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Structure-based design of agrochemicals Magnus W. Walter* Eli Lilly, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey, UK GU20 6PH. E-mail: [email protected] Received (in Cambridge) 1st February 2002 First published as an Advance Article on the web 25th March 2002 Covering: up to the present This review covers recent advances in the structure-based design of agrochemicals. The three main sectors of agrochemistry, i.e. herbicides, insecticides, and fungicides are covered. The literature in this eld to the present date is reviewed and 88 references are cited. 1 Introduction 1.1 Lead generation in agrochemistry 1.2 Structure-based design (SBD) 2 Herbicides 3 Insecticides 4 Fungicides 5 In vitro to in vivo translation of activity 6 Conclusions and outlook 7 Acknowledgements 8 References 1 Introduction Chemical crop protection plays a vital role in ensuring sucient food supply to a growing world population. In the face of ever more stringent demands with regard to ecacy and environ- mental safety, the discovery of new agrochemicals has become a dicult and resource-intensive undertaking. Whilst most of the compounds listed in the current Pesticide Manual were dis- covered through random screening programmes, the call for a more rational approach to the design of agrochemicals has been made for some considerable time. 1–5 After two decades of phenomenal developments in molecular biology and structural biology it seems timely to review the impact of structure-based design on agrochemical lead generation. Magnus W. Walter was born in Sweden and grew up in Germany. He received his rst degree (Diplomchemiker) from the Uni- versity of Bonn in 1993. From 1993 to 1997 he studied at the University of Oxford in Professor Sir Jack Baldwin’s research group in the Dyson Perrins Laboratory. In 1997 he obtained a DPhil for his work on resistance against β-lactam antibiotics. In 1998 he joined Jealott’s Hill Research Station (formerly Zeneca Magnus W. Walter Agrochemicals, now Syngenta) as team leader. After initial work in the lead-follow-up group, he moved into structure-based design. Since 2001 he has worked in Eli Lilly’s neuroscience research division as medicinal chemistry team leader. His main research interests are the rational design and synthesis of biologically active compounds. In addition to his industrial research career, he is lecturer and director of studies at St Hugh’s College, University of Oxford. 1.1 Lead generation in agrochemistry The aim of lead generation both in agrochemical and in pharmaceutical research is the discovery of molecules which cause a specic biological eect (Fig. 1). In the case of agrochemicals this means control of weeds (herbicides), fungi (fungicides) or insects (insecticides). Lead generation is the earliest stage in the discovery process of agrochemicals. Although terminology diers from company to company, in the context of this review a lead is understood to be a chemical compound with dened biological activity against an agrochemically important target species. Pharma- ceutical leads are usually dened as having inhibitory activity against an enzyme or (ant)agonistic activity against a receptor, i.e. in vitro activity. Agrochemical leads were historically and still are expected to show activity against the (living) target organism, i.e. in vivo activity. The emphasis on in vivo activity reects the fact that agrochemicals can be screened directly against the target organism. There is no need for the lengthy evaluation required before pharmaceutical leads can be tested in their target organism, i.e. in humans. However, before dis- missing the agrochemical lead discovery process as “easier” than the pharmaceutical one, several aspects need to be con- Fig. 1 Lead generation of agrochemicals and pharmaceuticals. 278 Nat. Prod. Rep., 2002, 19, 278–291 DOI: 10.1039/b100919m This journal is © The Royal Society of Chemistry 2002 Published on 25 March 2002. Downloaded by Temple University on 26/10/2014 17:14:07. View Article Online / Journal Homepage / Table of Contents for this issue

Structure-based design of agrochemicals

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Page 1: Structure-based design of agrochemicals

Structure-based design of agrochemicals

Magnus W. Walter*

Eli Lilly, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey, UK GU20 6PH.E-mail: [email protected]

Received (in Cambridge) 1st February 2002First published as an Advance Article on the web 25th March 2002

Covering: up to the present

This review covers recent advances in the structure-based design of agrochemicals. The three main sectors ofagrochemistry, i.e. herbicides, insecticides, and fungicides are covered. The literature in this field to the present dateis reviewed and 88 references are cited.

1 Introduction1.1 Lead generation in agrochemistry1.2 Structure-based design (SBD)2 Herbicides3 Insecticides4 Fungicides5 In vitro to in vivo translation of activity6 Conclusions and outlook7 Acknowledgements8 References

1 Introduction

Chemical crop protection plays a vital role in ensuring sufficientfood supply to a growing world population. In the face of evermore stringent demands with regard to efficacy and environ-mental safety, the discovery of new agrochemicals has become adifficult and resource-intensive undertaking. Whilst most of thecompounds listed in the current Pesticide Manual were dis-covered through random screening programmes, the call fora more rational approach to the design of agrochemicals hasbeen made for some considerable time.1–5 After two decades ofphenomenal developments in molecular biology and structuralbiology it seems timely to review the impact of structure-baseddesign on agrochemical lead generation.

Magnus W. Walter was born in Sweden and grew up in Germany.He received his first degree (Diplomchemiker) from the Uni-versity of Bonn in 1993. From 1993 to 1997 he studied at theUniversity of Oxford in Professor Sir Jack Baldwin’s researchgroup in the Dyson Perrins Laboratory. In 1997 he obtained aDPhil for his work on resistance against β-lactam antibiotics. In1998 he joined Jealott’s Hill Research Station (formerly Zeneca

Magnus W. Walter

Agrochemicals, now Syngenta) asteam leader. After initial work inthe lead-follow-up group, he movedinto structure-based design. Since2001 he has worked in Eli Lilly’sneuroscience research division asmedicinal chemistry team leader.His main research interests are therational design and synthesis ofbiologically active compounds. Inaddition to his industrial researchcareer, he is lecturer and directorof studies at St Hugh’s College,University of Oxford.

1.1 Lead generation in agrochemistry

The aim of lead generation both in agrochemical and inpharmaceutical research is the discovery of molecules whichcause a specific biological effect (Fig. 1). In the case of

agrochemicals this means control of weeds (herbicides), fungi(fungicides) or insects (insecticides).

