plants

Review Current Status and Future Prospects in Herbicide Discovery

Franck E. Dayan

Department of Bioagricultural Sciences and Pest Management, 1177 Campus Delivery, Colorado State University, Fort Collins, CO 80523, USA; [email protected]; Tel.: +1-662-816-6214



Received: 21 August 2019; Accepted: 9 September 2019; Published: 11 September 2019


Abstract: Herbicides represent about 60% of the pesticides (by volume) used worldwide. The success of herbicides can be attributed in part to a relatively steady discovery of one unique mechanisms of action (MOA) every two years from the early 1950s to the mid-1980s. While this situation changed dramatically after the introduction of glyphosate-resistant crops, evolution of resistance to glyphosate has renewed the agrichemical industry interest in new chemistry interacting with novel target sites. This review analyses recent characterization of new herbicide target sites, the chemical classes developed to inhibit these target sites, and where appropriate the innovative technologies used in these discovery programs.

Keywords: amino acid biosynthesis; lipid biosynthesis; mechanism of action; plastoquinone biosynthesis; pyrimidine biosynthesis; target site

1. Introduction The first synthetic herbicide was discovered in the early 1940s [1] and its efficacy and selectivity caused a paradigm change in agricultural weed management practices. New herbicide mechanisms of action (MOA) were discovered at a relatively steady rate of one unique MOA every two years from the early 1950s to the mid-1980s. Today, herbicides represent about 60% of the pesticides used worldwide, and most large-scale crop production systems rely extensively on synthetic herbicides to manage weeds. This has led to the relatively slow but steady evolution of many herbicide-resistant (HR) biotypes. The introduction of glyphosate-resistant (GR) crops in the last 25 years has compounded the selection pressure imposed by the repeated application of herbicides over larger areas. Managing these HR plants is problematic and the lack of control threatens farm profitability while challenging environmentally beneficial farming practices (e.g., no-till) [2,3]. The emergence and spread of HR weeds will require farmer cooperation to successfully control them [4]. While a tremendous commercial success, GR crops have been detrimental to herbicide discovery programs, causing a lapse in innovative research and development programs and a dearth of new chemistry with novel mechanisms of action (MOA) [5,6]. Factors that have contributed to a reduced investment in herbicide research and development are multifold and have been discussed elsewhere but include [7]:

The success of GR crops that revolutionized weed management [8]. • The increased cost of R&D programs for production of a single new active ingredient from $184 • million in 2000 to nearly $286 million in 2016 [9]. The increased barriers imposed by toxicological and environmental regulations that must be • fulfilled to ensure safety of the products [10].

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The severe attrition of the Agchem industry from more than 100 R&D companies to a few • dominating companies [11]. This may in part be due to late stage failures (duPont), expense of liabilities and the depth of intellectual property.

Readers are encouraged to read Gerwick’s review on the problems facing the agchem industry and Duke’s review on why no new herbicide modes of action have been commercialized in recent years to have a broader analysis of these problems [6,12]. So what are the current status and future prospects in herbicide discovery? Herbicides are small (usually <500 MW) molecules that tend to target plant-specific processes. Generally speaking, herbicides can be grouped into three main categories: 1) herbicides that target biochemical pathways and physiological processes involved with photosynthesis, 2) herbicides that inhibit the formation of biological building blocks (i.e., sugars, amino acids and fatty acids) or their assembly into macromolecules, and 3) herbicides with other modes of action (Table1). The many di fferent active ingredients can be categorized based on their physicochemical properties [13] or organized around their respective mechanisms of action (Table1). This low number of mechanisms of action is somewhat surprising, considering the thousands of potential molecular target sites that exist in plants and the hundreds of thousands of molecules screened for herbicidal activity every year. This review will not cover the known mechanisms of action, and readers interested in the topic are referred to the original reports and several reviews on that topic for more information (e.g., [14,15]). However, a good knowledge of herbicide target sites and their mechanisms of action is crucial to decipher the way new herbicides may exert their action.

Table 1. Classification of mechanisms of action for current and potential new commercial herbicides discussed in this review.

Group Type Mechanism/Target Light reaction Photosystem II Photosystem I Carotenoid Deoxyxylulose-5-phosphate synthase Biochemical pathways and Phytoene desaturase physiological processes involved with photosynthesis Plastoquinone p-Hydroxyphenylpyruvate dioxygenase Homogentisate solanesyltransferase1 Solanyl diphosphate synthase1 Chlorophylls Protoporphyrinogen oxidase Uncouplers Oxidative (photo)phosphorylation 5-enolpyruvylshikimate-3-phosphate Amino acids (EPSP) Synthase Acetolactate synthase Glutamine synthetase Dihydroxy-acid dehydratase1 Lipids Acetyl-CoA carboxylase Formation of biological building Fatty acid thioesterase1 blocks or their assembly into Very long chain fatty acid elongases macromolecules Cell walls Cellulose synthase and others Microtubule assembly α- and/or β-Tubulin Microtubule organization Microtubule organizing centers Folates Dihydropteroate synthetase Dihydrofolate reductase1 Nucleic acids DNA gyrase1 Dihydroorotate dehydrogenase1 Protein synthesis Peptide deformylase 1 Other processes Protein regulation Serine-threonine protein phosphatases Hormone Synthetic auxins Auxin-transport inhibition 1 bold and italics indicates recently described or potentially new mechanisms or targets. Plants 2019, 8, 341 3 of 18

As mentioned above, no herbicides with truly new molecular targets had been introduced in the past 30 years. Yet, the need for new tools is more dire than ever, especially to combat HR weeds, and in particular those that have evolved resistance to glyphosate [16]. Though there does not appear to be a ‘silver bullet’ coming down the Agchem pipeline, there has been a recent flurry of reports of new mechanisms of action. So what has changed? The dominance of glyphosate has been a destabilizing force affecting other agchem companies’ decisions to move forward with new chemistry discovered through their own R&D programs. As mentioned before, the potential market shares of new compounds were not sufficient to justify the cost of developing these products. While the Agchem market is still relying on glyphosate for weed control in all the major row crops, the emergence of GR weeds has begun to impact the current usefulness and future prospect of glyphosate. Indeed, farmers are already returning to older chemistry to control GR weeds. In this new environment, companies may be projecting that the time is ripe for introducing new chemistry and new MOA. This seems to be reflected with recent presentations at the 2019 International Union of Pure and Applied Chemistry (IUPAC) congress on plant protection in Ghent, Belgium [17] and the Agrochemical Division of American Chemical Society’s program at their 2019 national meeting in San Diego, CA. Additionally, several new startup companies have developed innovative technologies to explore new chemical spaces and/or facilitate the elucidation of their target sites. For example, MoA technology uses in vivo high throughput platforms, proprietary bioinformatics and Artificial Intelligence (AI) tools to discover novel herbicides with novel MOAs. Enko Chem Inc. aims to become a leading innovator in crop protection chemistry by utilizing a target-based discovery platform to produce high quality and novel small molecule starting points and a suite of tools and approaches to develop these into product candidates, and Agrematch develops AI and big-data tools for rational identification of molecules with desired biological activity and high potential to become crop protection products while significantly reducing R&D costs and accelerating time to market.

2. Novel Mechanisms of Action

2.1. Lipid Biosynthesis Lipid synthesis involves several biochemical pathways leading to the formation of important building blocks for membranes, cuticles and waxes necessary for plant survival. Consequently, it has been the target of several herbicide classes. Acetyl-coenzyme A carboxylase (ACCase) catalyzes the first committed step to fatty acid synthesis (Figure1A). Cyclohexanediones (e.g., sethoxydim) and aryloxyphenoxypropionates (e.g., diclofop-methyl) are the two major groups of herbicides targeting this enzyme. Inhibitors of ACCase are grass-selective because two forms of ACCase occur in plants. A prokaryotic form is insensitive to these herbicides and is found only in the plastids of dicotyledonous plants, whereas the herbicide-sensitive eukaryotic form is found in the cytoplasm of all plants and in the plastids of grasses. Non-grass plants are resistant because they can sustain lipid biosynthesis in the presence of such herbicides. While there are a host of enzymes catalyzing the many subsequent steps in fatty acid synthesis, the only other herbicide target site in this pathway are the very long chain fatty acid elongases (VLCFAE) (Figure1A). These enzymes are much further down the metabolic pathway, responsible for synthesis of waxes, cutins, and suberins. VLFCAEs are the targets of several classes of herbicides, including the chloroacetanilides (e.g., alachlor), the thiocarbamates (e.g., EPTC), and the oxyacetamides (e.g., flufenacet). Inhibition of VLFCAEs by these herbicides results in decreased growth and leaf curling or twisting [14]. Plants 2019, 8, 341 4 of 18 Plants 2019, 8, x FOR PEER REVIEW 4 of 18

cinmethylin

A B

FigureFigure 1. 1.(A ()A Overview) Overview of of fatty fatty acid acid biosynthesis biosynthesis and and herbicide herbicide targets targets in in plant plant cells. cells. InhibitionInhibition of of acetyl-CoA carboxylasecarboxylase (ACCase)(ACCase) bybyhaloxyfopmethyl haloxyfopmethyl or or tepraloxydim tepraloxydim disrupts disrupts early early fatty fatty acid acid biosynthesis.biosynthesis. Cinmethylin Cinmethylin prevents prevents the the release release of of both both unsaturatedunsaturated andand saturated fatty fatty acids acids from from the theplastids plastids through through inhibi inhibitiontion of of fatty fatty acid acid thioesterase thioesterase (FAT) (FAT) A Aand and B, B,respectively. respectively. Inhibitors Inhibitors of very- of very-long-chainlong-chain fatty fatty acid acid (VLCFA) (VLCFA) biosynthesis biosynthesis act atat thethe endoplasmic endoplasmic reticulum. reticulum. ACP: ACP: acyl acyl carrier carrier protein;protein; CoA: CoA: Coenzyme Coenzyme A. FromA. From [18 ][18] with with permission. permission. (B) ( StructureB) Structure of cinmethylin.of cinmethylin.

