SecA ATPase is a critical member of the Sec system, which is important in the translocation of membrane and secreted polypeptides/proteins in bacteria. Small molecule inhibitors can be very useful research tools as well as leads for future antimicrobial agent development. Based on previous virtual screening work, we optimized the structures of two hit compounds and obtained SecA ATPase inhibitors with IC50 in the single digit micromolar range. These represent the first low micromolar inhibitors of bacterial SecA and will be very useful for mechanistic studies. Post synthetic modification is an important and efficient way of DNA functionalization especially in DNA aptamer selection. In this research, the feasibility of norbornene (Neo) modified thymidine triphosphate incorporation was described. Besides, substituted tetrazines have been found to undergo facile inversed electron demand Diels-Alder reactions with "tunable" reaction rates. This finding paves the way to utilize tetrazine conjugation reactions for not only DNA but also other labeling work.

Type-1 diabetes is caused by the autoimmune destruction of insulin-producing beta cells in the pancreas. Beta-cell apoptosis involves a complex set of signaling cascades initiated by \(interleukin-1\beta (IL-1\beta)\), \(interferon-\gamma (IFN-\gamma)\), and \(tumor necrosis factor-\alpha (TNF-\alpha)\). \(IL-1\beta\) and \(TNF-\alpha\) induce \(NF\kappa B\) expression, while \(IFN-\gamma\) induces STAT1 activation. These cytokines lead to a decrease of beta-cell function. The goal of this thesis is to identify small-molecule suppressors of cytokine-induced beta-cell apoptosis using high-throughput screening approach. Using the rat INS-1E beta-cell line, I developed an assay to measure cellular viability after 48 hours of cytokine treatment. I screened 29,760 compounds for their ability to suppress the negative effects of the cytokines. I identified several compounds to be suppressors of beta-cell apoptosis. These efforts led to the discovery of \(GSK-3\beta\) and HDAC3 as novel targets for suppressing beta-cell apoptosis. I also followed up on BRD0608, a novel suppressor that increased ATP levels and decreased caspase activity in the presence of cytokines. To follow up this compound, 35 analogs related to BRD0476 were synthesized using solid-phase synthesis and tested for their protective effects in the presence of cytokines. A structurally related analog, BRD0476, was found to be more potent and active in human islets, decreasing caspase activation and increasing insulin secretion after a 6-day treatment. I performed gene-expression profiling of INS-1E cells treated with the cytokine cocktail in the absence or presence of \(10\mu M\) BRD0476. Gene-set enrichment analysis revealed that the gene sets most significantly changed by BRD0476 involved cellular responses to \(IFN-\gamma\). I therefore assessed the effects of BRD0476 on STAT1 transcriptional activity. Cytokine treatment increased the reporter-gene luciferase activity, while co-treatment with BRD0476 reduced this activity significantly. To identify the intracellular target(s) of BRD0476, I collaborated with the Proteomics Platform in Broad Institute using SILAC (stable isotope labeling by amino acids in cell culture). SILAC is a mass spectrometry-based method to identify proteins that bind a small molecule attached to a bead. Deubiquitinase USP9X was pulled down by BRD0476. Knock-down of USP9X by siRNA phenocopied the protective effects of BRD0476. Binding assays were performed to identify interactions between BRD0476 and USP9X. / Chemistry and Chemical Biology

