Small Molecule Fluorescent Ligands for the Atypical Chemokine Receptor 3 (ACKR3).

The atypical chemokine receptor 3 (ACKR3) is a receptor that induces cancer progression and metastasis in multiple cell types. Therefore, new chemical tools are required to study the role of ACKR3 in cancer and other diseases. In this study, fluorescent probes, based on a series of small molecule ACKR3 agonists, were synthesized. Three fluorescent probes, which showed specific binding to ACKR3 through a luminescence-based NanoBRET binding assay (pKd ranging from 6.8 to 7.8) are disclosed. Due to their high affinity at the ACKR3, we have shown their application in both competition binding experiments and confocal microscopy studies showing the cellular distribution of this receptor.

Spermine and spermidine bind CXCR4 and inhibit CXCR4- but not CCR5-tropic HIV-1 infection.

Semen is an important vector for sexual HIV-1 transmission. Although CXCR4-tropic (X4) HIV-1 may be present in semen, almost exclusively CCR5-tropic (R5) HIV-1 causes systemic infection after sexual intercourse. To identify factors that may limit sexual X4-HIV-1 transmission, we generated a seminal fluid-derived compound library and screened it for antiviral agents. We identified four adjacent fractions that blocked X4-HIV-1 but not R5-HIV-1 and found that they all contained spermine and spermidine, abundant polyamines in semen. We showed that spermine, which is present in semen at concentrations up to 14 mM, binds CXCR4 and selectively inhibits cell-free and cell-associated X4-HIV-1 infection of cell lines and primary target cells at micromolar concentrations. Our findings suggest that seminal spermine restricts sexual X4-HIV-1 transmission.

Small-Molecule Fluorescent Ligands for the CXCR4 Chemokine Receptor.

The C-X-C chemokine receptor type 4, or CXCR4, is a chemokine receptor found to promote cancer progression and metastasis of various cancer cell types. To investigate the pharmacology of this receptor, and to further elucidate its role in cancer, novel chemical tools are a necessity. In the present study, using classic medicinal chemistry approaches, small-molecule-based fluorescent probes were designed and synthesized based on previously reported small-molecule antagonists. Here, we report the development of three distinct chemical classes of fluorescent probes that show specific binding to the CXCR4 receptor in a novel fluorescence-based NanoBRET binding assay (pKD ranging 6.6-7.1). Due to their retained affinity at CXCR4, we furthermore report their use in competition binding experiments and confocal microscopy to investigate the pharmacology and cellular distribution of this receptor.

CXCR4 as a novel target in immunology: moving away from typical antagonists.

CXCR4 has been a target of interest in drug discovery for numerous years. However, so far, most if not all studies focused on finding antagonists of CXCR4 function. Recent studies demonstrate that targeting a minor allosteric pocket of CXCR4 induces an immunomodulating effect in immune cells expressing CXCR4, connected to the TLR pathway. Compounds binding in this minor pocket seem to be functionally selective with inverse agonistic properties in selected GPCR signaling pathways (Gi activation), but additional signaling pathways are likely to be involved in the immunomodulating effects. In depth research into these CXCR4-targeted immunomodulators could lead to novel treatment options for (auto)-immune diseases.

A nuclear export sequence promotes CRM1-dependent targeting of the nucleoporin Nup214 to the nuclear pore complex.

Nup214 is a major nucleoporin on the cytoplasmic side of the nuclear pore complex with roles in late steps of nuclear protein and mRNA export. It interacts with the nuclear export receptor CRM1 (also known as XPO1) via characteristic phenylalanine-glycine (FG) repeats in its C-terminal region. Here, we identify a classic nuclear export sequence (NES) in Nup214 that mediates Ran-dependent binding to CRM1. Nup214 versions with mutations in the NES, as well as wild-type Nup214 in the presence of the selective CRM1 inhibitor leptomycin B, accumulate in the nucleus of Nup214-overexpressing cells. Furthermore, physiological binding partners of Nup214, such as Nup62 and Nup88, are recruited to the nucleus together with Nup214. Nuclear export of mutant Nup214 can be rescued by artificial nuclear export sequences at the C-terminal end of Nup214, leading also to a correct localization of Nup88. Our results suggest a function of the Nup214 NES in the biogenesis of the nuclear pore complex and/or in terminal steps of CRM1-dependent protein export.

