Biology subjects

Leurs, R.

Publications and source records attributed to Leurs, R..

12 recordsLinked to original sources

bioRxiv2026

The FDA-approved CDK4/6 inhibitor Abemaciclib is a potent agonist of the β-arrestin-biased atypical chemokine receptor ACKR3

Atypical chemokine receptor 3 (ACKR3/CXCR7) is an intrinsically {beta}-arrestin-biased family-A GPCR that controls chemokine availability by scavenging CXCL12 and CXCL11, and consequently plays a key role in cancer, chronic inflammation, autoimmunity, cardiovascular disease, and CNS repair. Despite its pathophysiological significance, the chemical space available to interrogate and therapeutically modulate this receptor remains limited. Here, by using a drug-repurposing strategy to screen 2,342 FDA-approved compounds in a NanoBRET-based fluorescent chemokine-binding competition assay, we identified the clinically used CDK4/6 inhibitor abemaciclib (Verzenios(R)) as a nanomolar ACKR3 ligand. Abemaciclib inhibited CXCL12 binding to ACKR3 with comparable affinity to the ACKR3-reference agonist VUF15485, whereas the related clinically used CDK4/6 inhibitors palbociclib, ribociclib, dalpiciclib and trilaciclib were inactive on this receptor. Binding and functional profiling across classical and atypical chemokine receptors revealed selectivity for ACKR3, including lack of detectable activity at the CXCL12 receptors CXCR4 and ACKR5. Abemaciclib acted as a potent ACKR3 agonist, recruiting both {beta}-arrestin-1 and 2 and promoting rapid receptor internalization with potencies and efficacies, like the reference ACKR3 agonist VUF15485. Major CYP3A4-derived abemaciclib metabolites retained ACKR3 binding and agonist activity, albeit with reduced potency. Methionine 13C-HSQC NMR confirmed direct receptor engagement and revealed a small-molecule-like ACKR3 conformational signature distinct from the CXCL12-bound state. Cryo-EM of the abemaciclib-ACKR3-CID24 complex at 3.2 [A] uncovered a unique, inverted U-shaped ligand binding pose in the orthosteric pocket and activation-associated rearrangements of TM6, TM7 and helix 8. Functionally, abemaciclib blocked ACKR3-mediated uptake of CXCL11 and CXCL12 in vitro. Moreover, oral administration of abemaciclib in mice resulted in a time-dependent increase in plasma CXCL12 levels, indicative of in vivo ACKR3 target engagement. These findings establish this FDA-approved kinase inhibitor as a potent ACKR3 modulator and reveal an unexpected pharmacological link of this CDK4/6 inhibitor to atypical chemokine receptor biology.

molecular biology↗

bioRxiv2026

Chemokine and opioid peptide scavenging through constitutive and ligand-induced release of ACKR3-bearing extracellular vesicles

Atypical chemokine receptors (ACKRs) are non-signaling GPCRs that regulate ligand availability, with ACKR3 functioning as a dual scavenger of chemokines and opioid peptides. Here, we demonstrate that following ligand stimulation, besides the canonical internalization, ACKR3 is released on extracellular vesicles (EVs). ACKR3 was also found on EVs released under basal conditions, although to a lesser extent. These observations were confirmed across multiple cellular contexts, including endogenous systems. Mechanistically, basal and ligand-induced EV release are independent of GRKs and {beta}-arrestin but each relies on distinct trafficking routes and C-terminal determinants. Ligand-induced EV release is associated with plasma membrane localization and receptor recycling pathways. In contrast, basal EV release is governed by intracellular sorting processes and influenced by receptor ubiquitination and RAMP3. Functionally, EV-associated ACKR3 retains high-affinity ligand binding, enabling sequestration of CXCL12 and opioid peptides and thereby attenuating their signaling through CXCR4 and MOR. We also show that the release on EVs, in particular under basal conditions, is observed for other receptors such as KOR, CXCR4 and several ACKRs. Collectively, these findings establish EVs as regulators in chemokine and opioid systems and as a previously underappreciated dimension of ACKR3 and more broadly GPCR biology.

