10th German Pharm-Tox Summit / 91. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (DGPT)

Prof. Dr.

Chair

The third intracellular loop of muscarinic acetylcholine receptors M2 and M4 dictates differential β-arrestin2 complex formations and functional outcomes

G-protein coupled receptors

BR01

The Gi-coupled muscarinic acetylcholine receptors, M2 and M4, and their signaling are important drug targets for heart diseases and neural pathologies respectively. However, the molecular mechanisms underlying the GRK/arrestin-dependent regulation of these receptors are still incompletely understood. Because of purported contributions of the intracellular loop 3 (ICL3) to this regulation, these receptors have become model receptors to understand the nature of arrestin-receptor interactions and their effect on subsequent function.We have shown that M2 and M4 interact with β-arrestin2 (βArr2), depending on cytosolic GRK2/3 overexpression. Our fluorescence microscopy data indicates that M2 sequesters cytosolic βArr2 significantly more than the M4 does. This difference is supported using bystander BRET assays, which reveal that M2 internalizes together with βArr2 into early endosomes, whereas the M4 receptor does not induce βArr2 translocation to endosomes. Corresponding ICL3 chimeric receptors induce a complete switch in arrestin internalization behavior with both techniques, highlighting the importance of this loop in regulatory processes. Next, we determined the βArr2 conformational fingerprint using intramolecular NanoLuc/FlAsH-based biosensors to dissect the differences in arrestin conformations that create these receptor-specific phenotypes. To our surprise, interaction to M2 or M4 (-ICL3) induce almost identical response patterns in the βArr2 N-domain but differential responses in the βArr2 C-domain. This contradicts the current assumption that receptor binding to arrestin will always lead to arrestin activation with βArr2 C-domain rotation as a hallmark. The nature of this receptor-specific interaction and its functional outcome is analyzed in detail using βArr2 lacking the finger loop region (FLR) or the phosphatidylinositol 4,5-bisphosphate (PIP2) interaction site or contains mutations in the C-lobe base (CLB). Here, we detected again major differences between the individual mutations of βArr2 and their effect on M2 or M4 internalization.In summary, we are able to show that the different interactions with βArr2 are shaped by the interacting ICL3. This difference influences the function of βArr2 and may explain the marked differences in internalization behavior. These results provide valuable insights into ICL3-dependent GRK/βArr2-mediated receptor regulation and open doors for further investigation in the field of GPCRs with ICL3 phosphorylation.

Basic Research

Short talk

Investigations of G protein activation at the EP1 receptor highlight knew insights in G12/13 protein selectivity

The EP1 receptor is a member of the G protein-coupled-receptor (GPCRs) class and belongs to the prostanoid-receptors. It gets activated through its endogenous agonists prostaglandin-E1, -E2 and -E3 (PGE1-3) as well as the prostacyclin analog Iloprost and plays important physiological roles e.g. in smooth muscle contraction and neuronal cell death.EP1 receptor signaling has so far only been investigated through Ca2+ mobilization assays. To gain further insights into its molecular pharmacology and function we started to characterize the receptor by performing bioluminescent-resonance energy transfer (BRET)-based G protein activation assays in multicell recordings at a microplate reader using the agonists PGE1, PGE2, Iloprost and the PGE2-metabolite 15-Keto-PGE2.The data revealed a direct activation of Gq proteins through the EP1 receptor but no activation for the Go/i- and Gs protein families. Concentration-response curves showed EC50-values for Gq activation of PGE1 = 111 nM, PGE2 = 12 nM and Iloprost = 27 nM and for 15-keto PGE2 = 1.3 µM. Surprisingly, we demonstrated that the EP1 receptor selectively activates G12 proteins, but not G13 proteins, even though they belong to the same G protein family (concentration-response curves showing EC50-valus of G12 activation: PGE1 = 20 nM, PGE2 = 3 nM, Iloprost = 4 nM, 15-Keto PGE2 = 363 nM). This G12 protein preference was confirmed by additional BRET-based assays, including nano-BRET measurements of the G13-p115 interaction. Furthermore, the EC50-values for all agonists tested indicate a stronger preference of the EP1 receptor for G12 proteins.In summary, these results show that the EP1 receptor can activate Gq and G12 proteins, but not G13 proteins or proteins of the Gi/o or Gs family. Additionally, we demonstrated for the first time that a receptor predominantly activates G12 proteins, which could offer new insights into the selectivity of G proteins in general.

