The CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3) are seven transmembrane receptors that are involved in numerous pathologies, including several types of cancers. Both receptors bind the same chemokine, CXCL12, leading to significantly different outcomes. While CXCR4 activation generally leads to canonical GPCR signaling, involving Gi proteins and β‐arrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via β‐arrestin‐dependent signaling pathways. Understanding the dynamics and kinetics of their activation in response to their ligands is of importance to understand how signaling proceeds via these two receptors. In this thesis, different Förster resonance energy transfer (FRET)‐based approaches have been combined to individually investigate the early events of their signaling cascades. In order to investigate receptor activation, intramolecular FRET sensors for CXCR4 and ACKR3 were developed by using the pair of fluorophores cyan fluorescence protein and fluorescence arsenical hairpin binder. The sensors, which exhibited similar functional properties to their wild‐type counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RET‐based receptor sensors in the field of chemokine receptors. Additional FRET‐based settings were also established to investigate the coupling of receptors with G proteins, rearrangements within dimers, as well as G protein activation. On one hand, CXCR4 showed a complex activation mechanism in response to CXCL12 that involved rearrangements in the transmembrane domain of the receptor followed by rearrangements between the receptor and the G protein as well as rearrangements between CXCR4 protomers, suggesting a role of homodimers in the activation course of this receptor. This was followed by a prolonged activation of Gi proteins, but not Gq activation, via the axis CXCL12/CXCR4. In contrast, the structural rearrangements at each step of the signaling cascade in response to macrophage migration inhibitory factor (MIF) were dynamically and kinetically different and no Gi protein activation via this axis was detected. These findings suggest distinct mechanisms of action of CXCL12 and MIF on CXCR4 and provide evidence for a new type of sequential signaling events of a GPCR. Importantly, evidence in this work revealed that CXCR4 exhibits some degree of constitutive activity, a potentially important feature for drug development. On the other hand, by cotransfecting the ACKR3 sensor with K44A dynamin, it was possible to increase its presence in the plasma membrane and measure the ligand‐induced activation of this receptor. Different kinetics of ACKR3 activation were observed in response to CXCL12 and three other agonists by means of using the receptor sensor developed in this thesis, showing that it is a valuable tool to study the activation of this atypical receptor and pharmacologically characterize ligands. No CXCL12‐induced G protein activation via ACKR3 was observed even when the receptor was re-localized to the plasma membrane by means of using the mutant dynamin. Altogether, this thesis work provides the temporal resolution of signaling patterns of two chemokine receptors for the first time as well as valuable tools that can be applied to characterize their activation in response to pharmacologically relevant ligands. / Der CXC Chemokin‐Rezeptor 4 (CXCR4) und der atypische Chemokin‐Rezeptor 3 (ACKR3) sind heptatransmembranäre Rezeptoren, die in zahlreichen Krankheitsbildern eine Rolle spielen, wie in einigen Krebsarten. Beide Rezeptoren werden zwar von dem gleichen Chemokin CXCL12 aktiviert, allerdings mit unterschiedlichen Signalweiterleitungsmustern. Die Aktivierung von CXCR4 führt zu kanonischer GPCR Signaltransduktion über Gi‐Proteine und β‐Arrestine. Die Signalweiterleitung des Rezeptors ACKR3 hingegen, welcher hauptsächlich in intrazellulären Vesikeln vorliegt, erfolgt über ß‐Arrestinabhängige Signalwege. Es ist von großer Wichtigkeit die Dynamik und Kinetik dieser beiden Rezeptoren hinsichtlich der Aktivierung durch ihre Liganden und der Signalweiterleitung zu verstehen. In dieser Arbeit wurden verschiedene Förster‐Resonanzenergietransfer (FRET) Anwendungen kombiniert, um die frühen Phasen der Signal‐Kaskade von CXCR4 und ACKR3 zu untersuchen. Zur genaueren Aufklärung der Rezeptoraktivierung wurden intramolekulare FRET‐Sensoren entwickelt, hierzu wurden die Fluorophore Cyan‐fluoreszierendes Protein und engl. fluorescence arsenical hairpin binder verwendet. Die generierten Sensoren zeigten ähnliche funktionelle Eigenschaften wie die unveränderten Rezeptoren. Liganden‐induzierte Änderungen der Rezeptorkonformation können mittels dieser Sensoren beobachtet werden und stellen die ersten RET‐basierten Sensoren auf dem Forschungsgebiet der Chemokin‐Rezeptoren dar. Weitere FRET‐basierte Methoden wurden zur Untersuchung von Interaktionen zwischen Rezeptor und G‐Protein, Neuanordnung von Dimeren, sowie der G‐Protein Aktivierung eingesetzt und für beide Chemokin‐Rezeptoren etabliert. CXCR4 zeigte einen komplexen Aktivierungsmechanismus nach Stimulation durch CXCL12, bei welchem zunächst eine Neuordnung der Rezeptor‐Transmembrandomäne gefolgt von Neuordnungen zwischen Rezeptor und G‐Protein und zuletzt eine Neuordnung zwischen CXCR4 Protomeren erfolgte. Dies impliziert, dass im Aktivierungsprozess des Rezeptors Homodimere eine Rolle spielen. Zudem wurde eine verlängerte Gi ‐Protein Aktivierung gegenüber der Gq‐Protein Aktivierung bei CXCL12 stimuliertem CXCR4 beobachtet. Hingegen zeigte eine Stimulierung mit dem Macrophage Migration Inhibitory Factor (MIF) bei jedem Schritt der frühen Singal‐Kaskade veränderte Dynamiken und Kinetiken im Vergleich zu CXCL12. Darüber hinaus konnte keine Gi ‐Protein Aktivierung festgestellt werden. Dieser Befund zeigt individuelle Mechanismen für MIF und CXCL12 am CXCR4‐Rezeptor und liefert Belege für eine neuer Art von sequenziellen Signalweiterleitungen an GPCRs. Eine wichtige Beobachtung dieser Arbeit für eine potentielle Medikamentenentwicklung ist das CXCR4 ligandenunabhängige Aktivität zeigt. Um die Aktivierung des ACKR3 Sensors messen zu können wurde durch eine Co‐Transfektion mit K44A Dynamin eine höhere Membranständigkeit erreicht. CXCL12 und drei weiteren Agonisten zeigten am hier entwickelten ACKR3‐Sensor unterscheidbare Kinetiken. Mit diesem wertvollen Werkzeug können Liganden an diesem atypischen Rezeptor pharmakologisch charakterisiert werden. Es konnte keine CXCL12‐induzierte G‐Protein Aktivierung gemessen werden, trotz der stärkeren Präsenz an der Plasmamembran mit Hilfe der Dynamin‐Mutante. In Summe liefert diese Arbeit zum ersten Mal eine zeitliche Auflösung von Signalweiterleitungsmustern von zwei Chemokin‐Rezeptoren sowie wertvolle Werkzeuge zur Charakterisierung der frühen Phase der Signal‐Kaskade durch andere pharmakologisch relevanten Liganden.