Small molecules are important not only for treating human diseases but also for studying disease-related biological processes. This dissertation focuses on the effects of small molecules on pancreatic ductal adenocarcinoma cells. Here, I describe the discovery of two small-molecule tool compounds and their applications for interrogating the biological processes related to two distinct diseases in the human pancreas. First, BRD4770 was identified as a histone methyltransferase inhibitor through a target-based biochemical approach, and was used as a probe to study the function of methyltransferases in cancer cells. Second, BRD7552 was discovered as an inducer of Pdx1 using a cell-based phenotypic screening approach, and was used to induce the expression of Pdx1, a master regulatory transcription factor required for \(\beta\)-cell transdifferentiation. This compound is particularly interesting for the study of type-1 diabetes (T1D). The histone methyltransferase G9a catalyzes methylation of lysine 9 on histone H3, a modification linked to aberrant silencing of tumor-suppressor genes. The second chapter describes the collaborative effort leading to the identification of BRD4770 as a probe to study the function of G9a in human pancreatic cancer cells. BRD4770 induces cellular senescence and inhibits both anchorage-dependent and -independent proliferation in PANC-1 cell line, presumably mediated through ATM-pathway activation. Chapter three describes the study of a natural product gossypol, which significantly enhances the BRD4770 cytotoxicity in p53-mutant cells through autophagic cell death. The up-regulation of BNIP3 might be responsible for the synergistic cell death, suggesting that G9a inhibition may help overcome drug resistance in certain cancer cells. Ectopic overexpression of Pdx1, Ngn3, and MafA can reprogram pancreatic exocrine cells to insulin-producing cells in mice, which sheds light on a new avenue for treating T1D. The fourth chapter focuses on a gene expression-based assay using quantitative real-time PCR technique to screen >60,000 compounds for induction of one or more of these three transcription factors. A novel compound BRD7552 which up-regulated Pdx1 mRNA and protein levels in PANC-1 cells was identified. BRD7552 induces changes of the epigenetic markers within the Pdx1 promoter region consistent with transcriptional activation. Furthermore, BRD7552 partially complements Pdx1 in cell culture, enhancing the expression of insulin induced by the introduction of the three genes in PANC-1 cells. In summary, the central theme of my dissertation is to identify novel bioactive small molecules using different screening approaches, as well as to explore their effects in pancreatic ductal cells. / Chemistry and Chemical Biology

No description available.

The charge-density-wave (CDW) material orthorhombic tantalum trisulfide (TaS3) is a quasi-one dimensional material that forms long ribbon shaped crystals, and exhibits unique physical behavior. We have measured the dependence of the hysteretic voltage-induced torsional strain (VITS) in TaS3, which was first discovered by Pokrovskii et. al. in 2007, on temperature and applied torque. Our experimental results shows that the application of torque to the crystal could also change the VITS time constant, magnitude, and sign. This suggests that the VITS is a consequence of residual torsional strain originally present in the sample which twists the polarizations of the CDW when voltage is applied. This polarization twist then results in torque on the crystal. Another group of materials that may attract interest is that of small-molecule soluble organic semiconductors. Due to their assumed small phonon thermal conductivities and higher charge carrier mobilities, which will increase their seebeck coefficients with doping as compared to polymers, the small-molecule organic materials are promising for thermoelectric applications. In our experiments, we have measured the interlayer thermal conductivity of rubrene (C42H28), using ac-calorimetry. For rubrene, we find that the interlayer thermal conductivity, ≈ 0.7 mW/cm·K, is several times smaller than the (previously measured) in-plane value. Also, we have measured the interlayer and in-plane thermal conductivities of 6,13-bis((triisopropylsilyl)ethynyl) pentacene (TIPS-Pn). The in-plane value is comparable to that of organic metals with excellent π-orbital overlap. The interlayer (c-axis) thermal diffusivity is at least an order of magnitude larger than the in-plane, and this unusual anisotropy implies very strong dispersion of optical modes in the interlayer direction, presumably due to interactions between the silyl-containing side groups. Similar values for both in-plane and interlayer conductivities have been observed for several other functionalized pentacene semiconductors with related structures.

