AutoClickChem: Click Chemistry in Silico

Academic researchers and many in industry often lack the financial resources available to scientists working in “big pharma.” High costs include those associated with high-throughput screening and chemical synthesis. In order to address these challenges, many researchers have in part turned to alternate methodologies. Virtual screening, for example, often substitutes for high-throughput screening, and click chemistry ensures that chemical synthesis is fast, cheap, and comparatively easy. Though both in silico screening and click chemistry seek to make drug discovery more feasible, it is not yet routine to couple these two methodologies. We here present a novel computer algorithm, called AutoClickChem, capable of performing many click-chemistry reactions in silico. AutoClickChem can be used to produce large combinatorial libraries of compound models for use in virtual screens. As the compounds of these libraries are constructed according to the reactions of click chemistry, they can be easily synthesized for subsequent testing in biochemical assays. Additionally, in silico modeling of click-chemistry products may prove useful in rational drug design and drug optimization. AutoClickChem is based on the pymolecule toolbox, a framework that may facilitate the development of future python-based programs that require the manipulation of molecular models. Both the pymolecule toolbox and AutoClickChem are released under the GNU General Public License version 3 and are available for download from http://autoclickchem.ucsd.edu

Exploring the interaction between asymmetric phosphonates and acetylcholinesterase. Probing the gorge and P-site via customized covalent modification

Organophosphates (OPs) react with acetylcholinesterases (AChEs) to form a covalent bond at a specific serine residue in the active gorge, thereby providing a highly precise modification of this enzyme and an opportunity to explore protein structure, function and mechanism. The goal of this study is: 1) to show how covalent modification of AChEs by chromophore-linked OPs can be used to probe the active and peripheral sites of the protein and its local environment; 2) to differentiate AChE stereoselectivity with asymmetric phosphonothiolates. Click chemistry was used as a key transformation method to prepare an array of chromophores linked to a reactive fluorophosphonate (FP) head group. Chromophore-linked FPs varying in length and chromophore were synthesized and computer-modeled to visualize and calculate positioning of the chromophore-FP-AChE relative to the protein active gorge. The inhibition rates of chromophore-FPs against recombinant mouse AChE (rMAChE) and electric eel AChE (EEAChE) were determined via colorimetric assay and the dansyl containing FPs were demonstrated to be the most potent. In addition, the binding effects of the chromophore and FP moiety to protein were evaluated and results demonstrated that the size and structure of chromophore and the length of the ligands mutually affect the inhibition potency. Dansyl, dabsyl and pyrene were the best chromophores with least interaction with AChEs. To examine the interactions of asymmetric analogs of the OP compounds within the steric confines of AChEs, the synthesis of phosphonothiolate enantiomers as anti-AChEs was conducted using a chiral auxiliary for separation. X-ray analysis and 31P NMR were used to show an exclusively separation of the two diastereomers. The kinetic parameters ki and KD for the inhibition of recombinant human AChE (rHuAChE) were determined. A 4-fold difference in anti-AChE potency was observed between Sp (ki = 1.7 x 103 M-1min-1) and Rp (ki = 9.0 x 103 M-1min-1). These enantiomers link to the chromophores via click reaction to form chromophore-linked asymmetric phosphonothiolates (CLAPs), which can be used to study AChE stereospecificity

Dynamique structurale de l'acetylcholinesterase et ses implications dans la conception de réactivateurs