Fatty2.1.1. Acid Fatty Thioesterase Acid Thioesterase (FAT) (FAT) AnAn exciting exciting new new development development has has been been the the discovery discovery of new of new target target sites sites in fatty in fatty acid synthesisacid synthesis of anof old an herbicide, old herbicide, cinmethylin cinmethylin (Figure (Figure1B). These 1B). areThese the are fatty the acid fatty thioesterases acid thioesterases (FAT) (Figure (FAT) 1(FigureB) [ 18 ].1B) FATs[18]. are FATs plastid are localized plastid localized enzymes enzymes that mediate that themediate release the of release fatty acids of fatty from acids its acyl from carrier its acyl protein carrier (ACP)protein which (ACP) is necessary which is for necessary FA export for out FA of export the chloroplast out of the andchloroplast transfer and to the transfer endoplasmic to the endoplasmic reticulum asreticulum fatty acyl-CoAs. as fatty acyl-CoAs. FATsFATs are are inhibited inhibited by cinmethylin, by cinmethylin, a natural a natural product-like product-like benzyl benzyl ether derivativeether derivative of 1,4-cineole of 1,4-cineole that wasthat first was described first described by Shell inby 1981 Shell and in commercialized 1981 and commercialized in 1989 [19]. in Plants 1989 treated [19]. Plants with cinmethylin treated with havecinmethylin reduced levels have ofreduced the saturated levels C14:0of the and saturated C16:0 fatty C14:0 acids, andindicating C16:0 fatty that acids, the herbicideindicating inhibits that the bothherbicide classes ofinhibits FAT proteins both classes [18]. The of FAT direct proteins interaction [18]. of The cinmethylin direct interaction with FAT proteinsof cinmethylin was confirmed with FAT byproteins fluorescence-based was confirmed thermal by shift fluorescence-based assays and co-crystallization thermal ofshift cinmethylin assays and within co-crystallization the FAT enzyme. of 2.2.cinmethylin Plastoquinone within Biosynthesis the FAT enzyme.

2.2.Plastoquinone Plastoquinone isBiosynthesis a lipid (prenyl) quinone with important biological functions. It is best known for its role as an electron acceptor in the light reaction of photosynthesis. Specifically, it accepts Plastoquinone is a lipid (prenyl) quinone with important biological functions. It is best known electrons from photosystem II and transfers them to the cytochrome b6 complex. Its importance for its role as an electron acceptor in the light reaction of photosynthesis. Specifically, it accepts in agrochemistry cannot be overstated. Some of the earliest commercial herbicides (e.g., triazines, electrons from photosystem II and transfers them to the cytochrome b6 complex. Its importance in ureas, and nitriles) inhibit photosynthesis by competing for the plastoquinone binding site on PSII. agrochemistry cannot be overstated. Some of the earliest commercial herbicides (e.g., triazines, ureas, Later on, interest in plastoquinone renewed with the discovery of triketone herbicides that inhibit and nitriles) inhibit photosynthesis by competing for the plastoquinone binding site on PSII. Later p-hydroxyphenylpyruvate dioxygenase (HPPD), a key enzyme in plastoquinone synthesis (Figure2). on, interest in plastoquinone renewed with the discovery of triketone herbicides that inhibit p- These herbicides cause bleaching of photosynthetic tissues because plastoquinone is required for hydroxyphenylpyruvate dioxygenase (HPPD), a key enzyme in plastoquinone synthesis (Figure 2). the activity of phytoene desaturase [20], a well-known target of herbicides inhibiting carotenoid These herbicides cause bleaching of photosynthetic tissues because plastoquinone is required for the biosynthesis (Figure2). Consequently, industry started focusing on the biosynthesis pathway of activity of phytoene desaturase [20], a well-known target of herbicides inhibiting carotenoid plastoquinone in hope of identifying additional herbicide target sites. Its biosynthesis involves the biosynthesis (Figure 2). Consequently, industry started focusing on the biosynthesis pathway of convergence of two pathways (Figure2). On one hand, the quinone head is derived from tyrosine plastoquinone in hope of identifying additional herbicide target sites. Its biosynthesis involves the and involves HPPD to form homogentisate. On the other hand, the lipophilic tail is derived from the convergence of two pathways (Figure 2). On one hand, the quinone head is derived from tyrosine 2-C-Methyl-D-erythritol 4-Phosphate (MEP)-derived terpenoid pathway (Figure2). and involves HPPD to form homogentisate. On the other hand, the lipophilic tail is derived from the 2-C-Methyl-D-erythritol 4-Phosphate (MEP)-derived terpenoid pathway (Figure 2).

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FigureFigure 2. Overview2. Overview of the of relationship the relationship between between carotenoid caro andtenoid prenyl and quinone prenyl biosynthesis.quinone biosynthesis. Biosynthesis ofBiosynthesis carotenoids andof carotenoids plastoquinone and requiresplastoquinone the MEP, requires terpenoid the MEP, and homogentisateterpenoid and pathways.homogentisate Older chemistrypathways. such Older as clomazone chemistry inhibitssuch as 1-deoxy-D-xyluloseclomazone inhibits 5-phosphate1-deoxy-D-xylulos synthasee 5-phosphate (DXS), the firstsynthase step in the(DXS), MEP the pathway; first step a numberin the MEP of chemicalpathway; classesa number inhibit of chemical carotenoid classes biosynthesis inhibit carotenoid by targeting biosynthesis phytoene desaturaseby targeting (PDS); thephytoene newer triketonedesaturase herbicides (PDS); inhibit the p-hydroxyphenylpyruvatenewer triketone herbicides dioxygenase inhibit (HPPD) p- involvedhydroxyphenylpyruvate in homogentisate dioxygenase biosynthesis. (HPPD) The twoinvolved newest in homogentisate target sites affect biosynthesis. solanyl diphosphate The two synthasenewest (SPS)target responsiblesites affect forsolanyl the synthesisdiphosphate of thesynthase terpenoid (SPS) tail responsible of plastoquinone for the synthesis or homogentisate of the solanesylterpenoid transferase tail of plastoquinone (HST), the enzymeor homogentisate combining solanesyl solanyl transferase diphosphate (HST), and the homogentisate enzyme combining to form a plastoquinonesolanyl diphosphate precursor. and homogentisate to form a plastoquinone precursor.

2.2.1.2.2.1. Solanyl Solanyl Diphosphate Diphosphate Synthase Synthase (SPS)(SPS) TheThe building building block block ofof the lipid tail tail of of plastoquinon plastoquinonee is solanyl is solanyl diphosphate. diphosphate. It is obtained It is obtained by the by theactivity activity of of solanyl solanyl diphosphate diphosphate synthase synthase (SPS) (SPS) which which catalyzes catalyzes the the sequential sequential addition addition of ofseven seven isopentenylisopentenyl diphosphate diphosphate to geranylto geranyl diphosphate diphosphat [21e]. [21]. A collaboration A collaboration between between the herbicide the herbicide discovery groupdiscovery of Bayer group CropScience of Bayer CropScience and Targenomix and Targenom recently discoveredix recently usingdiscovered a systems using biology a systems approach biology that aclonifenapproach (Figure that aclonifen3) causes (Figure bleaching 3) causes of treated bleaching plants of by treated inhibiting plants SPS. by inhi Aclonifenbiting SPS. is a Aclonifen relatively is old diphenylethera relatively old herbicide diphenylether whose MOAherbicide was whose unknown. MOA The was binding unknown. of aclonifen The binding to SPS of aclonifen was confirmed to SPS by was confirmed by crystallography. Phenylalanine residues within the catalytic domain of SPS are crystallography. Phenylalanine residues within the catalytic domain of SPS are involved in the binding involved in the binding of aclonifen. Plants possess three genes encoding SPS. Two of the genes of aclonifen. Plants possess three genes encoding SPS. Two of the genes encode for SPS1 and SPS2 encode for SPS1 and SPS2 proteins that are localized in the chloroplast and involved in plastoquinone proteins that are localized in the chloroplast and involved in plastoquinone synthesis [22]. The other synthesis [22]. The other gene encodes for the mitochondrial isoforms (SPS3) involved in ubiquinone gene encodes for the mitochondrial isoforms (SPS3) involved in ubiquinone synthesis [22]. SPS1 and synthesis [22]. SPS1 and SPS2 are sensitive to aclonifen, whereas SPS3 is insensitive to the herbicide. SPS2 are sensitive to aclonifen, whereas SPS3 is insensitive to the herbicide.

PlantsPlants 20192019,, 88,, 341x FOR PEER REVIEW 6 of 18 Plants 2019, 8, x FOR PEER REVIEW 6 of 18 Cl Cl H2N O H2N O - -O + O N+ N aclonifen O aclonifen . O .

