Computational studies of protein-ligand recognition

Molecular recognition between biomolecules and ligands is very specific in living cells. The functions of all biochemical processes and cell mechanisms are dependent upon complex but specific non-covalent intermolecular interactions. As essential building blocks in protein and nucleic acid, phosphate groups are commonly found in nucleic acids, proteins, and lipids. Nearly half of known proteins have been shown to interact with ligands containing a phosphate group. Binding of a phosphoryl group is fundamental to a range of biological processes including metabolism, biosynthesis, gene regulation, signal transduction, muscle contraction, and antibiotic resistance. Phosphorylation is one of the most common forms of reversible posttranslational modification of protein and, nearly 30% of all proteins are phosphorylated on at least one residue in cells. However, phosphate binding sites are less well defined and fundamental principles of why and how proteins recognize phosphate groups are not yet fully understood. Molecular modeling is a common tool for studying biomolecular structure, dynamics, interaction and function. Due to the complex electrostatics, high concentration of ions and intricate interactions with environment, however, the modeling and designing of highly charged drug-like molecules and nucleic acid derivatives are extremely difficult. This thesis will focus on the highly charged phosphate, including its different protonation states, and energetic and thermodynamic driving forces behind protein-phosphate recognition. This thesis work will also discuss the development of more sophisticated computational models, AMOEBA+, that are necessary for a better understanding and prediction of the physical properties of small organic molecules. Four projects will be discussed in this dissertation: two projects on force field development, and two on applying molecular dynamic simulations to understand biological processes. These projects have led to new insights into understanding of physical and chemical principles and mechanisms underlying highly protein-phosphate binding and nucleic acid stability. In addition, this thesis work will enhance the capability to develop and apply computational and theoretical frameworks to model, predict and design proteins, therapeutics, and diagnostic strategies targeting phosphates, phosphate-containing biomoleculesBiomedical Engineerin

Molecular dynamics and virtual screening approaches in drug discovery

Computer-aided drug discovery (CADD) methods are now routinely used in the preclinical phase of drug development. Powerful high-performance computing facilities and the extremely fast CADD methods constantly scale up the coverage of drug-like chemical space achievable in rational drug development. In this thesis, CADD approaches were applied to address several early-phase drug discovery problems. Namely, small molecule binding site detection on a novel target protein, virtual screening (VS) of molecular databases, and characterization of small molecule interactions with metabolic enzymes were studied. Various CADD methods, including molecular dynamics (MD) simulations in mixed solvents, molecular docking, and binding free energy calculations, were employed. Co-solvent MD simulations detected biologically relevant binding sites and provided guidance