Christa Elisabeth Müller

GPR55 is a lipid-activated G protein-coupled receptor (GPCR) implicated in metabolism, inflammation, and neurodegeneration. Lysophosphatidylinositol (LPI) species are proposed as endogenous agonists, but progress in understanding GPR55 function has still been limited by suboptimal chemical probes. Here, we present cryo-electron microscopy (cryo-EM) structures of GPR55 in both inactive and active states. The inactive-state structure bound to the inverse agonist CID16020046 facilitated the rational design of an improved in vitro tool compound, BI-8310. Surprisingly, mutation of a glycosylation site in extracellular loop 2 converted CID16020046 into a potent agonist, enabling co-structure determination of the active, G₁₃-bound receptor. Comparative analysis of both states bound to the same ligand provides insights into an atypical activation mechanism that diverges from the typical helix VI outward movement observed in other GPCRs. Functional mutagenesis, N-glycoproteomics, and hydrogen-deuterium exchange mass spectrometry (HDX-MS) substantiate the findings. These results establish a basis for targeted modulation of GPR55 in pathophysiological conditions.

Allosteric modulators offer opportunities for pathway-selective G protein–coupled receptor (GPCR) signaling, but the mechanisms enabling biased allosteric modulation remain poorly understood. We identify a helix 8–proximate allosteric site in the immune-metabolic receptor GPR84 and define how it achieves G _i -biased signaling. Cryo–electron microscopy structures of the GPR84-G _i complexes bound to the orthosteric agonist OX04539 alone or with the positive allosteric modulator PSB-16671 reveal a helix 8–proximate allosteric site for PSB-16671 formed between transmembrane helices 1 (TM1) and TM7. Molecular dynamics simulations and mutagenesis uncover a polar interaction network linking orthosteric and allosteric sites through conserved residues including Asp66 ^2.50 , Asn104 ^3.36 , and Asn362 ^7.45 , and disrupting this network enhances allosteric cooperativity. PSB-16671 stabilizes a receptor conformation with pronounced TM6 displacement that favors G _i coupling while disfavoring β-arrestin recruitment, sustaining macrophage phagocytosis of cancer cells without the desensitization induced by balanced agonists. Sequence analysis suggests that receptor-specific helix 8–proximate allosteric sites may be broadly targetable across class A GPCRs. These findings establish mechanistic principles for biased allosteric modulation applicable beyond GPR84.

The SARS-CoV-2 main protease (Mpro) is a key antiviral drug target due to its essential role in the pathogen’s replication cycle. The development of potent next-generation Mpro inhibitors is of great importance to ensure the availability of safe and effective COVID-19 therapeutics. Associated research necessitates robust and highly sensitive biochemical assays for the early kinetic characterization of Mpro inhibitor candidates. These assays often reach their limits when characterizing highly potent inhibitors, with a common bottleneck being the insufficient catalytic efficiency and competitive capacity of employed Mpro substrates. To optimize assays for highly active Mpro inhibitors by providing Mpro substrates with improved kinetic features, we created a library of fluorogenic substrates (2–8). These reporters were structurally derived from the well-established Mpro substrate Boc-Abu-Tle-Leu-Gln-AMC (1) and the clinically approved covalent-reversible Mpro inhibitor nirmatrelvir. Kinetic evaluation of 1–8 identified hit compound 6 with about ten-fold improved catalytic efficiency (kcat/Km = 21,400 M–1 s–1) compared to parent substrate 1 (kcat/Km = 2410 M–1 s–1). Substrate 6 was successfully applied for the kinetic characterization of three highly potent Mpro inhibitors. Comparative X-ray crystallographic analyses revealed high similarity in the molecular interactions of the C145A mutant Mpro with nirmatrelvir and with a peptide whose sequence was derived from a natural Mpro substrate. The tetrapeptidic AMC derivative 6 will serve as a valuable biochemical tool contributing to the optimization of SARS-CoV-2 Mpro-directed drug research.

Extracellular adenosine triphosphate (ATP) and diphosphate (ADP) act as key signalling molecules in the central nervous system (CNS) and regulate neuroinflammatory responses through purinergic receptors. Although astrocytes and neurons undergo profound changes in signalling and metabolism during inflammation, the contribution of specific purinergic pathways to inflammation-induced neurodegeneration remains unclear. Here we show that the ADP/ATP-activated Gq-coupled receptor P2Y ₁ drives astrocyte-mediated neurotoxicity in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Using plasma membrane-targeted luciferase reporter mice, we demonstrate that extracellular ATP levels are increased during acute EAE. This was accompanied by elevated astrocytic P2ry1 expression, which is also observed in inflammatory MS lesions. In vivo, pharmacological inhibition or astrocyte-specific deletion of P2Y ₁ reduced disease severity, astrocytosis, and neuronal loss, whereas neuron-specific deletion exerted only modest effects. Mechanistically, astrocytic P2Y ₁ signalling promoted cytokine-induced ERK activation, inflammatory gene expression, and metabolic reprogramming in vitro. In contrast to supernatants from stimulated P2Y ₁ -deficient astrocyte culture, supernatants derived from stimulated P2Y ₁ -proficient astrocytes reduced neuronal viability, demonstrating neurotoxic effects mediated by astrocyte-derived factors. In contrast, neuronal P2Y ₁ signalling primarily contributed to oxidative stress and mitochondrial dysfunction. Together, these findings identify astrocytic P2Y ₁ as a key regulator of neuroinflammatory damage and a potential therapeutic target.

Ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39) is a purinergic immune checkpoint protein and a potential biomarker in tumor immunotherapy. The enzyme can hydrolyze extracellular adenosine tri- and diphosphate (ATP, ADP) to generate adenosine monophosphate (AMP), which is subsequently hydrolyzed by ecto-5′-nucleotidase (CD73) to generate immunosuppressive adenosine (ADO). CD39 inhibition increases the extracellular concentration of immunostimulatory ATP, concomitantly reducing ADO production. In the tumor microenvironment, ATP can recruit a variety of antitumor immune cells, including T cells, dendritic cells, M1 macrophages, B cells, natural killer cells, and N1 neutrophils, to the tumor site, thereby suppressing various tumor activities. Here, we present non-nucleotidic and nucleotide-derived CD39 inhibitors, structural insights, current clinical studies, and strategies for combination therapy. We highlight the potential advantages of targeting CD39 in tumor immunotherapy and provide valuable insights to guide future drug development and therapeutic applications.

Ectonucleotidases catalyze the hydrolysis of extracellular nucleotides, maintaining the balance between proinflammatory ATP and immunosuppressive adenosine. In the present study, we developed potent competitive inhibitors of the main ATP-hydrolyzing ectoenzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39) based on 8-butylthio-AMP as a lead structure. Altogether, 88 purine nucleotides and analogs with broad structural modifications were synthesized, 78 of which are new compounds. 8-Substitution of the purine scaffold with bulky residues is essential for high potency and confers metabolic stability. 8-(1-Naphthylthio)-N⁶-(4-phenylbutyl)-AMP (42b, PSB-24379) is the most potent CD39 inhibitor of the series (K_i 77.4 nM), showing ancillary CD73 inhibition (K_i 240 nM). Docking into a human CD39 homology model rationalized key interactions. PSB-24379 reduced ATP hydrolysis in melanoma and breast cancer cell membranes and partially reverted ATP-mediated effects on T cell activation and proliferation in an ATP-rich environment. These CD39 inhibitors represent high-quality tool compounds with potential as drugs for immunotherapy of cancer.

The ATP-gated P2X7 receptor (P2X7R) activates inflammatory signaling pathways in the central nervous system. In particular, P2X7Rs drive chronic glia-mediated neuroinflammation, which is increasingly recognized as a key contributor to Alzheimer’s disease, a neurodegenerative disorder that lacks effective disease-modifying therapies. Here we identify a potent and selective negative allosteric modulator of P2X7Rs with therapeutic potential. We synthesize a series of small molecules based on a polycyclic scaffold and confirm blood-brain barrier penetration by testing a radiolabeled analogue using positron emission tomography imaging. Through a structure-guided medicinal chemistry campaign centered on our scaffold, we identify four promising P2X7R antagonists. Of these, UB-ALT-P2 exhibits the most favorable safety profile, high oral bioavailability and robust brain penetration. High-resolution cryo-EM structures of UB-ALT-P2 bound to human, mouse, and rat P2X7Rs reveal a conserved antagonist binding mode with steric features that favor prolonged binding to human receptors. In the 5xFAD mouse model of AD, oral UB-ALT-P2 blunts weight loss, improves short- and long-term memory, reduces amyloid-β plaque burden, lowers hyperphosphorylated tau, and diminishes oxidative and inflammatory markers. These results establish UB-ALT-P2 as a potent and safe P2X7R antagonist that can mitigate core AD pathologies, providing a compelling foundation for further development.

Ecto‐5’‐nucleotidase (CD73) is a potential new drug target for cancer immunotherapy. Its overexpression is associated with various aggressive cancers, including triple‐negative breast cancer (TNBC) and pancreatic cancer, making it a promising target for diagnostic imaging. Besides antibodies, small‐molecule CD73 inhibitors have been developed and are currently in clinical trials. This study aimed to develop and evaluate fluorine‐18 labeled high‐affinity CD73 inhibitors as tracers for the non‐invasive positron emission tomography (PET) imaging of CD73 expression in cancer. Two CD73 inhibitors were selected for radiolabeling based on their high potency (K _i values of ca. 1 nM) and favorable pharmacokinetic properties, yielding [ ¹⁸ F]PSB‐19427 ([ ¹⁸ F] 1 ) and [ ¹⁸ F]MRS‐4648 ([ ¹⁸ F] 2 ). Ex vivo imaging studies on human breast cancer tissues indicated specific binding of both radiotracers. Subsequent in vivo studies proved [ ¹⁸ F] 1 to be superior due to its long elimination half‐life and its accumulation in TNBC and pancreatic cancer tissues, suggesting its potential as a versatile PET tracer for imaging of various solid tumors. Compared to [ ¹⁸ F]FDG, [ ¹⁸ F] 1 was superior in visualizing TNBC, offering potential advantages over [ ¹⁸ F]FDG in terms of specificity and diagnostic accuracy. Thus, [ ¹⁸ F] 1 is a PET tracer with outstanding properties suitable for broad application in cancer diagnosis and personalized medicine.