Acetylcholinesterase (AChE), one of nature fastest enzyme, is the target of multiple toxics,including organophosphate nerve agents (OP). In the first part of this thesis I present thestructure-based development of a new uncharged reactivator, which showed characteristicsbetter than any molecule commercially available to date. The molecule has been rationallydesigned to present both affinity to the inhibited enzyme and good reactivation capabilities.The interactions between the lead molecule KM297 and AChE has been characterizedby means of flexible docking, molecular dynamics simulations and X-ray protein crystallography.The deeper understanding of its binding modes to both native and OP-inhibitedAChE has helped in developing a derivative, JDS207, whose binding mode at the peripheralsite of AChE is optimized. This derivative has also been studied by flexible docking and Xraycrystallography. The design of this family of reactivators taught us that a deep insightof the AChE dynamics is necessary to optimize ligands. The second part of the thesis isdevoted to the analysis of molecular dynamics simulations of AChE. At first, we assessedthat combining multiple short simulations is a fast and reliable method to characterizethe dynamics of the amino-acids side-chains. By comparing dynamics of the side-chainsfrom hAChE and TcAChE, we confirm that some key dynamical differences exist betweenthe two enzyme. The knowledge of the rotamers issued of MD simulation has lead us todevelop a new method to generate flexible receptors for docking, which is specific to eachsingle residue in the enzyme. This method has been validated by comparing its outputstructures with the ones found on the PDB database.L’acétylcholinestérase (AChE), une des enzymes les plus rapides dans la nature, est lacible d’un large nombre de toxiques, dont notamment les neurotoxiques organophosphorés.La première partie de ce manuscrit de thèse décrit le développement raisonné d’un nouveauréactivateur, qui présente des propriétés de réactivation supérieures aux moléculesactuellement sur le marché. Les interactions entre cette molécule, KM297, et l’AChE ontété étudiées par dynamique moléculaire, docking et cristallographie aux rayons X. La connaissancedes modes de liaison du KM297 dans l’AChE native ou inhibé par un OP ontpermis de développer la molécule JDS207, qui se lie de façon exclusive au site périphériquede l’AChE. La deuxième partie de la thèse est dédiée à l’analyse des simulations de laAChE par dynamique moléculaire. On observe que la combinaison de multiples trajectoiresgénérées avec des paramètres de vélocité initiale différents est une méthode fiablepour caractériser les conformations atteintes par les chaînes latérales des acides aminés. Encomparant la distribution des rotamères pour l’AChE humaine et celle du poisson Torpedocalifornica, on montre que des différences importantes existent entre les enzymes des deuxespèces. A partir de ces informations sur les conformations de résidus clés du site actif,une méthode a été développée pour générer des récepteurs utilisable pour des calcules dedocking flexible, de façon à prendre en compte la dynamique propre à chaque résidu del’enzyme. Cette méthode a été validé en comparent les résultats obtenues à des structurescristallographiques connues

Dynamique structurale de l'acetylcholinesterase et ses implications dans la conception de réactivateurs

Acetylcholinesterase (AChE), one of nature fastest enzyme, is the target of multiple toxics,including organophosphate nerve agents (OP). In the first part of this thesis I present thestructure-based development of a new uncharged reactivator, which showed characteristicsbetter than any molecule commercially available to date. The molecule has been rationallydesigned to present both affinity to the inhibited enzyme and good reactivation capabilities.The interactions between the lead molecule KM297 and AChE has been characterizedby means of flexible docking, molecular dynamics simulations and X-ray protein crystallography.The deeper understanding of its binding modes to both native and OP-inhibitedAChE has helped in developing a derivative, JDS207, whose binding mode at the peripheralsite of AChE is optimized. This derivative has also been studied by flexible docking and Xraycrystallography. The design of this family of reactivators taught us that a deep insightof the AChE dynamics is necessary to optimize ligands. The second part of the thesis isdevoted to the analysis of molecular dynamics simulations of AChE. At first, we assessedthat combining multiple short simulations is a fast and reliable method to characterizethe dynamics of the amino-acids side-chains. By comparing dynamics of the side-chainsfrom hAChE and TcAChE, we confirm that some key dynamical differences exist betweenthe two enzyme. The knowledge of the rotamers issued of MD simulation has lead us todevelop a new method to generate flexible receptors for docking, which is specific to eachsingle residue in the enzyme. This method has been validated by comparing its outputstructures with the ones found on the PDB database.L’acétylcholinestérase (AChE), une des enzymes les plus rapides dans la nature, est lacible d’un large nombre de toxiques, dont notamment les neurotoxiques organophosphorés.La première partie de ce manuscrit de thèse décrit le développement raisonné d’un nouveauréactivateur, qui présente des propriétés de réactivation supérieures aux moléculesactuellement sur le marché. Les interactions entre cette molécule, KM297, et l’AChE ontété étudiées par dynamique moléculaire, docking et cristallographie aux rayons X. La connaissancedes modes de liaison du KM297 dans l’AChE native ou inhibé par un OP ontpermis de développer la molécule JDS207, qui se lie de façon exclusive au site périphériquede l’AChE. La deuxième partie de la thèse est dédiée à l’analyse des simulations de laAChE par dynamique moléculaire. On observe que la combinaison de multiples trajectoiresgénérées avec des paramètres de vélocité initiale différents est une méthode fiablepour caractériser les conformations atteintes par les chaînes latérales des acides aminés. Encomparant la distribution des rotamères pour l’AChE humaine et celle du poisson Torpedocalifornica, on montre que des différences importantes existent entre les enzymes des deuxespèces. A partir de ces informations sur les conformations de résidus clés du site actif,une méthode a été développée pour générer des récepteurs utilisable pour des calcules dedocking flexible, de façon à prendre en compte la dynamique propre à chaque résidu del’enzyme. Cette méthode a été validé en comparent les résultats obtenues à des structurescristallographiques connues

