Strukturelle und thermodynamische Charakterisierung der Fragment und Liganden Bindung an Thrombin

ITC-Verdrängungstitrationen und direkte ITC-Titrationen bei niedrigen c-Werten können als zuverlässige Technik verwendet werden kann, um schwache Wechselwirkungen zu untersuchen, wie sie üblicherweise für die Bindung von Fragmenten an ein Zielprotein vorkommen. Hierfür müssen jedoch einige Grundbedingungen erfüllt werden. Zuallererst ist für ITC Verdrängungstitrationen wichtig, dass das untersuchte Fragment mit einem Teil der Proteinbindetasche interagieren muss, der mit dem Bindungsbereich des verwendeten Referenzliganden überlappt. Zweitens muss der ausgewählte Referenzligand eine signifikant höhere oder niedrigere Bindungsenthalpie besitzen, so dass eine Wärmedifferenz zu dem untersuchten Fragment gemessen werden kann. Ferner ist zu beachten, dass bei jeder ITC-Verdrängungstitration, alle Fehler, die für die Referenzliganden ermittelt wurden, einen Einfluss auf die Genauigkeit der für das Fragment erhaltenen Parameter haben. Schließlich kann, wenn kein geeigneter Referenzligand zur Verfügung steht und das Fragment enthalpisch genug bindet, eine direkte Titration bei niedrigen c-Werten als Alternative verwendet werden. In diesem Fall ist jedoch ein gewisses Vorwissen über die zu erwartende Bindungsaffinität des Fragments nötig, um die erforderliche Überschuss Konzentration des Fragments am Ende der Titration abschätzen zu können. Die Löslichkeit des Fragments und des Proteins sind ebenfalls wichtige Aspekte bei der Titration mit niedrigen c-Werten, da hohe Konzentrationen des Fragments in der Injektionsspritze erforderlichen sind, um in der Probenzelle eine Konzentration des Fragments zu erreichen, die den KD-Wert des Fragments überschreitet. Daher ist eine Messung des KD-Werts des Fragments in einem unabhängigen Experiment sehr zu empfehlen, um die erforderliche Konzentration des Fragments abzuschätzen. Letztlich ist er sehr wichtig, dass die unter den verschiedenen Versuchsbedingungen gemessen Bindungsenthalpien nicht quantitativ verglichen werden sollten, sondern nur relativ zueinander durch Verwendung eines sorgfältig ausgewählten Messprotokolls. Es ist bemerkenswert, dass mit verschiedenen Referenzliganden, die für die Verdrängung der Fragmente verwendet wurden, abweichende enthalpische Signale beobachtet wurden. Wahrscheinlich wird dies durch Unterschiede in der Wasserstruktur der verschiedenen Liganden in der Bindetasche verursacht. Die Betrachtung der relativen Enthalpiedifferenzen in den verschiedenen Verdrängungsexperimenten zeigte dagegen eine konsistente relative Differenz. Dies ermöglicht es, die untersuchten Fragmente relativ zueinander zu charakterisieren. Die detaillierte Interpretation der enthalpischen Signaturen erfordert jedoch einen Vergleich der Kristallstrukturen der Fragmente. Röntgenkristallstrukturen von sechs Fragmenten, die die S1 Tasche von Thrombin adressieren und der Referenzliganden wurden bestimmt und in Bezug auf die thermodynamischen Bindungsprofile analysiert. Die Bindungsaffinität eines Liganden wird nicht nur durch die Eigenschaften des gebildeten Protein-Ligand-Komplexes bestimmt, sondern auch durch die Unterschiede von Liganden in wässriger Lösung vor Proteinbindung. Konformativ eingeschränkte Liganden können einen signifikanten Bindungsvorteil gegenüber flexiblen Liganden haben, wenn in einem rigiden Ligandgrundgerüst eine Geometrie bereits präorganisiert ist, die dem Liganden in gebundenem Zustand ähnlich ist. Ein neuer vom 3-Amidinophenylalanin abgeleiteter Matriptase Inhibitor wurde in Thrombin kristallisiert und die Röntgenkristallstruktur eines repräsentativen, verwandten Inhibitortyps in Matriptase mit dieser überlagert. Aus der Überlagerung dieser Strukturen konnten Informationen hinsichtlich der in dieser Studie beobachteten Selektivitätsprofile zu anderen Serinproteasen gezogen werden. Durch die große Homologie der mit dem Inhibitor interagierenden Aminosäuren in der Bindetasche von Thrombin und Matriptase können zudem zuverlässige Vorhersagen über den möglichen Bindungsmodus in Matriptase getroffen werden