for screening potential protein-protein interaction inhibitors for a crucial protein of the SARS-CoV-2. VS with fragment- and negative image-based (F-NIB) models identified three active and structurally novel inhibitors of the putative drug target phosphodiesterase 10A. MD simulations and docking provided detailed insights on the effects of active site structural flexibility and variation on the binding and resultant metabolism of small molecules with the cytochrome P450 enzymes. The results presented in this thesis contribute to the increasing evidence that supports employment and further development of CADD approaches in drug discovery. Ultimately, rational drug development coupled with CADD may enable higher quality drug candidates to the human studies in the future, reducing the risk of financially and temporally costly clinical failure. KEYWORDS: Structure-based drug development, Computer-aided drug discovery (CADD), Molecular dynamics (MD) simulation, Virtual screening (VS), Fragmentand negative image-based (F-NIB) model, Structure-activity relationship (QSAR), Cytochrome P450 ligand binding predictionMolekyylidynamiikka- ja virtuaaliseulontamenetelmät lääkeaine-etsinnässä Tietokoneavusteista lääkeaine-etsintää käytetään nykyisin yleisesti prekliinisessä lääketutkimuksessa. Suurteholaskenta ja äärimmäisen nopeat tietokoneavusteiset lääkeaine-etsintämenetelmät mahdollistavat jatkuvasti kattavamman lääkkeenkaltaisten molekyylien kemiallisen avaruuden seulonnan. Tässä väitöskirjassa tietokonepohjaisia menetelmiä hyödynnettiin lääketutkimuksen prekliiniseen vaiheeseen liittyvissä tyypillisissä tutkimusongelmissa. Työhön kuului pienmolekyylien sitoutumisalueiden tunnistus uuden kohdeproteiinin rakenteesta, molekyylitietokantojen virtuaaliseulonta sekä pienmolekyylien ja metabolian entsyymien välisten vuorovaikutusten tietokonemallinnus. Työssä käytettiin useita tietokoneavusteisen lääkeaine-etsinnän menetelmiä, sisältäen molekyylidynamiikkasimulaatiot (MD-simulaatiot) vaihtuvissa liuottimissa, molekulaarisen telakoinnin ja sitoutumisenergian laskennan. Orgaanisen liuottimen ja veden sekoituksessa tehdyt MD-simulaatiot tunnistivat biologisesti merkittäviä sitoutumisalueita SARS-CoV-2:n tärkeästä proteiinista ja ohjasivat infektioon liittyvän proteiini-proteiinivuorovaikutuksen potentiaalisten estäjien etsintää. Virtuaaliseulonnalla tunnistettiin kolme rakenteellisesti uudenlaista tunnetun lääkekehityskohteen, fosfodiesteraasi 10A:n, estäjää hyödyntäen fragmentti- ja negatiivikuvamalleja. MD-simulaatiot ja telakointi tuottivat yksityiskohtaista tietoa sytokromi P450 entsyymien aktiivisen kohdan rakenteen jouston ja muutosten vaikutuksesta pienmolekyylien sitoutumiseen ja metaboliaan. Tämän väitöskirjan tulokset tukevat kasvavaa todistusaineistoa tietokoneavusteisen lääkeaine-etsinnän käytön ja kehityksen hyödyllisyydestä prekliinisessä lääketutkimuksessa. Tietokoneavusteinen lääkeaine-etsintä voi lopulta mahdollistaa korkeampilaatuisten lääkekandidaattien päätymisen ihmiskokeisiin, pienentäen taloudellisesti ja ajallisesti kalliin kliinisen tutkimuksen epäonnistumisen riskiä. AVAINSANAT: Rakennepohjainen lääkeainekehitys, Tietokoneavusteinen lääkeaine-etsintä, Molekyylidynamiikkasimulaatio (MD-simulaatio), Virtuaaliseulonta, Fragmentti- ja negatiivikuvamalli, Rakenne-aktiivisuussuhde, Sytokromi P450 ligandien sitoutumisen ennustu