Figure 3. Structure of aclonifen, an inhibitor of chloroplasticchloroplastic solanyl diphosphatediphosphate synthase (SPS).(SPS). Figure 3. Structure of aclonifen, an inhibitor of chloroplastic solanyl diphosphate synthase (SPS). 2.2.2. Homogentisate Solanesyl Transferase (HST) 2.2.2. Homogentisate Solanesyl Transferase (HST) 2.2.2. Homogentisate Solanesyl Transferase (HST) As mentioned above, plastoquinone biosynthesis involves the convergence of homogentisate As mentioned above, plastoquinone biosynthesis involves the convergence of homogentisate and terpernoidAs mentioned synthesis. above, Thisplastoquinone step is catalyzed biosynthesis by homogentisate involves the solanesyl convergence transferase of homogentisate (HST) [23]. and terpernoid synthesis. This step is catalyzed by homogentisate solanesyl transferase (HST) [23]. HSTand terpernoid catalyzes the synthesis. prenylation This and step decarboxylation is catalyzed by of homogentisate homogentisate solanesyl to form 2-methyl-6-solanesyl-1,4- transferase (HST) [23]. HST catalyzes the prenylation and decarboxylation of homogentisate to form 2-methyl-6-solanesyl- benzoquinol,HST catalyzes the the first prenylation intermediate and decarboxylation in plastoquinone-9 of homogentisate biosynthesis. (Figureto form2 2-methyl-6-solanesyl-). This enzyme was 1,4-benzoquinol, the first intermediate in plastoquinone-9 biosynthesis. (Figure 2). This enzyme was known1,4-benzoquinol, to be sensitive the first to inhibition intermediate by haloxydine in plastoqu [23inone-9]. Haloxydine biosynthesis. acts as a(Figure suicide 2). inhibitor This enzyme mimicking was known to be sensitive to inhibition by haloxydine [23]. Haloxydine acts as a suicide inhibitor homogentisateknown to be sensitive binding. to inhibition by haloxydine [23]. Haloxydine acts as a suicide inhibitor mimicking homogentisate binding. mimickingMitsui Chemicalhomogentisate Agro Inc.binding. reported the discovery and development of cyclopyrimorate (Figure4) Mitsui Chemical Agro Inc. reported the discovery and development of cyclopyrimorate (Figure as a newMitsui bleaching Chemical herbicide Agro Inc. inhibiting reported HST. the discov This herbicideery and development was discovered of cyclopyrimorate in a program aiming (Figure at 4) as a new bleaching herbicide inhibiting HST. This herbicide was discovered in a program aiming combining4) as a new the bleaching pharmacophore herbicide backbone inhibiting of HST. credazine This herbicide and pyridafol. was discovered Structure optimizationin a program againstaiming at combining the pharmacophore backbone of credazine and pyridafol. Structure optimization theat combining weeds Scirpus the juncoidespharmacophore(sedge) backbone and Sagittaria of cr trifoliaedazine(threeleaf and pyridafol. arrowhead) Structure demonstrated optimization that against the weeds Scirpus juncoides (sedge) and Sagittaria trifolia (threeleaf arrowhead) demonstrated theagainst cyclopropane the weeds ringScirpus and juncoides the methyl (sedge) group and at Sagittaria the ortho trifolia positions (threeleaf (2 and arrowhead) 6, respectively) demonstrated on the that the cyclopropane ring and the methyl group at the ortho positions (2 and 6, respectively) on the phenylthat the ring cyclopropane were critical ring for and activity. the methyl The hydrophobicitygroup at the ortho of thepositions moiety (2 at and position 6, respectively) 2 can modulate on the phenyl ring were critical for activity. The hydrophobicity of the moiety at position 2 can modulate activity,phenyl ring with were cyclopropane critical for beingactivity. optimal. The hydropho Additionally,bicity of a the hydroxy moiety group at position at position 2 can modulate 4 on the activity, with cyclopropane being optimal. Additionally, a hydroxy group at position 4 on the pyridazineactivity, with ring cyclopropane is important. being Extensive optimal. biochemical Additionally, work determineda hydroxy thatgroup plants at position treated with4 on thisthe pyridazine ring is important. Extensive biochemical work determined that plants treated with this herbicidepyridazine have ring decreasedis important. levels Extensive of chlorophyll, biochemical carotenoids work determined and plastoquinone, that plants treated and accumulate with this herbicide have decreased levels of chlorophyll, carotenoids and plastoquinone, and accumulate homogentisate.herbicide have decreased The effect levels of the of herbicide chlorophyll, was carotenoids strongly reversed and plastoquinone, by decyl plastoquinone and accumulate and homogentisate. The effect of the herbicide was strongly reversed by decyl plastoquinone and moderatelyhomogentisate. reversed The effect by homogentisate, of the herbicide suggesting was strongly that cyclopyrimorate reversed by decyl targeted plastoquinone HST. Further and moderately reversed by homogentisate, suggesting that cyclopyrimorate targeted HST. Further work workmoderately demonstrated reversed that by homogentisate, cyclopyrimorate suggesting was a proherbicide that cyclopyrimorate that needed targeted to be bioactivated HST. Further into work its demonstrated that cyclopyrimorate was a proherbicide that needed to be bioactivated into its des- des-morpholinocarbonyldemonstrated that cyclopyrimorate cyclopyrimorate was a (DMC) proherbicide metabolite that (Figureneeded4 )to to be inhibit bioactivated HST [ 24 into]. In its planta des- morpholinocarbonyl cyclopyrimorate (DMC) metabolite (Figure 4) to inhibit HST [24]. In planta metabolismmorpholinocarbonyl of cyclopyrimorate cyclopyrimorate into DMC (DMC) releasing metabolit thee free(Figure hydroxy 4) to groupinhibit onHST position [24]. In 4 ofplanta the metabolism of cyclopyrimorate into DMC releasing the free hydroxy group on position 4 of the pyridazinemetabolism ring of cyclopyrimorate is critical for bioactivation into DMC releasing of this herbicide. the free hydroxy The I50 valuesgroup foron position cyclopyrimorate 4 of the pyridazine ring is critical for bioactivation of this herbicide. The I50 values for cyclopyrimorate and andpyridazine DMC on ring HST is critical were 3.9 for and bioactivation 561 µM, respectively. of this herbicide. DMC The is a I50 competitive values for cyclopyrimorate inhibitor of HST and for DMC on HST were 3.9 and 561 μM, respectively. DMC is a competitive inhibitor of HST for homogentisateDMC on HST butwere uncompetitive 3.9 and 561 toward μM, respectively. the prenyl diphosphate DMC is a (Figurecompetitive2). inhibitor of HST for homogentisate but uncompetitive toward the prenyl diphosphate (Figure 2). homogentisate but uncompetitive toward the prenyl diphosphate (Figure 2).

Figure 4.4. Cyclopyrimorate and itsits bioactivebioactive metabolitemetabolite des-morpholinocarbonyldes-morpholinocarbonyl cyclopyrimoratecyclopyrimorate Figure 4. Cyclopyrimorate and its bioactive metabolite des-morpholinocarbonyl cyclopyrimorate (DMC).(DMC). (DMC). Bleaching symptomssymptoms are are the the results results of aof dramatic a dramatic decrease decrease in plastoquinone in plastoquinone levels levels in treated in treated plants. Bleaching symptoms are the results of a dramatic decrease in plastoquinone levels in treated Sinceplants. this Since target this site target is downstream site is downstream enzyme ofen HPPDzyme of in HPPD the plastoquinone in the plastoquinone biosynthesis biosynthesis pathway, plants. Since this target site is downstream enzyme of HPPD in the plastoquinone biosynthesis activitypathway, of activity cyclopyrimorate of cyclopyrimorate is enhanced is enhanced in tank mix in tank with mix 4-HPPD with 4-HPPD inhibitors. inhibitors. This herbicide This herbicide will be pathway, activity of cyclopyrimorate is enhanced in tank mix with 4-HPPD inhibitors. This herbicide developedwill be developed for weed for management weed management in rice paddies, in rice including paddies, acetolactate including synthase acetolactate (ALS) synthase resistant (ALS) weed will be developed for weed management in rice paddies, including acetolactate synthase (ALS) speciesresistant with weed projected species commercializationwith projected commercialization in 2020. in 2020. resistant weed species with projected commercialization in 2020. 2.3. Amino Acid Biosynthesis and Protein Regulation 2.3. Amino Acid Biosynthesis and Protein Regulation

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2.3. Amino Acid Biosynthesis and Protein Regulation

2.3.1.Plants Dihydroxy-Acid 2019, 8, x FOR PEER Dehydratase REVIEW (DHAD) 7 of 18 2.3.1.A large Dihydroxy-Acid number of Dehydratase herbicides inhibit(DHAD) branched chain amino acid biosynthesis by targeting acetolactateA large synthase, number the of first herbicides step committed inhibit inbranched this pathway chain (Figureamino 5acid)[ 14 ].biosynthesis In light of theby commercialtargeting successacetolactate of this chemistry,synthase, industrythe first hasstep searchedcommitted for in chemicals this pathway that could (Figure inhibit 5) [14]. this pathwayIn light byof otherthe means.commercial A number success of inhibitors of this chemis of acetohydroxytry, industry acid has isomeroreductase searched for chemicals have been that discovered could inhibit but this none of thempathway have by been other developed means. A intonumber commercial of inhibitors products. of acetohydroxy acid isomeroreductase have been discoveredAn innovative but none resistant-gene-directed of them have been developed discovery into approach commercial led to products. the discovery of a new herbicide target siteAn in innovative the branched resistant-gene-directed chain amino acid pathway discov [25ery]. Aspterricapproach acidled isto a naturalthe discovery herbicide of produceda new byherbicide the soil fungus target siteAspergillus in the branched terreus (Figure chain 5amino). A research acid pathway group [25]. at University Aspterric acid of California is a natural Los Angelesherbicide analyzed produced the microbialby the soil gene fungus cluster Aspergillus involved terreus in the (Figure biosynthesis 5). A research of this microbialgroup at University phytotoxin. Theyof California discovered Los that Angeles that the analyzed cluster the also microbial included gene a paralog cluster form involved of dihydroxy in the biosynthesis acid dehydratase of this (DHAD),microbial the phytotoxin. last common They enzyme discovered of the that branched that the chain cluster amino also included acid biosynthesis a paralog form pathway of dihydroxy (Figure5 ). Furtheracid dehydratase work demonstrated (DHAD), that the last aspterric common acid enzyme targets of DHAD, the branched and the chain DHAD amino paralog acid biosynthesis present in the genepathway cluster (Figure was aspterric 5). Further acid-resistant, work demonstrated its target th enzymeat aspterric [25 ].acid Aspterric targets DHAD, acid is aand relatively the DHAD weak phytotoxinparalog present which mayin the not gene rise cluster to a successful was aspterric commercial acid-resistant, herbicide, its target but itenzyme might [25]. serve Aspterric as a structural acid is a relatively weak phytotoxin which may not rise to a successful commercial herbicide, but it might backbone to elaborate new herbicide classes with improved physicochemical properties. As well, serve as a structural backbone to elaborate new herbicide classes with improved physicochemical it is not clear whether DHAD is a good target site for herbicide to control weeds under agronomic properties. As well, it is not clear whether DHAD is a good target site for herbicide to control weeds conditions, and more work must be carried out to validate it as a desirable target site. Nevertheless, under agronomic conditions, and more work must be carried out to validate it as a desirable target one of the advantages of this microbial gene co-clustering analysis is that it can lead to the discovery of site. Nevertheless, one of the advantages of this microbial gene co-clustering analysis is that it can genes involved in a phytotoxin biosynthesis, the identification of its target site, and the isolation of a lead to the discovery of genes involved in a phytotoxin biosynthesis, the identification of its target herbicide-resistantsite, and the isolation form of of a this herbicide-resistant target site [26]. form of this target site [26].