Investigations on lin-Benzopurines With Respect to Dissociation Behavior, Pocket Cross-Talk, Targeting Resistance Mutants, Residual Mobility, and Scaffold Optimization

The present thesis deals with the characterization and improvement of selective antibiotics targeting the enzyme tRNA–guanine transglycosylase. Recently, the lin-benzopurine scaffold was introduced as promising starting point for the structure-based design of TGT inhibitors. Two classes, namely the lin-benzoguanines and lin-benzohypoxanthines, were found to inhibit the target enzyme in the nanomolar range. However, the analyzed molecules did not show ideal drug metabolism and pharmacokinetic features since firstly they showed poor permeation through cell membranes and secondly their attached 2-substituents were poorly defined in the difference electron density in crystal structures complicating the establishment of a structure–activity relationship. In a comprehensive study the protonation inventory of lin-benzopurines is addressed. Initial ITC measurements performed with lin-benzoguanine-type ligands suggested the uptake of one proton by the ligand. This protonation takes place at the basic guanidine moiety of the aminopyrimidinone structure of the scaffold. The lin-benzohypoxanthines do not show this behavior. pKa Calculations support the observations. While the lin-benzohypoxanthines bind to a TGT conformation closely similar to the apo enzyme interacting with only one aspartate within the G34 recognition site, addition of the exocyclic amino functional group in case of the lin-benzoguanines induces the rotation of a second aspartate towards the binding pocket. The negatively charged environment of both aspartates in short distance provokes a pKa shift in case of the lin-benzoguanines strong enough to induce the uptake of a proton. The hypothesis is proofed by studies using site-directed mutagenesis. Considering the binding affinities across the series of lin-benzopurines, a rather flat structure–activity relationship is observed. Therefore, additional insight into the driving forces of binding was gained by factorizing the free binding energy into enthalpy–entropy contribution. As expected, bindings of both series were found to be enthalpy-driven. Thereby, the lin-benzohypoxanthines exhibit a less pronounced enthalpic term due to their missing interaction to Asp102, which can be partly compensated by a crystallographically conserved water cluster located at the bottom of the G34 recognition site. While the thermodynamic profiles of the lin-benzohypoxanthines remain nearly unchanged, data for the lin-benzoguanines are found to be quite diverse. Obviously, the structural changes triggered by Asp102 in case of the lin-benzoguanines enable a cross-talk between U33 subpocket addressed by the 2-substituent and the G34 recognition site occupied by the parent scaffold. Based on elevated temperature factors, a high flexibility of the 2-substituent of the lin-benzoguanines was assumed. Therefore, we tested whether a binder with high residual mobility can avoid a loss in binding affinity in case of resistant mutations compared to a binder adopting one ordered binding mode. After identification of an appropriate mutation site, different mutants were expressed and crystallized. The derived binding affinities of various 2-amino-lin-benzoguanines could be related to the binding of the parent scaffold inducing disorder of the protein in proximal distance to the mutation site rather than to the different 2-substituents. Only marginal differences could be ascribed to the properties of the substitution pattern, most likely due to electrostatic attractions and repulsions, respectively. In further experiments MD-simulations were used to predict the binding modes of extended 2-amino-lin-benzoguanines. Subsequent crystal structure analyses unravelled novel aspects important for the further design and characterization of TGT inhibitors: Firstly, we were able to spot the 2-subsituent of the extended 2-amino-lin-benzoguanines, which bind to the ribose-32 subpocket that has never been occupied before. Secondly, the results emphasize the importance of the applied crystallization conditions. Poorly defined electron density does not always indicate ligands or substituents exhibiting high mobility in the protein-bound state. The applied crystallization protocol takes a major impact on the derived difference electron density and has to be considered in the discussion of the obtained structures. The class of 5-azacytosines was investigated as a novel scaffold to inhibit TGT. Similarly as the lin-benzopurines, also the 5-azacytosines are deduced from the natural substrate guanine and establish similar binding features, however, the key interaction to Asp102 that was proven to be of utmost importance for substrate recognition, ligand protonation, and pocket crosstalk is poorly established. Different from all known TGT inhibitors, they do not bind to the protein in a planar fashion. In consequence, the compared to lin-benzoguanines low pKa cannot be shifted into a window appropriate for ligand protonation and binding affinity drops