Computational studies of damaged DNA : an investigation of DNA O-linked adducts formed due to exposure to phenolic carcinogens

This thesis systematically develops a computational model to identify the conformational and base-pairing preferences of PhOdG, 4-Cl-PhOdG, DCP-OdG, TCP-OdG, and PCP-OdG by gradually increasing the size of the system also structural properties of unsubstituted O-linked. All adducts at nucleoside level adopted syn conformation. Moreover, effect of protonation at N3 and N7 site on the structural properties and deglycosilation barrier of adducted guanosine was probed. It was highly desirable to include O-linked phenolic as well as C8-dG adducts into a DNA strand in order to understand the detrimental effect of them and the conformational distortion of double helix duplex the desired modified base into NarI DNA duplex through the employment of molecular dynamic simulation (MD) was assessed. The anti-conformation against cytosine is preferred with this model for all adducts and syn conformer for all unsubstituted O-linked and ortho and para Clinked structures against guanine mismatch is the lowest energy structure.University of Lethbridg

In silico studies of the effect of phenolic compounds from grape seed extracts on the activity of phosphoinositide 3-kinase (PI3K) and the farnesoid x receptor (FXR)

In silico studies of the effect of phenolic compounds from grape seed extracts on the activity of phosphoinositide 3-kinase (PI3K) and farnesoid X receptor (FXR)Montserrat Vaqué Marquès En aquesta tesis es pretén aplicar metodologies computacionals (generació de farmacòfors i docking proteïna lligand) en l'àmbit de la nutigenòmica (ciència que pretén entendre, a nivell molecular, com els nutrients afecten la salut). S'aplicaran metodologies in silico per entendre a nivell molecular com productes naturals com els compostos fenòlics presents en la nostra dieta, poden modular la funció d'una diana comportant un efect en la salut. Aquest efecte es creu que podria ser degut a la seva interacció directa amb proteïnes de vies de senyalització molecular o bé a la modificació indirecta de l'expressió gènica. Donat que el coneixement de l'estructura del complex lligand-receptor és bàsic per entendre el mecanisme d'acció d'aquests lligands s'aplica la metodologia docking per predir l'estructura tridimensional del complex. En aquest sentit, un dels programes de docking és AutoGrid/AutoDock (un dels més citats). No obstant, l'automatització d'AutoGrid/AutoDock no és trivial tan per (a) la cerca virtual en una llibreria de lligands contra un grup de possibles receptors, (b) l'ús de flexibilitat, i (c) realitzar un docking a cegues utilitzant tota la superfície del receptor. Per aquest motiu, es dissenya una interfície gràfica de fàcil ús per utilitzar AutoGrid/AutoDock. Blind Docking Tester (BDT) és una aplicació gràfica que s'executa sobre quatre programes escrits en Fortran i que controla les condicions de les execucions d'AutoGrid i AutoDock. BDT pot ser utilitzat per equips d'investigadors en el camp de la química i de ciències de la vida interessats en dur a terme aquest tipus d'experiments però que no tenen suficient habilitats en programació. En la modulació del metabolisme de la glucosa, treballs in vivio i in vitro en el nostre grup de recerca s'han atribuït els efectes beneficiosos de l'extracte de pinyol de raïm en induir captació de glucosa (punt crític pel manteniment de l'homeostasis de la glucosa). No obstant alguns compostos fenòlics no tenen efecte en la captació de la glucosa, d'altres l'inhibeixen reversiblement. En alguns casos aquesta inhibició és el resultat de la competició dels compostos fenòlics amb ATP pel lloc d'unió de l'ATP de la subunitat catalítica de la fosfatidil inositol 3-kinasa (PI3K). Estudis recents amb inhibidors específics d'isoforma han identificat la p110α (la subunitat catalítica de PI3Kα) com la isoforma crucial per la captació de glucosa estimulada per insulina en algunes línies cel·lulars. Els programes computacionals han estat aplicats per tal de correlacionar l'activitat biològica dels compostos fenòlics amb informació estructural per obtenir una relació quantitativa estructura-activitat (3D-QSAR) i obtenir informació dels requeriments estructura-lligand per augmentar l'afinitat i/o selectivitat amb la diana (proteïna). Tot hi haver-se demostrat que l'adició d'extractes de compostos fenòlics en l'aliment pot tenir en general un benefici per la salut, s'ha de tenir en compte que l'estudi 3D-QSAR (construït a partir d'inhibidors sintètics de p110α) prediu que algunes d'aquestes molècules poden agreujar la resistència a la insulina en individus susceptibles dificultant la capatació de glucosa en múscul i teixit adipós i, per tant, produir un efecte secundari indesitjat. Resultats en el nostre grup de recerca han demostrat que compostos fenòlics presents en extractes de llavor de raïm