A B

FigureFigure 5. (5.A )(A Key) Key enzymes enzymes involved involved in in branched branched chain chain aminoamino acidacid biosynthesis. DHAD DHAD isis the the most most recentrecent putative putative herbicide herbicide target target site site in in this this pathway. pathway. ((BB)) StructureStructure of of the the microbial microbial metabolite metabolite aspterric aspterric acid,acid, an an inhibitor inhibitor of of DHAD. DHAD. ALS: ALS: acetolactate acetolactate synthase;synthase; KARI: acetohydroxy acid acid isomeroreductase; isomeroreductase; DHAD:DHAD: dihydroxy dihydroxy acid acid dehydratase. dehydratase.

2.3.2.2.3.2. 3-Dehydroquinate 3-Dehydroquinate Synthase Synthase TheThe shikimate shikimate pathway pathway is is one one of of the the central central pathwayspathways associated in in plant plant metabolism, metabolism, providing providing thethe carbon carbon skeletons skeletons for for the the aromatic aromatic amino amino acidsacids lL-tryptophan,-tryptophan, Ll-phenylalanine,-phenylalanine, and and L-tyrosinel-tyrosine (Figure 6) and many important secondary metabolites (e.g., chlorogenic acid, alkaloids, glucosinolates, auxin, tannins, suberin, lignin and lignan, and tocopherols) [27]. It is estimated that

Plants 2019, 8, 341 8 of 18

(Figure6) and many important secondary metabolites (e.g., chlorogenic acid, alkaloids, glucosinolates, auxin,Plants 2019 tannins,, 8, x FOR suberin, PEER REVIEW lignin and lignan, and tocopherols) [27]. It is estimated that at least8 of 30% 18 of all fixed carbon is directed through this pathway to support the flux required to produce these plantat least components. 30% of all fixed carbon is directed through this pathway to support the flux required to produceSince these mammals plant components. cannot synthesize these amino acids, this pathway is particularly desirable as a potentialSince targetmammals for herbicides.cannot synthesize To date, these glyphosate amino acids, is the this only pathway herbicide is particularly targeting this desirable pathway as bya actingpotential as an target irreversible for herbicides. inhibitor To of date, 5-enolpyruvylshikimate-3-phospate glyphosate is the only herbicide targeting synthase (Figurethis pathway6). While by glyphosateacting as an slowly irreversible depletes inhibitor the pools of of 5- aromaticenolpyruvylshikimate amino acids, its-3- herbicidalphospate synthase activity is (Figure associated 6). While witha deregulationglyphosate slowly of the shikimatedepletes the pathway, pools of leading aromatic to accumulationamino acids, its of herbicidal high levels activity of shikimate-3-phosphate is associated with anda deregulatio shikimaten and of the siphoning shikimate of carbonpathway, and leading phosphate to accumulation from other pathways, of high levels disrupting of shikimate more than-3- justphosphate the shikimate and shikimate pathway and [14 ].siphoning of carbon and phosphate from other pathways, disrupting more than just the shikimate pathway [14].

3-dehydroshikimate- O O - SD O O O shikimate HO - O DHQD O OH 3-dehydroquinate HO OH SK - OH O O OH O OH shikimate- OH O 3-phosphate O OH OH O HO P O - OH HO HO P O DQS O OH HO EPSPS O OH O HO - OH OH phosphoenol O pyruvate O 7-deoxysedoheptulose DAHPS HO O + HO P OH O OH - O OH O O 5-enolpyruvyl P O HO O Phenylalanine shikimate-3- HO 3-deoxy-D-arabino- Tryptophan OH O phosphate heptulosonate-7- - D-erythrose P O phosphate Tyrosine HO O O 4-phosphate HO OH O

- - O O O CS O O - - O O + - NH3 O O O - O O O OH O arogenate OH PAT CM chorismate prephenate OH

FigureFigure 6. 6.Shikimate Shikimate pathwaypathway andand aromaticaromatic amino acid biosynthesis showing showing the the metabolites metabolites formed formed atat each each step step catalyzedcatalyzed byby thethe enzymesenzymes abbreviatedabbreviated in bold.bold. The The target target site site of of glyphosate glyphosate is is EPSPS EPSPS (green).(green). TheThe targettarget site of the the antimetabolite antimetabolite 7 7-deoxysedoheptulose-deoxysedoheptulose (red) (red) is DQS is DQS (orange). (orange). DAHPS DAHPS:: 3- 3-deoxy-deoxy-Dd--arabino-heptulosonate-7-phosphatearabino-heptulosonate-7-phosphate synthase, synthase, DQS DQS:: 3 3-dehydroquinate-dehydroquinate synthase, synthase, DHQD/SD DHQD/SD:: 3-dehydroquinatede3-dehydroquinatede hydratase,hydratase, SK:SK: shikimateshikimate kinase,kinase, EPSPS:EPSPS: 5-enolpyruvylshikimate5-enolpyruvylshikimate 3 3-phosphate-phosphate synthase,synthase, CS: CS: chorismate chorismate synthase,synthase, CM:CM: chorismatechorismate mutase,mutase, PAT:PAT: prephenateprephenate aminotransferase.aminotransferase.

Recently,Recently, aa group from from Tübingen Tübingen University University (Germany) (Germany) has focus has focuseded on antimetabolites on antimetabolites as novel as novelstructural structural backbone backboness to discover to discover inhibitors inhibitors affectinga newffecting target new sites. target Antimetaboli sites. Antimetabolitestes are interesting are interestingmolecules as molecules they inhibit as theyenzymes inhibit by mimicking enzymes by their mimicking physiological their substrates. physiological Their substrates. study identified Their studythe rare identified sugar 7-deoxy the rare-sedoheptulose sugar 7-deoxy-sedoheptulose (7dSh) as an inhibitor (7dSh) of 3- asdehydroquinate an inhibitor of synthase 3-dehydroquinate (Figure 6), synthasea key enzyme (Figure of6 the), a shikimate key enzyme pathway. of the 7d shikimateSh is active pathway. at low micromolar 7dSh is active range at[28] low. The micromolar growth of rangeplants [ 28treated]. The with growth 25 μM of plants7dSH treatedwas inhibited with 25 toµ Mthe 7dSHsame wasdegree inhibited as an equivalent to the same amount degree of as anglyphosate. equivalent However, amount of treatment glyphosate. with However, 7dSH caused treatment an withaccumulation 7dSH caused of 3 an-deoxy accumulation-D-arabino of- 3-deoxy-heptulosonated-arabino-heptulosonate-7-phosphate,-7-phosphate, the substrate of the3-dehydroquinate substrate of 3-dehydroquinate synthase (Figure synthase 6), (Figurewhereas6), whereasglyphosate glyphosate caused causeda rapid a rapidaccumulation accumulation of shikimate, of shikimate, a well a-known well-known biomarker biomarker of 5 of- 5-enolpyruvylshikimateenolpyruvylshikimate 3- 3-phosphatephosphate synthase synthase inhibition inhibition [29] [29. ].Not Not surprisingly, surprisingly, plants plants treated treated with with 7dSh7dSh have have lower lower levelslevels ofof freefree aromaticaromatic aminoamino acidsacids (tyrosine,(tyrosine, phenylalanine,phenylalanine, and and tryptophan). tryptophan). This This apparently deregulates the biosynthesis of other amino acids, resulting in accumulation of the branched chain amino acids (valine, leucine, and isoleucine), as well as arginine. 7dSh did not have any preemergence activity. However, it controlled velvetleaf (Abutilon theophrasti) when applied as a postemergence herbicide at a rate of 2 kg ha-1. The addition of an adjuvant was required to obtain this

Plants 2019, 8, 341 9 of 18 apparently deregulates the biosynthesis of other amino acids, resulting in accumulation of the branched chain amino acids (valine, leucine, and isoleucine), as well as arginine. 7dSh did not have any Plants 2019, 8, x FOR PEER REVIEW 9 of 18 preemergence activity. However, it controlled velvetleaf (Abutilon theophrasti) when applied as a -1 postemergence herbicideherbicide atat aa raterate ofof 22 kgkg haha-1. The The addition addition of of an an adjuvant adjuvant was was required required to obtain this level of activity.activity. OnOn thethe otherother hand,hand, 7dSh7dSh had no activityactivity on greengreen foxtailfoxtail ((SetariaSetaria viridisviridis)) suggestingsuggesting selectivity for broadleaf weedweed control.control. 2.3.3. Serine/Threonine Protein Phosphatases (PPs) 2.3.3. Serine/Threonine Protein Phosphatases (PPs) More than 70% of all proteins have multiple phosphorylation sites and many of these proteins’ More than 70% of all proteins have multiple phosphorylation sites and many of these proteins’ activities are regulated via phosphorylation. This is achieved by the concerted action of protein activities are regulated via phosphorylation. This is achieved by the concerted action of protein kinases and phosphatases, that account for between 2–4% of the protein-encoding genes of most kinases and phosphatases, that account for between 2–4% of the protein-encoding genes of most plants [30,31]. The specificity of protein kinases is based on primary sequence recognition, whereas plants [30,31]. The specificity of protein kinases is based on primary sequence recognition, whereas protein phosphatases tend to be non-discriminate. However, studies across many eukaryote systems protein phosphatases tend to be non-discriminate. However, studies across many eukaryote systems confirmed that the phosphatases are not involved in generic dephosphorylation but are in fact as confirmed that the phosphatases are not involved in generic dephosphorylation but are in fact as highly regulated as their kinase counterparts. Phosphoprotein phosphatases represent a large group of highly regulated as their kinase counterparts. Phosphoprotein phosphatases represent a large group proteins, that include a sub-class called serine/threonine phosphatases (PPs) [32]. of proteins, that include a sub-class called serine/threonine phosphatases (PPs) [32]. As their names imply, protein serine/threonine phosphatases (PPs) remove phosphate groups As their names imply, protein serine/threonine phosphatases (PPs) remove phosphate groups bound to serine and threonine residues. PPs are categorized into three subclasses—phosphoprotein bound to serine and threonine residues. PPs are categorized into three subclasses—phosphoprotein phosphatases, metal-dependent protein phosphatases, and aspartate-based phosphatases. The PPs in phosphatases, metal-dependent protein phosphatases, and aspartate-based phosphatases. The PPs in plants belongs to the phosphoprotein phosphatase sub-class [32]. plants belongs to the phosphoprotein phosphatase sub-class [32]. PPs are the target of endothall (Figure7), an old herbicide that was first commercialized in the PPs are the target of endothall (Figure 7), an old herbicide that was first commercialized in the 1950s. Endothall induces severe growth inhibition [15]. Endothall is a structural analog of cantharidin, 1950s. Endothall induces severe growth inhibition [15]. Endothall is a structural analog of a natural product from the blister beetle (Epicauta spp.) and the Spanish fly (Lytta vesicatoria) (Figure7). cantharidin, a natural product from the blister beetle (Epicauta spp.) and the Spanish fly (Lytta Both of these molecules cause similar symptoms on plants [33]. vesicatoria) (Figure 7). Both of these molecules cause similar symptoms on plants [33].