Prof.

Dynamics shaping GPCR functionality

Over one-third of approved drugs target G protein-coupled receptors (GPCRs), but the mechanisms enabling GPCR signaling still need to be fully understood. GPCRs are highly dynamic entities that can translate extracellular stimuli into intracellular signaling cascades. Thereby, the receptors do not act as simple on-off switches but as highly dynamic, allosteric machines assessing different conformational states corresponding to specific signaling outcomes. The conformational states and corresponding signaling vary and depend on many factors, including the ligand, the receptor"s microenvironment, and cell type specific expression pattern.Breakthroughs in protein structure determination and prediction methods have accelerated the progress of 3D structural information in recent years. However, static structures hide dynamic details that shape the functional outcome.Here, we use molecular dynamic (MD) simulations to elucidate structural mechanisms at different GPCRs. We show three case studies where the combination of MD simulations and dynamic 3D pharmacophore analysis enabled a mechanistic understanding of subtle differences in the dynamic ligand-GPCR interplay.1Our first study focuses on binding mode ensembles as a potential mechanism for partial receptor activation of muscarinic receptors. The dynamic interaction patterns correlate with the agonistic efficiency, contributing to the mechanistic understanding of partial agonism. Our second application gives mechanistic insight into peptide binding to the atypical chemokine receptor 3 (ACKR3). The reported opioid scavenging function renders ACKR3 a potential analgesic target without typical opioid-related side effects.2 In the third approach, we focus on subtype-specific binding site characteristics of the sphingosine-1-phosphate receptor family. We analyzed differences in binding mode dynamics to rationalize the selectivity profile of marketed drugs like Siponimod and Ozanimod.3In these three case studies we demonstrate the advantage of analyzing receptor-ligand interactions in a dynamic manner to get insights into specific mechanisms. Understanding these sophisticated mechanisms can foster GPCR research and open new avenues for developing new functionally selective drugs.1) Bock, A. et al., J. biol. chem., 2016. 2) Meyrath, M. et al., Nat. Commun., 2020. 3) Wunsch, F. et al., Arch. Pharm., 2023.

Chemogenetic G protein-ligand pairs for causal investigation of cellular biology in vitro and in vivo

Signal transduction and second messengers

BR03

Nature has always inspired the development of pharmaceutical agents, with acetylsalicylic acid being among the most famous examples. Similarly, many compounds used in basic science are either natural products or derived from naturally occurring scaffolds. One such example is the natural depsipeptide FR900359 (FR), which inhibits heterotrimeric Gq, G11, and G14 proteins specifically and potently and is, therefore, widely used as pharmacological tool to decipher signaling downstream of G protein-coupled receptors.FR is produced by the obligate bacterial endosymbiont of an ornamental plant and is concentration-dependently lethal when ingested by insects. We hypothesize that insect lethality is causally linked to inhibition of insect Gq. To investigate this hypothesis, we used the fruit fly, Drosophila melanogaster, which also dies when exposed to FR, as in vivo model. We rationally designed and engineered Drosophila Gαq (DGq) proteins with wildtype-like signaling behavior but reduced FR-sensitivity. Pan-neuronal expression of these modified Gαq variants rescued FR-dependent lethality of the animals without any obvious side effects.The specificity of FR effects can be harnessed for chemogenetic applications of Gαq variant-FR pairs in Drosophila melanogaster, as well as other model organisms accessible to (chemo-)genetic approaches. We, therefore, transferred the promising modifications that we had introduced into DGq to the murine Gαq protein and thereby developed fully functional mGαq variants that are entirely refractory to FR-inhibition.With our results, we show that FR-induced lethality in Drosophila melanogaster is caused by Gq-inhibition, suggesting that this natural product may have evolved as an antiherbivory compound that specifically inhibits several species" Gαq function in vivo. Moreover, we qualify Gq protein-FR pairs for causal investigation of cellular biology in vitro and in vivo.