Cell Biology↗

bioRxiv2024

Inhibition of constitutive activity of the atypical chemokine receptor ACKR3 by the small-molecule inverse agonist VUF16840

The atypical chemokine receptor 3 (ACKR3) has emerged as a promising drug target for the treatment of cancer, cardiovascular, and autoimmune diseases. In this study, we present the pharmacological characterization of VUF16840, the first small-molecule inverse agonist of ACKR3. VUF16840 effectively displaces CXCL12 binding to ACKR3 and inhibits chemokine-induced {beta}-arrestin2 recruitment in a concentration-dependent manner. Furthermore, VUF16840 stabilizes the inactive conformation of ACKR3, as demonstrated by its ability to suppress constitutive {beta}-arrestin2 recruitment. This inverse agonism alters ACKR3 constitutive trafficking, leading to receptor enrichment at the plasma membrane and inhibition of intracellular CXCL12 uptake. Importantly, VUF16840 exhibits high selectivity for ACKR3 over a broad panel of human chemokine receptors. These findings establish VUF16840 as a potent and selective ACKR3 inverse agonist capable of modulating constitutive and chemokine-induced signaling and internalization events. As such, VUF16840 represents a valuable pharmacological tool for exploring the molecular and translational roles of ACKR3 in both physiological and pathological contexts.

pharmacology and toxicology↗

bioRxiv2024

Characterization of AZ12464237 as a high affinity, non-nucleotide antagonist radioligand for the P2Y12 receptor

The purinergic receptor P2Y12 (P2Y12R) is a well-recognized target for anti-thrombotic agents. This receptor is also expressed in microglia, where it plays a key role in neuroinflammation and microglia activation. To investigate P2Y12R-mediated actions in the central nervous system (CNS), the development of novel brain-penetrant ligands is essential, along with further in vitro studies. A radiolabeled, easily accessible tool compound would significantly advance such drug discovery efforts. Herein, we describe the 3H-labeling of a non-nucleotide P2Y12R antagonist AZ12464237, and its in vitro binding properties to the receptor in membrane preparations form transfected cells, as well as on mouse brain tissues. The radioligand shows high affinity toward both the human and rat P2Y12R in transfected cells, with Kd values of 3.12 {+/-} 0.70 nM (human) and 16.6 {+/-} 3.4 nM (rat), as determined by saturation binding studies. The binding kinetics of [3H]AZ12464237 are rapid with a short target residence time ([~]1 min). We further confirmed the selectivity of the radioligand by performing competitive displacement studies, in which reported P2Y12R ligands and other P2Y receptors ligands were tested for binding against [3H]AZ12464237. Additionally, the radioligand proved useful for in vitro autoradiography studies on mouse brain tissues, although a small amount of off-target binding was observed in P2Y12R knock-out mice. This could be traced to glycogen synthase kinase 3 . Considering the growing interest in P2Y12R as a biomarker for microglia activation, [3H]AZ12464237 represents a promising tool for in vitro studies, including screening assays aimed at identifying novel P2Y12R ligands for CNS applications.

pharmacology and toxicology↗

bioRxiv2024

ACKR5/GPR182 is a scavenger receptor for the atypical chemokine CXCL17, GPR15L and various endogenous peptides

GPR182/ACKR5, the most recently deorphanized chemokine receptor, is mainly expressed on endothelial cells and was proposed to act as a scavenger regulating the availability of a large set of chemokines. In this study, we first established the exact profiles and ranking of the chemokines binding to the human and mouse GPR182. We confirmed the high promiscuity of GPR182 towards XC, CC and CXC chemokines and a clear difference in the chemokine repertoires of the human and mouse orthologues. We next demonstrated that, beyond classical chemokines, GPR182 exhibits potent binding to the chemoattractant protein GPR15L/C10orf99, the atypical chemokine CXCL17 and various endogenous peptides, mainly from the opioid, apelin, and PACAP families. We also showed that these newly identified ligands engage GPR182 through varied binding modes. While GPR15L, just like classical chemokines, predominantly engages GPR182 via its N terminus, conversely to the C terminus-dependent binding to its cognate receptor GPR15, CXCL17 exhibits a more complex interaction, relying on both the N and C terminus. The binding mode of the newly identified peptide ligands also differ from the interactions with their cognate receptors. Our findings establish the first scavenger receptor for CXCL17 and GPR15L and advance the understanding of GPR182 ligand interactions, suggesting a regulatory role beyond chemokines.