Biophysical Properties of DNA Minor Groove Binding by Heterocyclic Cations of Varying Structures

Small heterocyclic cations that bind to DNA are interesting systems to study due to their structural diversity, pharmaceutical potential, and characteristic target recognition patterns. Clinically, such compounds offer an attractive therapeutic approach as inhibitors of protein-DNA interactions implicated in disease. Due to their typical intrinsic fluorescence, these compounds also have potential as convenient biotechnological probes for studying DNA. Finally, from a biophysics perspective, an intricate understanding of the factors driving DNA binding by these compounds can extend our understanding of DNA targeting more broadly. In this thesis, the electrostatics and hydration properties of DNA binding by eight of these heterocyclic cations in complex with various DNA sequences are investigated

An in-silico study: Investigating small molecule modulators of bio-molecular interactions

Small molecule inhibitors are commonly used to target protein targets that assist in the spread of diseases such as AIDS, cancer and deadly forms of influenza. Despite drug companies spending millions on R&D, the number of drugs that pass clinical trials is limited due to difficulties in engineering optimal non-covalent interactions. As many protein targets have the ability to rapidly evolve resistance, there is an urgent need for methods that rapidly identify effective new compounds. The thermodynamic driving force behind most biochemical reactions is known as the Gibbs free energy and it contains opposing dynamic and structural components that are known as the entropy (ΔS°) and enthalpy (ΔH°) respectively. ΔG° = ΔH° - TΔS°. Traditionally, drug design focussed on complementing the shape of an inhibitor to the binding cavity to optimise ΔG° favourability. However, this approach neglects the entropic contribution and phenomena such as Entropy-Enthalpy Compensation (EEC) often result in favourable bonding interactions not improving ΔG°, due to entropic unfavorability. Similarly, attempts to optimise inhibitor entropy can also have unpredictable results. Experimental methods such as ITC report on global thermodynamics, but have difficulties identifying the underlying molecular rationale for measured values. However, computational techniques do not suffer from the same limitations. MUP-I can promiscuously bind panels of hydrophobic ligands that possess incremental structural differences. Thus, small perturbations to the system can be studied through various in silico approaches. This work analyses the trends exhibited across these panels by examining the dynamic component via the calculation of per-unit entropies of protein, ligand and solvent. Two new methods were developed to assess the translational and rotational contributions to TΔS°, and a protocol created to study ligand internalisation. Synthesising this information with structural data obtained from spatial data on the binding cavity, intermolecular contacts and H-bond analysis allowed detailed molecular rationale for the global thermodynamic signatures to be derived

Inhibitor Synthesis and Biophysical Characterization of Protein–Ligand–Solvent Interactions An Analysis of the Thermodynamics and Kinetics of Ligand Binding to Thermolysin