incrementen l'activitat del receptor "farnesoid x receptor" (FXR) de manera dosi depenent quan el lligand natural de FXR (CDCA) és present. Les metodologies in silico, docking i 3D-QSAR, han estat aplicades juntament amb dades biològiques d'agonistes no esteroidals de FXR que s'uneixen a un lloc d'unió proper però diferent al lligand esteroidal 6CDCA. Els resultats han mostrat que els compostos fenòlics no són capaços d'activar FXR per ells mateixos però poden afegir noves interaccions que estabilitzarien la conformació activa de FXR en presència del lligand natural CDCA. Els compostos fenòlics podrien induir canvis conformacionals específics que augmentarien l'activitat de FXR. In silico studies of the effect of phenolic compounds from grape seed extracts on the activity of phosphoinositide 3-kinase (PI3K) and farnesoid X receptor (FXR)Montserrat Vaqué Marquès This thesis was written with the aim of applying computational methods that have already been developed for molecular design and simulation (i.e. pharmacophore generation and protein-ligand docking) to nutrigenomics. So, in silico tools that are routinely used by the pharmaceutical industry to develop drugs have been used to understand, at the molecular level, how natural products such as phenolic compounds (i.e. molecules that are commonly found in fruits and vegetables) can improve health and prevent diseases. Therefore, we first focused on predicting the structure of protein-ligand complexes. The docking algorithms can use the individual structures from receptor and ligand to predict (1) whether they can form a complex and (2) if so, the structure of the resulting complex. This prediction can be made, for instance, with AutoGrid/AutoDock, the most cited docking software in the literature. The automation of AutoGrid/AutoDock is not trivial for tasks such as (1) the virtual screening of a library of ligands against a set of possible receptors; (2) the use of receptor flexibility and (3) making a blind-docking experiment with the whole receptor surface. Therefore, in order to circumvent these limitations, we have designed BDT (i.e. blind-docking tester; http://www.quimica.urv.cat/~pujadas/BDT), an easy-to-use graphic interface for using AutoGrid/AutoDock. BDT is a Tcl/Tk graphic front-end application that runs on top of four Fortran programs and which controls the conditions of the AutoGrid and AutoDock runs. As far as the modulation of the glucose metabolism is concerned, several in vivo and in vitro results obtained by our group have shown that grape seed procyanidin extracts (GSPE) stimulate glucose uptake in 3T3-L1 adipocytes and thus help to maintain their glucose homeostasis. In contrast, it is also well known that although some phenolic compounds do not affect glucose uptake, others reversibly inhibit it in several cell lines. Moreover, for at least some of these phenolic compounds, this inhibition is the result of their competition with ATP for the ATP-binding site in p110α (i.e. the α isoform of the catalytic subunit of phosphoinositide 3-kinase or PI3Kα). Furthermore, recent studies with isoform-specific inhibitors have identified p110α as the crucial isoform for insulin-stimulated glucose-uptake in some cell lines. Therefore, although it has been proved that the addition of phenolic compound extracts to food can have an overall benefit on health, it should be taken into account that some of these molecules may exacerbate insulin resistance in susceptible individuals via impaired glucose uptake in muscle and adipose tissues and, therefore, produce an undesirable side effect. In this context, we have applied computational approaches (i.e. protein-ligand docking and 3D-QSAR) to predict the IC50 (i.e. the concentration that reduces the p110α activity to 50%). Our results agree with previous experimental results and predict that some compounds are potential inhibitors of this enzyme. Recent results in our research group have demonstrated that the phenolic compounds in GSPE increase the activity of the farnesoid X receptor (i.e. FXR) in a dose-dependent way when the natural ligand of FXR (i.e. CDCA) is also present. The phenolic compounds might induce specific conformational changes that increase FXR activity and then contribute to cardioprotection through mechanisms that are independent of their intrinsic antioxidant capacities but that involve direct interaction with FXR to modulate gene expression. Taking into account this hypothesis a 3D-QSAR analysis was made in an attempt to understand how phenolic compounds activate FXR. So, our results explain why phenolic compounds cannot activate FXR by themselves and how they can add new interactions to stabilize the active conformation of FXR when its natural ligand (i.e. CDCA) is present. Therefore, we proposed a mechanism of FXR activation by dietary phenolic compounds in which they may enhance bile acid-bound FXR activity

