Biology subjects

Chevigne, A.

Publications and source records attributed to Chevigne, A..

16 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

De novo macrocyclic peptide discovery produces potent class A GPCR ligands with diverse pharmacology

G protein-coupled receptors (GPCRs) represent one of the largest protein families of therapeutic importance, as over one third of all currently approved drugs target GPCRs. A growing proportion of GPCR drugs are peptides. However, class A GPCRs (over 80% of clinically studied targets) are underexploited as peptide targets, presumably since their only structured domain is the core, seven-helix, integral membrane fold presenting a limited solvent-exposed surface area. Screening libraries of de novo macrocyclic peptides have allowed for the targeting of otherwise intractable proteins, but their potential to generate selective ligands for class A GPCRs has not been demonstrated. Here, we used random nonstandard peptides integrated discovery (RaPID) to identify de novo macrocyclic peptide ligands targeting the chemokine receptor CXCR4. CXCR4 regulates cell migration, immune surveillance, and inflammation, making it an important therapeutic target for multiple diseases. We discovered novel, potent CXCR4-selective antagonists binding within the orthosteric pocket with nanomolar affinity. The most active peptides effectively inhibited receptor signaling, suppressed chemokine-driven cell migration, and blocked CXCR4-driven HIV entry. Our results show that even the structurally minimal class A GPCRs are viable targets for de novo cyclic peptide discovery, highlighting the potential for uncovering ligands towards other members of the GPCR superfamily.

pharmacology and toxicology↗

bioRxiv2026

Programming Targeted Immune Cell Migration Using Orthogonal Chemokine-Receptor Pairs

Chemokines orchestrate the migration of immune cells throughout the body, but harnessing chemokine signaling to engineer targeted cell trafficking is challenging because of their overlapping interactions and broad expression patterns. Here we used structure-guided protein design to engineer orthogonal chemokine-receptor pairs with minimal crosstalk to native systems. T cells engineered with an ortho-receptor selectively infiltrated tissues producing the ortho-chemokine, in vitro and in vivo. By engineering T cells that both sense and secrete the ortho-chemokine, we generated collective swarming behavior in which pioneer cells attract follower cells via an ortho-chemokine gradient self-organized by positive feedback. By providing orthogonal genetic control over both the generation and sensing of chemokine gradients, this system enables the construction of autonomous spatial targeting programs in engineered cells.

synthetic 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↗

bioRxiv2026

Atypical GPCR Activation Resolved by Nanobody Engineering

Abstract/ Summary ParagraphG protein-coupled receptors (GPCRs) are the largest family of clinically targeted proteins, yet most therapeutics target a narrow subset of structurally well-behaved receptors. The atypical chemokine receptor ACKR3 defies canonical models, displaying broad ligand recognition, high basal activity, and resistance to inhibition. Using engineered nanobodies, cryo-EM, NMR, and structure-guided pharmacology, we uncover an unconventional activation mechanism in ACKR3 that challenges established paradigms of GPCR activation. We find that receptor activity is controlled by changes in extracellular pocket volume rather than conformational rearrangements in conserved microswitches, and an expanded aromatic cluster at the intracellular transducer binding pocket stabilizes the active state. These findings redefine how GPCRs can be modulated and open new strategies for targeting pharmacologically intractable receptors.

biochemistry↗

bioRxiv2025

Atypical activation and molecular glue-like dimerization mechanism of an intrinsically-biased chemokine receptor