Individuals with high levels of plasma triglycerides are at high risk to develop cardiovascular disease (CVD), currently one of the major causes of death worldwide. Recent epidemiological studies show that loss-of-function mutations in the APOC3 gene lower plasma triglyceride levels and reduce the incidence of coronary artery disease. The APOC3 gene encodes for apolipoprotein (APO) C3, known as an inhibitor of lipoprotein lipase (LPL) activity. Similarly, a common gain-of-function mutation in the LPL gene is associated with reduced risk for CVD. LPL is central for the metabolism of lipids in blood. The enzyme acts at the endothelial surface of the capillary bed where it hydrolyzes triglycerides in circulating triglyceride-rich lipoproteins (TRLs) and thereby allows uptake of fatty acids in adjacent tissues. LPL activity has to be rapidly modulated to adapt to the metabolic demands of different tissues. The current view is that LPL is constitutively expressed and that the rapid modulation of the enzymatic activity occurs by some different controller proteins. Angiopoietin-like protein 4 (ANGPTL4) is one of the main candidates for control of LPL activity. ANGPTL4 causes irreversible inactivation through dissociation of the active LPL dimer to inactive monomers. Other proteins that have effects on LPL activity are the APOCs which are surface components of the substrate TRLs. APOC2 is a well-known LPL co-factor, whereas APOC1 and APOC3 independently inhibit LPL activity. Given the important role of LPL for triglyceride homeostasis in blood, the aim of this thesis was to find small molecules that could increase LPL activity and serve as lead compounds in future drug discovery efforts. Another aim was to investigate the molecular mechanisms for how APOC1 and APOC3 inhibit LPL activity. Using a small molecule screening library we have identified small molecules that can protect LPL from inactivation by ANGPTL4 during incubations in vitro. Following a structure-activity relationship study we have synthesized lead compounds that more efficiently protect LPL from inactivation by ANGPTL4 in vitro and also have dramatic triglyceride-lowering properties in vivo. In a separate study we show that low concentrations of fatty acids possess the ability to prevent inactivation of LPL by ANGPTL4 under in vitro conditions. With regard to APOC1 and APOC3 we demonstrate that when bound to TRLs, these apolipoproteins prevent binding of LPL to the lipid/water interface. This results in decreased lipolysis and in an increased susceptibility of LPL to inactivation by ANGPTL4. We demonstrate that hydrophobic amino acid residues that are centrally located in the APOC3 molecule are critical for attachment of this protein to lipid emulsion particles and consequently for inhibition of LPL activity. In summary, this work has identified a lead compound that protects LPL from inactivation by ANGPTL4 in vitro and lowers triglycerides in vivo. In addition, we propose a molecular mechanism for inhibition of LPL activity by APOC1 and APOC3.

Molecular and functional analysis of small molecule binding to protein can provoke insights into cellular signaling and regulatory systems as well as facilitate pharmaceutical drug discovery. In label free small molecule detection the displacement assay format can be applied. This assay format comprises the displacement of receptor molecules bond to immobilized ligand by a competition reaction with ligand in solution. This is beneficial because displacement of high molecular receptors is detected compared to low molecular ligand as in classical binding analysis therefore potentially lowering the method detection limit. It was hypothesized that with choosing appropriate measuring methods and theoretical modeling reaction rate constants can be determined separately in every kinetic stage of the assay format. Herein elucidating the dominant valence of antibody antigen binding in the established assay was of great importance. Using the Influenza Hemagglutinin (HA) peptid binding to mono or bivalent Anti-Hemagglutinin peptide antibody displacement assay formats could be established. The exact time resolved analysis of binding and dissolution of ligand HA and Anti-Hemagglutinin peptide antibody was achieved with surface plasmon resonance (SPR) spectroscopy. Mathematical models could be developed from kinetic equations of ligand binding to mono or bivalent antibody. With this, an accurate simulation of the SPR results was reached. The simulation plot had to be exactly adjusted to the SPR results to determine all kinetic rate constants defining ligand and receptor binding kinetics. Large variations in receptor concentration gave almost identical rate constants in binding; this proves the quality of SPR measurements and demonstrates consistence between measurement, simulation, and binding model. Maximum decline of SPR response could be used to determine ligand concentrations in analyte. Displacement dependence from antigen concentration was found to be exponential and was explained by rebinding. Kinetic data and models could be transferred for the simulation of almost stationary displacement assay formats realized with impedance and fluorescence spectroscopy. With the obtained results it was possible to detect the displacement of the bacterial signaling autoinducer AI-2 by a displacement assay format using periplasmic binding protein LuxP as receptor. Concluding it can be said that the hypothesis could be proved and the obtained results can facilitate the use of displacement assay formats in biosensing. Displacement assay formats should be especially interesting in small molecule detection and in compact integrated mass sensitive sensor designs suitable as mobile sensors