Lead generation is the earliest stage in the discovery processof agrochemicals. Although terminology differs from companyto company, in the context of this review a lead is understood tobe a chemical compound with defined biological activityagainst an agrochemically important target species. Pharma-ceutical leads are usually defined as having inhibitory activityagainst an enzyme or (ant)agonistic activity against a receptor,i.e. in vitro activity. Agrochemical leads were historically andstill are expected to show activity against the (living) targetorganism, i.e. in vivo activity. The emphasis on in vivo activityreflects the fact that agrochemicals can be screened directlyagainst the target organism. There is no need for the lengthyevaluation required before pharmaceutical leads can be testedin their target organism, i.e. in humans. However, before dis-missing the agrochemical lead discovery process as “easier”than the pharmaceutical one, several aspects need to be con-

Fig. 1 Lead generation of agrochemicals and pharmaceuticals.

278 Nat. Prod. Rep., 2002, 19, 278–291 DOI: 10.1039/b100919m

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sidered. Early in vivo activity studies of agrochemical leads aredone under glass house conditions or even in highly artificial invivo high-throughput-screening (HTS) set-ups. The transitionto field tests represents a significant hurdle which a large pro-portion of leads fail even after optimisation work. In additionto activity in the field, the (biochemical) mode of action ofagrochemical leads has to be established as part of the regis-tration for new agrochemical products. This may require isol-ation and characterisation of the molecular target, i.e. enzymeor receptor. Hence, it could be argued that in vitro activitystudies are carried out at a later stage with in vivo activity beingused as an initial ‘filter’.

Given the importance of in vivo activity the agrochemicalindustry has heavily invested in in vivo HTS technology. It isnow possible to test large numbers of compounds (�100000 peryear) for whole organism activity against a variety of targetspecies from plants, fungi and insects. The emphasis ofpharmaceutical lead discovery has been on in vitro HTS. Bothin vitro and in vivo tests require large numbers of compoundsand produce relatively few hits. As it is the case in pharma-ceutical lead discovery, agrochemical lead discovery uses bothrational and random methods to generate compounds forscreening. Compounds for “random screening” can comefrom historic compound collections, combinatorial chemistry,natural products or even imitative approaches (“me-too-chemistry”). Rational design of compounds for screening refersto methods which pre-select or design compounds on the basisof information about the target site of action—structure-baseddesign—or on the basis of structural similarity to known activecompounds. The enormous capacity of HTS to test compoundstogether with the ability of combinatorial chemistry to deliverlarge numbers of compounds in a time- and cost-effectivemanner has led to a strong emphasis of non-rational methodsfor lead generation over the last decade. More recently it hasbeen realised that the most efficient way of harnessing theadvances in technology could lie in a combination of rationaland non-rational methods, i.e. combinatorial exploration oflead templates derived from structural data.6–8

1.2 Structure-based design (SBD)

SBD is a term used to describe a process in which structuralinformation about binding of a molecule (usually an inhibitor)to an enzyme (very often at or near the active site) is used todesign other molecules with improved binding properties in aniterative process. SBD is routinely used in medicinal chemistryand has made significant contributions to the discovery of anumber of marketed drugs.8–11 At the start of an SBD project asuitable target enzyme (or receptor) has to be chosen. In orderto justify the significant investment required by SBD it is essen-tial that the target is “validated”. This means there has to besubstantial experimental evidence that binding to the target willhave the desired (lethal) effect on the organism. In the case ofagrochemicals, commercial pesticides with a known mode ofaction validate a molecular target most convincingly. New tar-gets are, of course, considerably more interesting from a com-mercial point of view, but validation of such new targets can bedifficult.12 As genomic and proteomic data becomes increas-ingly available, methods to establish the role of enzymes inagrochemical target organisms and their suitability as targetsfor agrochemicals will be further developed. In addition to suchgeneral considerations, there are additional requirements whichare specific for SBD. Foremost amongst those is readily access-ible, three-dimensional structural data of enzyme–inhibitorcomplexes. This largely rules out (at least at present) membraneproteins or large protein assemblies as targets for SBD. Themost accurate structural data is provided by high resolutionx-ray crystallographic images and NMR data of protein–inhibitor or protein–substrate complexes. Prior availability ofsuch data constitutes an ideal case. Thanks to rapid advances in

protein isolation, purification and crystallization, the numberof protein structures in the public domain (PDB database) 13 iscurrently greater than 15000 and growing rapidly.14 In theabsence of a structure of the enzyme with inhibitor bound,SBD can be based on an enzyme–inhibitor complex model.Since the difficulty of modelling structures accurately increasesdramatically with molecule size it is usually easier to dock themodel of an inhibitor into a pre-existing x-ray structure of theenzyme. Docking of inhibitors into enzymes is a computation-ally demanding process whose success depends to a large extenton the quality of the scoring function which is used to assess thebinding energy of each inhibitor pose. If the structure of theprotein of interest is not available models can sometimes bederived from homologous enzymes whose structure is known(homology modelling). The fewer experimentally determinedstructural data are available, the more carefully the modellingresults have to be interpreted and the more difficult SBDbecomes.

SBD is a resource intensive approach to lead generationrequiring pure protein in relatively large quantities, access tox-ray facilities, and appropriate computational and modellingtools. It is also a multi-disciplinary endeavour which relieson efficient communication between chemists, biochemists,biophysicists and computational chemists. Whilst SBD iscommonly used by medicinal chemists in the design of newpharmaceuticals there are far fewer examples for its applicationin the search for new agrochemicals. The aim of this review is togive an overview of such attempts and document the changingrole of SBD in agrochemistry from an exotic outsider to aroutine method of lead generation. The three traditional sec-tors of agrochemistry (herbicides, insecticides, and fungicides)will be reviewed separately before concluding with a briefdiscussion on the impact of emerging technologies, such asgenomics and proteomics, on the lead discovery process inagrochemistry and SBD within it.

SBD is one of several rational approaches to the design ofpesticides. Others include 3D-QSAR studies, model- ortoxophore-based design and “biochemically inspired” designmethods. These methods have been used in agrochemistry forsome time and have been reviewed elsewhere.1–5,15–17 An exhaust-ive coverage of these approaches is beyond the scope of thisreview which will be restricted to studies involving structuraldata of target enzymes or binding sites.

Only references and material in the public domain have beenused for this article. In an area of great interest to industrialresearch, such as SBD, it is very likely that a lot more materialhas not been made publicly accessible by companies employingthis approach.