Figure 7. Structure of endothall, cantharidin and its dicarboxylic acid analog. Figure 7. Structure of endothall, cantharidin and its dicarboxylic acid analog. Endothall and cantharidin both inhibit plant serine/threonine protein phosphatases in a time-dependentEndothall and manner, cantharidin suggesting both that inhibit these plant compounds serine/threonine act as slow, protein irreversible phosphatases inactivators in a oftime- the serinedependent/threonine manner, protein suggesting phosphatase that these activities compounds [33]. The act catalytic as slow, domain irreversible of all PP inactivators highly conserved of the acrossserine/threonine animals, plantsprotein and phosphatas protozoans.e activities Inhibitors, [33]. The such catalytic as cantharidin domain of and all endothall,PP highly conserved bind to a hydrophobicacross animals, pocket plants of theand PP protozoans. active site. EndothallInhibitors, issuch a very as effectivecantharidin herbicide and endothall, to manage bind weeds to ina aquatichydrophobic environments pocket of [34 the]. PP active site. Endothall is a very effective herbicide to manage weeds in aquatic environments [34]. 2.4. Pyruvate Dehydrogenase Complex (PDHc) 2.4. Pyruvate Dehydrogenase Complex (PDHc) Pyruvate dehydrogenase complex (PDHc) catalyzes the oxidative decarboxylation of pyruvate to formPyruvate acetate and dehydrogenase its subsequent complex acetylation (PDHc) of coenzyme catalyzes A the (CoA) oxidative to produce decarboxylation acetyl-CoA [ 35of]. pyruvate As such itto isform a critically acetate important and its subsequent for cellular acetylation processes. of The coenzyme complex A consists (CoA) ofto threeproduce enzymes acetyl-CoA and a number[35]. As ofsuch cofactors. it is a critically Pyruvate important dehydrogenase for cellular E1 is processes. a thiamine The diphosphate- complex consists and Mg of2 +three-dependent enzymes enzyme and a catalyzingnumber of thecofactors. first step Pyruvate of the multistep dehydrogen processase E1 associated is a thiamine with PDHc diphosphate- [35]. and Mg2+-dependent enzymeThe catalyzing Institute of the Pesticide first step and of Organicthe multistep Chemistry process of associated Central China with NormalPDHc [35]. University recently reportedThe novelInstitute cyclic of Pesticide methylphosphonates and Organic Chemistry (Figure8) that of Central target pyruvateChina Normal dehydrogenase University complex recently (PDHc)reported using novel molecular cyclic methylphos docking andphonates three-dimensional (Figure 8) that quantitative target pyruvate structure-activity dehydrogenase relationship complex studies(PDHc) [using36]. Early molecular acetylphosphinates docking and andthree-dimensiona acetylphosphonatesl quantitative analogs structure-activity had relatively low relationship herbicidal studies [36]. Early acetylphosphinates and acetylphosphonates analogs had relatively low herbicidal activity, but these structures served as the basis for structural optimization to generate 1-(substituted phenoxyacetoxy)alkylphosphonate derivatives with notably higher herbicidal activities (Figure 8) [36]. Herbicidal activity is proportional to inhibition of PDHc E1.

Plants 2019, 8, 341 10 of 18 activity, but these structures served as the basis for structural optimization to generate 1-(substituted phenoxyacetoxy)alkylphosphonate derivatives with notably higher herbicidal activities (Figure8)[ 36]. Plants 2019,, 8,, xx FORFOR PEERPEER REVIEWREVIEW 10 of 18 Herbicidal activity is proportional to inhibition of PDHc E1. Recent development reported that these PDHc inhibitors were most effective against broadleaf Recent development reported that these PDHc inhibitors were most effective against broadleaf weeds and active at rates ranging from 50 and 300 ai g/ha, whereas they had no effect on maize and rice weeds and active at rates ranging from 50 and 300 ai g/ha, whereas they had no effect on maize and even at 900 1200 ai g/ha. Some of these compounds also had activity on sedge weeds when applied at rice even at− 900−1200 ai g/ha. Some of these compounds also had activity on sedge weeds when 225 375 ai g/ha [37]. applied− at 225−375 ai g/ha [37].

FigureFigure 8. Structure 8. Structure of a potentof a potent 1-(substituted 1-(substituted phenoxyacetoxy)alkylphosphonate phenoxyacetoxy)alkylphosphonate that targets that targets pyruvate dehydrogenase complex (PDHc).pyruvate dehydrogenase complex (PDHc). 2.5. Imadazoleglycerol Phosphate Dehydratase (IGPD) 2.5. Imadazoleglycerol Phosphate Dehydratase (IGPD) Imadazoleglycerol phosphate dehydratase (IGPD) catalyzes an important step in histidine Imadazoleglycerol phosphate dehydratase (IGPD) catalyzes an important step in histidine biosynthesis in plants and microorganisms. It has been studied for many years as a potential target biosynthesis in plants and microorganisms. It has been studied for many years as a potential target for herbicides, since this enzyme does not exist in animals. A class of phloem-mobile herbicides for herbicides, since this enzyme does not exist in animals. A class of phloem-mobile herbicides (the (the triazole-phosphonates) act as potent inhibitors of IGPD [38]. Syngenta has been working on triazole-phosphonates) act as potent inhibitors of IGPD [38]. Syngenta has been working on this target this target site for many years. The triazole phosphonate inhibitor 2-hydroxy-3-(1,2,4-triazol-1-yl) site for many years. The triazole phosphonate inhibitor 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphonate (Figure9) is structurally similar to the proposed diazafulvene intermediate in propylphosphonate (Figure 9) is structurally similar to the proposed diazafulvene intermediate in IGPD catalysis [39]. Several triazole phosphonate inhibitors have activities similar to glyphosate [40] IGPD catalysis [39]. Several triazole phosphonate inhibitors have activities similar to glyphosate [40] Structurally, triazole phosphonate inhibitors consists of three parts, the triazole head, an Structurally, triazole phosphonate inhibitors consists of three parts, the triazole head, an hydroxylated linker and a phosphate mimick. The position of the hydroxy group alters the binding of hydroxylated linker and a phosphate mimick. The position of the hydroxy group alters the binding the molecules to the catalytic domain of IGPD forming either 5- or 6-membered ring chelates with one of the molecules to the catalytic domain of IGPD forming either 5- or 6-membered ring chelates with of the Mn atom [41]. one of the Mn atom [41].

Figure 9. Structure of 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphonate, an herbicidal inhibitor of Figure 9. Structure of 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphonate, an herbicidal inhibitor of imadazoleglycerol phosphate dehydratase (IGPD). imadazoleglycerolimadazoleglycerol phosphphosphate dehydratase (IGPD). 2.6. Dihydroorotate Dehydrogenase (DHODH) 2.6. Dihydroorotate Dehydrogenase (DHODH) De novo pyrimidine nucleotide biosynthesis (also known as the orotate pathway) consists of De novo pyrimidine nucleotide biosynthesis (also known as the orotate pathway) consists of six six enzymaticDe novo pyrimidine steps leading nucleotide to the formation biosynthesis of uridine (also known monophosphate as the orotate from pathway) carbamoylphosphate, consists of six enzymatic steps leading to the formation of uridine monophosphate from carbamoylphosphate, aspartate,enzymatic and steps 5-phosphoribosyl-1-pyrophosphate. leading to the formation of uridine Because monophosphate of the central rolefrom of nucleotides,carbamoylphosphate, inhibition aspartate, and 5-phosphoribosyl-1-pyrophosphate. Because of the central role of nucleotides, ofaspartate, this pathway and 5-phosphoribosyl-1-pyrophosphate. is lethal to most organisms. The Because fourth stepof the is catalyzedcentral role by dihydroorotateof nucleotides, inhibition of this pathway is lethal to most organisms. The fourth step is catalyzed by dihydroorotate dehydrogenaseinhibition of this (DHODH), pathway is which lethal carries to most out organi the ubiquinone-mediatedsms. The fourth step is oxidation catalyzed of by dihydroorotate dihydroorotate to dehydrogenase (DHODH), which carries out the ubiquinone-mediated oxidation of dihydroorotate orotatedehydrogenase [42]. (DHODH), which carries out the ubiquinone-mediated oxidation of dihydroorotate to orotate [42]. to orotateAll plant [42]. DHODHs are flavoproteins located on the outer surface of the inner mitochondrial All plant DHODHs are flavoproteins located on the outer surface of the inner mitochondrial membrane.All plant Plant DHODHs DHODHs are haveflavoproteins different substratelocated on specificity the outer and surface inhibition of the from inner the mitochondrial animal form membrane. Plant DHODHs have different substrate specificity and inhibition from the animal form ofmembrane. this enzyme Plant [43 DHODHs]. FMC Agricultural have different Solutions substrate recently specificity announced and inhibition a new herbicide from the chemical animal classform of this enzyme [43]. FMC Agricultural Solutions recently announced a new herbicide chemical class (arylof this pyrrolidinone enzyme [43]. anilide) FMC Agricultural targeting DHODH. Solutions The recently common announced chemical a name new herbicide of the flagship chemical molecule class (aryl pyrrolidinone anilide) targeting DHODH. The common chemical name of the flagship molecule currently(aryl pyrrolidinone being developed anilide) wastargeting provisionally DHODH. approved The common as tetflupyrolimet chemical name (Figure of the flagship 10). This molecule potent currently being developed was provisionally approved as tetflupyrolimet (Figure 10). This potent herbicidecurrently isbeing selective developed for grass was control provisionally in rice. Sensitiveapproved plants as tetflupyrolimet treated with tetflupyrolimet(Figure 10). This have potent no herbicide is selective for grass control in rice. Sensitive plants treated with tetflupyrolimet have no chlorosisherbicide but is selective develop afor unique grass stuntingcontrol in phenotype rice. Sens suggestingitive plants that treated they arewith lacking tetflupyrolimet a key molecule have forno chlorosis but develop a unique stunting phenotype suggesting that they are lacking a key molecule for growth (pyrimidine). The target site was discovered using a combination of forward genetic screens and metabolomics approaches and confirmed by determining intrinsic affinities of specific analogs using biochemical methods [17]. Structure-activity studies determined that the 3S-4R enantiomer is the active form of this aryl pyrrolidinone anilide, and the 3R-4S enantiomerenantiomer hadhad nono