Glucocorticoids induce a maladaptive epithelial stress response to aggravate acute kidney injury

Nuclear receptors, enzymes and other targets

BR04

Acute kidney injury (AKI) is a frequent and challenging clinical condition associated with high morbidity and mortality and represents a common complication in critically ill patients with COVID-19. In AKI, renal tubular epithelial cells (TECs) are a primary site of damage, and recovery from AKI depends on TEC plasticity. However, the molecular mechanisms underlying adaptation and maladaptation of TECs in AKI remain largely unclear. Here, our study of an autopsy cohort of patients with COVID-19 provided evidence that injury of TECs by myoglobin, released as a consequence of rhabdomyolysis, is a major pathophysiological mechanism for AKI in severe COVID-19. Analyses of human kidney biopsies, mouse models of myoglobinuric and gentamicin-induced AKI, and mouse kidney tubuloids showed that TEC injury resulted in activation of the glucocorticoid receptor by endogenous glucocorticoids, which aggravated tubular damage. The detrimental effect of endogenous glucocorticoids on injured TECs was exacerbated by the administration of a widely clinically used synthetic glucocorticoid, dexamethasone, as indicated by experiments in mouse models of myoglobinuric- and folic acid–induced AKI, human and mouse kidney tubuloids, and human kidney slice cultures. Mechanistically, studies in mouse models of AKI, mouse tubuloids, and human kidney slice cultures demonstrated that glucocorticoid receptor signaling in injured TECs orchestrated a maladaptive transcriptional program to hinder DNA repair, amplify injury-induced DNA double-strand break formation, and dampen mTOR activity and mitochondrial bioenergetics. This study identifies glucocorticoid receptor activation as a mechanism of epithelial maladaptation, which is functionally important for AKI.

Feasibility analysis for the implementation of an environmental information and classification system in Germany ("Arzneimittelindex Umwelt")

Pharmacological education

ED-Pharm

Pharmaceuticals are released into the environment through multiple pathways and can be found in significant concentrations, already exceeding threshold concentrations in some areas. There are numerous ongoing initiatives along the pharmaceutical life cycle trying to reduce the input of pharmaceuticals into the environment. A high impact in this respect can be achieved at the beginning of the life cycle, as well as by reducing the number of prescriptions / dispensed drugs or optimizing them into an eco-directed pharmaceutical prescribing / dispensing. An environmental information and classification system for pharmaceuticals, as implemented e.g. in Sweden and Finland, is a tool that can help choose more environmentally friendly pharmaceuticals. The aim of our study is to understand how an implementation of a similar system could look like in Germany.An environmental information and classification system should ideally be easily implementable, hostable and updatable for the hosting site. From the perspective of healthcare actors, it should also aim to be practical, clear and capable of integration into existing guidelines. To understand how to achieve this goal, a literature review of existing systems in Sweden, Finland and Scotland was performed and supplemented by interviews with relevant stakeholders. Furthermore, a concept for an environmental classification of pharmaceuticals was created. In a following step, conclusions were drawn and a draft concept for Germany was developed and presented to a project advisory board. Feedback was used to further develop the concept.A concept for this pharmaceutical index environment ("Arzneimittelindex Umwelt") was developed based on the three pillars (i) information, (ii) classification, and (iii) dissemination system. The information will be based on the public results from European environmental risk assessments according to European Medicines Agency´s technical guideline. The concept includes a new developed classification system. All information should be hosted on a website of the German Environment Agency. The dissemination includes integration into existing pharmaceutical and medical guidelines, pharmacy and medical practice software, prioritization lists for OTC drugs, and medicine lists of pharmaceutical commissions in hospitals. Furthermore, the information shall be made available to scientists, universities, water suppliers and health insurances (e.g. to implement them into contract tenders).

Short talk 13
G-Protein-coupled receptors

Appointment

Chair

Programme

Short talk 13G-Protein-coupled receptors

Appointment

Chair

Programme

Short talk 13
G-Protein-coupled receptors