pharmacology and toxicology↗

bioRxiv2024

Design, synthesis and pharmacological characterization of the first photoswitchable small-molecule agonist for the Atypical Chemokine Receptor 3

1.Photopharmacology offers the promise of optical modulation of cellular signaling in a spatially and temporally controlled fashion with light-sensitive molecules. This study presents the first small-molecule photoswitchable agonist for an atypical G protein-coupled receptor (GPCR), the atypical chemokine receptor 3 (ACKR3). Inspired by a known benzylpiperidine-based ACKR3 agonist scaffold, 12 photoswitchable azobenzene-containing analogs were synthesized and characterized for their interaction with ACKR3. After analysis of Structure-Photochemistry and Structure-Affinity Relationships (SAR), compound 3e was selected as the best photoswitchable ACKR3 agonist in the series. Compound 3e can be effectively switched from its thermodynamically stable trans state to the less active cis-isomer with a PhotoStationary State of 96 %. The thermodynamically less stable cis-3e only slowly switches back to the trans state (t1/2,37 {degrees}C = 15 days), and trans-3e binds and activates ACKR3 at 10-fold lower concentrations compared to its cis-isomer. Compound 3e demonstrates selectivity for ACKR3 within in a panel of chemokine receptors. Using the recently published ACKR3 cryo-EM structures in computational studies, a binding mode for trans-3e is proposed and is perfectly in line with the observed SAR and the loss of interaction with ACKR3 upon photoswitching. ACKR3 agonist 3e (VUF25471) is the first photoswitchable ligand for an atypical GPCR and will be a useful tool to investigate the role of ACKR3 in biological settings.

pharmacology and toxicology↗

bioRxiv2024

Molecular basis of ligand promiscuity, structural mimicry, and atypical dimerization in the chemokine receptors

Selectivity of natural agonists for their cognate receptors is one of the hallmarks of the members of GPCR family, and it is crucial for the specificity of downstream signal-transduction. However, this selectivity often breaks down in the chemokine receptor subfamily, wherein a high degree of promiscuity is observed with one receptor recognizing multiple chemokines and one chemokine binding to multiple receptors. The molecular determinants of such a striking promiscuity for natural ligands in the chemokine-chemokine receptor system remain mostly elusive and represent an important knowledge gap in our current understanding. Here, we carry out a comprehensive transducer-coupling analysis, testing all known C-X-C chemokines on every C-X-C type chemokine receptor, to generate a global fingerprint of the selectivity and promiscuity encoded within this system. Taking lead from our finding, we determined cryo-EM structures of the most promiscuous receptor, CXCR2, in complex with every interacting chemokine, and deciphered the conserved molecular signatures and distinct binding modalities. While most chemokines position themselves on the receptor as a dimer, CXCL6 exhibits a monomeric binding pose induced by a previously unanticipated reorientation of its carboxyl-terminal -helix, leading to disruption of the dimer interface. Surprisingly, one of the chemokines, CXCL5, induces a ligand-swapped dimer of CXCR2, the first of its kind observed in class A GPCRs, wherein each protomer of the ligand engages its own receptor without any discernible receptor-receptor interface. These unique observations provide a possible structural mechanism for inherent functional specialization encoded in chemokines despite their convergence to a common receptor. Furthermore, we also determined cryo-EM structures of CXCR3 in complex with G-protein-biased and {beta}-arrestin-biased small molecule agonists that elucidate distinct allosteric modulations in the receptor driving their divergent transducer-coupling bias. Guided by structural analysis and experimental validation, we discover that in contrast to previously held notion, small molecule agonists of CXCR3 display robust agonism at CXCR7, an intrinsically biased, {beta}-arrestin-coupled receptor, making them first-in-class dual agonists for chemokine receptors with exclusive {beta}arr-bias at CXCR7. Taken together, our study provides molecular insights into ligand promiscuity and signaling bias at the chemokine receptors, and also demonstrates a proof of principle that naturally encoded structural mimicry can be recapitulated using synthetic pharmacophores with potential implications for developing novel therapeutics.