Cholinesterase Research

This collection of 10 papers includes original as well as review articles focused on the cholinesterase structural aspects, drug design and development of novel cholinesterase ligands, but also contains papers focused on the natural compounds and their effect on the cholinergic system and unexplored effects of donepezil

Carbonic Anhydrase II: A Model System for Artificial Copper Center Design, Protein-guided Cycloadditions, Tethering Screenings and Fragment-based Lead Discovery

Im Rahmen dieser Dissertation wurde eine Auswahl recht verschiedene Ansätze zur Fragment-basierten Leitstruktursuche (fragement-based lead discovery) mit dem Zielprotein Carboanhydrase II durchgeführt. Die verschiedenen Projekte wurden entscheidend durch die Protein Kristallographie unterstützt und methodisch gelenkt. Durch Verbesserung von Strahlungsquellen und der Rechenleistung von Computern hat sich die Protein-Kristallographie in den letzten Jahren zu einem Analytik Instrument entwickelt, welches routinemäßig zur Strukturaufklärung eingesetzt werden kann. Circa 200 bis 250 Datensätze wurden im Rahmen dieser Arbeit gesammelt um Fragestellungen der verschiedenen ambitionierten Projekte mit Focus auf die Strukturbiologie zu beantworten. Wie kann die Wechselwirkung zwischen Proteinen und kleinen Substanzen für Wirkstoffentwicklungen ausgenutzt werden? Ist es möglich, z.B. Reaktionen die in situ ablaufen, durch Protein-Kristallographie zu verfolgen? Kann die Erfolgsquote von Fragment-basierter Kristallographie durch einen Tethering-Ansatz, welcher schwach bindende Fragmente am Protein fixiert, erhöht werden? Die Verfügbarkeit großer Mengen an CA II ist die Grundvoraussetzung um solch eine Bandbreite sehr verschiedener Projekte durchführen zu können. Daher wurde das CA II Gen in das GST gene fusion System kloniert, ein System welches Gen Expression auf sehr hohem Niveau ermöglicht. Das Expressionssystem wurde hinsichtlich der Proteinausbeute optimiert und stellt 30 mg Protein aus 1 L Zellkultur bereit. Etliche CA II Mutanten wurden durch ortsgerichtete Mutation hergestellt und eine Quecksilber-freie Protein-Kristallisation für den Wildtyp und die Mutanten erstellt. Die Durchführung einer prominenten Click-Chemie-Reaktion, der Huisgen Reaktion – eine Cu+ katalysierte 2+3 dipolare Cycloaddition – in der Bindetasche der CA II war das anfängliche Ziel des ersten Projektes dieser Dissertation. Dafür sollte ein künstliches Kupfer-Zentrum an der Oberfläche der CA II eingeführt, und dieses neue katalytische Zentrum für die Bildung von Triazolen aus Alkinen und Aziden ausgenutzt werden. Leider verblieben diese Versuche eines rationalen Designs erfolglos. Dies zeigt auf, dass unser derzeitig noch begrenztes Verständnis über den Zusammenhang zwischen Aminosäure-Mutationen und Proteinarchitektur in komplexen biologischen Systemen, wie z.B. in hoch entwickelten Proteinen, unvorhersehbaren Auswirkungen mit sich bringen kann. Schließlich, eher durch einen glücklichen Zufall als gezieltes Design, konnte die Bildung eines Kupfer-Zentrums an der Oberfläche der CA II zustande gebracht werden. Neben dem zuvor beschriebenen Design eines Metall-Zentrums, wurde das Click-Chemie-Projekt ebenso durch einen neuartigen Tethering-unterstützten Ansatz vorangetrieben. In diesem Ansatz wird eine Reaktionskomponente kovalent an die Proteinoberfläche gebunden, während die zweite Komponente durch einen Sulfonamid-Anker an das Zink-Ion im aktiven Zentrum bindet. Die Reaktion konnte entweder durch Cu+ Ionen oder aber durch die Proteinumgebung ausgelöst werden. Bemerkenswerterweise erfolgte die Protein-induzierte Triazolbildung mit ausgeprägter Regio- sowie Stereoselektivität. Aus dem racemischen Alkingemisch wurde selektiv das 1,5-S-Triazol gebildet. Die Tethering Methode wurde abgesehen von diesem Click-Chemie-Ansatz ebenso für ein Fragment-Screening in Hinblick auf die CA II angewandt. Der Tethering-Ansatz ermöglicht die Identifizierung von Fragmenten mit eher schwacher Affinität zum Zielprotein. Durch einen computerbasierten Docking-Ansatz wurde eine virtuelle Bibliothek von Fragmenten getestet um so eine überschaubare Anzahl an Substanzen für die Synthese auszuwählen. Durch HPLC-MS Messungen konnten viele Fragmente mit einer vielversprechenden Affinität zu CA II identifiziert werden. Einige dieser Substanzen wurden im kovalenten Komplex mit CA II kristallographisch untersucht und zeigten, dass eine Saccharin-Bindetasche an der Protein-Oberfläche mit diesem Tethering-Ansatz erfolgreich untersucht werden kann. Zusätzlich konnte eine zweite Bindetasche an der Oberfläche, bekannt für die Bindung von CA II Aktivatoren, untersucht werden. In einem weiteren Ansatz zur Fragment-basierten Leitstruktursuche wurden vier neue Zink-koordinierende Kopfgruppen an CA Isoenzymen getestet. Eine Komplexstruktur von CA II mit 1,2-HOPTO zeigte einen neuartigen Zn2+ koordinierenden Bindungsmodus mit Wechselwirkungen zu Thr199 und Thr200. Das Fragment, welches zusätzlich perfekt durch ein Wassernetzwerk koordiniert ist, verspricht eine neue Klasse von CA Inhibitoren. Durch die Addition von Substituenten, die mit den hydrophilen und lipophilen Bereichen der CA II Bindetasche wechselwirken, kann die Bindungsaffinität verbessert werden