Plants 2019, 8, 341 11 of 18 growth (pyrimidine). The target site was discovered using a combination of forward genetic screens andPlants metabolomics 2019, 8, x FOR PEER approaches REVIEW and confirmed by determining intrinsic affinities of specific analogs11 of 18 Plants 2019, 8, x FOR PEER REVIEW 11 of 18 using biochemical methods [17]. Structure-activity studies determined that the 3S-4R enantiomer herbicidal activity. Additionally, the presence of the electron withdrawing groups (fluorine) on the isherbicidal the active activity. form ofAdditionally, this aryl pyrrolidinone the presence anilide, of the electron and the withdrawing 3R-4S enantiomer groups had (fluorine) no herbicidal on the two benzyl rings and the alkylation (methyl group) of the γ-lactam heterocycle are required for activity.two benzyl Additionally, rings and thethe presence alkylation of the(methyl electron group) withdrawing of the γ-lactam groups heterocycle (fluorine) on are the required two benzyl for herbicidal activity. Tetflupyrolimet competes for the quinone binding site on DHODH. The activity ringsherbicidal and the activity. alkylation Tetflupyrolimet (methyl group) competes of the γfor-lactam the quinone heterocycle binding are requiredsite on DHODH. for herbicidal The activity activity. of tetflupyrolimet was about 10-fold greater on the foxtail DHODH enzyme (I50 = 3 nM) compared to Tetflupyrolimetof tetflupyrolimet competes was about for 10-fold the quinone greater binding on the foxtail site on DHODH DHODH. enzyme The activity (I50 = 3 of nM) tetflupyrolimet compared to rice (I50 = 33 nM). However, selectivity for rice is much greater than 10-fold, suggesting that wasrice about(I50 = 10-fold33 nM). greater However, on the selectivity foxtail DHODH for rice enzyme is much (I50 =greater3 nM) comparedthan 10-fold, to rice suggesting (I50 = 33 nM).that differential metabolism may also contribute to tolerance in rice. Additional work demonstrated that However,differential selectivity metabolism for ricemay is also much contribute greater thanto tole 10-fold,rance suggestingin rice. Additional that diff erentialwork demonstrated metabolism maythat tetflupyrolimet was much less active on animal DHODH. Commercialization of this product is alsotetflupyrolimet contribute to was tolerance much inless rice. active Additional on animal work DHODH. demonstrated Commercialization that tetflupyrolimet of this was product much less is projected to be in 2024. activeprojected on animalto be in DHODH. 2024. Commercialization of this product is projected to be in 2024.

Figure 10. Structure of tetflupyrolimet, an aryl pyrrolidinone anilide targeting Dihydroorotate Figure 10.10. StructureStructure of of tetflupyrolimet, tetflupyrolimet, an aryl pyrrolidinone anilide targetingtargeting DihydroorotateDihydroorotate dehydrogenase, a key enzyme in pyrimidine biosynthesis. dehydrogenase, aa keykey enzymeenzyme inin pyrimidinepyrimidine biosynthesis.biosynthesis. 2.7. Peptide Deformylase 2.7. Peptide Deformylase In higher plants, synthesis of plastidplastid encoded proteins is initiated with N-formylmethionine. In higher plants, synthesis of plastid encoded proteins is initiated with N-formylmethionine. Removal of the N-formyl group by a peptide deformylasedeformylase and the methionine by methionine amino Removal of the N-formyl group by a peptide deformylase and the methionine by methionine amino peptidase isis necessarynecessary to to produce produce the the mature mature protein. protein. The The initiator initiator methionine methionine is sometimes is sometimes retained retained [44]. peptidase is necessary to produce the mature protein. The initiator methionine is sometimes retained Peptide[44]. Peptide deformylase deformylase is the targetis the of target actinonin, of actinonin, an hydroxamic an hydroxamic acid microbial acid metabolite microbial produced metabolite by [44]. Peptide deformylase is the target of actinonin, an hydroxamic acid microbial metabolite soilproduced actinomycetes by soil actinomycetes (Figure 11)[45 (Figure]. This unique11) [45]. MOA This unique has received MOA a has lot ofreceived interest a andlot of the interest herbicidal and produced by soil actinomycetes (Figure 11) [45]. This unique MOA has received a lot of interest and activitythe herbicidal of actinonin activity has beenof actinonin patented, has but nobeen commercial patented, product but no has commercial been developed. product Plants has treated been the herbicidal activity of actinonin has been patented, but no commercial product has been withdeveloped. actinonin Plants are treated stunted with with actinonin bleached are foliage stunted which with ultimatelybleached foliage develop which necrotic ultimately lesions. develop It has developed. Plants treated with actinonin are stunted with bleached foliage which ultimately develop provednecrotic e lesions.ffective onIt has many proved important effective weed on speciesmany important [46,47]. weed species [46,47]. necrotic lesions. It has proved effective on many important weed species [46,47].

Figure 11. FigureStructure 11. Structure of actinonin, of actinonin, a microbial a microbial metabolite metabolite targeting targeting chloroplastic chloroplastic peptide peptide deformylase. Figure 11. Structure of actinonin, a microbial metabolite targeting chloroplastic peptide deformylase. 2.8. DNA Gyrase deformylase. 2.8. DNADNA Gyrase gyrases are prokaryotic Type II topoisomerases that were thought to be absent from most 2.8. DNA Gyrase eukaryotes. However, ancestral forms of DNA gyrases may be present in certain organelles of plants and DNA gyrases are prokaryotic Type II topoisomerases that were thought to be absent from most apicomplexans,DNA gyrases although are prokaryotic their exact Type functions II topoisomerases in replication arethat not were well thought understood. to be absent A research from group most eukaryotes. However, ancestral forms of DNA gyrases may be present in certain organelles of plants fromeukaryotes. the University However, of Western ancestral Australia forms of investigated DNA gyrases DNA may gyrase be present as a potential in certain herbicide organelles target of siteplants by and apicomplexans, although their exact functions in replication are not well understood. A research testingand apicomplexans, the activity of although a number their of compounds exact functions likely in to replication interact with are not this well enzyme. understood. Several A molecules, research group from the University of Western Australia investigated DNA gyrase as a potential herbicide includinggroup from the the antimicrobial University ciprofloxacin of Western (FigureAustralia 12 ),investigated were herbicidal DNA by gyrase inhibiting as thea potential function herbicide of gyrase target site by testing the activity of a number of compounds likely to interact with this enzyme. intarget higher site plants by testing [48]. Threethe activity genes (ofATGYRA a number, ATGYRB1 of compounds, ATGYRB2 likely) encoding to interact for plantwith gyrasesthis enzyme. were Several molecules, including the antimicrobial ciprofloxacin (Figure 12), were herbicidal by inhibiting identifiedSeveral molecules, in Arabidopsis including thaliana the. Forwardantimicrobial genetic ciprofloxacin approaches (Figure led to the12), discovery were herbicidal of a point by inhibiting mutation the function of gyrase in higher plants [48]. Three genes (ATGYRA, ATGYRB1, ATGYRB2) encoding intheATGYRA functionthat of gyrase confers in resistance higher plants to ciprofloxacin, [48]. Three thusgenes confirming (ATGYRA that, ATGYRB1 this gene, ATGYRB2 encodes a) functional encoding for plant gyrases were identified in Arabidopsis thaliana. Forward genetic approaches led to the organelle-localizedfor plant gyrases were DNA identified gyrase that in is Arabidopsis the target of thaliana quinolone. Forward antimalarial genetic drugs approaches [49]. led to the discovery of a point mutation in ATGYRA that confers resistance to ciprofloxacin, thus confirming discovery of a point mutation in ATGYRA that confers resistance to ciprofloxacin, thus confirming that this gene encodes a functional organelle-localized DNA gyrase that is the target of quinolone that this gene encodes a functional organelle-localized DNA gyrase that is the target of quinolone antimalarial drugs [49]. antimalarial drugs [49]. Subsequent work exploring the activity of ciprofloxacin analogs on DNA gyrase led to the Subsequent work exploring the activity of ciprofloxacin analogs on DNA gyrase led to the characterization of the pharmacophore scaffold required for activity and the discovery of structures characterization of the pharmacophore scaffold required for activity and the discovery of structures

Plants 2019, 8, x FOR PEER REVIEW 12 of 18

with improved herbicidal efficacy and diminished antibacterial activity, relative to ciprofloxacin. The optimized experimental analog 44 (Figure 12) had an ethyl side chain and a piperidine ring instead of a cyclopropyl side chain and a piperazine ring attached to the fluoroquinolone scaffold. This molecule was slightly less herbicidal than ciprofloxacin, but its specificity for plant DNA gyrase was Plantssuperior,2019, 8, 341leading to a 600-fold increase in selectivity for plants relative to other organisms [50]. 12 of 18

PlantsSubsequent 2019, 8, x FOR workPEER REVIEW exploring the activity of ciprofloxacin analogs on DNA gyrase led12 to of the18 characterization of the pharmacophore scaffold required for activity and the discovery of structures withwith improved improved herbicidal herbicidal eefficacyfficacy andand diminisheddiminished antibacterialantibacterial activity, relative to to ciprofloxacin. ciprofloxacin. The The optimizedoptimized experimental experimental analog analog 44 44 (Figure (Figure 12 12)) had had an an ethyl ethyl side side chain chain and and a a piperidine piperidine ring ring instead instead of aof cyclopropyl a cyclopropyl side chainside chain and a and piperazine a piperazine ring attached ring attached to the fluoroquinoloneto the fluoroquinolone scaffold. scaffold. This molecule This wasmolecule slightly was less slightly herbicidal less herbicidal than ciprofloxacin, than ciprofloxacin, but its specificity but its specificity for plant for DNA plant gyrase DNA was gyrase superior, was leadingsuperior, to aleading 600-fold to a increase 600-fold in increase selectivity in selectivity for plants for relative plants to relative other organisms to other organisms [50]. [50]. Figure 12. Structure of the fluoroquinolone ciprofloxacin and structure-optimized analog 44 with increased specificity against plants DNA gyrase and better herbicidal profile.