Kinetic Analysis of the Early Signaling Steps of the Human Chemokine Receptor CXCR4.

G protein-coupled receptors (GPCRs) are biologic switches that transduce extracellular stimuli into intracellular responses in the cell. Temporally resolving GPCR transduction pathways is key to understanding how cell signaling occurs. Here, we investigate the kinetics and dynamics of the activation and early signaling steps of the CXC chemokine receptor (CXCR) 4 in response to its natural ligands CXC chemokine ligand (CXCL) 12 and macrophage migration inhibitory factor (MIF), using Förster resonance energy transfer-based approaches. We show that CXCR4 presents a multifaceted response to CXCL12, with receptor activation (≈0.6 seconds) followed by a rearrangement in the receptor/G protein complex (≈1 seconds), a slower dimer rearrangement (≈1.7 seconds), and prolonged G protein activation (≈4 seconds). In comparison, MIF distinctly modulates every step of the transduction pathway, indicating distinct activation mechanisms and reflecting the different pharmacological properties of these two ligands. Our study also indicates that CXCR4 exhibits some degree of ligand-independent activity, a relevant feature for drug development. SIGNIFICANCE STATEMENT: The CXC chemokine ligand (CXCL) 12/CXC chemokine receptor (CXCR) 4 axis represents a well-established therapeutic target for cancer treatment. We demonstrate that CXCR4 exhibits a multifaceted response that involves dynamic receptor dimer rearrangements and that is kinetically embedded between receptor-G protein complex rearrangements and G protein activation. The alternative endogenous ligand macrophage migration inhibitory factor behaves opposite to CXCL12 in each assay studied and does not lead to G protein activation. This detailed understanding of the receptor activation may aid in the development of more specific drugs against this target.

CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes.

G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of ß-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels.

Modulators of CXCR4 and CXCR7/ACKR3 Function.

The two G protein-coupled receptors (GPCRs) C-X-C chemokine receptor type 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are part of the class A chemokine GPCR family and represent important drug targets for human immunodeficiency virus (HIV) infection, cancer, and inflammation diseases. CXCR4 is one of only three chemokine receptors with a US Food and Drug Administration approved therapeutic agent, the small-molecule modulator AMD3100. In this review, known modulators of the two receptors are discussed in detail. Initially, the structural relationship between receptors and ligands is reviewed on the basis of common structural motifs and available crystal structures. To date, no atypical chemokine receptor has been crystallized, which makes ligand design and predictions for these receptors more difficult. Next, the selectivity, receptor activation, and the resulting ligand-induced signaling output of chemokines and other peptide ligands are reviewed. Binding of pepducins, a class of lipid-peptides whose basis is the internal loop of a GPCR, to CXCR4 is also discussed. Finally, small-molecule modulators of CXCR4 and ACKR3 are reviewed. These modulators have led to the development of radio- and fluorescently labeled tool compounds, enabling the visualization of ligand binding and receptor characterization both in vitro and in vivo. SIGNIFICANCE STATEMENT: To investigate the pharmacological modulation of CXCR4 and ACKR3, significant effort has been focused on the discovery and development of a range of ligands, including small-molecule modulators, pepducins, and synthetic peptides. Imaging tools, such as fluorescent probes, also play a pivotal role in the field of drug discovery. This review aims to provide an overview of the aforementioned modulators that facilitate the study of CXCR4 and ACKR3 receptors.