In the pre-clinical development stages of most drug design campaigns, the equilibrium binding affinity of a prospective lead candidate, in the form of an IC50, Kd or ΔG° value, is the most commonly employed benchmark parameter for its effectiveness as a putative drug. Hydrogen bonding, van der Waals and electrostatic interactions, as well as hydrophobic effects are among the most prominent factors that contribute to binding. In structure based design approaches, these interactions can routinely be linked to a structural motif of a drug molecule, which can greatly assist in the construction of compounds with a desired set of properties. Equilibrium binding affinity can also be expressed in terms of kinetics, were the steady-state constant Kd is defined as the ratio of the rate constants of dissociation (kd) and association (ka). The thermodynamic expression ΔG° can be subdivided into an enthalpic (ΔH°) and an entropic (–TΔS°) term. In either case, the molecular mechanisms that define the kinetics of binding or the compensation of enthalpic and entropic contributions are not fully understood. The goal of this dissertation is the in-depth investigation of the molecular processes that drive protein–ligand interactions. A special focus is set on the partitioning of thermodynamic and kinetic parameters into their respective microscopic elements. For this, the metalloprotease thermolysin (TLN) is used as a model system. This protein is well characterized and represents a robust system with excellent crystallographic properties and a thoroughly documented inhibitor class. The first publication (Chapter 2) presents an improved strategy for the synthesis and purification of phosphonamidate peptides that are known as potent inhibitors of TLN. Due to the inherent instability of the phosphorous–nitrogen bond, the introduction of polar functional groups into the inhibitor scaffold is quite challenging. Here, a synthetic strategy is presented that minimizes the amount of hydrolysis during peptide coupling, deprotection and purification through the use of an allyl-based protection system and a solid-phase extraction (SPE) protocol for the final purification step. This allows the synthesis of highly pure TLN inhibitors incorporating a variety of functional groups for use in biophysical experiments. In the second publication (Chapter 3), a strategy for the design of inhibitors is highlighted, which relies on the targeted design of water networks that are formed around a protein–ligand complex. Based on information from a previous study, the shape of a hydrophobic portion of a TLN ligand is altered in a way that allows a beneficial stabilization of water molecules in the first solvation layer of the complex. Supported by molecular dynamics simulations, a series of diastereomeric inhibitors is synthesized and the binding process is characterized by X-ray crystallography, isothermal titration calorimetry (ITC) and surface plasmon resonance spectroscopy (SPR). The optimization of the hydrophobic P2’ moiety results in a 50-fold affinity enhancement compared to the original methyl substituted ligand. This improvement is mainly driven by a favorable enthalpic term that originates from the stabilization of water polygons in the solvation shell. In the follow-up study in Chapter 4, the binding signature of a series of inhibitors that place a charged and polar moiety in the solvent exposed S2’ pocket of TLN is investigated. Here, a partially hydrated ammonium group is gradually retracted deeper into the hydrophobic protein environment. From the crystal structures it is evident that the polar ligands do not recruit an increased amount of water molecules into their solvation layer when compared to related analogues that feature a purely aliphatic residue at the solvent interface. The penalty for the partial desolvation of the charged functional group, in combination with the lack of a strongly ordered water network, results in a severe affinity decrease that is driven by an unfavorable enthalpic term. The deep, hydrophobic S1’ pocket of TLN determines the substrate specificity of the protease and is commonly addressed by high affinity inhibitors. Experimental evidence from previous studies suggests, however, that this apolar crevice is only poorly solvated in the absence of an interaction partner. With the study in Chapter 5, an attempt for the experimental analysis of the hydration state of the S1’ pocket is presented. For this, a special inhibitor is designed that transforms the protein pocket into a cavity, while simultaneously providing enough empty space for the accommodation of several water molecules. A detailed analysis of an experimentally phased electron density map reveals that the cavity remains completely unsolvated and thus, vacuous. As an intriguing prospect for the exploitation of such poorly hydrated protein pockets in drug design, the placement of an iso-pentyl moiety in the ligand’s P1’ position results in a dramatic, enthalpically driven gain in affinity by a factor of 41 000. With a detailed structural analysis of a series of chemically diverse TLN inhibitors, the kinetics of the protein–ligand binding process are investigated in Chapter 6. From the SPR derived kinetic information, it becomes apparent that the nature of the functional group in the P2’ position of a thermolysin inhibitor has a significant impact on its dissociation kinetics. This property can be linked to the interaction between the respective functionality of a ligand and Asn112, a residue that lines the active site of the protease and is commonly believed to align a substrate for proteolytic cleavage. This residue undergoes a significant conformational change when the protein transitions from its closed state to its open form, from which a ligand is released. Interference with this retrograde induced-fit mechanism through strong hydrogen-bonding interactions to an inhibitor results in a pronounced deceleration of the dissociation process. The case of the known inhibitor ZFPLA demonstrates that a further restriction of the rotation of Asn112 by a steric barrier in the P1 position of a ligand, can reduce the rate constant of dissociation by a factor of 74 000. Fragment-based lead discovery has become a popular method for the generation of prospective drug molecules. The weak affinity of fragments and the necessity for high concentrations, however, can result in false-positive signals from the initial binding assays that routinely plague fragment-based screening. The pursuit of such a “red herring” can lead to a significant loss of time and resources. In Chapter 7, a molecule that emerged as one of the most potent binders from an elaborate fragment screen against the aspartic protease endothiapepsin is identified as a false-positive. Detailed crystallographic, HPLC and MS experiments reveal that the affinity detected in multiple assays can in fact be attributed to another compound. This entity is formed from the initially employed molecule in a reaction cascade that results in a major rearrangement of its heterocyclic core structure. Supported by quantum chemical calculations and NMR experiments, a mechanism for the formation of the elusive compound is proposed and its binding mode analyzed by X-ray crystallography