2.9. Dihydrofolate Reductase (DHFR) The biosynthesis of folate has been the target for pharmaceutical and agrochemical discovery (Figure 13). Folate is an important metabolite required for the synthesis of numerous compounds necessary for plant growth and development. To date, asulam (Figure 13) is only one commercial herbicide to inhibit this pathway by targeting 7,8-dihydropteroate synthetase [51]. This carbamate herbicide is an analogue of 4-aminobenzoate, one of the substrates of 7,8-dihydropteroate synthase, Figure 12. Structure of the fluoroquinolone ciprofloxacin and structure-optimized analog 44 with andFigure its selectivity 12. Structure is based of theon differentialfluoroquinolone metabolic ciprofloxa degradation.cin and structure-optimized analog 44 with increased specificity against plants DNA gyrase and better herbicidal profile. increasedAnother specificity enzyme againstin this plantspathway, DNA dihydrofolgyrase and atebetter reductase herbicidal (DHFR) profile. (Figure 12) has already 2.9.received Dihydrofolate some interest Reductase as (DHFR)a target for drug development due to its essential role in the synthesis of 2.9. Dihydrofolate Reductase (DHFR) DNA precursors and some amino acids. A recent study by a group at the University of Western The biosynthesis of folate has been the target for pharmaceutical and agrochemical discovery AustraliaThe biosynthesis identified DHFR of folate as a haspotential been newthe target target forfor pharmaceuticalherbicides based and on theagrochemical herbicidal activity discovery of (Figure 13). Folate is an important metabolite required for the synthesis of numerous compounds (Figureantimalarial 13). Folate compounds is an important such as pyrimethamine metabolite re quiredand cycloguanil for the synthesis (Figure 13)of numerous[52]. The requirement compounds necessary for plant growth and development. To date, asulam (Figure 13) is only one commercial necessaryof two of for the plant three growth isoforms and of development.DHFR for seed To development date, asulam was (Figure identified 13) is byonly knockout one commercial mutant herbicide to inhibit this pathway by targeting 7,8-dihydropteroate synthetase [51]. This carbamate herbicideanalysis. toValidation inhibit this of pathwaythis enzyme by targeting as a new 7,8-dihydropteroate target site was confirmed synthetase by [51].screening This carbamate mutated herbicide is an analogue of 4-aminobenzoate, one of the substrates of 7,8-dihydropteroate synthase, herbicideArabidopsis is anthaliana analogue seeds of for 4-aminobenzoate, resistance to these one antimalarial of the substrates compounds. of 7,8-dihydropteroate A G137D mutation synthase, in the and its selectivity is based on differential metabolic degradation. andisoform its selectivity 1 of DHFR is basedand a. on differential metabolic degradation. Another enzyme in this pathway, dihydrofolate reductase (DHFR) (Figure 12) has already received some interest as a target for drug development due to its essential role in the synthesis of DNA precursors and some amino acids. A recent study by a group at the University of Western Australia identified DHFR as a potential new target for herbicides based on the herbicidal activity of antimalarial compounds such as pyrimethamine and cycloguanil (Figure 13) [52]. The requirement of two of the three isoforms of DHFR for seed development was identified by knockout mutant analysis. Validation of this enzyme as a new target site was confirmed by screening mutated Arabidopsis thaliana seeds for resistance to these antimalarial compounds. A G137D mutation in the isoform 1 of DHFR and a.

Figure 13. Simplified biosynthesis of folate in higher plants and structures of herbicidal compounds targeting this pathway. HPPK: 6-hydroxymethyldihydropterin pyrophosphokinase; DHPS: dihydropteroate synthase; DHFS: dihydrofolate synthetase; DHFR: dihydrofolate reductase; FPGS: folylpolyglutamate synthetase; ADCS: aminodeoxychorismate synthase; ADCL: aminodeoxychorismate lyase.

Another enzyme in this pathway, dihydrofolate reductase (DHFR) (Figure 12) has already received some interest as a target for drug development due to its essential role in the synthesis of DNA

Plants 2019, 8, 341 13 of 18 precursors and some amino acids. A recent study by a group at the University of Western Australia identified DHFR as a potential new target for herbicides based on the herbicidal activity of antimalarial compounds such as pyrimethamine and cycloguanil (Figure 13)[52]. The requirement of two of the Plants 2019, 8, x FOR PEER REVIEW 13 of 18 three isoforms of DHFR for seed development was identified by knockout mutant analysis. Validation of thisFigure enzyme 13. asSimplified a new target biosynthesis site was of folate confirmed in higher by plants screening and structures mutated Arabidopsisof herbicidal thalianacompoundsseeds for resistancetargeting to these this antimalarial pathway. compounds.HPPK: 6-hydroxymethyldihydropterin A G137D mutation in the pyrophosphokinase; isoform 1 of DHFR DHPS: and a A71V mutationdihydropteroate in isoform 2 synthase; of DHFR DHFS: imparted dihydrofolate resistance, synthetase; confirming DHFR: that dihydrof the herbicidalolate reductase; activity FPGS: associated with thefolylpolyglutamate antimalarial molecules synthetase; were dueADCS: to inhibition aminodeoxychorismate of DHFR. This discovery synthase; sets the stageADCL: for high throughputaminodeoxychorismate screening of chemical lyase. libraries to identify molecules with better herbicidal profile.

3.3. New New Insight Insight on on Known Known MechanismsMechanisms ofof ActionAction

3.1.3.1. New New Insight Insight on on Glufosinate Glufosinate MechanismMechanism ofof ActionAction TheThe MOA MOA of glufosinateof glufosinate has beenhas been studied studied extensively. extensively. While theWhile inhibition the inhibition of glutamine of glutamine synthetase andsynthetase subsequent and accumulation subsequent accumulation of ammonia, disruptionof ammonia, of aminodisruption acid balance,of amino and acid reduction balance, of and both thereduction light and of dark both reaction the light of and photosynthesis dark reaction are of wellphotosynthesis documented, are these well diddocumented, not account these for thedid rapidnot desiccationaccount for of the the rapid foliage desiccation induced by of glufosinate. the foliage Newinduced insight by glufosinate. on the factors New contributing insight on to the the factors contact activitycontributing of glufosinate to the contact has been activity reported of glufosinate by the Weed has Researchbeen reported Laboratory by the atWeed Colorado Research State Laboratory University. Glufosinateat Colorado triggers State University. a rapid and Glufosinate massive triggers production a rapid of reactiveand massive oxygen production species of (ROS) reactive driving oxygen the catastrophicspecies (ROS) lipid driving peroxidation the catastrophic of the cell lipid membranes peroxidation and rapidof the cellcell deathmembranes (Figure and 14A) rapid [53]. cell The death effect was(Figure proportional 14A) [53]. to The absorption effect was of proportional the herbicide. to Interestingly,absorption of youngthe herbicide. leaves wereInterestingly, less sensitive young to glufosinate.leaves were While less sensitive older leaves to glufosinate. absorbed While more glufosinateolder leaves thanabsorbed younger more tissues glufosinate (Figure than 14B), younger similar levelstissues of glutamine(Figure 14B), synthetase similar levels inhibition of glutamine and ammonia synthetase accumulation inhibition were and observedammonia (Figureaccumulation 14C,D), indicatingwere observed that ammonia (Figure accumulation14C, D), indicating was not that responsible ammonia foraccumulation the toxicity was of this not herbicide. responsible In contrast,for the glufosinatetoxicity of inducedthis herbicide. a rapid In andcontrast, massive glufosinate accumulation induced of a ROS rapid in and older massive tissue accumulation and almost no of ROS ROS in youngerin older tissue tissue (Figure and almost 14E), no which ROS correlatedin youngerdirectly tissue (Figure with the 14E), level which of injury. correlated directly with the level of injury.

A

FigureFigure 14. 14.New New insight insight intointo thethe mechanismsmechanisms of action (MOA) of glufosinate. glufosinate. (A (A) )Older Older leaves leaves are are more more sensitivesensitive to to glufosinate glufosinate than than meristematicmeristematic tissuetissue and younger leaves. leaves. ( (BB) )Absorption Absorption of of glufosinate. glufosinate. (C()C Inhibition) Inhibition of of glutamine glutamine synthetase synthetase (GS). (GS). ((DD)) AmmoniaAmmonia accumulation. ( (EE)) Reactive Reactive oxygen oxygen species species (ROS)(ROS) accumulation. accumulation. Reproduced Reproduced fromfrom TakanoTakano etet al.al. 2019 with permission.