Kinetic and Thermodynamic Characterization of the Bacterial Lectin FimH

One fundamental aim of drug discovery is the development of new molecular entities that have a considerably advantage over already existing therapies. Urinary tract infections (UTIs) urgently require an alternative to the conventional antibiotic therapy as resistance rates for antibiotics are increasing. The development of an anti-adhesive UTI treatment strategy with the bacterial lectin FimH as target is a promising approach to remedy such alarming tendencies. FimH is presented by uropathogenic E. coli (UPEC) strains on the tip of type 1 pili and mediates adhesion to mannosylated residues on the urothelium. This interaction prevents the clearance of UPECs during micturition and enables internalization of the pathogens by urothelial cells. Mannoside-derived FimH antagonists are under development and are considered as promising treatment option for UTIs. In contrast to antibiotics, FimH antagonists do not necessarily exert resistance mechanisms against drugs because they block the adhesion of bacteria to the urothelium without killing them or inhibiting their growth. ________ In the present thesis, FimH and its interaction with mannose-based antagonists were biophysically characterized. Additionally, new methodical approaches are introduced, which are relevant not only for a strategic development of FimH antagonists but also for drugs of other therapeutic areas. The following aspects were investigated: ________ Publication 2: The publication “KinITC – One method supports both thermo-dynamic and kinetic SARs” (Chemistry, 2018,24(49), 13049-13057) comments on kinITC-ETC, a new method based on ITC data to reveal the kinetic fingerprint of a drug–target interaction. In this study, kinITC-ETC was independently validated for the first time. Moreover, structural properties of FimH antagonists could be correlated with kinetic parameters of FimH–antagonist interactions. ________ Manuscript 1: The development of an off-rate screening approach is presented in the study “Off-rate screening by surface plasmon resonance – The search for promising lead structures targeting low-affinity FimH”. The method is subsequently applied to screen a mannose-based compound library against full-length FimH. The assay allows classification of structurally diverse FimH antagonist in order to spot chemical classes exhibiting long dissociative half-lives. ________ Publication 3: The lectin domain is conformationally rigid and needs the pilin domain for allosteric propagation. However, the crosstalk between allosteric sites within the lectin domain takes also place in the absence of the pilin domain as demonstrated in the publication “Conformational switch of the bacterial adhesin FimH in the absence of the regulatory domain – Engineering a minimalistic allosteric system” (J. Biol. Chem., 2018, 293(5), 1835-1849). Mutants of the isolated lectin domain, FimHLD R60P and V27C/L34C, exhibited a low-affinity state and mimic full-length FimH regarding its conformational transition upon mannoside binding. ________ Publication 4: The publication “Target-directed dynamic combinatorial chemistry: A study on potentials and pitfalls as exemplified on a bacterial target” (Chemistry, 2017, 23, 11570-11577) illustrates a target-directed dynamic combinatorial chemistry (tdDCC) approach employing reversible acylhydrazone formation with FimH full-length as target. Optimal sample preparation and data procession are discussed in detail. Finally, the results of the tdDCC assay were subsequently compared with the affinity of library constituents by SPR. ________ Publication 5: In the publication “Comparison of affinity ranking by target-directed dynamic combinatorial chemistry and surface plasmon resonance” larger FimH antagonist libraries were screened using the tdDCC method established in publication 3. The comparison of amplification rates of library substituents with respective binding affinities determined by SPR revealed a linear association. Furthermore, the hazardous acylhydrazone moiety could be replaced by various bioisosteres without changing the affinity of the parent compound. ________ Manuscript 2: The hydrogen bond network formed between mannose derivates and the CRD of FimH is extensively elucidated in the manuscript ”High-affinity carbohydrate–lectin interaction: How nature makes it possible”. Computational methods and structural prediction in combination with binding data revealed that the hydrogen bond network forms a unified whole. The removal of only a single hydroxyl group leads to a disruption of the cooperative interplay within the network and consequently results in a dramatic loss in binding affinity. ________ Manuscript 3: In the study “The tyrosine gate of the bacterial adhesion FimH – An evolutionary remnant paves the way for drug discovery”, ITC measurements demonstrated the influence of the tyrosine gate on binding affinity between FimH and natural ligands. While the tyrosine gate is exploited to form optimal hydrophobic interactions with aryl aglycones of synthetic FimH antagonists in order to increase their binding affinity, the tyrosine gate has only a marginal impact on the KD of natural ligands. In contrast to wild-type FimH, mutants that partially or completely lack the tyrosine gate exhibited a comparable binding affinity to dimannoside. ________ Publication 6: The publication “Improvement of aglycone π-stacking yields nanomolar to sub-nanomolar FimH antagonists” displays that fluorination of biphenyl mannosides further improved π-π stacking with the tyrosine gate, reaching nanomolar affinities with FimHFL and even picomolar affinities with FimHLD. It also could be shown that ligand binding to FimHFL occurs with a highly favorable enthalpic and a considerably unfavorable entropic contribution. ________ Publication 7: In the publication “Enhancing the enthalpic contribution of hydrogen bonds by solvent shielding” microcalorimetric studies of FimH could reveal that conformational adaptions of the binding site can establish a solvent-free cavity. Shielding the solvent results in a lower dielectric environment, in which the formation of hydrogen bonds has a considerable enthalpic contribution to the binding free energy. In the case of FimH approximately -13 kJ mol-1 for mannoside binding

Characterization of Biaryl Torsional Energetics and its Treatment in OPLS All-Atom Force Fields

The frequency of biaryl substructures in a database of approved oral drugs has been analyzed. This led to designation of 20 prototypical biaryls plus 10 arylpyridinones for parametrization in the OPLS all-atom force fields. Bond stretching, angle-bending, and torsional parameters were developed to reproduce the MP2 geometries and torsional energy profiles. The transferability of the new parameters was tested through their application to three additional biaryls. The torsional energetics for the 33 biaryl molecules are analyzed and factors leading to preferences for planar and nonplanar geometries are identified. For liquid biphenyl, the computed density and heat of vaporization at the boiling point (255 °C) are also reported

Characterization of Biaryl Torsional Energetics and its Treatment in OPLS All-Atom Force Fields