in outdoor screening. / Die Analyse des Bindungsverhaltens niedermolekularer Liganden an Proteine ist für die Aufklärung von biologischen Regulationssystemen oder bei der Suche neuer medizinischer Wirkstoffe von Wichtigkeit. Ein markierungsfreies Detektions¬prinzip zur Erfassung niedermolekularer Liganden ist die Displacement- oder Replacement-Methode. Bei dieser tritt die Bindung des Rezeptors an den immobilisierten Liganden mit der Bindung an freien Liganden in Konkurrenz, sodass anstelle der niedermolekularen Liganden die hochmolekularen Rezeptoren detektiert werden können. In dieser Arbeit wurde von der Hypothese ausgegangen, dass durch die Auswahl geeigneter Messverfahren und der zugeordneten Modellierung die einzelnen kinetischen Stadien des Displacements separat zur Bestimmung der kinetischen Konstanten der Displacementprozesse genutzt werden können. Dabei sollte unter anderem auch eine Aussage über die dominierende Valenz der Antigen-Antikörper-Bindung erreicht werden. Hierzu wurden auf der Basis des Modellsystems Hämagglutinin-Peptid/ Hämagglutinin-Antikörper Displacement-Assays mit mono- und bivalenten Anti-körpern entwickelt, anhand derer eine genaue zeitaufgelöste Analyse des Bindungs- und Ablösungsverhaltens vom Liganden HA an den Anti-HA-Antikörper (Rezeptor) mittels Oberflächenplasmonenresonanz(SPR)-Spektroskopie erzielt wurde. Ausgehend von den Reaktionsgleichungen zwischen Liganden und mono- und bivalenten Rezeptoren wurden mathematische Modelle entwickelt, die eine exakte Simulation der SPR-Ergebnisse ermöglichten. Durch genaues Anpassen der Simulationsplots an die Messplots konnten alle Ratenkonstanten, die die Kinetik der Reaktionen zwischen Liganden, Rezeptoren und ihren Komplexen bestimmen, ermittelt werden. Da auch für eine große Variation der Rezeptorkonzentrationen in der Analytlösung nahezu identische Werte für die Ratenkonstanten erhalten wurden, ergeben Messungen und Simulationen ein konsistentes Bild der Anbindungskinetik und bestätigen die Qualität der Messungen. Aus Messungen des maximalen Responsabfalles kann die Konzentration der freien Antigene beim Displacement ermittelt werden. Man findet eine exponentielle Abhängigkeit des Displacements von der Konzentration der freien Antigene, die sich durch den sogenannten „Rebindingeffekt“ erklären lässt. Die gewonnenen kinetischen Daten und entwickelten Modellierungsverfahren konnten zur Simulation quasistationärer Detektionsverfahren, die mit Fluoreszenz- und Impedanzspektroskopie durchgeführt wurden, erfolgreich angewandt werden. Die erzielten Erkenntnisse konnten auf ein wissenschaftlich herausforderndes biologisches System (LuxP/AI2) angewandt werden, bei dem das niedermolekulare Signalmolekül AI2 über ein Displacementassay detektiert wurde. Dieses System ermöglicht einen Einblick in die Intra- und Interspezieskommunikation bei Bakterien. Insgesamt zeigt sich, dass die hier formulierte Hypothese als bewiesen angesehen werden kann. Die in dieser Arbeit gewonnenen Erkenntnisse eröffnen verschiedene Einsätze der Displacementmethode in der Biosensorik. Insbesondere lassen sich damit kleine Moleküle markierungsfrei quantitativ bestimmen, ohne hoch präzise Analysengeräte einsetzen zu müssen. Damit ergibt sich die Möglichkeit, sehr kompakte integrierte massensensitive Sensoren, die nicht die Empfindlichkeit hochempfindlicher SPR-Spektrometer erreichen, zur Detektion kleiner Moleküle einzusetzen. Dies ist besonders für mobile Anwendungen von Interesse.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disease resulting in episodic but progressive extraskeletal bone formation. FOP is caused by missense mutations in the cytoplasmic domain of the type I bone morphogenetic protein (BMP) receptor ACVR1, leading to dysregulated activation. Currently there are no available drug treatments and the structural mechanism of mutant activation is still poorly characterised. To address this, a number of BMP and TGFβ receptors, including FOP mutants of ACVR1 were cloned, expressed and purified for both structural and biophysical experiments. The arginine at the site of most recurrent FOP mutation, R206H, is common across all type I receptors except BMPR1A and BMPR1B which have a lysine at this site. The novel structure of BMPR1B differed to wild-type ACVR1 showing some of the conformational changes expected of the active conformation. However, a variety of disease related ACVR1 mutant structures, including ACVR1 R206H, revealed a surprisingly persistent inactive conformation in the kinase domain. Some conformational changes suggestive of activation were observed in the mutant Q207D affecting the ATP pocket, the β4–β5 hairpin and the activation loop. Additionally, the structure of the Q207E mutant showed a slight release of the regulatory glycine-serine rich domain from its inhibitory position. These subtle changes suggest that the mutant inactive conformation is destabilised and potentially more dynamic. In agreement, all of the ACVR1 mutants showed reduced binding to the inhibitory protein FKBP12. However, mutant phosphorylation of the substrate Smad1 was not constitutive, but dependent on the co-expression of the partner ACVR2, consistent with recent evidence from transgenic knock-out mice. A novel 2-aminopyridine inhibitor scaffold with favourable specificity for ACVR1 was identified using a fluorescence-based thermal shift assay. Further derivatives were characterised with improved potency and selectivity. The crystal structures of ACVR1 bound to these inhibitors showed exquisite shape complementarity, contributing to their favourable specificity. This work has increased the understanding of FOP-associated mutant activation and provided a novel starting scaffold for potential drug development.