biochemistry↗

bioRxiv2023

Pharmacological characterization of seven human histamine H3 receptor isoforms

The histamine H3 receptor (H3R) regulates as a presynaptic G protein-coupled receptor the release of histamine and other neurotransmitters in the brain, and is consequently a potential therapeutic target for neuronal disorders. The human H3R encodes for seven splice variants that vary in the length of intracellular loop 3 and/or the C-terminal tail but are all able to induce heterotrimeric Gi protein signaling. The last two decades H3R drug discovery and lead optimization has been exclusively focused on the 445 amino acids-long reference isoform H3R-445. In this study, we pharmacologically characterized for the first time all seven H3R isoforms by determining their binding affinities for reference histamine H3 receptor agonists and inverse agonists. The H3R-453, H3R-415, and H3R-413 isoforms display similar binding affinities for all ligands as the H3R-445. However, increased agonist binding affinities were observed for the three shorter isoforms H3R-329, H3R-365, and H3R-373, whereas inverse agonists such as the approved anti-narcolepsy drug pitolisant (Wakix(R)) displayed significantly decreased binding affinities for the latter two isoforms. This opposite change in binding affinity of agonist versus inverse agonists on H3R-365 and H3R-373 is associated with their higher constitutive activity in a cAMP biosensor assay as compared to the other 5 isoforms. The observed differences in pharmacology between longer and shorter H3R isoforms should be considered in future drug discovery programs.

pharmacology and toxicology↗

bioRxiv2023

Conformational dynamics underlying Atypical Chemokine Receptor 3 activation

Atypical Chemokine Receptor 3 (ACKR3) is a G protein-coupled receptor that does not signal through G proteins. It is known as a chemokine scavenger involved in various pathologies, making it an appealing yet intriguing therapeutic target. Indeed, the structural properties that govern ACKR3 functional selectivity and the overall conformational dynamics of ACKR3 activation are poorly understood. Here we combined Hydrogen/Deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics simulations to examine the binding mode and mechanism of action of various small-molecule ACKR3 ligands of different efficacy for {beta}-arrestin recruitment. Our results show that activation or inhibition of ACKR3 is largely governed by intracellular conformational changes of helix 6, intracellular loop 2 and helix 7, while the DRY motif becomes protected during both processes. Moreover, HDX-MS identifies the binding sites and the allosteric modulation of ACKR3 upon {beta}-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small-molecule ligands, the overall activation dynamics of ACKR3, the binding mode of {beta}-arrestin 1 and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.

biochemistry↗

bioRxiv2023

Pharmacological characterization and radiolabeling ofVUF15485, a high-affinity small-molecule agonist for theatypical chemokine receptor ACKR3

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this {beta}-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a BRET-based {beta}-arrestin2 recruitment assay VUF15485 acts as an ACKR3 agonist with high potency (pEC50 = 7.6) and shows a similar extent of receptor activation compared to CXCL12 when using a newly developed, FRET-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485 was tested against a (atypical) chemokine receptor panel (agonist and antagonist mode) and proves to be selective for ACKR3. VUF15485 was subsequently labeled with tritium at one of its methoxy groups affording [3H]VUF15485. The small-molecule agonist radioligand binds saturably and with high affinity to human ACKR3 (Kd = 8.2 nM). [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (RT < 2 min) for its binding to ACKR3. Displacement of [3H]VUF15485 binding to membranes of HEK293T cells, transiently expressing ACKR3, with a number of CXCR3, CXCR4 or ACKR3 small-molecule ligands confirmed the ACKR3 profile of the [3H]VUF15485 binding site. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose VUF15485 to bind in the major and the minor binding pocket of ACKR3.

pharmacology and toxicology↗

bioRxiv2022

Adhesion of Crithidia fasciculata promotes a rapid change in developmental fate driven by cAMP signaling