Exploring the role of conformational dynamics in the regulation of tyrosine kinases.

Tyrosine kinases (TKs) are a family of signalling proteins of great pharmaceutical im- portance, as they are involved in the regulation of most cellular pathways. TKs catalytic activity is strictly regulated by conformational changes and post-translational modifi- cations, and their deregulation is involved in numerous human diseases, ranging from cancer to autoimmune diseases. Among tyrosine kinases, Abl and Src are of particular interest for cancer research. The Abl domain in the BCR-Abl fusion protein is the main cause of chronic myeloid leukemia, and it was the target of the first successful anti- leukemic therapy, the powerful kinase inhibitor imatinib. We now know that imatinib effectively inhibits BCR-Abl, as well as Kit and Lck kinases, by binding to a specific inactive state, in which the conserved Asp-Phe-Gly motif (DFG) assumes a peculiar "out" conformation. Still, there are many questions on its mode of action. For instance, other TKs with an extended identity with Abl (such as Src, which has 45% sequence identity) bind much less strongly to imatinib, in spite of very similar binding mode. Moreover, the mode of action of drug-resistant mutations that induce imatinib resis- tance and cause an increasing number of relapses in patients under treatment, is still poorly understood. Understanding the molecular mechanisms responsible for the ob- served differences in imatinib activity, is essential for the development of new selective anticancer drugs. In this thesis, by using computational and experimental approaches, I have investigated the reasons leading to drug resistance and the differential binding affinity in homologous TKs. A combination of enhanced sampling molecular dynam- ics simulations (such as parallel tempering metadynamics or PTmetaD) were used to reconstruct and compare the free energy landscape associated with the relevant con- formational changes. Mutagenesis and isothermal titration calorimetry were used to validate the computational results

Fine-tuning spermidine binding modes in the putrescine binding protein PotF

A profound understanding of the molecular interactions between receptors and ligands is important throughout diverse research, such as protein design, drug discovery, or neuroscience. What determines specificity and how do proteins discriminate against similar ligands? In this study, we analyzed factors that determine binding in two homologs belonging to the well-known superfamily of periplasmic binding proteins, PotF and PotD. Building on a previously designed construct, modes of polyamine binding were swapped. This change of specificity was approached by analyzing local differences in the binding pocket as well as overall conformational changes in the protein. Throughout the study, protein variants were generated and characterized structurally and thermodynamically, leading to a specificity swap and improvement in affinity. This dataset not only enriches our knowledge applicable to rational protein design but also our results can further lay groundwork for engineering of specific biosensors as well as help to explain the adaptability of pathogenic bacteria

The Development and Assessment of Computational Approaches to the Thermodynamics and Kinetics of Binding

Molecular recognition refers to the interaction between two or more molecules through complementary noncovalent bonding, for example, via hydrogen bonding, electrostatic interactions, van der Waals forces or hydrophobic forces. Molecular recognition plays an important role in biology and mediates interactions between receptors and ligands, antigens and antibodies, nucleic acids and proteins, proteins and proteins, enzymes and substrates, and nucleic acids with each other. Many cellular processes are governed by a group of proteins acting in a coordinated manner; such complicated mechanisms are closely regulated: changes in the populations of particular complexes or changes in concentrations of the products of protein mediated reactions can switch cells from one state to another (from replication to apoptosis, for example). These small variations in molecular populations are caused by very delicate differences in the thermodynamics or kinetics of reactions. This implies that in order to understand not only biological systems in terms of their molecular components, but also to be able to predict and model system response to stimuli (whether it is a natural substrate or a drug), characterisation of the thermodynamic and kinetic components of the binding process is of paramount importance. This combined computational-experimental project was focused on the development of new computational approaches able to predict the enthalpic component of ligand binding, using quantum mechanics. A concept of ‘theoceptors’ was developed, which are theoretical receptors constructed by computing the optimal geometry of ligands binding in the receptor. This project was supported by AstraZeneca, and it included an industrial placement in the Structural and Biophysical Sciences area, where the experimental data was generated to characterise the thermodynamics and kinetics of binding of a range of ligands to two biological targets, using two experimental techniques, isothermal titration calorimetry and surface plasmon resonance. The findings contribute greatly to the process currently underway of expanding our understanding of the relevance of both of these aspects of biochemistry to drug discovery