Abstract Macrophages found in the tumor microenvironment play a crucial role in initiating an immunosuppressive tumor microenvironment that negatively impacts immunotherapy efficacy and aids tumor progression and metastasis. Constituting the most abundant immune cell in tumor microenvironment (TME), tumor associated macrophages (TAMs) have emerged as an attractive approach for anti-cancer therapy. However, two major challenges need to be overcome for successfully utilizing macrophages for immunotherapy. First, tumors repolarize the TAMs predominantly to M2 tumor-aiding phenotype by secreting various immunosuppressive cytokines. Second, cancer cells overexpress a membrane protein CD47 that interacts with signal-regulating protein alpha (SIRPalpha) expressed on macrophages. This crosstalk provides a downregulatory signal in the form of activation of SHP1/2 that inhibits cancer cell phagocytosis, and CD47, therefore, functions as a “don’t-eat-me” signal. We rationalized that these challenges can be overcome by engineering a nanoparticle system that can deliver a rationale combination of immunomodulatory agents to the TAMs that can both repolarize the M2 macrophages to M1 phenotype efficiently and concurrently block CD47-SIRPalpha interactions by inhibiting SHP2 signaling. Herein, we designed a lipid nanoparticle (LNP) system loaded with amphiphilic R848-cholesterol in its hydrophobic lipid bilayer, while SHP099 gets encapsulated in the hydrophilic core. Our previous studies have shown that the conjugation of cholesterol to the inhibitor stabilizes the lipid bilayer at a high inhibitor concentration. The LNPs showed high optimal drug loading, size, and stability. In vitro studies showed that the M2 macrophages treated with the LNPs system repolarized to M1 phenotype and expressed co-stimulatory molecules while having enhanced phagocytic potential. In vivo efficacy studies in 4T1 tumor-bearing mice showed that LNPs exhibit superior anti-tumor efficacy compared to other treatments. We evaluated the effect of MARCO-targeted LPNs by the conjugating anti-MARCO antibody on the LPN surface. However, no comparable difference in treatment efficacy was observed between the targeted MARCO-LNPs and the non-targeted LNPs. These results demonstrate that the MARCO targeting system designed in this study is largely ineffective in the targeted delivery of its drug cargo specifically to TAMs. Thus, the lipid nanoparticle-mediated co-delivery of a rational combination of TLR7/8 agonist and SHP2 inhibitor in the TAMs increases M2 to M1 repolarization and phagocytosis potential of macrophages. Recommended Citation Malik, V., Ramesh, A. and Kulkarni, A.A. (2021), TLR7/8 Agonist and SHP2 Inhibitor Loaded Nanoparticle Enhances Macrophage Immunotherapy Efficacy. Adv. Therap., 4: 2100086. https://doi.org/10.1002/adtp.202100086

Molecular and functional analysis of small molecule binding to protein can provoke insights into cellular signaling and regulatory systems as well as facilitate pharmaceutical drug discovery. In label free small molecule detection the displacement assay format can be applied. This assay format comprises the displacement of receptor molecules bond to immobilized ligand by a competition reaction with ligand in solution. This is beneficial because displacement of high molecular receptors is detected compared to low molecular ligand as in classical binding analysis therefore potentially lowering the method detection limit. It was hypothesized that with choosing appropriate measuring methods and theoretical modeling reaction rate constants can be determined separately in every kinetic stage of the assay format. Herein elucidating the dominant valence of antibody antigen binding in the established assay was of great importance. Using the Influenza Hemagglutinin (HA) peptid binding to mono or bivalent Anti-Hemagglutinin peptide antibody displacement assay formats could be established. The exact time resolved analysis of binding and dissolution of ligand HA and