The frequency of biaryl substructures in a database of approved oral drugs has been analyzed. This led to designation of 20 prototypical biaryls plus 10 arylpyridinones for parametrization in the OPLS all-atom force fields. Bond stretching, angle-bending, and torsional parameters were developed to reproduce the MP2 geometries and torsional energy profiles. The transferability of the new parameters was tested through their application to three additional biaryls. The torsional energetics for the 33 biaryl molecules are analyzed and factors leading to preferences for planar and nonplanar geometries are identified. For liquid biphenyl, the computed density and heat of vaporization at the boiling point (255 °C) are also reported

Biophysical characterization of carbohydrate-lectin interactions

Improved knowledge of the biological role of lectins has raised the demand for carbohydrate-based therapeutics in recent years. The potential market is estimated to be greater than 20 billion dollars. However, lectins are challenging drug targets due to the unique binding properties of their extensively hydroxylated carbohydrate ligands. Hydroxyl groups provide directionality and therefore specificity, but are penalized with high desolvation costs. Consequently, monovalent carbohydrate-lectin interactions tend to be rather weak, often in the millimolar range. Moreover, the polar character of carbohydrates creates large obstacles for drug application regarding oral availability and long-lasting plasma levels. The key to the successful development of carbohydrate-based drugs is the simultaneous optimization of carbohydrate lead structures in terms of pharmacodynamics and pharmacokinetics. To further enhance the success rate of carbohydrate-based drug candidates, the understanding of carbohydrate-lectin interactions on a molecular basis has to be improved. For this purpose, we combined structural information (X-ray crystallography and nuclear magnetic resonance spectroscopy), binding data (isothermal titration calorimetry, microscale thermophoresis, and fluorescence polarization assay) and computational methods (quantum mechanical calculations and molecular dynamics simulations) to explore the lectins FimH and E-selectin and their interaction with carbohydrates and mimetics thereof. FimH is a virulence factor of uropathogenic E. coli located at the tip of the bacterial type 1 pili. It interacts with the mannosylated glycoprotein uroplakin 1a in the urothelial mucosa and thereby mediates adhesion to the bladder wall as the initial step of urinary tract infections (UTI). In manuscript 1 we investigated the energy contribution to binding of the hydroxyl groups mediating the interaction between FimH and the carbohydrate moiety of its ligands. The rigidity of this bacterial lectin was demonstrated in manuscript 2, where could show that the affinity of a septanose as a mannose mimic is reduced by a factor of 10, mainly due to its flexibility in solution and the consequent conformational restrictions upon binding. In manuscript 3 we analyzed interactions between the tyrosine gate motif of FimH and the aglycones of different ligand classes. This motif (Tyr48, Tyr137) forms the entrance of the binding pocket and significantly contributes to binding affinity. In manuscript 4 we explored 2-C-branched mannosides as a novel family of FimH antagonists. In manuscript 5, a pharmacodynamically and pharmacokinetically optimized FimH antagonist was explored by oral application in a mouse model for UTI, resulting in a 1000-fold reduction of the bacterial load in the bladder. Finally, in manuscript 6 we reanalyzed ITC results from our previous studies with the novel analytical tool kinITC, allowing the determination of kinetics in addition to the thermodynamics of binding. The hydrophobic aglycone turned out to be mainly responsible for guiding the antagonist to its binding site whereas the hydrogen bond network between the mannose moiety and the protein had predominantly an impact on the off-rate. E-selectin is a lectin expressed on the surface of vascular endothelial cells and is involved in the recruitment of leukocytes to the site of inflammation. By interacting with the tetrasaccharide epitope sialyl Lewis(x), E-selectin establishes the initial contact and enables leukocytes to roll along the endothelial surface. Whereas this process is a defense mechanism in case of infections and injuries, excessive extravasation of leukocytes can have deleterious consequences in case of numerous diseases with an inflammatory component, e.g. asthma, psoriasis or stroke. Thus, blocking the interaction of E-selectin with its physiological ligands is a promising strategy to suppress the inflammatory response at the beginning of the cascade. For reliable and materially efficient affinity measurements, we developed and evaluated a novel assay for E-selectin based on microscale thermophoresis technology in manuscript 7. In the subsequent manuscripts, we applied the microscale thermophoresis assay. In manuscript 8 we performed a competitive library screen, whereby four promising small-molecule fragments were identified for further development towards a non-carbohydrate E-selectin antagonist. Finally, in manuscript 9 we were able to improve the affinity of a sialyl Lewis(x) mimic to E-selectin by pre-organizing the acid in its bioactive conformation