Anne Steen

Biased and g protein-independent signaling of chemokine receptors.

Biased signaling or functional selectivity occurs when a 7TM-receptor preferentially activates one of several available pathways. It can be divided into three distinct forms: ligand bias, receptor bias, and tissue or cell bias, where it is mediated by different ligands (on the same receptor), different receptors (with the same ligand), or different tissues or cells (for the same ligand–receptor pair). Most often biased signaling is differentiated into G protein-dependent and β-arrestin-dependent signaling. Yet, it may also cover signaling differences within these groups. Moreover, it may not be absolute, i.e., full versus no activation. Here we discuss biased signaling in the chemokine system, including the structural basis for biased signaling in chemokine receptors, as well as in class A 7TM receptors in general. This includes overall helical movements and the contributions of micro-switches based on recently published 7TM crystals and molecular dynamics studies. All three forms of biased signaling are abundant in the chemokine system. This challenges our understanding of “classic” redundancy inevitably ascribed to this system, where multiple chemokines bind to the same receptor and where a single chemokine may bind to several receptors – in both cases with the same functional outcome. The ubiquitous biased signaling confers a hitherto unknown specificity to the chemokine system with a complex interaction pattern that is better described as promiscuous with context-defined roles and different functional outcomes in a ligand-, receptor-, or cell/tissue-defined manner. As the low number of successful drug development plans implies, there are great difficulties in targeting chemokine receptors; in particular with regard to receptor antagonists as anti-inflammatory drugs. Un-defined and putative non-selective targeting of the complete cellular signaling system could be the underlying cause of lack of success. Therefore, biased ligands could be the solution.

Biased and Constitutive Signaling in the CC-chemokine Receptor CCR5 by Manipulating the Interface between Transmembrane Helices 6 and 7

Background: Information about the structure-function relationship and activation mechanism of 7TM receptors is needed. Results: Single mutations in CCR5 induce biased signaling with increased activation through Gαi but decreased β-arrestin recruitment. Conclusion: The TM6/7 interface controls the G protein-dependent and -independent activity state of CCR5. Significance: Knowledge about specific 7TM receptor regions targeted by pathway-selective (biased) ligands is vital for future drug design. The equilibrium state of CCR5 is manipulated here toward either activation or inactivation by introduction of single amino acid substitutions in the transmembrane domains (TMs) 6 and 7. Insertion of a steric hindrance mutation in the center of TM7 (G286F in position VII:09/7.42) resulted in biased signaling. Thus, β-arrestin recruitment was eliminated, whereas constitutive activity was observed in Gαi-mediated signaling. Furthermore, the CCR5 antagonist aplaviroc was converted to a full agonist (a so-called efficacy switch). Computational modeling revealed that the position of the 7TM receptor-conserved Trp in TM6 (Trp-248 in position VI:13/6.48, part of the CWXP motif) was influenced by the G286F mutation, causing Trp-248 to change orientation away from TM7. The essential role of Trp-248 in CCR5 activation was supported by complete inactivity of W248A-CCR5 despite maintaining chemokine binding. Furthermore, replacing Trp-248 with a smaller aromatic amino acid (Tyr/Phe) impaired the β-arrestin recruitment, yet with maintained G protein activity (biased signaling); also, here aplaviroc switched to a full agonist. Thus, the altered positioning of Trp-248, induced by G286F, led to a constraint of G protein active, but β-arrestin inactive and thus biased, CCR5 conformation. These results provide important information on the molecular interplay and impact of TM6 and TM7 for CCR5 activity, which may be extrapolated to other chemokine receptors and possibly to other 7TM receptors.

Allosteric and Orthosteric Sites in CC Chemokine Receptor (CCR5), a Chimeric Receptor Approach

Background: Characterization of 7TM biology and chemistry is needed generally and within chemokine receptors. Results: A CCR5-CCR2 receptor chimera was constructed by transferring all extracellular regions of CCR2 to CCR5. CCR2 chemokine binding was maintained and so was small molecule CCR5 agonists and antagonists. Conclusion: Orthosteric and allosteric sites could be structurally separated and still act together. Significance: New basic knowledge to be used in drug development. Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2-like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units.

Determination of the binding mode for the cyclopentapeptide CXCR4 antagonist FC131 using a dual approach of ligand modifications and receptor mutagenesis.

The cyclopentapeptide FC131 (cyclo(‐L‐Arg1‐L‐Arg2‐L‐2‐Nal3‐Gly4‐D‐Tyr5‐)) is an antagonist at the CXC chemokine receptor CXCR4, which plays a role in human immunodeficiency virus infection, cancer and stem cell recruitment. Binding modes for FC131 in CXCR4 have previously been suggested based on molecular docking guided by structure–activity relationship (SAR) data; however, none of these have been verified by in vitro experiments.