Metabotropic glutamate receptor 5 (mGluR5) is a key regulator of numerous brain functions including memory, cognition, and motor behavior. Dysregulation of mGluR5 signaling is evident in Huntington's disease (HD) neuropathology, an inherited, neurodegenerative disease characterized with progressive deterioration in motor, cognitive, and psychiatric functions. In this context, two cellular proteins draw particular interest for this thesis: vesicular glutamate transporter 3 (VGLUT3) and optineurin (OPTN). VGLUT3 modulates glutamate release from selected neurons that are affected by HD, while OPTN is a mGluR5-interacting protein and contributes to neuronal vulnerability in HD. However, current evidence on their involvement in mGluR5 signaling and HD pathogenesis is still lacking. Using VGLUT3 knockout (VGLUT3⁻ᐟ⁻) mice, we showed that this transporter dynamically regulated glutamate receptor densities in different brain regions. Of note, VGLUT3 deletion upregulated mGluR5 in the cerebral cortex and the striatum, unlike the hippocampus which exhibited reduced mGluR5 cell surface densities. We then crossed VGLUT3⁻ᐟ⁻ mice with the zQ175 knock-in mouse model of HD (zQ175:VGLUT3⁻ᐟ⁻) to assess the impact of VGLUT3 transmission loss on HD progression. The longitudinal behavioral assessment revealed that VGLUT3 ablation rescued the deficits in motor coordination and short-term memory in both male and female zQ175 mice throughout 15 months of age. Furthermore, VGLUT3 deletion rescued striatal cell loss likely via activation of Akt and ERK1/2 cellular pathways, with no impact on total mutant huntingtin aggregation or the associated microgliosis. To delineate the role of OPTN in mGluR5 signaling, we employed a CRISPR/Cas9 OPTN-deficient cell line and global OPTN knockout mice. We demonstrated that OPTN was essential for mGluR5-mediated canonical signaling and ERK1/2 activation in both the striatal cell line, STHdh^Q7/Q7, and acute hippocampal slices. We then showed that OPTN deletion impaired autophagic machinery via GSK3β/ZBTB16 and mTOR/ULK1 signaling pathways downstream of mGluR5. This work offers novel insights into the molecular roles of VGLUT3 transmission and OPTN in mGluR5 signaling and provides a rationale for their targeting to therapeutically mitigate pathological mGluR5 signaling in HD.

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京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24558号 / 薬科博第175号 / 新制||薬科||19(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 土居 雅夫, 教授 竹島 浩, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

When applied to drug discovery, modern computational systems can provide insight into the highly complex systems underlying drug activity and predict compounds or targets of interest. Many tools have been developed for computer aided drug discovery (CADD), focusing on small molecule ligands, protein targets, or both. The aim of this thesis is the improvement of CADD tools for describing small molecule properties and application of CADD to several stages of drug discovery regarding two targets for the treatment of obesity and related diseases: the neuropeptide Y4 receptor (Y4R) and the melanocortin-4 receptor (MC4R). In the first chapter, the major categories of CADD are outlined, including descriptions for many of the popular tools and examples where these tools have directly contributed to the discovery of new drugs. Following the introduction, several improvements for encoding stereochemistry and signed property distribution are introduced and tested in scenarios meant to simulate applications in virtual high-throughput screening. Y4R and MC4R are both class A G-protein coupled receptors (GPCRs) with endogenous peptide ligands that play critical roles in the signaling of satiety and energy metabolism. So far, no structures from either receptor family have been experimentally elucidated. CADD was combined with high-throughput screening (HTS) to discover the first small molecule positive allosteric modulators (PAMs) of Y4R. Secondly, CADD techniques were used to model the interaction of Y4R and pancreatic polypeptide based on experimental results that elucidate specific binding contacts. Similar SB-CADD approaches were used to model the interaction of MC4R with its high affinity peptide agonist α-MSH. Due to its role in monogenic forms of obesity, these models were used to predict which residues directly participate in binding and correlate mutated residues with their potential role in the binding site.