Context-Dependent Signaling of CXC Chemokine Receptor 4 and Atypical Chemokine Receptor 3.

G protein-coupled receptors (GPCRs) are regulated by complex molecular mechanisms, both in physiologic and pathologic conditions, and their signaling can be intricate. Many factors influence their signaling behavior, including the type of ligand that activates the GPCR, the presence of interacting partners, the kinetics involved, or their location. The two CXC-type chemokine receptors, CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3), both members of the GPCR superfamily, are important and established therapeutic targets in relation to cancer, human immunodeficiency virus infection, and inflammatory diseases. Therefore, it is crucial to understand how the signaling of these receptors works to be able to specifically target them. In this review, we discuss how the signaling pathways activated by CXCR4 and ACKR3 can vary in different situations. G protein signaling of CXCR4 depends on the cellular context, and discrepancies exist depending on the cell lines used. ACKR3, as an atypical chemokine receptor, is generally reported to not activate G proteins but can broaden its signaling spectrum upon heteromerization with other receptors, such as CXCR4, endothelial growth factor receptor, or the α 1-adrenergic receptor (α 1-AR). Also, CXCR4 forms heteromers with CC chemokine receptor (CCR) 2, CCR5, the Na+/H+ exchanger regulatory factor 1, CXCR3, α 1-AR, and the opioid receptors, which results in differential signaling from that of the monomeric subunits. In addition, CXCR4 is present on membrane rafts but can go into the nucleus during cancer progression, probably acquiring different signaling properties. In this review, we also provide an overview of the currently known critical amino acids involved in CXCR4 and ACKR3 signaling.

CXCR4/ACKR3 Phosphorylation and Recruitment of Interacting Proteins: Key Mechanisms Regulating Their Functional Status.

The C-X-C motif chemokine receptor type 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3/CXCR7) are class A G protein-coupled receptors (GPCRs). Accumulating evidence indicates that GPCR subcellular localization, trafficking, transduction properties, and ultimately their pathophysiological functions are regulated by both interacting proteins and post-translational modifications. This has encouraged the development of novel techniques to characterize the GPCR interactome and to identify residues subjected to post-translational modifications, with a special focus on phosphorylation. This review first describes state-of-the-art methods for the identification of GPCR-interacting proteins and GPCR phosphorylated sites. In addition, we provide an overview of the current knowledge of CXCR4 and ACKR3 post-translational modifications and an exhaustive list of previously identified CXCR4- or ACKR3-interacting proteins. We then describe studies highlighting the importance of the reciprocal influence of CXCR4/ACKR3 interactomes and phosphorylation states. We also discuss their impact on the functional status of each receptor. These studies suggest that deeper knowledge of the CXCR4/ACKR3 interactomes along with their phosphorylation and ubiquitination status would shed new light on their regulation and pathophysiological functions.

Modulation of the Allosteric Communication between the Polo-Box Domain and the Catalytic Domain in Plk1 by Small Compounds.

The Polo-like kinases (Plks) are an evolutionary conserved family of Ser/Thr protein kinases that possess, in addition to the classical kinase domain at the N-terminus, a C-terminal polo-box domain (PBD) that binds to phosphorylated proteins and modulates the kinase activity and its localization. Plk1, which regulates the formation of the mitotic spindle, has emerged as a validated drug target for the treatment of cancer, because it is required for numerous types of cancer cells but not for the cell division in noncancer cells. Here, we employed chemical biology methods to investigate the allosteric communication between the PBD and the catalytic domain of Plk1. We identified small compounds that bind to the catalytic domain and inhibit or enhance the interaction of Plk1 with the phosphorylated peptide PoloBoxtide in vitro. In cells, two new allosteric Plk1 inhibitors affected the proliferation of cancer cells in culture and the cell cycle but had distinct phenotypic effects on spindle formation. Both compounds inhibited Plk1 signaling, indicating that they specifically act on Plk1 in cultured cells.