2 Herbicides

Adenylosuccinate synthetase (EC 6.3.4.4) is a crucial enzyme inpurine biosynthesis, catalyzing the GTP-dependent conversionof IMP and aspartic acid into AMP.18 AdSS has been validatedas target for herbicides by the herbicidal activity of two naturalproducts which inhibit AdSS: hydantocidin 1 and hadacidin 2(Fig. 2).19

Hydantocodin 1 is believed to be a pro-herbicide which isphoshorylated in planta to give hydantocidin monophosphate(HMP, 3), the actual inhibitor. Fig. 3 shows a view of the AdSSactive site with HMP bound (derived from PDB ID: 1SOO).20

HMP acts as a mimic of the natural substrate IMP and adoptsa very similar position in the enzyme active site (Fig. 4). AdSShas a relatively extended active site which accommodates bothHMP and the co-substrate aspartate. Hadacidin has beenshown to act as a competitive inhibitor of aspartic acid andbinds in the same region of the active site. Several structures ofthe binary AdSS–hadacidin complex have been reported (PDBID: 1CG0,21 PDB ID: 1CG1,21 PDB ID: 1CG3,21 PDB ID:1CH8,22 PDB ID: 1CIB,22 PDB ID: 1GIM,23 PDB ID:

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1GIN 23). Fig. 5 shows HMP and hadacidin bound to theenzyme active site (PDB ID: 1JUY).24 Hadacidin occupies theaspartate binding site of the enzyme without distortion ofthe overall geometry of the active site. Hanessian et al. havereported an elegant design approach in which hybrid AdSSinhibitors were obtained by combining the structures ofhydacidin and hydantocidin.25 Conceptually similar approacheshave been successfully used in medicinal chemistry to designmore potent hybrid inhibitors which incorporate structuralfeatures of both “fragment inhibitors”. If both fragments canbe combined without distortion of the overall binding geo-metry, the binding potency of the resulting hybrid inhibitorscan be increased by several orders of magnitude. As a resultfragments can have relatively low binding affinity and methods

Fig. 2 Adenylosuccinate synthetase (EC 6.3.4.4) and inhibitors.

Fig. 3 Hydantocidin mono-phosphate (HMP, 3) bound in the activesite of adenylosuccinate synthase (AdSS). View derived from PDBentry 1S00. Carbon atoms of HMP are coloured in purple.†

† Technical note: Colour plates were generated from structures asdeposited in the PDB 13,14 using InsightII (Accelrys, San Diego, USA).Spheres of 5–7 Å around the inhibitor were extracted. Occasionallyresidues were removed for greater clarity.

amenable to high-throughput mode, such as protein NMRbinding assays, can be used for their identification.26

Hanessian designed and prepared compounds 4 and 5 toinvestigate whether hybrids would be more potent enzymeinhibitors (Fig. 6). The linker had been chosen on the basis ofmodelling studies. Screening confirmed that hybrids 4 and 5

Fig. 4 6-Phosphoryl-inosin mono-phosphate (IMP) and hadacidinbound in the active site of AdSS. View derived from PDB entry 1CG0.Carbon atoms of IMP and Hadacidin are coloured in grey.

Fig. 5 HMP and hadacidin bound in the active site of AdSS. Viewderived from PDB entry 1JUY. Carbon atoms of HMP and Hadacidinare coloured in grey.

Fig. 6 Design of hybrid inhibitors of AdSS.

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were significantly more active than either of the natural inhib-itors. Structural proof was obtained by x-ray crystallographicanalyses (PDB ID: 1QF4, PDB ID: 1QF5).25 Fig. 7 shows 4bound to the active site. The overlay of the ADSS–4 com-plex with both the binary ADSS–HMP and the ternary com-plex ADSS–HMP–hydantocidin complex (Fig. 8) confirms that4 acts as a hybrid with the same major interactions betweeninhibitor and enzyme.

Amino acid biosynthesis is an important target for severalcommercial classes of herbicides.27,28 Certain genes coding forenzymes involved in amino acid biosynthesis occur in bacteria,plants and fungi, but not in animals and humans. Hence, inhib-itors of these amino acid biosynthetic enzymes are less likelyto have toxic side effects than compounds that interfere withubiquitous biological processes, such as protein biosynthesis.

Tryptophan synthase (TRPS, EC 4.2.1.20) catalyzes the finaltwo steps in the biosynthesis of tryptophan.29 The initial stepconsists of an acid–base-catalyzed indole–indolenine tauto-merisation. This sets up a β-hydroxyimine intermediate whichundergoes a retro-aldol reaction to give indole and (R)-glyceraldehyde-3-phosphate (α-reaction, Fig. 9). The indolereacts in a pyridoxalphophate-catalyzed reaction with serine togive tryptophane and water (β-reaction). The α- and β-reactionsites of TRPS are connected through a 25 Å long hydrophobictunnel which is assumed to “direct” the indole nucleus to thesite of the β-reaction.30 Several structures of the enzyme incomplex with different inhibitors have been deposited in the

Fig. 7 Inhibitor 4 bound in the active site of AdSS. View derived fromPDB entry 1QF5. Carbon atoms of 4 are coloured in orange.

Fig. 8 Overlay of 4 and HMP/Hadacidin bound to AdSS active site.View derived from PDB entries 1QF5 and 1JUY. Carbon atoms of 4 arecoloured in orange.

PDB (PDB IDs: 1A50,31 1A5A,32 1A5B,32 1A5S,31 1BEU,33

1BKS,34 1C29,30 1C8V,30 1C9D,30 1CW2,30 1CX9,30 1FUY,35

1GEQ,36 1QOP,37 1QOQ,37 1TTP,38 TTQ,38 1UBS,38 2TRS,38

2TYS,39 2WSY 31).A rational design approach to TRPS inhibitors as herbicides

based on the structure of the (previously known) 32 inhibitorindole-3-propanol-phosphate (6) in complex with the enzymehas been described by Sachpatzidis et al. (Fig. 10).30 Compound