Lead optimization for new antimalarials and Successful lead identification for metalloproteinases: A Fragment-based approach Using Virtual Screening

Lead optimization for new antimalarials and Successful lead identification for metalloproteinases: A Fragment-based approach Using Virtual Screening Computer-aided drug design is an essential part of the modern medicinal chemistry, and has led to the acceleration of many projects. The herein described thesis presents examples for its application in the field of lead optimization and lead identification for three metalloproteins. DOXP-reductoisomerase (DXR) is a key enzyme of the mevalonate independent isoprenoid biosynthesis. Structure-activity relationships for 43 DXR inhibitors are established, derived from protein-based docking, ligand-based 3D QSAR and a combination of both approaches as realized by AFMoC. As part of an effort to optimize the properties of the established inhibitor Fosmidomycin, analogues have been synthesized and tested to gain further insights into the primary determinants of structural affinity. Unfortunately, these structures still leave the active Fosmidomycin conformation and detailed reaction mechanism undetermined. This fact, together with the small inhibitor data set provides a major challenge for presently available docking programs and 3D QSAR tools. Using the recently developed protein tailored scoring protocol AFMoC precise prediction of binding affinities for related ligands as well as the capability to estimate the affinities of structurally distinct inhibitors has been achieved. Farnesyltransferase is a zinc-metallo enzyme that catalyzes the posttranslational modification of numerous proteins involved in intracellular signal transduction. The development of farnesyltransferase inhibitors is directed towards the so-called non-thiol inhibitors because of adverse drug effects connected to free thiols. A first step on the way to non-thiol farnesyltransferase inhibitors was the development of an CAAX-benzophenone peptidomimetic based on a pharmacophore model. On its basis bisubstrate analogues were developed as one class of non-thiol farnesyltransferase inhibitors. In further studies two aryl binding and two distinct specificity sites were postulated. Flexible docking of model compounds was applied to investigate the sub-pockets and design highly active non-thiol farnesyltransferase inhibitor. In addition to affinity, special attention was paid towards in vivo activity and species specificity. The second part of this thesis describes a possible strategy for computer-aided lead discovery. Assembling a complex ligand from simple fragments has recently been introduced as an alternative to traditional HTS. While frequently applied experimentally, only a few examples are known for computational fragment-based approaches. Mostly, computational tools are applied to compile the libraries and to finally assess the assembled ligands. Using the metalloproteinase thermolysin (TLN) as a model target, a computational fragment-based screening protocol has been established. Starting with a data set of commercially available chemical compounds, a fragment library has been compiled considering (1) fragment likeness and (2) similarity to known drugs. The library is screened for target specificity, resulting in 112 fragments to target the zinc binding area and 75 fragments targeting the hydrophobic specificity pocket of the enzyme. After analyzing the performance of multiple docking programs and scoring functions forand the most 14 candidates are selected for further analysis. Soaking experiments were performed for reference fragment to derive a general applicable crystallization protocol for TLN and subsequently for new protein-fragment complex structures. 3-Methylsaspirin could be determined to bind to TLN. Additional studies addressed a retrospective performance analysis of the applied scoring functions and modification on the screening hit. Curios about the differences of aspirin and 3-methylaspirin, 3-chloroaspirin has been synthesized and affinities could be determined to be 2.42 mM; 1.73 mM und 522 μM respectively. The results of the thesis show, that computer aided drug design approaches could successfully support projects in lead optimization and lead identification. fragments in general, the fragments derived from the screening are docke

