Mutational analysis of the extracellular disulphide bridges of the atypical chemokine receptor ACKR3/CXCR7 uncovers multiple binding and activation modes for its chemokine and endogenous non-chemokine agonists.

The atypical chemokine receptor ACKR3/CXCR7 plays crucial roles in numerous physiological processes but also in viral infection and cancer. ACKR3 shows strong propensity for activation and, unlike classical chemokine receptors, can respond to chemokines from both the CXC and CC families as well as to the endogenous peptides BAM22 and adrenomedullin. Moreover, despite belonging to the G protein coupled receptor family, its function appears to be mainly dependent on ß-arrestin. ACKR3 has also been shown to continuously cycle between the plasma membrane and the endosomal compartments, suggesting a possible role as a scavenging receptor. So far, the molecular basis accounting for these atypical binding and signalling properties remains elusive. Noteworthy, ACKR3 extracellular domains bear three disulphide bridges. Two of them lie on top of the two main binding subpockets and are conserved among chemokine receptors, and one, specific to ACKR3, forms an intra-N terminus four-residue-loop of so far unknown function. Here, by mutational and functional studies, we examined the impact of the different disulphide bridges for ACKR3 folding, ligand binding and activation. We showed that, in contrast to most classical chemokine receptors, none of the extracellular disulphide bridges was essential for ACKR3 function. However, the disruption of the unique ACKR3 N-terminal loop drastically reduced the binding of CC chemokines whereas it only had a mild impact on CXC chemokine binding. Mutagenesis also uncovered that chemokine and endogenous non-chemokine ligands interact and activate ACKR3 according to distinct binding modes characterized by different transmembrane domain subpocket occupancy and N-terminal loop contribution, with BAM22 mimicking the binding mode of CC chemokine N terminus.

Development of Mimokines, chemokine N terminus-based CXCR4 inhibitors optimized by phage display and rational design.

The chemokine receptor CXCR4 (C-X-C chemokine receptor type 4 also known as fusin or CD184 (cluster of differentiation 184)) is implicated in various biological and pathological processes of the hematopoietic and immune systems. CXCR4 is also one of the major coreceptors for HIV-1 entry into target cells and is overexpressed in many cancers, supporting cell survival, proliferation, and migration. CXCR4 is thus an extremely relevant drug target. Among the different strategies to block CXCR4, chemokine-derived peptide inhibitors hold great therapeutic potential. In this study, we used the N-terminus of vCCL2/vMIPII, a viral CXCR4 antagonist chemokine, as a scaffold motif to engineer and select CXCR4 peptide inhibitors, called Mimokines, which imitate the chemokine-binding mode but display an enhanced receptor affinity, antiviral properties, and receptor selectivity. We first engineered a Mimokine phage displayed library based on the first 21 residues of vCCL2, in which cysteine 11 and 12 were fully randomized and screened it against purified CXCR4 stabilized in liposomes. We identified Mimokines displaying up to 4-fold higher affinity for CXCR4 when compared to the reference peptide and fully protected MT-4 cells against HIV-1 infection. These selected Mimokines were then subjected to dimerization, D-amino acid, and aza-ß3-amino acid substitution to further enhance their potency and selectivity. Optimized Mimokines exhibited up to 120-fold enhanced CXCR4 binding (range of 20 nM) and more than 200-fold improved antiviral properties (≤ 1 µM) compared to the parental Mimokines. Interestingly, these optimized Mimokines also showed up to 25-fold weaker affinity for ACKR3/CXCR7 and may therefore serve as lead compounds for further development of more selective CXCR4 peptide inhibitors and probes.

Different contributions of chemokine N-terminal features attest to a different ligand binding mode and a bias towards activation of ACKR3/CXCR7 compared with CXCR4 and CXCR3.

BACKGROUND AND PURPOSE: Chemokines and their receptors form an intricate interaction and signalling network that plays critical roles in various physiological and pathological cellular processes. The high promiscuity and apparent redundancy of this network makes probing individual chemokine/receptor interactions and functional effects, as well as targeting individual receptor axes for therapeutic applications, challenging. Despite poor sequence identity, the N-terminal regions of chemokines, which play a key role in their activity and selectivity, contain several conserved features. Thus far little is known regarding the molecular basis of their interactions with typical and atypical chemokine receptors or the conservation of their contributions across chemokine-receptor pairs. EXPERIMENTAL APPROACH: We used a broad panel of chemokine variants and modified peptides derived from the N-terminal region of chemokines CXCL12, CXCL11 and vCCL2, to compare the contributions of various features to binding and activation of their shared receptors, the two typical, canonical G protein-signalling receptors, CXCR4 and CXCR3, as well as the atypical scavenger receptor CXCR7/ACKR3, which shows exclusively arrestin-dependent activity. KEY RESULTS: We provide molecular insights into the plasticity of the ligand-binding pockets of these receptors, their chemokine binding modes and their activation mechanisms. Although the chemokine N-terminal region is a critical determinant, neither the most proximal residues nor the N-loop are essential for binding and activation of ACKR3, as distinct from binding and activation of CXCR4 and CXCR3. CONCLUSION AND IMPLICATIONS: These results suggest a different interaction mechanism between this atypical receptor and its ligands and illustrate its strong propensity to activation.

Human herpesvirus 8-encoded chemokine vCCL2/vMIP-II is an agonist of the atypical chemokine receptor ACKR3/CXCR7.

