Review):
The paper contains a continued investigation of previously described dihydrodipicolinate synthase (DHDPS) inhibitor (MBDTA-2) to determine if the MBDTA-2 could be activated in planta to a more potent inhibitor. The hypothesis of potential demethylation of the methoxy was solid and the authors clearly showed that the hydroxy analog has lower affinity for inhibition of both forms of Arabidopsis DHDPS. The authors show diligence looking at MBDTA-2 inhibition of other plant enzymes to explain the previously described in vivo data. Dose response curves for MBDTA-2 inhibition of the second enzyme in the lysine biosynthesis pathway, dihydrodipicolinate reductase (DHDPR), show that MBDTA-2 provides about 10-fold greater inhibition for both forms of Arabidopsis DHDPR than it did for DHDPS with IC50 values for inhibition of DHDPR in the single micromolar concentration. This rate of inhibition suggests more relevance for translation to whole plant growth inhibition.
It is unfortunate that the co-crystallization attempts with DHDPR and MBDTA-2 were not successful as the physical interaction would be very useful for additional analog synthesis and structure-activity relationship (SAR) evaluation. Use of binding models is common in rational drug design so it is understandable for the authors to pursue a binding model for MBDTA-2. It is difficult to assess the utility of the docking model for SAR development without a better understanding of how many docking conformation predictions the software provided and/or a measure of the docking score. The increase in IC50 values under saturated substrate and co-factor concentrations does help support that MBDTA-2 is a competitive inhibitor with respect to either the substrate and/or the co-factor. The measure of the apparent Km values for the substrate and co-factor with MBDTA-2 at the sub-saturated IC50 values (6.92 and 8.58 micromolar) would help better understand the potential interaction between MBDTA-2 and the substrate and co-factor at the binding site.
The translation of enzyme inhibition to whole organism inhibition is a common barrier in ration drug design. The use of the model dicot plant Arabidopsis (previous publication) and, the agronomically important monocot weed, Lolium rigidum to assess potential translation of inhibition to whole plant activity is key to understanding the potential of lysine biosynthesis inhibition to be a herbicidal target site. The authors utilized a unique method to assess the growth inhibition of Lolium with multiple applications directly to the Lolium seed. Interpretation of the whole plant data for such an application would be clearer with the inclusion of the application rate and whole plant data for the positive control, chlorosulfuron PESTANAL.
Novel herbicidal target site are desperately needed and this paper has identified new opportunities to investigate. A key discussion point in this paper is that a dual-target enzyme inhibitor as a commercial herbicide would be beneficial, especially for the potential prevention of target-site based resistance in weeds. As the authors state, this has been addressed through the use of mixtures of herbicide active ingredient with different modes of action (MoA) targeting the same weed. One of the largest challenges in developing novel MoA herbicides, other than identifying novel herbicidal MoA, is the translation of in vitro activity to whole plant control in field applications for a single-target herbicide. It would be interesting to get the authors' perspectives on opportunities to utilize the binding data for MBDTA-2 on DHDPS and the docking model data for MBDTA-2 on DHDPR to identify new analogs that could have increased affinity for both enzymes with the goal to increase the whole plant activity.
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Reviewer #3 (Public Review):
The authors provide further information on the mode action of a lysine synthesis inhibitor (MBTA-2) that is a potential herbicide. The authors determine that MBDTA-2 is not a proherbicide for DHDPS, but do not show that it is not a proherbicide for DHDPR. This should be done, especially since the findings support a very unusual conclusion: inhibition of consecutive enzymes of the lysine synthesis pathway by the same compound through binding an allosteric site for one enzyme and as a competitive inhibitor of the other. Having two molecular targets in the same pathway could hinder evolution of target site-base herbicide resistance. The bioassay of activity of MBDTA-2 on Lolium rigidum was done in such a way that it is difficult to determine if the activity is sufficient to be considered a herbicide.
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