Anti-Hemagglutinin peptide antibody was achieved with surface plasmon resonance (SPR) spectroscopy. Mathematical models could be developed from kinetic equations of ligand binding to mono or bivalent antibody. With this, an accurate simulation of the SPR results was reached. The simulation plot had to be exactly adjusted to the SPR results to determine all kinetic rate constants defining ligand and receptor binding kinetics. Large variations in receptor concentration gave almost identical rate constants in binding; this proves the quality of SPR measurements and demonstrates consistence between measurement, simulation, and binding model. Maximum decline of SPR response could be used to determine ligand concentrations in analyte. Displacement dependence from antigen concentration was found to be exponential and was explained by rebinding. Kinetic data and models could be transferred for the simulation of almost stationary displacement assay formats realized with impedance and fluorescence spectroscopy. With the obtained results it was possible to detect the displacement of the bacterial signaling autoinducer AI-2 by a displacement assay format using periplasmic binding protein LuxP as receptor. Concluding it can be said that the hypothesis could be proved and the obtained results can facilitate the use of displacement assay formats in biosensing. Displacement assay formats should be especially interesting in small molecule detection and in compact integrated mass sensitive sensor designs suitable as mobile sensors in outdoor screening.:Zusammenfassung i Summery iii Inhaltsverzeichnis v 1. Problemstellung 1 2. Kinetisches Modell für Displacement-Assays mit monovalenten Antikörpern 5 2.1 Kinetisches Modell 6 2.1.1 Grundgleichungen 6 2.1.2 Analytische Näherungslösung 8 2.1.3 Numerische Lösung 10 2.2 Vergleich mit experimentellen Beispielen 18 2.2.1 Surface-Plasmonen-Resonanzspektroskopie eines Hämagglutinin- Peptid/Hämagglutinin-Antikörper-Displacement-Assays 18 2.2.2 Fluoreszenzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 36 2.2.3 Analyse eines LuxP/AI2-Displacementassays mittels SPR-Spektroskopie 52 3. Kinetisches Modell für Displacement-Assays mit bivalenten Antikörpern 70 3.1 Kinetisches Modell 70 3.1.1 Grundgleichungen 70 3.1.2 Numerische Lösung 72 3.2 Vergleich mit experimentellen Beispielen 80 3.2.1 Surface-Plasmonen-Resonanzspektroskopie eines Hämagglutinin- Peptid/Hämagglutinin-Antikörper-Displacement-Assays 80 3.2.2 Impedanzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 88 4. Konklusionen und Ausblick 92 5. Anhänge 98 A1: Oberflächenplasmonenresonanzspektroskopie 98 A2: Aufbau der Messschichten und Messreihen eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 105 A3: Schichtaufbau zur Analyse eines LuxP/AI2-Displacementassays mittels SPR-Spektroskopie 119 A4: Aufbau zur Fluoreszenzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 125 A5: Impedanzspektroskopie 128 A6: Transferratenkonstanten 139 A7: Abkürzungsverzeichnis 141 6. Literatur 146 7. Danksagung 155 8. Selbständigkeitserklärung 157 / Die Analyse des Bindungsverhaltens niedermolekularer Liganden an Proteine ist für die Aufklärung von biologischen Regulationssystemen oder bei der Suche neuer medizinischer Wirkstoffe von Wichtigkeit. Ein markierungsfreies Detektions¬prinzip zur Erfassung niedermolekularer Liganden ist die Displacement- oder Replacement-Methode. Bei dieser tritt die Bindung des Rezeptors an den immobilisierten Liganden mit der Bindung an freien Liganden in Konkurrenz, sodass anstelle der niedermolekularen Liganden die hochmolekularen Rezeptoren detektiert werden können. In dieser Arbeit wurde von der Hypothese ausgegangen, dass durch die Auswahl geeigneter Messverfahren und der zugeordneten Modellierung die einzelnen kinetischen Stadien des Displacements separat zur Bestimmung der kinetischen Konstanten der Displacementprozesse genutzt werden können. Dabei sollte unter anderem auch eine Aussage über die dominierende Valenz der Antigen-Antikörper-Bindung