Kinetoplastids are single-celled parasites responsible for human and animal disease. For the vast majority of kinetoplastids, colonization of an insect host is required for transmission. Stable attachment to insect tissues via the single flagellum coincides with differentiation and morphological changes. Although this process is essential for the generation of infectious forms, the molecular mechanism driving differentiation following adherence is not well understood. To study this process, we elaborate upon an in vitro model in which flagellated swimming cells of the kinetoplastid Crithidia fasciculata rapidly differentiate following adhesion to artificial substrates. Manipulation of culture parameters revealed that growth phase and time had a strong influence on the proportion of adherent cells. Live imaging of cells transitioning from swimming to an attached cell fate show parasites undergoing a defined sequence of events including an initial adhesion near the base of the flagellum, immediately followed by flagellar shortening, cell rounding, and the formation of a hemidesmosome-like structure between the tip of the shortened flagellum and the substrate. We have also assayed the role of the cyclic AMP (cAMP) signaling pathway in differentiation of C. fasciculata. Pharmacological inhibition of cAMP phosphodiesterases eliminated the ability of swimming cells to attach without affecting their growth rate. Further, treatment with inhibitor did not affect the growth rate of established attached cells, indicating its effect is limited to a critical window of time during the early stages of adhesion. Finally, in swimming parasites we have shown that a receptor adenylate cyclase localizes to the distal portion of the flagellum. In attached cells it is absent from the shortened flagellum and instead localizes to the cell body. Similarly, a putative phosphodiesterase, found along the length of the flagellum, also relocalizes to the cell body in attached parasites. These data suggest that in C. fasciculata cAMP signaling is required for adherence, that cAMP flux in the flagellum of swimming cells is spatially restricted, and that signaling domains may be reorganized during differentiation and attachment. These studies contribute to our understanding of the flagellum as a multi-functional organelle integrating processes related to motility, signaling, attachment, and differentiation and further develop C. fasciculata as a model kinetoplastid. Author SummaryParasites from the order Kinetoplastida are transmitted by insects and cause diseases such as Leishmaniasis, Chagas disease, and Human African Trypanosomiasis. A key aspect of the life cycle of these parasites is their ability to adhere to surfaces within the insect and subsequently differentiate into forms that are transmissable to their next host. Here, we explore the molecular mechanisms underpinning both adherence and differentiation using the mosquito parasite Crithidia fasciculata. This organism is closely related to pathogenic species but grows to high densities in culture and adheres robustly in vitro. We identified factors affecting the rate of adherence and defined morphological stages of this process including rapid shortening of the cells single flagellum. Using electron microscopy, we confirmed that adhesion to artificial substrates results in the formation of a filament-rich adhesive plaque at the tip of the flagellum that resembles the attachment to insect tissue. We also demonstrate the key role played by the cyclic AMP signaling pathway during adherence and differentiation. Specifically, pharmacological inhibition of enzymes that degrade cyclic AMP completely blocks adherence, and tagging of several proteins in this pathway show that their localization along the flagellum changes following differentiation to the attached form. These data provide insights into processes critical for all kinetoplastid life cycles.

microbiology↗

bioRxiv2022

Genetically encoded sensors for measuring histamine release both in vitro and in vivo

Histamine (HA) is a key biogenic monoamine involved in a wide range of physiological and pathological processes in both the central nervous system and the periphery. Because the ability to directly measure extracellular HA in real-time will provide important insights into the functional role of HA in complex circuits under a variety of conditions, we developed a series of genetically encoded G protein-coupled receptor activation-based (GRAB) HA (GRABHA) sensors. These sensors produce a robust increase in fluorescence upon HA application, with good photostability, sub-second kinetics, nanomolar affinity, and high specificity. Using these GRABHA sensors, we measured electrical stimulation-evoked HA release in acute brain slices with high spatiotemporal resolution. Moreover, we recorded HA release in the preoptic area of the hypothalamus and in the medial prefrontal cortex during the sleep-wake cycle in freely moving mice, finding distinct patterns of HA release in these specific brain regions. Together, these in vitro and in vivo results show that our GRABHA sensors have high sensitivity and specificity for measuring extracellular HA, thus providing a robust new set of tools for examining the role of HA signaling in both health and disease.

neuroscience↗