6 has no in vivo herbicidal activity. This is probably due to themodest levels of enzyme inhibition (20 µM) and the instabilityof the phosphate ester under in vivo conditions. Replacement ofthis moiety by a metabolically more stable phosphonate groupas in 7 gave only similar levels of activity and did not lead to asignificant increase in whole plant herbicidal activity. Theauthors then prepared and tested a series of 4-aryl-thiobutylphosphonates. The rationale for their design was as follows:sulfur mimics the sp3-hydridised intermediate of the naturalreaction intermediate and acts as a “transition-state analogue”.A hydrogen bond donor as ortho-substituent replaces the indolnitrogen. Despite opening of the heteroaromatic ring and theresulting increase of rotational freedom (unfavourable entropiceffects) 8 and 9 had significantly increased in vitro activity.Substitution of the aromatic ring or introduction of a doublebond in the side chain did not lead to any significant change inactivity.30 The structures of 8 (PDB ID: 1C29,30 Fig. 11), 9(PDB ID: 1CX9,30 Fig. 12) and 10 (PDB ID: 1C9D,30 Fig. 13)bound to TRPS confirmed the design hypotheses. The phos-phonate inhibitors bind to the α-site of the enzyme. Thearomatic ring makes extensive hydrophobic contacts whereasthe ortho-substituent interacts with an aspartate residue (D60)which has been implicated as base in the first catalytic step. Thebutyl side chain positions the phosphonate group in a hydro-philic polar environment with close contacts to serine residue(S235) and water molecules. Replacement of the hydroxylgroup in the ortho-position by an amino group gave the mostpotent compound (9: IC50 = 178 nM). As expected the phos-phonate moiety in 8 to 10 also increased the stability of theinhibitors and as a result they showed whole-plant activity. Themode of action of these herbicides was confirmed by “reversalstudies”, which showed that plants grown in the presence oftryptophan did not show any signs of herbicidal effects. Fig. 14illustrates the excellent fit between the enzyme inhibitor com-

Fig. 9 α-Reaction of tryptophan biosynthesis.

Fig. 10 Design of inhibitors tryptophan synthase (TRPS, EC 4.2.1.20).

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plexes of 8, 9 and 10. The presence of the relatively small fluor-ine substituent in 10 has no influence on the binding mode inthe active site. The overlay of 8, 9, and 10 in the active siteshows how similarly inhibitors of this type bind (Fig. 14). Onthe basis of their structural work the authors suggest severalstarting points for SBD to increase in vitro potency. Thus, newstructural platforms could result from attachment of the butyl-phosphonate side chain to an indolene nucleus. This wouldmaintain the sp3-hybridisation at the C-3 position of thenatural substrate whilst avoiding the entropic penalty due toring-opening of the indole nucleus. Finn et al. have describedvery similar inhibitors on the basis of a biochemical designapproach also starting from mechanistic considerations.40

Fig. 11 Inhibitor 8 bound in the active site of tryptophan synthase(TRPS). View derived from PDB entry 1C29. Carbon atoms of 8 arecoloured in cyan.

Fig. 12 Inhibitor 9 bound in the active site of TRPS. View derivedfrom PDB entry 1CX9. Carbon atoms of 9 are coloured in green.

Fig. 13 Inhibitor 10 bound in the active site of TRPS. View derivedfrom PDB entry 1C9D. Carbon atoms of 9 are coloured in green.

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) is a keyenzyme in branched-chain amino acid biosynthesis, anotheragrochemically important pathway in amino acid biosyn-thesis.1,41 Two commercially important classes of herbicides(sulfonyl ureas and imidazolinone) target AHAS and validatethe pathway as an effective mode of action for herbicides.27

Since acetohydroxyacid reductoisomerase (AHRI, E.C.1.1.1.86)catalyzes the subsequent step in this biosynthetic pathway it hasalso attracted interest as a potential target of herbicides. Thestructure and biochemistry of AHRI has recently beenreviewed (PDB IDs of AHRI and AHRI–inhibitor complexes:1QMG,42 1YVE 43). N-Hydroxy-N-isopropyloxamate 11 (Ip-OHA) and 2-dimethylphosphinoyl-2-hydroxyacetic acid 12(Hoe704) are known inhibitors of AHRI (Fig. 15). Both com-

pounds are assumed to be transition-state mimics. They bindcompetitively with the natural substrate and show time-dependent inhibition of the enzyme. The structure of IpOHAbound to the enzyme has been reported (PDB ID: 1YVE).43

The active site is buried in the enzyme and shows the presenceof only 12 water molecules, 5 of which are co-ordinating twomagnesium cations in the active site which have been shown tobe essential for enzyme activity. The inhibitor also co-ordinatesto both metal centres. More recently, the structure of AHRI hasbeen reported in complex with 2,3-dihydroxy-3-methylvalerateand a reaction mechanism has been proposed (PDB ID:1QMG).42 Despite high in vitro potency both IpOHA andHoe704 are poor herbicides and high concentrations arerequired to achieve lethality in plants. It has been suggested

Fig. 14 Overlay of 8, 9, and 10 bound to TRPS active site. Viewderived from PDB entries 1C29 (carbon atoms in cyan), 1CX9 (carbonatoms in green), and 1C9D (carbon atoms in grey), respectively.

Fig. 15 Branched-chain amino acid biosynthesis: (a) Threoninedehydratase (TD) [E.C. 4.2.1.16]; (b) Acetohydroxyacid synthase(AHAS) [E.C. 4.1.3.18]; (c) acetohydroxyacid reductoisomerase(AHRI) [E.C. 1.1.1.86].

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that in vivo active compounds would have to have a higherassociation rate or be non-competitive. Although such non-competitive inhibitors were identified through a HTS approach,their in vivo potency was low and in vitro activity was lacking. Itcan only be a matter of time before SBD approaches to novelacetohydroxyacid reductoisomerase inhibitors with improved invivo activity will be reported.

The biosynthesis of the basic amino acid lysine has alsoreceived some attention as a potential target for herbicides.44

Fig. 16 gives a simplified overview of the biosynthetic pathway

to lysine. The structures of aspartate-semialdehyde dehydro-genase (ASADH, E.C. 1.2.1.11, PDB ID: 1BRM),45 dihydro-dipicolinate synthetase (DPS, E.C. 4.2.1.52, PDB ID: 1DHP),46

and diaminopimelate dehydrogenase (DAPD, E.C. 1.4.1.16,PDB ID: 1DAP) 47 have been reported. Several enzyme–inhibitor complexes have been reported (PDB ID: 1ARZ) 47 andrenewed interest in this pathway from an SBD point of view canbe anticipated.