CXCR7, also known as atypical chemokine receptor 3 (ACKR3), is a naturally-biased, {beta}-arrestin-coupled seven transmembrane receptor (7TMR) that lacks productive coupling with heterotrimeric G-proteins. Despite a critical involvement in cancer metastasis, cardiovascular pathophysiology, and inflammatory disorders, the molecular basis of non-canonical activation and functional divergence of CXCR7 remains elusive. Here, we present a complete landscape of CXCR7 activation using a series of cryo-EM structures, and discover an atypical activation mechanism that is distinct from prototypical GPCRs. CXCR7 is maintained in a basal conformation by a unique tripartite ionic-lock involving TM5-TM6, in contrast to a broadly conserved TM3-TM6 ionic-lock in GPCRs, which is disrupted upon receptor activation. Importantly, activation of CXCR7 results in a constricted pocket and distinct surface topology on the intracellular side compared to prototypical GPCRs. Serendipitously, we capture novel dimeric arrangements of CXCR7 with an inter-protomer stitching by a native phospholipid serving as a molecular glue, and identify previously unanticipated extrahelical allosteric sites on the receptor. Surprisingly, in an intermediate state structure of CXCR7, the second extracellular loop (ECL2) displays a self-blocking conformation, in stark contrast to ECL2-mediated self-activating mechanism reported recently for some orphan GPCRs. Finally, we unequivocally establish CXCR7 as an atypical opioid receptor via a large peptide library screening and structure elucidation in complex with distinct opioid peptides imparting full receptor activation. In summary, our study elucidates an atypical mechanism of CXCR7 activation, and establishes it as an alternative, non-canonical opioid receptor target with potential for novel pain therapeutics.

biochemistry↗

bioRxiv2025

GPR15LG binds CXCR4 and synergistically modulates CXCL12-induced cell signaling and migration

GPR15LG, a chemokine-like ligand for the G-protein coupled receptor 15 (GPR15), is abundantly expressed in the gastrointestinal mucosa and inflamed skin. Emerging evidence suggests its involvement in inflammatory disorders and cancers. This study investigates the effects of GPR15LG on the signaling and downstream functions of C-X-C chemokine receptor type 4 (CXCR4), which plays a critical role in immune cell trafficking and cancer metastasis. The results demonstrate that GPR15LG binds to the orthosteric site of CXCR4, modulating downstream signaling in a context-dependent manner. Specifically, GPR15LG enhances CXCL12-mediated CXCR4 signaling synergistically, promoting wound healing and cell migration across various cell types, including CD4+ T cells and cancer cells. These findings underscore the role of GPR15LG in inflammation and metastasis, offering potential therapeutic avenues for CXCR4-mediated diseases. TeaserGPR15LG binds CXCR4 thereby modulating CXCL12/CXCR4 signaling and immune and cancer cell trafficking.

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

Physical interaction with Ephrin B1 promotes CXCR4 intracellular localization and oncogenic potential

Chemokine receptor 4 (CXCR4) is a member of the chemokine receptor exclusively activated by the chemokine CXCL12. While CXCR4 regulates numerous physiological processes associated with cell migration and embryogenesis, its overexpression has been involved in various cancer types. Studies suggest that intracellular CXCR4 expression rather than CXCR4-operated signaling underlies its pro-tumorigenic functions. Given the role of GPCR interacting proteins in their trafficking and subcellular localization, we characterized the CXCR4 interactome using an affinity purification coupled to mass spectrometry (AP-MS) strategy. The most abundant protein identified in the CXCR4 interactome is Ephrin B1, a member of the Ephrin protein family that shares several functions with CXCR4, such as the regulation of cell migration and proliferation. Further studies showed that interaction between CXCR4 and Ephrin B1 is direct and enhanced by treating cell with CXCL12. They also indicated that Ephrin B1 prevents CXCR4 N-glycosylation, decreases CXCR4 cell surface expression and consistently inhibits CXCL12-induced CXCR4 coupling to Gi1-3 and the recruitment of {beta}-arrestins 1 and 2. Conversely, Ephrin B1 signals to Erk1,2 through CXCR4 activation and mediates the decrease in Death Receptor 5 expression elicited by intracellular CXCR4. Collectively, these findings identify Ephrin B1 as a key mediator of CXCR4 tumorigenic signaling.

biochemistry↗

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

Targeting the Atypical Chemokine Receptor 2 (ACKR2) improves the benefit of anti-PD-1 immunotherapy in melanoma