Chemical, molecular pharmacology and neuroprotective properties of the essential oil derived from Aloysia citrodora Palau

Essential oils derived from dried and fresh leaves of Aloysia citrodora were obtained by hydrodistillation, and were investigated for a range of pharmacological properties: receptor binding, in vitro acetylcholinesterase (AChE) inhibitory, antioxidant activities, and neuroprotection properties relevant to neurodegenerative diseases. Fresh leaf A. citrodora essential oil inhibited [3H] nicotine binding to well washed rat forebrain membranes, with mean apparent IC50 of 0.0018 mg/ml. No significant binding activity was observed for A. citrodora essential oil derived from fresh or dried leaves, for GABAAR and NMDARs. A. citrodora essential oil, both dried and fresh, exhibited radical scavenging activity (up to 100%, IC50 < 0.0001 mg/ml) and iron (II) chelating properties (approx. IC50 = 0.05 mg/ml), and showed neuroprotective characteristics against the toxic effects of H2O2 (100%, 0.001 mg/ml) and β-amyloid (approx. 50%, 0.01 mg/ml) in CAD neuronal cell culture. Both EOs from dried and fresh leaves also displayed effective AChE inhibitory activity, with the dried leaves oil displaying more clear AChE inhibitory activity than fresh oil, which could be related to the higher respective levels of caryophyllene oxide. Recombinant human anticholinesterase enzyme was used for structure based in silico screening of A. citrodora essential oil constituents for AChE Inhibitors, and the top scoring hits with highest pharmacophore fit values showed common interactions with residues at the active site of that of donepezil. The top seven hits in order of fit score, were β-curcumene, curcumene bisabolene, trans-calamenene, caryophyllene oxide, β-sesquiphellandrene and geranyl acetate. This indicates that plants may yield novel effective and safe AChE inhibitors, other than alkaloids. To begin to identify the chemicals underpinning the pharmacological properties of A. citrodora, GC/MS analysis of the chemical composition of the essential oil from leaves of A. citrodora identified eighty three major chemicals, including the presence of terpenoids, monoterpenes and sesquiterpenes, and 6-methyl-5-hepten-2-one, the main constituents being limonene, caryophyllene oxide, curcumene, spathulenol, 1,8-cineole constituting 47% of the total oil. Finally, a simple, inexpensive solid phase extraction method was developed for fractionation of essential oils. Collectively, this thesis provides a better understanding of the pharmacology of the Aloysia essential oil and its constituents relating to its potential use in the treatment neurodegenerative disease

Exploring heterocyclic scaffolds in the development of multi-target anti-Alzheimer and multi-trypanosomatid compounds

