Analyse ligandeninduzierter Konformationsänderungen von Proteinen

Der Estrogenrezeptor gehört zur Proteinfamilie der Kernrezeptoren. Diese ligandenkontrollierten Transkriptionsfaktoren regulieren spezifisch die Transkription ihrer Zielgene. Die Aktivität der Kernrezeptoren wird durch die Bindung kleiner lipophiler Liganden, wie Steroidhormone oder therapeutische Wirkstoffe, reguliert, welche an die Ligandenbindungsdomäne des Rezeptors binden und Konformationsänderungen induzieren. Dadurch wird die Interaktion des Rezeptors mit einer Vielzahl an Koregulatorproteinen sowie der DNA kontrolliert. Kernrezeptoren spielen bei der Vermittlung einer Vielzahl physiologischer Prozesse, aber auch bei der Entstehung von Erkrankungen, wie Diabetes, Krebs, Asthma oder Hormonresistenz, eine entscheidende Rolle und stellen daher interessante Zielstrukturen für die moderne Wirkstoff-Forschung dar. In der vorliegenden Arbeit wird die Entwicklung des fluoreszenzbasierten direkten Bindungsassays FLiN (Fluorescent Labels in Nuclear receptors) beschrieben, der in der Lage ist, spezifische Konformationsänderungen in der Ligandenbindungsdomäne des humanen Estrogenrezeptors beta zu detektieren. Der FLiN-Assay basiert auf der Einführung eines Cystein-Rests innerhalb der flexiblen Helix 12 in der Ligandenbindungsdomäne des Rezeptors und anschließender Modifikation mit einem Thiol-reaktiven solvatochromen Fluorophor. Durch die Bindung agonistischer oder antagonistischer Liganden werden unterschiedliche Konformationen der Helix 12 stabilisiert, wodurch es zu Veränderungen in der Polarität der Mikroumgebung des Fluorophors kommt. Diese Veränderungen verursachen unterschiedliche Verschiebungen des Emissionsspektrums des Fluorophors, anhand derer die verschiedenen Konformationen unterschieden werden können und sich Bindungsaffinitäten der Liganden ermitteln lassen. Zur Etablierung des FLiN-Assays wurden in einem strukturbasierten Ansatz verschiedene Proteinkonstrukte der Ligandenbindungsdomäne entworfen, heterolog exprimiert, aufgereinigt, mit einem Fluorophor modifiziert und ihre Eignung als Sensoren für die unterschiedlichen Konformationsänderungen des ER analysiert. Nach einer Optimierung des Assays, bei der unterschiedliche Fluorophore und Puffersysteme getestet wurden, wurden die Ergebnisse des FLiN-Assays mit Hilfe eines orthogonalen Assay-Systems validiert. Dabei konnte gezeigt werden, dass der FLiN-Assay für den Einsatz im Hochdurchsatzformat geeignet ist und, im Gegensatz zu anderen hochdurchsatzfähigen in vitro Testsystemen, zur gezielten Suche nach neuen Liganden die eine aktive oder inaktive Konformation des Rezeptors stabilisieren, eingesetzt werden kann. Zusätzlich wurden zeitaufgelöste FLiN-Messungen zur Analyse der Kinetik der Ligandenbindung an den Estrogenrezeptor durchgeführt. Im Rahmen eines Kooperationsprojektes wurde die Bindung verschiedener Inhibitoren an die p38α MAP-Kinase mit Hilfe des direkten Bindungsassays FLiK sowie strukturbiologischer Studien analysiert. Dabei wurden Komplexkristallstrukturen der Kinase mit einem Benzosuberon-Inhibitor (1) sowie zwei Dibenzoxepinon-Verbindungen (2 und 3) generiert. Die Inhibitoren induzierten einen, für diese Verbindungsklasse charakteristischen, Glycin-flip innerhalb der Scharnierregion. Sowohl die strukturbiologischen Untersuchungen als auch die Analyse mittels FLiK zeigten, dass die untersuchten Inhibitoren jedoch nicht in der Lage waren, einen reinen Typ-II Bindungsmodus zu induzieren.The estrogen receptor belongs to the nuclear receptors, a protein family of ligand-dependent multidomain transcription factors that specifically regulate the expression of their target genes. The activity of NRs is under control of lipophilic small-molecule ligands, like hormones and drugs, that bind to the ligand-binding domain of the receptor, where they account for conformational changes and interactions with a variety of transcriptional co-regulator proteins and DNA. Nuclear receptors play a fundamental role in the control of physiological functions. Moreover, they are involved in pathological processes, such as diabetes, cancer, asthma, and hormone resistance syndromes. Therefore, they are important targets for current drug discovery efforts. This thesis describes the development of FLiN (Fluorescent Labels in Nuclear receptors), a fluorescence-based direct binding assay that can monitor conformational changes within the ligand-binding domain of the human estrogen receptor β. The assay technology is based on the introduction of a cysteine residue within the flexible helix 12 in the ligand-binding domain of the receptor and subsequent labeling with a thiol-reactive solvatochromic fluorophore. Binding of agonistic or antagonistic ligands stabilizes distinct conformations of helix 12 and leads to changes in the polarity of the microenvironment of the fluorophore. These changes translate into alterations of the emission spectrum that can be used to distinguish different conformations and to determine binding affinities of the ligands. In order to establish the FLiN-assay, a series of protein constructs of the receptors’ ligand-binding domain was designed in a structure-guided approach. The protein constructs were produced by heterologous expression, purified, labelled with a solvatochromic fluorophore and their suitability to report on conformational changes was assessed. After optimization of buffer-conditions and testing of different fluorophores, the results of FLiN were validated with an orthogonal assay system. The FLiN assay produces robust data, suitable for high-throughput screening, and allows for screening for new scaffolds that are able to stabilize active or inactive conformations of the receptor, which is not possible with conventional competition assay systems. Furthermore, time-resolved FLiNmeasurements were used to monitor the kinetics of ligand-binding to the receptor. Within the framework of a cooperation project, the binding of different inhibitors to p38α MAP kinase was analyzed using the FLiK-direct binding assay and structural biology studies. Three crystal structures of the kinase in complex with a benzosuberone (1) and two dibenzoxepinone inhibitors (2 and 3) were solved, revealing a characteristic binding mode with an induction of the so-called glycine-flip within the hinge region that provides selectivity over other protein kinases. Structural biology studies and FLiK-measurements both showed that the compounds were not able to stabilize a complete type II binding mode