erreicht werden. Hierzu wurden auf der Basis des Modellsystems Hämagglutinin-Peptid/ Hämagglutinin-Antikörper Displacement-Assays mit mono- und bivalenten Anti-körpern entwickelt, anhand derer eine genaue zeitaufgelöste Analyse des Bindungs- und Ablösungsverhaltens vom Liganden HA an den Anti-HA-Antikörper (Rezeptor) mittels Oberflächenplasmonenresonanz(SPR)-Spektroskopie erzielt wurde. Ausgehend von den Reaktionsgleichungen zwischen Liganden und mono- und bivalenten Rezeptoren wurden mathematische Modelle entwickelt, die eine exakte Simulation der SPR-Ergebnisse ermöglichten. Durch genaues Anpassen der Simulationsplots an die Messplots konnten alle Ratenkonstanten, die die Kinetik der Reaktionen zwischen Liganden, Rezeptoren und ihren Komplexen bestimmen, ermittelt werden. Da auch für eine große Variation der Rezeptorkonzentrationen in der Analytlösung nahezu identische Werte für die Ratenkonstanten erhalten wurden, ergeben Messungen und Simulationen ein konsistentes Bild der Anbindungskinetik und bestätigen die Qualität der Messungen. Aus Messungen des maximalen Responsabfalles kann die Konzentration der freien Antigene beim Displacement ermittelt werden. Man findet eine exponentielle Abhängigkeit des Displacements von der Konzentration der freien Antigene, die sich durch den sogenannten „Rebindingeffekt“ erklären lässt. Die gewonnenen kinetischen Daten und entwickelten Modellierungsverfahren konnten zur Simulation quasistationärer Detektionsverfahren, die mit Fluoreszenz- und Impedanzspektroskopie durchgeführt wurden, erfolgreich angewandt werden. Die erzielten Erkenntnisse konnten auf ein wissenschaftlich herausforderndes biologisches System (LuxP/AI2) angewandt werden, bei dem das niedermolekulare Signalmolekül AI2 über ein Displacementassay detektiert wurde. Dieses System ermöglicht einen Einblick in die Intra- und Interspezieskommunikation bei Bakterien. Insgesamt zeigt sich, dass die hier formulierte Hypothese als bewiesen angesehen werden kann. Die in dieser Arbeit gewonnenen Erkenntnisse eröffnen verschiedene Einsätze der Displacementmethode in der Biosensorik. Insbesondere lassen sich damit kleine Moleküle markierungsfrei quantitativ bestimmen, ohne hoch präzise Analysengeräte einsetzen zu müssen. Damit ergibt sich die Möglichkeit, sehr kompakte integrierte massensensitive Sensoren, die nicht die Empfindlichkeit hochempfindlicher SPR-Spektrometer erreichen, zur Detektion kleiner Moleküle einzusetzen. Dies ist besonders für mobile Anwendungen von Interesse.:Zusammenfassung i Summery iii Inhaltsverzeichnis v 1. Problemstellung 1 2. Kinetisches Modell für Displacement-Assays mit monovalenten Antikörpern 5 2.1 Kinetisches Modell 6 2.1.1 Grundgleichungen 6 2.1.2 Analytische Näherungslösung 8 2.1.3 Numerische Lösung 10 2.2 Vergleich mit experimentellen Beispielen 18 2.2.1 Surface-Plasmonen-Resonanzspektroskopie eines Hämagglutinin- Peptid/Hämagglutinin-Antikörper-Displacement-Assays 18 2.2.2 Fluoreszenzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 36 2.2.3 Analyse eines LuxP/AI2-Displacementassays mittels SPR-Spektroskopie 52 3. Kinetisches Modell für Displacement-Assays mit bivalenten Antikörpern 70 3.1 Kinetisches Modell 70 3.1.1 Grundgleichungen 70 3.1.2 Numerische Lösung 72 3.2 Vergleich mit experimentellen Beispielen 80 3.2.1 Surface-Plasmonen-Resonanzspektroskopie eines Hämagglutinin- Peptid/Hämagglutinin-Antikörper-Displacement-Assays 80 3.2.2 Impedanzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 88 4. Konklusionen und Ausblick 92 5. Anhänge 98 A1: Oberflächenplasmonenresonanzspektroskopie 98 A2: Aufbau der Messschichten und Messreihen eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 105 A3: Schichtaufbau zur Analyse eines LuxP/AI2-Displacementassays mittels SPR-Spektroskopie 119 A4: Aufbau zur Fluoreszenzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 125 A5: Impedanzspektroskopie 128 A6: Transferratenkonstanten 139 A7: Abkürzungsverzeichnis 141 6. Literatur 146 7. Danksagung 155 8. Selbständigkeitserklärung 157