[eng] The aim of this PhD thesis consists of the synergistic combination of both highly efficient synthetic approaches and molecular modelling tools for the structure-based drug design and synthesis of novel bioactive heterocyclic compounds. The work carried out has followed two main research lines, namely the development of novel disease-modifying anti-Alzheimer agents and still unexplored chemical entities for the treatment of Neglected Tropical Diseases (NTDs). The results obtained have been presented as a compendium of publications and draft manuscripts. In the framework of the anti-Alzheimer research line, first the hit-to-lead optimization of a practically inactive propidium-related compound easily accessed via a Povarov multicomponent reaction (MCR) approach (Di Pietro O. et al. Eur. J. Med. Chem., 2014, 73, 141), and the subsequent molecular hybridization with a 6-chlorotacrine unit through a molecular dynamics-driven tether length optimization, overall led to one of the most potent non-covalent dual binding site acetylcholinesterase inhibitor (AChEI) ever described in the literature (Di Pietro O. et al. Eur. J. Med. Chem., 2014, 84, 107). Second, the combined recourse to the highly versatile click-chemistry strategy, through the well-known Cu-catalyzed azide-alkyne cycloaddition reaction, and convenient computational chemistry tools, allowed the rational design and synthesis of a novel series of 1,4-disubstituted triazole-based propargylamines as irreversible MAO-B inhibitors (draft manuscript) with the perspective to be further linked to a second pharmacophoric moiety to derive novel MTDLs as potential anti-Alzheimer drug candidates. Furthermore, an extensive computation of the BACE-1 apo conformational ensemble by means of combined molecular dynamics technique and Principal Component Analysis (PCA) method, allowed to carry out an exhaustive study of a secondary transient druggable pocket (draft manuscript) and a virtual screening of 500,000 commercially available fragments for further drug discovery purposes. Finally, in the framework of the NTDs research line, 2−4-step sequences involving as the key step an initial Povarov MCR gave easy access to a small library of quinolones and tricyclic heterofused quinolines, which were subjected to phenotypic whole-cell screenings, leading to the individuation of several low micromolar multi-trypanosomatid hit compounds (Di Pietro et al. Eur. J. Med. Chem. 2015, accepted with minor revision)

Structure, function, evolution and inhibition studies of the organophosphate detoxifying enzyme αE7

Insecticide resistance is a global concern that threatens human health and agricultural productivity. Understanding the molecular basis of resistance will help to manage future insecticide use to ensure that effective, safe and inexpensive pest control is available. In the Australian sheep blowfly Lucilia cuprina, a single mutation (Gly137Asp) in the αE7 carboxylesterase gives rise to resistance by converting the enzyme into an organophosphate (OP) hydrolase. This emergence of new activity provides a unique opportunity to investigate the molecular basis for enzyme evolution. In this thesis, I investigated the structure, function, evolution and inhibition of αE7. Chapter two describes the role of structural diversity in the function of wild type αE7. I applied new methods for extracting information about structural diversity from X-ray diffraction data to explore the changes in structure that accompany high affinity OP binding in αE7. In chapter three, I investigated the molecular basis for the evolution of catalytic OP detoxification in the blowfly. I determined the structure of the Gly137Asp variant by X-ray crystallography, which, along with molecular dynamics simulations and enzyme activity assays, revealed the role of Asp137 in the new catalytic mechanism. The new sidechain is disordered, and potentially only displays a fraction of its catalytic potential. Chapter four explores this catalytic potential through the laboratory-directed evolution of αE7 for increased OP hydrolase activity. I performed detailed kinetic and structural analysis of the evolutionary trajectory and characterized the structural changes responsible for the 8000-fold increase in OP hydrolase activity. The analysis unmasked a hidden, catalytically relevant, conformation of the active site. Furthermore, the results revealed the role of conformational diversity in the evolutionary optimization of αE7 and highlight the challenges to satisfying the competing demands of substrate binding and catalysis in the tightly packed environment of an enzyme’s active site. This work establishes that only a fraction of the evolutionary potential of αE7 has been explored in nature. In chapter five, I combined structural knowledge of αE7 with a computational screen to discover new potent and selective inhibitors of αE7. These compounds, based on a boronic acid scaffold, act as synergists to reduce the amount of OP required to kill L. cuprina by up to 16-fold, and abolish resistance. The broad-spectrum potential for the compounds as a new class of synergist was demonstrated by their low toxicity to animals and their ability to potentiate OP insecticides against another common insect pest, the peach-potato aphid Myzus persicae. These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy. More broadly, this thesis makes contributions to characterizing structural protein heterogeneity using X-ray diffraction, to understanding the molecular basis of enzyme evolution and to the use of in silico screens for the discovery of enzyme inhibitors. The results from this thesis will assist the of control insect pests and the management of insecticide resistance