Effects of GPR139 agonism on effort expenditure for food reward in rodent models: Evidence for pro-motivational actions

Apathy, deficiency of motivation including willingness to exert effort for reward, is a common symptom in many psychiatric and neurological disorders, including depression and schizophrenia. Despite improved understanding of the neurocircuitry and neurochemistry underlying normal and deficient motivation, there is still no approved pharmacological treatment for such a deficiency. GPR139 is an orphan G protein-coupled receptor expressed in brain regions which contribute to the neural circuitry that controls motivation including effortful responding for reward, typically sweet gustatory reward. The GPR139 agonist TAK-041 is currently under development for treatment of negative symptoms in schizophrenia which include apathy. To date, however, there are no published preclinical data regarding its potential effect on reward motivation or deficiencies thereof. Here we report in vitro evidence confirming that TAK-041 increases intracellular Ca2+ mobilization and has high selectivity for GPR139. In vivo, TAK-041 was brain penetrant and showed a favorable pharmacokinetic profile. It was without effect on extracellular dopamine concentration in the nucleus accumbens. In addition, TAK-041 did not alter the effort exerted to obtain sweet gustatory reward in rats that were moderately food deprived. By contrast, TAK-041 increased the effort exerted to obtain sweet gustatory reward in mice that were only minimally food deprived; furthermore, this effect of TAK-041 occurred both in control mice and in mice in which deficient effortful responding was induced by chronic social stress. Overall, this study provides preclinical evidence in support of GPR139 agonism as a molecular target mechanism for treatment of apathy

Optimized Target Residence Time: Type I1/2I^{1}/_2 Inhibitors for p38α\alpha MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R-Spine