Immune checkpoint blockade (ICB) therapies, targeting PD-1 or PD-L1, have transformed cancer treatment, particularly for aggressive cancers. However, many patients fail to benefit from ICBs due to tumor characteristics, including a non-inflammatory tumor microenvironment (TME) that impedes immune cell infiltration. This study investigated the potential of targeting the Atypical Chemokine Receptor 2 (ACKR2), known for scavenging CXCR3-related chemokines crucial for lymphocyte recruitment to tumors. Genetic targeting of ACKR2 in melanoma cells increased the release of essential chemokines associated with the inflamed TME. In mouse models, ACKR2 inhibition suppressed tumor growth, improved survival, and enhanced activated immune cell infiltration into the TME. Moreover, ACKR2 targeting synergized with anti-PD-1 therapy, overcoming resistance to anti-PD-1 and improving its efficacy. Analysis of melanoma patient data from The Cancer Genome Atlas (TCGA) revealed that patients with high levels of chemokines scavenged by ACKR2 had significantly better survival rates, with increased expression of NK cell and CD8 T cell markers indicating their presence in the TME. Notably, even in patients with high CD8 expression, those expressing low ACKR2 survived better than those expressing high ACKR2. This study emphasizes the clinical importance of targeting ACKR2 as an attractive strategy for the development of combination immunotherapies to treat cold tumors, which are clinically stratified to not be eligible for ICB-based therapy.

cancer biology↗

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 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↗

bioRxiv2021

Computationally designed GPCR quaternary structures bias signaling pathway activation

Communication across membranes controls critical cellular processes and is achieved by receptors translating extracellular signals into selective cytoplasmic responses. While receptor tertiary structures can now be readily characterized, receptor associations into quaternary structures are very challenging to study and their implications in signal transduction remain poorly understood. Here, we report a computational approach for predicting membrane receptor self-associations, and designing receptor oligomers with various quaternary structures and signaling properties. Using this approach, we designed chemokine receptor CXCR4 dimers with reprogrammed stabilities, conformations, and abilities to activate distinct intracellular signaling proteins. In agreement with our predictions, the designed CXCR4s dimerized through distinct conformations and displayed different quaternary structural changes upon activation. Consistent with the active state models, all engineered CXCR4 oligomers activated the G protein Gi, but only a few specific dimer structures also recruited {beta}-arrestins. Overall, we demonstrate that quaternary structures represent an important unforeseen mechanism of receptor biased signaling and reveal the existence of a conformational switch at the dimer interface of several G protein-coupled receptors including CXCR4, mu-Opioid and type-2 Vasopressin receptors that selectively control the activation of G proteins vs {beta}-arrestin-mediated pathways. The approach should prove useful for predicting and designing receptor associations to uncover and reprogram selective cellular signaling functions.

biophysics↗

bioRxiv2020

CXCR4-binding PET tracers link monocyte recruitment and endothelial injury in murine atherosclerosis

Viral macrophage inflammatory protein 2 (vMIP-II/vCCL2) binds to multiple chemokine receptors, and vMIP-II based PET tracer (64Cu-DOTA-vMIP-II: vMIP-II tracer) accumulates at atherosclerotic lesions in mice. The magnitude of 64Cu-DOTA-vMIP-II accumulation correlated with monocyte recruitment, as Apoe-/- mice treated with AAV-mApoE showed PET signal declining as monocyte recruitment subsided. Unexpectedly, monocytes themselves were not the major target of the 64Cu-DOTA-vMIP-II tracer. Using fluorescence-tagged vMIP-II tracer, competitive receptor blocking with CXCR4 antagonists, CXCR4-specific tracer 64Cu-DOTA-FC131, or CXCR4 staining during disease progression and regression, endothelial cell expression of CXCR4 proved to be the main target of 64Cu-DOTA-vMIP-II imaging. Expression of CXCR4 was low in non-plaque areas, but strongly detected on endothelium at the edges of progressing plaques, corresponding to a population of proliferating endothelium and to the location in plaques where monocyte recruitment occurred. Thus, endothelial injury status of plaques is marked by CXCR4 expression and that this injury correlates with the tendency of such plaques to recruit monocytes. Furthermore, our findings suggest PET tracers that, through binding CXCR4, may be useful to monitor plaque injury status.

pathology↗