Development of Computational Methods to Predict Protein Pocket Druggability and Profile Ligands using Structural Data

This thesis presents the development of computational methods and tools using as input three-dimensional structures data of protein-ligand complexes. The tools are useful to mine, profile and predict data from protein-ligand complexes to improve the modeling and the understanding of the protein-ligand recognition. This thesis is divided into five sub-projects. In addition, unpublished results about positioning water molecules in binding pockets are also presented. I developed a statistical model, PockDrug, which combines three properties (hydrophobicity, geometry and aromaticity) to predict the druggability of protein pockets, with results that are not dependent on the pocket estimation methods. The performance of pockets estimated on apo or holo proteins is better than that previously reported in the literature (Publication I). PockDrug is made available through a web server, PockDrug-Server (http://pockdrug.rpbs.univ-paris-diderot.fr), which additionally includes many tools for protein pocket analysis and characterization (Publication II). I developed a customizable computational workflow based on the superimposition of homologous proteins to mine the structural replacements of functional groups in the Protein Data Bank (PDB). Applied to phosphate groups, we identified a surprisingly high number of phosphate non-polar replacements as well as some mechanisms allowing positively charged replacements. In addition, we observed that ligands adopted a U-shape conformation at nucleotide binding pockets across phylogenetically unrelated proteins (Publication III). I investigated the prevalence of salt bridges at protein-ligand complexes in the PDB for five basic functional groups. The prevalence ranges from around 70% for guanidinium to 16% for tertiary ammonium cations, in this latter case appearing to be connected to a smaller volume available for interacting groups. In the absence of strong carboxylate-mediated salt bridges, the environment around the basic functional groups studied appeared enriched in functional groups with acidic properties such as hydroxyl, phenol groups or water molecules (Publication IV). I developed a tool that allows the analysis of binding poses obtained by docking. The tool compares a set of docked ligands to a reference bound ligand (may be different molecule) and provides a graphic output that plots the shape overlap and a Jaccard score based on comparison of molecular interaction fingerprints. The tool was applied to analyse the docking poses of active ligands at the orexin-1 and orexin-2 receptors found as a result of a combined virtual and experimental screen (Publication V). The review of literature focusses on protein-ligand recognition, presenting different concepts and current challenges in drug discovery.Tässä väitöskirjassa esitetään tietokoneavusteisia menetelmiä ja työkaluja, jotka perustuvat proteiini-ligandikompleksien kolmiulotteisiin rakenteisiin. Ne soveltuvat proteiini-ligandikompleksien rakennetiedon louhimiseen, optimointiin ja ennustamiseen. Tavoitteena on parantaa sekä mallinnusta että käsitystä proteiini-liganditunnistuksesta. Väitöskirjassa työkalut kuvataan viitenä eri alahankkeena. Lisäksi esitetään toistaiseksi julkaisemattomia tuloksia vesimolekyylien asemoinnista proteiinien sitoutumistaskuihin. Kehitin PockDrugiksi kutsumani tilastollisen mallin, joka yhdistää kolme ominaisuutta – hydrofobisuuden, geometrian ja aromaattisuuden – proteiinitaskujen lääkekehityskohteeksi soveltuvuuden ennustamista varten siten, että tulokset ovat riippumattomia sitoutumistaskun sijoitusmenetelmästä. Apo- ja holoproteiinien taskujen ennustaminen toimii paremmin kuin alan kirjallisuudessa on aiemmin kuvattu (Julkaisu I). PockDrug on vapaasti käyttäjien saatavilla PockDrug-verkkopalvelimelta (http://pockdrug.rpbs.univ-paris-diderot.fr), jossa on lisäksi useita työkaluja proteiinin sitoutumiskohdan analyysiin ja karakterisointiin (Julkaisu II). Kehitin myös muokattavissa olevan tietokoneavusteisen prosessin, joka perustuu samankaltaisten proteiinien päällekkäin asetteluun, louhiakseni Protein Data Bankista (PDB) toiminnallisten ryhmien rakenteellisia korvikkeita. Tätä fosfaattiryhmiin soveltaessani tunnistin yllättävän paljon poolittomia fosfaattiryhmän korvikkeita ja joitakin positiivisesti varautuneita korvikkeita mahdollistavia mekanismeja. Lisäksi havaitsin, että ligandit omaksuivat U muotoisen konformaation fylogeneettisesti riippumattomien proteiinien nukleotidien sitoutumistaskuissa (Julkaisu III). Tutkin PDB:n proteiini-ligandikompleksien suolasiltojen yleisyyttä viidelle emäksiselle toiminnalliselle ryhmälle. Suolasiltojen yleisyys vaihteli guanidinium-ionin 70 prosentista tertiääristen ammoniumkationien 16 prosenttiin. Jälkimmäisessä tapauksessa suolasiltojen vähäisyys vaikuttaa riippuvan siitä, että vuorovaikuttaville ryhmille on vähemmän tilaa. Mikäli tarkastellut emäksiset ryhmät eivät osallistuneet vahvoihin karboksylaattivälitteisiin suolasiltoihin, niiden ympäristössä vaikutti olevan runsaasti happamia toiminnallisia ryhmiä, kuten hydroksi- ja fenoliryhmiä sekä vesimolekyylejä (Julkaisu IV). Lopuksi kehitin työkalun, joka mahdollistaa telakoinnista saatujen sitoutumisasentojen analyysin. Työkalu vertaa telakoitua ligandisarjaa sitoutuneeseen vertailuligandiin, joka voi olla eri molekyyli. Graafisena tulosteena saadaan diagrammi ligandien muotojen samankaltaisuudesta ja molekyylivuorovaikutusten sormenjälkiin perustuvasta Jaccard-pistemäärästä. Työkalua sovellettiin oreksiini-1- ja oreksiini-2-reseptoreille yhdistetyllä virtuaalisella ja kokeellisella seulonnalla löydettyjen aktiivisten ligandien sitoutumisasentojen analyysiin (Julkaisu V).Cette thèse présente le développement de méthodes et d’outils informatiques basés sur la structure tridimensionnelle des complexes protéine-ligand. Ces différentes méthodes sont utilisées pour extraire, optimiser et prédire des données à partir de la structure des complexes afin d’améliorer la modélisation et la compréhension de la reconnaissance entre une protéine et un ligand. Ce travail de thèse est divisé en cinq projets. En complément, une étude sur le positionnement des molécules d’eau dans les sites de liaisons a aussi été développée et est présentée. Dans une première partie un modèle statistique, PockDrug, a été mis en place. Il combine trois propriétés de poches protéiques (l’hydrophobicité, la géométrie et l’aromaticité) pour prédire la druggabilité des poches protéiques, si une poche protéique peut lier une molécule drug-like. Le modèle est optimisé pour s’affranchir des différentes méthodes d’estimation de poches protéiques. La qualité des prédictions, est meilleure à la fois sur des poches estimées à partir de protéines apo et holo et est supérieure aux autres modèles de la littérature (Publication I). Le modèle PockDrug est disponible sur un serveur web, PockDrug-Server (http://pockdrug.rpbs.univ-paris-diderot.fr) qui inclus d’autres outils pour l’analyse et la caractérisation des poches protéiques. Dans un second temps un protocole, basé sur la superposition de protéines homologues a été développé pour extraire des replacements structuraux de groupements chimiques fonctionnels à partir de la Protein Data Bank (PDB). Appliqué aux phosphates, un grand nombre de remplacements non-polaires ont été identifié pouvant notamment être chargés positivement. Quelques mécanismes de remplacements ont ainsi pu être analysé. Nous avons, par exemple, observé que le ligand adopte une configuration en forme U dans les sites de liaison des nucléotides indépendamment de la phylogénétique des protéines (Publication III). Dans une quatrième partie, la prévalence des ponts salins de cinq groupements chimiques basiques a été étudié dans les complexes protéine-ligand. Ainsi le pourcentage de pont salin fluctue de 70% pour le guanidinium à 16% pour l’amine tertiaire qui a le plus faible volume disponible autour de lui pour accueillir un group pouvant interagir. L’absence d’acide fort comme l’acide carboxylique pour former un pont salin est remplacé par un milieu enrichis en groupement chimiques fonctionnels avec des propriétés acides comme l’hydroxyle, le phénol ou encore les molécules d’eau (Publication IV). Dans un dernier temps un outil permettant l’analyse des poses de ligand obtenues par une méthode d’ancrage moléculaire a été développé. Cet outil compare ces poses à un ligand de référence, qui peut être une molécule différente en combinant l’information du chevauchement de forme de la pose et du ligand de référence et un score de Jaccard basé sur une comparaison des empreintes d’interaction moléculaires du ligand de référence et de la pose. Cette méthode a été utilisé dans l’analyse des résultats d’ancrage moléculaires pour des ligands actifs pour les récepteurs aux orexine 1 et 2. Ces ligands actifs ont été trouvés à partir de résultats combinant un criblage virtuel et expérimental. La revue de la littérature associée est focalisée sur la reconnaissance moléculaire d’un ligand pour une protéine et présente diffèrent concepts et challenges pour la recherche de nouveaux médicaments