Skepinone-L was recently reported to be a p38$\alpha$ MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, this class of compounds still act as fully ATP-competitive Type I binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first Type  $I^{1}/_2$ binders for p38$\alpha$ MAP kinase.  $I^{1}/_2$ formula inhibitors interfere with the R-spine, inducing a glycine flip and occupying both hydrophobic regions I and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X-ray crystallography

Targeting the Hinge Glycine Flip and the Activation Loop: Novel Approach to Potent p38α Inhibitors

The p38 MAP kinase is a key player in signaling pathways regulating the biosynthesis of inflammatory cytokines. Small molecule p38 inhibitors suppress the production of these cytokines. Therefore p38 is a promising drug target for novel anti-inflammatory drugs. In this study, we report novel dibenzepinones, dibenzoxepines, and benzosuberones as p38α MAP kinase inhibitors. Previously reported dibenzepinones and dibenzoxepines were chemically modified by introduction of functional groups or removal of a phenyl ring. This should result in targeting of the hydrophobic region I, the “deep pocket”, and the hinge glycine flip of the kinase. Potent inhibitors with IC₅₀ values in the single digit nanomolar range (up to 3 nM) were identified. Instead of targeting the “deep pocket” in the DFG-out conformation, interactions with the DFG-motif in the in-conformation could be observed by protein X-ray crystallography

Dibenzosuberones as p38 Mitogen-Activated Protein Kinase Inhibitors with Low ATP Competitiveness and Outstanding Whole Blood Activity

p38α mitogen-activated protein (MAP) kinase is a main target in drug research concerning inflammatory diseases. Nevertheless, no inhibitor of p38α MAP kinase has been introduced to the market. This might be attributed to the fact that there is no inhibitor which combines outstanding activity in biological systems and selectivity. Herein an approach to the development of such inhibitors on the basis of the highly selective molecular probe Skepinone-L is described. Introduction of a “deep pocket” moiety addressing the DFG motif led to an increased activity of the compounds. Hydrophilic moieties, addressing the solvent-exposed area adjacent to hydrophilic region II, conserved a high activity of the compounds in a whole blood assay. Combined with their outstanding selectivity and low ATP competitiveness, these inhibitors are very interesting candidates for use in biological systems and in therapy

Design, Synthesis, and Biological Evaluation of Novel Type I¹/₂ p38α MAP Kinase Inhibitors with Excellent Selectivity, High Potency, and Prolonged Target Residence Time by Interfering with the R‑Spine

We recently reported <b>1a</b> (skepinone-L) as a type I p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, as a type I inhibitor, it is entirely ATP-competitive and shows just a moderate residence time. Thus, the scope was to develop a new class of advanced compounds maintaining the structural binding features of skepinone-L scaffold like inducing a glycine flip at the hinge region and occupying both hydrophobic regions I and II. Extending this scaffold with suitable residues resulted in an interference with the kinase’s R-Spine. By synthesizing 69 compounds, we could significantly prolong the target residence time with one example to 3663 s, along with an excellent selectivity score of 0.006 and an outstanding potency of 1.0 nM. This new binding mode was validated by cocrystallization, showing all binding interactions typifying type I¹/₂ binding. Moreover, microsomal studies showed convenient metabolic stability of the most potent, herein reported representatives

Targeting Drug Resistance in EGFR with Covalent Inhibitors: A Structure-Based Design Approach

Receptor tyrosine kinases represent one of the prime targets in cancer therapy, as the dysregulation of these elementary transducers of extracellular signals, like the epidermal growth factor receptor (EGFR), contributes to the onset of cancer, such as non-small cell lung cancer (NSCLC). Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686, which takes advantage of increased residence time at EGFR by alkylating Cys797 and thereby preventing toxic effects. Here, we present a structure-based approach, rationalized by subsequent computational analysis of conformational ligand ensembles in solution, to design novel and irreversible EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against approximately 1500 compounds. Using protein X-ray crystallography, we deciphered the binding mode in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M, which constituted the basis for further rational design approaches. Chemical synthesis led to further compound collections that revealed increased biochemical potency and, in part, selectivity toward mutated (L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and kinetic studies were performed to substantiate our initial findings. Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed the alkylation of Cys797