3.2.3.2. New New Insight Insight on on Slow-Binding Slow-Binding PropertiesProperties ofof HPPDHPPD Inhibitors AsAs mentioned mentioned inin SectionSection 2.2,2.2, inhibition inhibition of of plastoquinone plastoquinone biosynthesis biosynthesis has has been been recognized recognized as an as anexcellent excellent target target for fornew new herbicide herbicide research research since sincethe discovery the discovery that triketone that triketone herbicides herbicides inhibit HPPD. inhibit HPPD.These herbicides These herbicides bind slowly bind but slowly very tightly but very (nearly tightly irreversibly) (nearly irreversibly) to the catalytic to site the by catalytic coordinating site by with Fe atom involved in catalysis [54]. A research group based at Central China Normal University provided new molecular insights into the mechanism of 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibition using enzyme kinetics, X-ray crystallography of Arabidopsis thaliana HPPD complexed with herbicides, and computational simulations approaches. This work dissected the interaction between ligand and receptor to discover a novel quinazoline-2.4-dione herbicide

Plants 2019,, 8,, 341x FOR PEER REVIEW 14 of 18 benquitrione (Y13161) (Figure 15) [55]. Their analysis suggests that the slow binding properties of coordinating with Fe atom involved in catalysis [54]. A research group based at Central China HPPD inhibitors may be related to steric hindrance requiring a conformational change on the enzyme Normal University provided new molecular insights into the mechanism of 4-hydroxyphenylpyruvate upon herbicide binding. Benquitrione has excellent herbicidal activity that compares favorably with dioxygenase (HPPD) inhibition using enzyme kinetics, X-ray crystallography of Arabidopsis thaliana that of mesotrione. This molecule also demonstrated selectivity on corn and sorghum, whereas the HPPD complexed with herbicides, and computational simulations approaches. This work dissected mesotrione caused injury to sorghum. Finally, the structural features of benquitrione can also serve Plantsthe interaction 2019, 8, x FOR between PEER REVIEW ligand and receptor to discover a novel quinazoline-2.4-dione herbicide14 of 18 as a template to develop the next generation of high performance HPPD-inhibiting herbicides. benquitrione (Y13161) (Figure 15)[55]. Their analysis suggests that the slow binding properties of benquitrioneHPPD inhibitors (Y13161) may be (Figure related 15) to steric[55]. Their hindrance analys requiringis suggests a conformational that the slow changebinding on properties the enzyme of HPPDupon herbicide inhibitors binding. may be related Benquitrione to steric has hindrance excellent requiring herbicidal a conf activityormational that compares change favorablyon the enzyme with uponthat of herbicide mesotrione. binding. This Benquitrione molecule also has demonstrated excellent herbicidal selectivity activity on corn that and compares sorghum, favorably whereas with the thatmesotrione of mesotrione. caused injuryThis molecule to sorghum. also Finally, demonstrated the structural selectivity features on corn of benquitrione and sorghum, can alsowhereas serve the as mesotrionea template tocaused develop injury the nextto sorghum. generation Finally, of high the performance structural features HPPD-inhibiting of benquitrione herbicides. can also serve as a template to develop the next generation of high performance HPPD-inhibiting herbicides. Figure 15. Structure of novel HPPD inhibitor Y13161 (benquitrione).

4. Promising New Chemistry

4.1. Isoxazolopyridine Herbicides

BASF recently reported a new class of herbicides based on an isoxazolopyridine (OXP) backbone Figure 15. Structure of novel HPPD inhibitor Y13161 (benquitrione). (Figure 16). Some ofFigure the compounds 15. Structure ofhave novel selectivity HPPD inhibitor on monocotyledonous Y13161 (benquitrione). crops while providing 4.excellent Promising post-emergence New Chemistry control of dicot weeds and good activity on some grasses. Sensitive plants 4.treated Promising with OXPNew Chemistryherbicides develop necrosis on the foliage and the compounds appear to have 4.1.systemic Isoxazolopyridine activity, with Herbicides phloem-mobility in dicots but limited translocation in monocots. 4.1. IsoxazolopyridineBiochemical/physiological Herbicides studies excluded known MOA. Therefore, isoxazolopyridines may BASF recently reported a new class of herbicides based on an isoxazolopyridine (OXP) backbone have a novel mechanism of action on plant photosystems that involves carbohydrate metabolism (FigureBASF 16). recently Some of reported the compounds a new class have of herbicides selectivity based on monocotyledonous on an isoxazolopyridine crops while(OXP) providing backbone within the chloroplast. Cellular thermal shift assays suggest that light harvesting protein may be the (Figureexcellent 16). post-emergence Some of the compounds control of dicot have weeds selectivity and good on monocotyledonous activity on some grasses. crops while Sensitive providing plants potential target. Interestingly, a L218V mutation on the D1 protein of photosystem II from excellenttreated with post-emergence OXP herbicides control develop of dicot necrosis weeds onand the good foliage activity and on the some compounds grasses. Sensitive appear to plants have metamitron tolerant Chenopodium album (Common lambsquarters) provided partial resistance to treatedsystemic with activity, OXP with herbicides phloem-mobility develop necrosis in dicots on but the limited foliage translocation and the compounds in monocots. appear to have systemicOXPs, whereas activity, the with well-known phloem-mobility S264G mutation in dicots did but not limited protect translocation plants against in monocots. the activity of OXPs. Biochemical/physiological studies excluded known MOA. Therefore, isoxazolopyridines may have a novel mechanism of action on plant photosystems that involves carbohydrate metabolism within the chloroplast. Cellular thermal shift assays suggest that light harvesting protein may be the potential target. Interestingly, a L218V mutation on the D1 protein of photosystem II from metamitron tolerant Chenopodium album (Common lambsquarters) provided partial resistance to OXPs, whereas the well-known S264G mutation did not protect plants against the activity of OXPs.

Figure 16. Structural features of the chemical classes mentioned in Section4. Figure 16. Structural features of the chemical classes mentioned in Section 4. Biochemical/physiological studies excluded known MOA. Therefore, isoxazolopyridines may have4.2. Isoxazoline-Substituted a novel mechanism Uracil of action Herbicides on plant photosystems that involves carbohydrate metabolism withinSinochem the chloroplast. Agrochemicals Cellular thermalR&D Co. shift Ltd. assays develo suggestped novel that light uracil harvesting herbicides protein containing may be thean isoxazolopyrimidine ring (Figure 16). These compounds effectively control a number of economically

Figure 16. Structural features of the chemical classes mentioned in Section 4.

4.2. Isoxazoline-Substituted Uracil Herbicides Sinochem Agrochemicals R&D Co. Ltd. developed novel uracil herbicides containing an isoxazolopyrimidine ring (Figure 16). These compounds effectively control a number of economically

Plants 2019, 8, 341 15 of 18 potential target. Interestingly, a L218V mutation on the D1 protein of photosystem II from metamitron tolerant Chenopodium album (Common lambsquarters) provided partial resistance to OXPs, whereas the well-known S264G mutation did not protect plants against the activity of OXPs.

4.2. Isoxazoline-Substituted Uracil Herbicides Sinochem Agrochemicals R&D Co. Ltd. developed novel uracil herbicides containing an isoxazolopyrimidine ring (Figure 16). These compounds effectively control a number of economically important monocot and dicots weeds and appears to be safe to wheat, corn and rice. One of the most promising molecules (SYP-1604) can be used alone or in combination with other herbicides. SYP-1604 spectrum of activity outperforms saflufenacil when applied post and compared favorably to flumioxazin when applied pre. Little is known about its MOA, but it appears to act as an inhibitor of protoporphyrinogen oxidase.

4.3. Benzoxaboroles Herbicides Benzoxaboroles (Figure 16) are derivatives of boronic acids that were first described over 50 years ago. Interest in these compounds renewed in 2006, after reports of the sugar-binding properties of certain benzoxaboroles were made public. Consequently, most of benzoxaboroles have been described over the last decade [56]. Interest in these structures widened due to their range of biological activities, including the commercialization of two pharmaceutical products. Benzoxaboroles have recently been considered as starting points for new herbicides. Scientists at Corteva explored the chemical space occupied by benzoxaboroles and uncovered a promising area for new herbicide discovery. A limiting factor is the relatively high pka of boronic acid (8.8), but structures with constricted rings, such as the benzoxaboroles, can be designed with lower pka values A number of benzoxaboroles were identified herbicidal hits in initial screens, and plants treated with this type of chemistry developed unique symptoms. However, rates required for activity were high (1–4 kg ha-1). Symptoms of plants treated with this class of chemistry varied from stunting to bleaching and necrosis on the leaf margin. While no target site has been identified or reported to date, structure-activity work demonstrated that adding a methyl group enhanced the activity. Replacing this methyl group with cyano group was even more potent. Interestingly, increasing the steric bulk by adding a phenyl ring was advantageous, but decoration with electron withdrawing groups (i.e., chlorophenyl) has a negative impact. Finally, adding alkyl spacer to the phenyl ring positively modulated activity. While no herbicides have arisen from this chemical class, isoxaboroles may lead to new scaffolds to develop new phloem-mobile molecules.

5. Conclusions The time for innovative MOA and chemistry targeting these is overdue. The current crisis experienced by farmers facing difficulties managing weeds that have evolved resistance to many of the existing MOA must be addressed, and new weed management tools are necessary. While the current renewed interest in research and development programs observed in the agrichemical industry, as well as academic and governmental institutions is a positive development, there is no silver bullet chemistry ready to enter the marketplace. Many of the MOA and associated chemistries described in this review are at least 5 years away from commercialization or will fail to reach commercialization due to the many hurdles facing such a process. The aggregation of the agrichemical industry is certain to continue, further limiting diversity in creativity and discovery. One may hope that the few startup companies using truly innovative approaches to herbicide discovery will provide platforms to explore new chemical spaces and biochemical processes. It may also be time to incorporate non-chemical means of weed control, such as mechanical seed destruction (e.g., Harrington seed destructor), robotic weed management, and precision farming.

Funding: This work was funded by the USDA National Institute of Food and Agriculture, Hatch Project 1016591, COL00785. Plants 2019, 8, 341 16 of 18

Acknowledgments: The author thanks presenters at the Herbicide Mechanisms of Action and Resistance session at the 2019 IUPAC congress in Ghent, Belgium and the New Herbicides and Their Modes of Action symposium at the 2019 ACS national meeting in San Diego for sharing some of their most recent discoveries. Conflicts of Interest: The author declares no conflict of interest.

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