Biotin is an essential cofactor for enzymes involved in carb-oxylation, transcarboxylation and decarboxylation reactions.Since only plants, bacteria and some fungi carry the genes forbiotin biosynthesis this pathway has been targeted for thedevelopment of herbicides. The details of the pathway—including over-expression and structural determination ofenzymes—have been largely established in bacteria and recentlyreviewed.48

Dethiobiotin synthetase (DTBS, E.C. 6.3.3.3) is the penultim-ate enzyme biotin biosynthesis.49 It catalyzes the formation of acyclic urea precursor of biotin from diaminopelargonic acid,carbon dioxide and ATP (Fig. 17). The reaction mechanism is

presumed to involve initial formation of a carbamate which isthen phosphorylated. Ring-closure of this intermediate leads tothe cyclic urea in DTB. Since only scant information is availableabout the plant pathway, Rendina et al. used the bacterialenzyme as model for the design and synthesis of DTBS inhib-

Fig. 16 Key steps in lysine biosynthesis: (a) aspartate kinase (AK)[E.C. 2.7.2.4]; (b) aspartate-semialdehyde dehydrogenase (ASADH)[E.C. 1.2.1.11]; (c) dihydrodipicolinate synthetase (DPS) [E.C. 4.2.1.52];(d) diaminopimelate dehydrogenase (DAPD) [E.C. 1.4.1.16].

Fig. 17 (a) Dethiobiotin synthetase of DTBS (E.C. 6.3.3.3) andinhibitors.

itors as herbicides.50 Diamino acid derivative 13 was preparedas substrate mimic, but only showed poor levels of inhibition.Attempts to mimic the carbamate intermediate of the DTBScatalyzed reaction led to the preparation of carboxylates andphosphonates, for example 14 and 15. These compounds alsoshowed poor levels of enzyme inhibition. Unsurprisingly, invivo activity of all compounds was also reported to be weak.Indeed, phosphonate 15 was found to be a substrate of theenzyme. Co-crystallisation of 15 with the enzyme led to thestructure of the enzyme–inhibitor complex which was verysimilar to a previously reported structure of an ATP analoguebound in the active site of DTBS (PDB ID: 1DTS).50 The sub-strate analogue binds relatively close to the surface of theenzyme in a solvent exposed region. This observation mayaccount for the weak levels of inhibition. The authors do notreport any attempts to use the structural data for the design ofmore potent inhibitors.

Glyphosate 16 (Fig. 18) is arguably the most important

herbicide in current use. Although its discovery was not theresult of a rational or even structure-based design discoveryprogramme, the recently published structure of glyphosatebound to EPSP synthase warrants mention.51

Despite its deceptively simple structure, glyphosate is a highlyeffective and safe weed control agent. Inhibition of EPSPblocks biosynthesis of aromatic amino acids via the shikimatepathway (Fig. 18). This pathway is only present in plants andbacteria, where it is of high importance. It has been estimatedthat more than a third of plant dry mass consists of aromaticcompounds derived from shikimate. Interference with this vitalbiosynthetic pathway is lethal to plants. The exact bindingmode of glyphosate has now been elucidated by high-resolutionx-ray structures of EPSP bound with its natural substrateshikimate-3-phosphate (S3P) (PDB ID: 1G6T) 51 and in thepresence of S3P and glyphosate (PDB ID: 1G6S).51 Binding ofglyphosate does not significantly alter the structure of theinhibitor-free binary EPSP–S3P complex. In the latter a phos-phate and formate ion (from the buffer) occupy the position ofglyphosate. The authors propose an ordered mechanism inwhich S3P binding occurs prior to glyphosate binding. S3Ptriggers an induced-fit mechanism which converts the enzymefrom an open into a closed form. This domain closure can onlyoccur in the presence of a negatively charged molecule whichcompensates for the accumulation of positively charged proteinresidues in the closed form. Even with the new structural data inhand it seems unlikely that a second glyphosate will be found,but it might provide starting points for structure-based designof inhibitors interfering for example with the induced-fit mech-anism of EPSP. Furthermore, the structural data may help tounderstand the emerging resistance against glyphosate.52

3 Insecticides

Acetylcholinesterase (AChE, E.C. 3.1.1.7) is the most import-ant target of commercial insecticides. All organophosphate(OP) and carbamate insecticides are believed to exert their

Fig. 18 (a) 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPsynthase) [E.C. 2.5.1.19].

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activity by irreversible inhibition of AChE. Despite the provenefficaciousness of AChE inhibitors as insecticides, OPs andcarbamates are relatively non-selective and highly toxic to non-target species, including humans. Interest in selective AChEinhibitors has also been fuelled by the discovery that AChEinhibition relieves the symptoms of Alzheimer’s disease(AD).53,54 AChE belongs to the ubiquitous class of serinehydrolases with which it shares a common mechanism. Thestructure of AChE from Torpedo californica was reported in1991.55 The active site of the enzyme is buried at the end of anarrow gorge which is lined with aromatic residues. The struc-tures of several enzyme–inhibitor complexes and their use inthe SBD of anti-AD drugs have been reported since, and a totalof 46 structures have been deposited in the PDB. As a result ofthese extensive studies, interactions between aromatic AChEresidues and the quaternary ammonium ion of the natural sub-strate or its replacement in the inhibitor have been recognised ascrucial for potent binding of inhibitors. Recently Lewis et al.described an SBD approach to the development of AChEinhibitors as new insecticides.56 The authors set out to designreversible inhibitors with a view to developing insecticides withan improved selectivity for target species.

The inhibitors were designed to combine binding features oftwo previously known classes of AChE inhibitors (Fig. 19).

Tacrine 17 was the first treatment for some of the symptoms ofAlzheimer’s disease. Tacrine and trifluoromethyl ketone 18 arenanomolar inhibitors of AChE. Enzyme inhibitor complexesshow that tacrine makes stacking interactions with aromaticresidues F330 and Y84 (PDB ID: 1ACJ, Fig. 20) 57 whilst tri-

fluoromethyl ketone 18 binds to a different region of the activesite (PDB ID: 1AMN, Fig. 21).58 The trifluoromethyl ketoneforms a covalent hemiketal with active site serine S200 whichacts as a transition state mimic. Viner et al.’s design of revers-ible inhibitors was based on the assumption that a hybrid

Fig. 19 Design of reversible AChE inhibitors.