Analysis, design and "in silico" evaluation of e-selectin antagonists

E-selectin, is member of a family of cell-adhesion proteins, which plays a crucial role in many physiological processes and diseases [1], and in particular, in the early phases of the inflammatory response. Its role is to promote the tethering and the rolling of leukocytes along the endothelial surface [2]. These steps are then followed by integrin-mediated firm adhesion and final transendothelial migration. Therefore, control of the leukocyte-endothelial cell adhesion process may be useful in cases, where excessive recruitment of leukocytes can contribute to acute or chronic diseases such as stroke, reperfusion injury, psoriasis or rheumatoid arthritis [3]. In this work, efforts to develop in silico-based protocols to study the interaction between E-selectin and its ligands, are presented. Hence, different protocols had to be developed and validated. In particular, a new procedure for the analysis of the conformational preferences of E-selectin antagonists was established and the results compared to those obtained with the MC(JBW)/SD approach, which had already demonstrated its validity in the past [161,168]. Thus, the comparison between the two protocols permitted to recognize a different conformational preference of the two methods for the orientation of the sialic acid moiety of sLex (3) (torsions Φ3 and Ψ3, Figure A), which reflects the contrasting opinions existing for the conformation adopted by sLex (3) in solution [150–168]. A more detailed analysis revealed that probably both approaches deliver only a partially correct view and that in reality, in solution, sLex (3) exists as a mixture of low energy conformers and not as supposed to date [150–154,161–163] as a population of a single conformer. In addition, a docking routine was established and the impact of different partialcharge methods and of explicit solvation on the binding mode studied. MD simulations enabled to gain an insight into the dynamical character of the protein-ligand interactions. In particular, the observations done in an atomic-force microscopy study [350], describing the interactions between the carboxylic group of sLex and Arg97, and between the 3– and 4–hydroxyls of fucose and the calcium ion, as the two main energy barriers for the dissociation process of the protein-ligand complex, found confirmation in our MD-investigations. Thus, these two contacts always lasted longer than any other in the MD simulation. QSAR-models with Quasar [270–272,351] and Raptor [315,316,335] were successfully derived and will permit a semi-quantitative in silico estimation of the binding affinity for the ligands that will be designed in the future. Finally, the developed protocols and models were applied for the development of new E-selectin antagonists. Unfortunately, to date, only few biological data is available to evaluate our design strategies. However, the impact of the ligand’s pre-organization on the binding affinity could be established at least for the Lexcore of sLex (3). Hence, the importance of the exo-anomeric effect, of the steric compression, and of the hydrophobic interaction between the methyl group of fucose and the β-face of galactose was clearly demonstrated