Development and validation of new approaches for quantitative intermolecular interaction analysis by MicroScale Thermophoresis under near-native experimental conditions

The analysis of molecular binding events is of great importance for both basic research and drug development. Several biophysical methods can be applied for the characterization of interactions, regarding affinity, kinetics or thermodynamic parameters. One commonly applied biophysical method is label-free MicroScale Thermophoresis (MST), which provides Kd determination under label-free and in-solution conditions. This method utilizes the intrinsic fluorescence of tryptophan, an amino acid incorporated in the vast majority of proteins. Up to now, label-free MST was restricted to quantification of interactions in which only one binding partner exhibits fluorescence in the detection wavelength. This excludes proteins as second binding partners, along with a significant number of small molecules or fragments. The reason for this is that preferred scaffolds used for the synthesis of small molecules and fragments often include indole or similar ring systems, which lead to fluorescence interference in label-free MST assays. Because of these reasons, the main goal of this thesis was to explore approaches which would enable a broader applicability of label-free MST and facilitate the quantification of intermolecular interactions under close-to-native MST-based experimental conditions. In a first approach, a modified emission filter was tested to potentially cut off any unwanted signal arising from interfering compounds to a higher extent compared to the established filter. The modified emission bandwidth indeed decreased the extent of fluorescence interference caused by compounds. However, as many compounds exhibit so-called privileged structures, such as indole motifs which are also present in tryptophan residues of proteins, the number of compounds that still interfere in label-free assays highly depends on the compound library used and remains hard to predict, as different chemical substituents can already drastically alter the emission spectrum of compounds. As this modification of the device´s optical system did not provide an overall solution for interfering compounds and in addition could not be used for the analysis of protein-protein interactions (PPIs), a second strategy was developed. Here, a composition gradient titration strategy in combination with data analysis based on a least-mean-square approximation was applied for the quantification of PPIs in a label-free MST approach. The obtained Kd values were in good agreement with data obtained from standard (= non-label-free) MST experiments. Although in general, this approach was suitable for the quantification of PPIs, simulations of various experimental conditions revealed several limitations and restrictions regarding proteins´ fluorescence intensities, Fnorm values and start concentrations in general. Due to the limitations of both strategies, a compromise strategy between preserving the proteins´ native structure as much as possible, while at the same time making use of the advantages of a fluorescent tag was applied. Therefore, target proteins were site-specifically labeled at their His6-tag using tris-NTA fluorophores, which was found to provide robust results for both, protein-small molecule and PPIs. Using such a site-specific labeling approach, the proteins´ native structure is highly preserved and interference of fluorophores with ligand binding is prevented. Among three different fluorophores tested, RED-tris-NTA proved to be the most suitable dye for this purpose. Furthermore, this approach offered the possibility to directly measure MST in crude cell lysate, which further increased the close-to-native format. In addition, such measurements wouldn´t be possible using label-free MST, which further highlights the advantages of the site-specific labeling approach

High-performance liquid chromatography analysis of mezlocillin, piperacillin, their degradation products, and of ioxitalamic acid in plasma and urine of healthy volunteers

In plasma and urine of 10 healthy volunteers after intravenous administration of 4 g mezlocillin and piperacillin, respectively, the parent compounds as well as degradation products were assayed by high-performance liquid chromatography. Ioxitalamic acid, a renal contrast medium, was administered simultaneously, in order to measure the glomerular filtration rate, and to control the collection of 24-h urine. As metabolite of mezlocillin the corresponding penicilloic acid only was found, whereas in the case of piperacillin a further degradation product was observed. Half of the doses given was recovered in the urine as unchanged drugs, and in addition 5-10% as metabolites. No differences were found in the pharmacokinetic behaviour of both antibiotics

Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain

Glucose homeostasis and growth essentially depend on the hormone insulin engaging its receptor. Despite biochemical and structural advances, a fundamental contradiction has persisted in the current understanding of insulin ligand-receptor interactions. While biochemistry predicts two distinct insulin binding sites, 1 and 2, recent structural analyses have resolved only site 1. Using a combined approach of cryo-EM and atomistic molecular dynamics simulation, we present the structure of the entire dimeric insulin receptor ectodomain saturated with four insulin molecules. Complementing the previously described insulin-site 1 interaction, we present the first view of insulin bound to the discrete insulin receptor site 2. Insulin binding stabilizes the receptor ectodomain in a T-shaped conformation wherein the membrane-proximal domains converge and contact each other. These findings expand the current models of insulin binding to its receptor and of its regulation. In summary, we provide the structural basis for a comprehensive description of ligand-receptor interactions that ultimately will inform new approaches to structure-based drug design.Peer reviewe

Development of a Microscale Thermophoresis-Based Method for Screening and Characterizing Inhibitors of the Methyl-Lysine Reader Protein MRG15

MRG15 is a transcription factor containing the methyl-lysine reader chromodomain. Despite its involvement in different physiological and pathological states, to date the role of this protein has not been fully elucidated due to the lack of a specific and potent chemical probe.In this work, we report the development of a microscale thermophoresis (MST)-based assay for the study of MRG15-ligand binding interactions. After the development, the assay was validated using a small focused library and UNC1215 as the reference compound, to yield the identification of 10 MRG15 ligands with affinities ranging from 37.8 nM to 59.1 µM. Hence, our method is robust, convenient, and fast and could be applied to other methylation reader domain-containing proteins for the identification of new chemical probes

α5β1-integrin and MT1-MMP promote tumor cell migration in 2D but not in 3D fibronectin microenvironments

Cell migration is a crucial event for physiological processes, such as embryonic development and wound healing, as well as for pathological processes, such as cancer dissemination and metastasis formation. Cancer cell migration is a result of the concerted action of matrix metalloproteinases (MMPs), expressed by cancer cells to degrade the surrounding matrix, and integrins, the transmembrane receptors responsible for cell binding to matrix proteins. While it is known that cell-microenvironment interactions are essential for migration, the role of the physical state of such interactions remains still unclear. In this study we investigated human fibrosarcoma cell migration in two-dimensional (2D) and three-dimensional (3D) fibronectin (FN) microenvironments. By using antibody blocking approach and cell-binding site mutation, we determined that α5β1-integrin is the main mediator of fibrosarcoma cell migration in 2D FN, whereas in 3D fibrillar FN, the binding of α5β1- and αvβ3-integrins is not necessary for cell movement in the fibrillar network. Furthermore, while the general inhibition of MMPs with GM6001 has no effect on cell migration in both 2D and 3D FN matrices, we observed opposing effect after targeted silencing of a membrane-bound MMP, namely MT1-MMP. In 2D fibronectin, silencing of MT1-MMP results in decreased migration speed and loss of directionality, whereas in 3D FN matrices, cell migration speed is increased and integrin-mediated signaling for actin dynamics is promoted. Our results suggest that the fibrillar nature of the matrix governs the migratory behavior of fibrosarcoma cells. Therefore, to hinder migration and dissemination of diseased cells, matrix molecules should be directly targeted, rather than specific subtypes of receptors at the cell membrane