Fig. 20 Tacrine 17 bound in the active site of AChE. View derivedfrom PDB entry 1ACJ. Carbon atoms of 17 are coloured in cyan.

inhibitor bridging the tacrine binding site with the active siteserine would have potent inhibitory properties (Fig. 19). Com-putational methods were employed to determine optimal lengthand position of the linker unit shown in Fig. 19. A series oftrifluoromethyl ketones was synthesised and these were foundto be potent inhibitors of the enzyme, e.g. 19 with a Ki of3nM. Support for the design concept came from compound 20without the trifluoromethyl moiety which was 3 orders ofmagnitude less potent than 19. X-ray crystallographic analysisconfirmed that inhibitor 19 was bridging the active serine andthe aromatic binding site.59 Fig. 22 shows a comparison of

predicted (carbon atoms coloured in magenta) and observed(carbon atoms coloured in green) binding mode of 19 in theAChE active site. Whilst the overlay is generally good for thetrifluoromethyl group the 4-aminoquinoline bicycle is flippedover in the predicted structure with respect to the observedstructure.

4 Fungicides

Fungi are of considerable importance both as human path-ogens and agricultural pests. Melanin biosynthesis (Fig. 23) isa well-established molecular target of agrochemically usefulfungicides. Melanin is an aromatic polymer of polyketide originderived from 1,3,6,8-tetrahydroxynaphthalene (4HN) by twosuccessive steps of oxidation and dehydration (Fig. 23). The

Fig. 21 Trifluoromethyl ketone 18 bound in the active site of AChE.View derived from PDB entry 1AMN. Carbon atoms of 18 are colouredin green.

Fig. 22 Comparison of predicted and observed binding mode of 19(see text).

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oxidation steps are catalyzed by NADPH-dependent reduct-ases whilst the dehydration step is carried out by scytalonedehydratase.

Melanin is located between the cell wall and cell membraneof specific fungal organelles which are involved in the fungalinfection process. Disruption of melanine biosynthesis causesfungi to lose their pathogenicity. Several commercial fungicidestarget melanin biosynthesis. Tricyclazole 21, phthalide 22 andpyroquilon 23 (Fig. 24) are commercial fungicides which requirerelatively high application rates of 1–2 kg hectare�1.

Jordan’s group have investigated the enzymes involved inmelanin biosynthesis with the aim of developing new fungi-cides.60–63 Their work on scytalone dehydratase and trihydroxy-naphthalene reductase is the most comprehensive example ofan SBD approach to the design of new agrochemicals. 1,3,8-Trihydroxynaphthalene reductase (3HNRase, E.C. 1.1.1.252)and 1,3,6,8-tetrahydroxynaphthalene reductase (4HNRase,E.C. 1.1.1.252) belong to the large family of short-chaindehydrogenases/reductases (SDR).64 The structures of 21 (PDBID: 1YBV),65 22 (PDB ID: 1G0N) 65 23 (PDB ID: 1G0O),65 andindenone 24 (Fig. 25: PDB ID: 1DOH) 65 bound in the activesite of 3HNRase have been reported. All compounds are potentinhibitors of the enzyme with Ki values in the lower nanomolarrange. Although of different resolution, all structures are gen-erally in good agreement with regard to the position and orient-ation of active site residues (Fig. 26 ). The active site volume isrelatively small (ca. 300 Å3) and rich in polar and hydrophilicresidues. The carbonyl groups of the inhibitors make inter-actions with enzyme residues Y178 and S164. The relative

Fig. 23 Fungal melanin pathway (a) 1,3,6,8-Tetrahydroxynaphthalenereductase (4HNRase) [E.C. 1.1.1.252]; (b) 1,3,8-trihydroxynaphtha-thalene reductase (3HNRase) [E.C. 1.1.1.252]; (c)/(d) Scytalonedehydratase (SDase) [E.C. 4.2.1.94].

Fig. 24 Inhibitors of fungal melanin biosynthesis.

orientation between the co-factor NADPH and the carbonylgroup of each inhibitor is similar (Fig. 27). The nicotinamidenucleus of NADPH is orientated parallel to the aromaticinhibitor at a distance of 3.3 to 3.6 Å. This allows π–π stackinginteractions. Mechanistically, the carbonyl groups of the inhib-itors mimic the carbonyl tautomer of the natural naphtholsubstrate of 3HNRase. Further stabilization of the inhibitor inthe active site is provided by π-stacking interactions with Y223.Although the orientation of all inhibitors within the active siteis very similar the overlay between individual atoms is poor

Fig. 25 Indenone 24 bound in the active site of 3HNRase. Viewderived from PDB entry 1DOH. 24 is coloured in magenta.

Fig. 26 Overlay of 3HNRase active sites with phthalide 22 andindenone 24. View derived from PDB entries 1G0N and 1DOH,respectively. Carbon atoms of 22 are coloured in green.

Fig. 27 NDAPH in 1DOH. Carbon atoms of NADPH are colouredin green.

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(Fig. 28). The aromatic stacking interactions appear to deter-mine the position of the inhibitors, but other interactionsbetween inhibitor and enzyme interactions vary for each struc-ture. Despite the detailed structural information new inhibitorshave not yet been designed. This may be due to the restrictedsize of the active site which does not have any unoccupiedareas that could be exploited by attaching appropriate residuesto the aromatic core structure of inhibitors 21, 22, 23, and 24.66

The discovery of indenone 24 as a 3HNRase inhibitor was theresult of an interesting “retrospective” screening strategy.67 Theauthors searched their company database for compounds within vivo activity against several fungal diseases. The initial hit setwas relatively large and had to be further reduced by elimin-ating unknown and undesirable structures‡ The remainderwas screened for activity against 3HNRase. This resulted inthe identification of 24 with a Ki value of 25 nM. The structureof the second reductase involved in melanin biosynthesis,1,3,6,8-tetrahydronaphthalene reductase (4HNRase) has beendescribed and compared to 3HNRase.65,68 4HNRase has a 30-fold lower affinity to pyroquilon 23 than 3HNRase. This differ-ence can be rationalised from the structure of 4HNRase on thebasis of unfavourable interactions between the carboxylate ofthe terminal I282 with the carbon skeleton of the inhibitor. Theinteractions caused by the terminal isoleucine residue have alsobeen invoked rationalising the substrate selectivity of 3HNRasefor 3HNR over 4HNR (ca. 300-fold preference).

Scytalone dehydratase (SDase, E.C.4.2.1.94) catalyzes thedehydration of scytalone to 3HN and vermilone to 2HN (seeFig. 23 above). The commercial fungicides carpropamid 25 anddicyclomet 26 target SDase. As SDase is a unique enzyme with-out functional or structural counterpart in plants or animals,inhibitors are less likely to exhibit undesired toxic side effects.