Reversed binding of a small molecule ligand in homologous chemokine receptors – differential role of extracellular loop 2

BACKGROUND AND PURPOSE The majority of small molecule compounds targeting chemokine receptors share a similar pharmacophore with a centrally located aliphatic positive charge and flanking aromatic moieties. Here we describe a novel piperidine‐based compound with structural similarity to previously described CCR8‐specific agonists, but containing a unique phenyl‐tetrazol moiety which, in addition to activity at CCR8 was also active at CCR1.

Differential CCR7 Targeting in Dendritic Cells by Three Naturally Occurring CC-Chemokines

The CCR7 ligands CCL19 and CCL21 are increasingly recognized as functionally different (biased). Using mature human dendritic cells (DCs), we show that CCL19 is more potent than CCL21 in inducing 3D chemotaxis. Intriguingly, CCL21 induces prolonged and more efficient ERK1/2 activation compared with CCL19 and a C-terminal truncated (tailless) CCL21 in DCs. In contrast, tailless-CCL21 displays increased potency in DC chemotaxis compared with native CCL21. Using a CCL21-specific antibody, we show that CCL21, but not tailless-CCL21, accumulates at the cell surface. In addition, removal of sialic acid from the cell surface by neuraminidase treatment impairs ERK1/2 activation by CCL21, but not by CCL19 or tailless-CCL21. Using standard laboratory cell lines, we observe low potency of both CCL21 and tailless-CCL21 in G protein activation and β-arrestin recruitment compared with CCL19, indicating that the tail itself does not improve receptor interaction. Chemokines interact with their receptors in a stepwise manner with ultimate docking of their N-terminus into the main binding pocket. Employing site-directed mutagenesis we identify residues in this pocket of selective CCL21 importance. We also identify a molecular switch in the top of TM7 important for keeping CCR7 in an inactive conformation (Tyr312), as introduction of the chemokine receptor-conserved Glu (or Ala) induces high constitutive activity. Summarized, we show that the interaction of the tail of CCL21 with polysialic acid is needed for strong ERK signaling, whereas it impairs CCL21-mediated chemotaxis and has no impact on receptor docking consistent with the current model of chemokine:receptor interaction. This indicates that future selective pharmacological targeting of CCL19 versus CCL21 should focus on a differential targeting of the main receptor pocket, while selective targeting of tailless-CCL21 versus CCL21 and CCL19 requires targeting of the glycosaminoglycan (GAG) interaction.

Gating function of isoleucine-116 in TM-3 (position III:16/3.40) for the activity state of the CC-chemokine receptor 5 (CCR5).

A conserved amino acid within a protein family indicates a significance of the residue. In the centre of transmembrane helix (TM)‐5, position V:13/5.47, an aromatic amino acid is conserved among class A 7TM receptors. However, in 37% of chemokine receptors – a subgroup of 7TM receptors – it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM‐3 for CCR5 activity.

Corrigendum: Differential CCR7 Targeting in Dendritic Cells by Three Naturally Occurring CC-Chemokines

[This corrects the article on p. 568 in vol. 7, PMID: 28018341.].

Molecular Pharmacology of CXCR4 Inhibition

In recent years, the chemokine receptor CXCR4 has been shown to be implemented in the mobilization of progenitor cells from the bone marrow. This finding has prompted a search for CXCR4 antagonists acting as stem cell mobilizing agents. In accordance, it is important to look into the molecular pharmacology of well-known CXCR4 antagonists in order to augment the potency and affinity and to increase the specificity of future CXCR4-targeting compounds. In this chapter, binding modes of CXCR4 antagonists that have been shown to mobilize stem cells are discussed. In addition, comparisons between results obtained from structure–function studies and findings from newly released crystal structures are drawn.

Inhibition of HIV Fusion by Small Molecule Agonists through Efficacy-Engineering of CXCR4

CXC chemokine receptor 4 (CXCR4) is involved in multiple physiological and pathological processes, notably as a coreceptor for human immunodeficiency virus (HIV) cell entry. Its broad expression pattern and vital biological importance make CXCR4 a troublesome drug target, as disruption of the interaction with its endogenous ligand, CXC chemokine ligand 12 (CXCL12), has severe consequences. In fact, only one CXCR4 drug, the bicyclam antagonist and HIV entry inhibitor AMD3100 (Plerixafor/Mozobil), has been approved for clinical use, however only for stem cell mobilization-a consequence of CXCR4 antagonism. Here, we report the engineering of an efficacy switch mutation in CXCR4-F292A7.43 in the middle of transmembrane helix 7-that converted the antagonists AMD3100 and AMD11070 into partial agonists. As agonists on F292A CXCR4, AMD3100 and AMD11070 were less disruptive to CXCR4 signaling while they remained efficient inhibitors of HIV fusion. This demonstrates that small molecule CXCR4 agonists can have a therapeutic potential as HIV entry inhibitors.