Jordan’s group reported an SBD approach to novel SDaseinhibitors on the basis of the x-ray structure of salicylamide 27bound in the active site (Fig. 29, PDB ID: 1STD).69 Despite thein vitro potency of 27 the presence of a phenolic hydroxyl groupis generally considered undesirable in herbicides. Acidicphenols can act as proton shuttles across membranes and dis-turb the pH gradient. This is generally referred to as uncouplingand can lead to non-specific phytotoxicity. Phenols are alsoreadily metabolised by conjugation. This prompted Jordan toinvestigate possible salicylamide replacements using an SBDapproach. Compound 27 binds in the active site of SDase via anextended hydrogen bond network. Due to a strong internalhydrogen bond between the aromatic hydroxyl group and the

Fig. 28 Overlay of 3HNRase active sites with 21, phthalide 22 andindenone 24. View derived from PDB entries 1YBV, 1G0N and 1DOH,respectively.

‡ Note: compound collections may contain compounds of unknownstructure, for example microbial extracts.

amide carbonyl oxygen salicylamides adopt a rigid flat bicyclicstructure. Based on a previous report that 4-aminoquinazolinesare bioisosteres of salicylamides 70 Jordan used heterocyclicscaffolds in his inhibitor design.71 The x-ray structure alsoshowed a water molecule bound in the vicinity of the ligand.The design strategy aimed at displacing this water molecule bysuitable extension of the inhibitor. Such displacements andtheir effects on inhibitor binding have previously been describedand used in SBD of pharmaceuticals.66 The gain in entropycaused by “liberating” a tightly bound structural water mole-cule to bulk solvent water has been estimated to be as high as2 kcal mol�1.72 This gain in entropy has to be offset againstenthalpic penalties caused by breaking hydrogen bonds. Inthe case of SDase the entropic gain was expected to exceedthe penalty for breaking the hydrogen bond between N-3 of thequinazoline inhibitor and the structural water molecule. Thedesign sequence adopted by Jordan et al. is shown in Fig. 30.

Quinazoline and benztriazine ring systems were chosen asmimics of the salicylamide moiety. Both heterocycles retain anitrogen atom as hydrogen bond acceptor in the 2-position ofthe heteroaromatic ring system. The direct analogues 28 and 29of salicylamide 27 had similar inhibitory potency. The chiralα-methyl bromobenzyl side chain could be replaced by 3,3-diphenylpropyl moiety resulting in an increase in activity. Theauthors suggest that the 3,3-diphenyl propyl substituent is con-formationally less mobile and this favours binding on entropicgrounds. Variation of the 2-substituent confirmed the import-ance of a hydrogen bond accepting group in this position: 32is two orders of magnitude less active than 30. Even moreimpressively, introduction of a nitrile group as in 33 increasedthe activity by four orders of magnitude. A similar trend wasobserved in the benztriazine series 29, 31, 34, and 35. Proof forthe validity of the design strategy was obtained from the crystalstructure of 32 bound to the active site of SDase. Fig. 31 showsa benztriazine inhibitor bound to the active site of SDase (PDBID: 5STD).71 The structural water molecule is shown as a redsphere. A nitrile group in the 2-position as in 35 displaces thestructural water molecule (Fig. 32, PDB ID: 3STD).71 Com-pounds 32 and 34 with a hydrogen atom in this position aresignificantly less active because the hydrogen is too small todisplace the water molecule. Since the CH group cannot formhydrogen bonds with the structural water, both entropic andenthalpic factors contribute to this dramatic loss of activity.The overlay of 32 and 35 bound to the active sites of theenzyme (Fig. 33 ) illustrates the role of the cyano-group inreplacing the water molecule.

In addition to the rational approach towards developingsalicylamide replacements, Jordan’s group has also investigateda combinatorial strategy (Fig. 34).73 (R)-α-Methyl-4-bromo-benzylamine and several other amines were reacted with a set of192 carboxylic acids. The acid components were chosen froman in-house compound collection on the basis of availabilityand structural and electronic similarity to known enzyme

Fig. 29 Commercially used inhibitors of Scytalone dehydratase(SDase) [E.C. 4.2.1.94].

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Fig. 30 Design of SDase inhibitors I.

inhibitors. Amide products were tested as mixtures and only themost active ones were deconvoluted. This led to the discoveryof 36 as picomolar inhibitor. The similarity of the carboxylicacid portion of 36 to the one in carpropamide 25 is noteworthy(Fig. 29).

The structure of 36 bound to the active site was solved at1.8 Å resolution. The authors state that the binding modecorresponded to other previously known inhibitors, but theco-ordinates appear not to have been deposited in the PDB.

Another approach towards the design of new SDase inhib-itors reported by Jordan’s group sought to replace the α-methylbromobenzyl side chain in salicylamide 37 by a phenyl orphenoxypropyl group. Coupling of the appropriate amine tothe carboxyl-component present in the known fungicides car-

Fig. 31 Benztriazine inhibitor bound in the active site of SDase. Viewderived from PDB entry 5STD. Inhibitor carbon atoms are coloured ingrey.

propamid 25 and dicyclomet 26 gave potent enzyme inhibitors(Fig. 35). A detailed SAR study was undertaken in both series.X-ray crystallographic analysis of cyanoacetamide inhibitor 39showed that the inhibitor is nearly completely buried in theactive site. Binding of the inhibitor to the active site appears tobe driven largely by entropic contributions or hydrophobicinteractions.66

Another design approach was based on the structure ofknown fungicide dicylomet analogue 40. Its aim was to developstructures in which new interactions between the carboxamideinhibitors and the active site would lead to greater inhibitorypotency.74 An overlay of several enzyme inhibitor complexesindicated that one of the aromatic residues in the enzyme activesite (F53) appeared to be relatively mobile. Further modellingwork suggested that a phenyl substituent in the amine side

Fig. 32 Inhibitor 35 bound in the active site of SDase. View derivedfrom PDB entry 3STD. Inhibitor carbon atoms are coloured in cyan.

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chain of 40 would be able to undergo favourable π–π stackinginteraction with F53. To compensate for the increased size ofthe phenyl group, the t-butyl substituent had to be replaced by asmaller group (Fig. 36). Compounds 41 to 43 were prepared andthe activity trend within the series confirmed the predictions.X-ray crystallographic analysis of ethyl-substituted 41 con-firmed that F532 was interacting with the newly introducedphenyl group in 41 via π–π stacking interactions.

Jordan’s work on SDase is arguably the most comprehensiveand successful SBD approach to new agrochemicals reported inthe literature yet. It is an excellent illustration not only of iter-ative inhibitor design on the basis of x-ray crystallographicdata, but also of its combination with combinatorial methods.75

Fig. 33 Overlay of benztriazine inhibitor in Fig. 31 and compound 35bound to active site of SDase. View derived from PDB entries5STDand 3STD, respectively.

Fig. 34 Design of SDase inhibitors II.

Fig. 35 Design of SDase inhibitors III.

Iterative design methods depend crucially on rapidly access-ible crystal structures of enzyme–inhibitor complexes. Whilstcrystallisation of soluble proteins and enzymes is becomingmore and more a routine procedure there is still only scantcrystallographic data for membrane-bound or multi-enzymecomplexes. Since a number of highly effective pesticides targetmembrane-bound proteins, such as ion channels in insects orfungal respiration, the complete lack or the difficulty of obtain-ing such structures limits the use of SBD in agrochemistry. Buteven if crystallographic data is not readily available a judiciouscombination of modelling methods and structural data can beuseful for the design of new agrochemicals.

Inhibition of mitochondrial respiration is an importantmode of action of fungicides.76 Fig. 37 shows the structuresof recently commercialised compounds which target themitochondrial bc1 complex. Syngenta’s (formerly ZenecaAgrochemicals’) azoxystrobin was the first commercialised res-piration inhibitor derived from naturally occurring strobilurinA (47).77 Azoxystrobin and strobilurine A contain the samemethoxyacrylate moiety which has been identified as the toxo-phore of this class of fungicides.78 Both 45 and 46 contain anoxime-ether which mimics the methoxyacrylate group instrobilurin and azoxystrobin whilst famoxadone has a differ-ent toxophore.79 The recently published x-ray crystallographicanalyses of bc1 complexed with different inhibitors raises theexciting prospect of a detailed understanding of the bindingmode of respiration inhibitors.80–83 This could lead to the devel-opment of new fungicides and to an improved understandingof molecular changes leading to resistance against respirationinhibitors. Very little of this work has found its way into thepublic domain, but a recent report by Jordan on the x-ray struc-ture of bc1 inhibitor famoxodone is an indication of the effortswhich are underway in leading industrial laboratories.84

5 In vitro to in vivo translation of activity

In vitro to in vivo translation of activity, or the question of whatmakes an enzyme inhibitor a pesticide, remains one of the mostdifficult problems in the design of new agrochemicals. Num-erous factors contribute to varying extent to this translation.A suitable physicochemical profile, such as logp, pKa and vol-atility, is essential to ensure uptake and translocation of pesti-cides amongst others. Empirical rules similar to Lipinsky’s ruleof five in drug design can help to classify chemical platforms assuitable or unsuitable for development.85 Those experienced inthe art of pesticide design may even be able to look at thestructure of a lead molecule and give an intuitive assessment ofwhether a particular structure will be active in the field. Giventhe resource intensive nature of SBD it is absolutely essentialthat such considerations are taken into account before anextensive design programme is undertaken. In fact, Jordan’swork on SDase shows that SBD can be useful in the develop-ment of replacements of groups which are known to be prob-lematic, such as phenols. Although SBD constitutes an in vitroapproach to lead design, in vivo data for structural platformsshould be obtained very early on in the design process. Unlessthere is a convincing rationale for the lack of in vivo activitythere will be considerable reluctance to work on in-vitro-only-leads.

Fig. 36 Design of SDase inhibitors IV.

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Fig. 37 Respiration inhibitors.

This underlines that in vitro and in vivo approaches to leaddesign must not be seen as mutually exclusive. A successfulstrategy has to rely on both, as outlined in Fig. 38.

The long-term goal, however, must be rational understandingof factors that govern in vivo to in vitro translation. A report byHuber et al. suggests that structural data can play a role,too.86 The authors solved the structures of herbicides in com-plex with their detoxifying enzyme glutathione-S-transferase.These ubiquitous enzymes catalyze the conjugation of gluta-thione with a wide variety of xenobiotics, including herbicides.Fig. 39 shows the structures of GST-I from maize in complexwith the herbicide atrazine. Fig. 40 shows the structure of GSTfrom Arabidopsis thaliana in complex with FOE4053. Theenzyme active site contains an herbicide-binding region (H site)and a glutathione-binding site (G site). The substrate specificityis governed by the shape of the H-site. The structural data

Fig. 38 Future lead discovery in agrochemistry?

indicate which enzyme residues are most important and howthe herbicides could be altered to make them less prone tometabolism.

6 Conclusions and outlook

As genetic sequencing of increasing numbers of organismsreaches completion, the question arises as to how to exploit thiswealth of data.87 Whilst genetic data-mining is only beginningto have an impact on the discovery of agrochemicals and drugs,the interest in understanding function and structure of geneproducts is focusing attention on high-throughput methods,such as automated x-ray crystallography and structural gen-omics.88 With recent progress in crystallisation techniques it isconceivable that structures of proteins may be determined

Fig. 39 Atrazine bound in the active site of glutathione-S-transferase(GST-I) [E.C. 2.5.1.18].

Fig. 40 FOE4053 bound in the active site of GST-I. View derivedfrom PDB entry 1BX9. Inhibitor carbon atoms are coloured in green.

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before their function is known. Much more readily accessiblestructural data will make structure-based inhibitor design aroutine approach to lead generation. Its success in agrochem-istry will depend on a fusion of the traditional random in vivoapproach with more rational in vitro approaches. These includeSBD and the use of combinatorial techniques, such as focusedlibraries to explore inhibitory platforms as well as randomscreening of compound collections or natural products.

Agrochemical lead discovery and lead optimisation is adifficult endeavour. SBD is no panacea, but the work covered inthis review is evidence that it has the potential to make animportant contribution to the development of new and betterpesticides.

7 Acknowledgements

I am grateful to my former colleagues at Jealott’s Hill ResearchStation, Dr Russell Viner, Dr Ewan Chrystal and Dr TerenceLewis for introducing me to the fascinating and challengingtopic of structure-based design of agrochemicals.

I am also grateful to Dr Rajesh Sangar for help with thecolour illustrations and to Dr Helene Rudyk and ProfessorChristopher J. Schofield for valuable comments.

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