The atypical chemokine receptor CXCR7/ACKR3 binds two endogenous chemokines, CXCL12 and CXCL11, and is upregulated in many cancers or following infection by several cancer-inducing viruses, including HHV-8. ACKR3 is a ligand-scavenging receptor and does not activate the canonical G protein pathways but was proposed to trigger ß-arrestin-dependent signaling. Here, we identified the human herpesvirus 8-encoded CC chemokine vCCL2/vMIP-II as a third high-affinity ligand for ACKR3. vCCL2 acted as partial ACKR3 agonist, inducing ß-arrestin recruitment to the receptor, subsequent reduction of its surface levels and its delivery to endosomes. In addition, ACKR3 reduced vCCL2-triggered MAP kinase and PI3K/Akt signaling through other chemokine receptors. Our data suggest that ACKR3 acts as a scavenger receptor for vCCL2, regulating its availability and activity toward human receptors, thereby likely controlling its function in HHV-8 infection. Our study provides new insights into the complex crosstalk between viral chemokines and host receptors as well as into the biology of ACKR3, this atypical and still enigmatic receptor.

Isolation of an HIV-1 neutralizing peptide mimicking the CXCR4 and CCR5 surface from the heavy-chain complementary determining region 3 repertoire of a viremic controller.

OBJECTIVES: The recent identification of neutralizing antibodies able to prevent viral rebound reemphasized the interest in humoral immune responses to control HIV-1 infection. In this study, we characterized HIV-1-inhibiting sequences from heavy-chain complementary determining region 3 (HCDR3) repertoires of a viremic controller. DESIGN AND METHODS: IgM and IgG-derived HCDR3 repertoires of a viremic controller presenting plasma-neutralizing activity and characterized by over 20 years of infection with a stable CD4 T-cell count were displayed on filamentous phage to identify HCDR3 repertoire-derived peptides inhibiting HIV-1 entry. RESULTS: Screening of phage libraries against recombinant gp120 led to the identification of an HCDR3-derived peptide sequence (LRTV-1) displaying antiviral properties against both X4 and R5 viruses. The interaction of LRTV-1 with gp120 was enhanced upon CD4 binding and sequence comparison revealed homology between LRTV-1 and the second extracellular loop of C-X-C chemokine receptor type 4 (CXCR4) (11/23) and the N-terminus of C-C chemokine receptor type 5 (CCR5) (7/23). Alanine scanning experiments identified different clusters of residues critical for interaction with the viral envelope protein. CONCLUSIONS: LRTV-1 peptide is to date the smallest human HCDR3 repertoire-derived peptide identified by phage display inhibiting HIV entry of R5 and X4 viruses. This peptide recognizes a CD4-dependent gp120 epitope critical for coreceptor binding and mimics the surface of CXCR4 and CCR5. Our data emphasize the potential of human HCDR3 immune repertoires as sources of small biologically active peptides for HIV cure.

Neutralising properties of peptides derived from CXCR4 extracellular loops towards CXCL12 binding and HIV-1 infection.

The chemokine receptor CXCR4 interacts with a single endogenous chemokine, CXCL12, and regulates a wide variety of physiological and pathological processes including inflammation and metastasis development. CXCR4 also binds the HIV-1 envelope glycoprotein, gp120, resulting in viral entry into host cells. Therefore, CXCR4 and its ligands represent valuable drug targets. In this study, we investigated the inhibitory properties of synthetic peptides derived from CXCR4 extracellular loops (ECL1-X4, ECL2-X4 and ECL3-X4) towards HIV-1 infection and CXCL12-mediated receptor activation. Among these peptides, ECL1-X4 displayed anti-HIV-1 activity against X4, R5/X4 and R5 viruses (IC50=24 to 76µM) in cell viability assay without impairing physiological CXCR4-CXCL12 signalling. In contrast, ECL2-X4 only inhibited X4 and R5/X4 strains, interfering with HIV-entry into cells. At the same time, ECL2-X4 strongly and specifically interacted with CXCL12, blocking its binding to CXCR4 and its second receptor, CXCR7 (IC50=20 and 100µM). Further analysis using mutated and truncated peptides showed that ECL2 of CXCR4 forms multiple contacts with the gp120 protein and the N-terminus of CXCL12. Chemokine neutralisation was mainly driven by four aspartates and the C-terminal residues of ECL2-X4. These results demonstrate that ECL2 represents an important structural determinant in CXCR4 activation. We identified the putative site for the binding of CXCL12 N-terminus and provided new structural elements to explain the recognition of gp120 and dimeric CXCR4 ligands.

Selection of a CXCR4 antagonist from a human heavy chain CDR3-derived phage library.

Phage display technology is a powerful selection approach to identify strong and specific binders to a large variety of targets. In this study, we compared the efficacy of a phage library displaying human heavy chain complementarity determining region 3 (HCDR3) repertoires with a set of conventional random peptide libraries for the identification of CXCR4 antagonists using a peptide corresponding to the second extracellular loop of the receptor CXCR4 as target. A total of 11 selection campaigns on this target did not result in any specific ligand from the random peptide libraries. In contrast, a single selection campaign with an HCDR3 library derived from the IgM repertoire of a nonimmunized donor resulted in nine specific peptides with lengths ranging from 10 to 19 residues. Four of these HCDR3 sequences interacted with native receptor and the most frequently isolated peptide displayed an affinity of 5.6 µm and acted as a CXCR4 antagonist (IC(50) = 23 µm). To comprehend the basis of the highly efficient HCDR3 library selection, its biochemical properties were investigated. The HCDR3 length varied from 3 to 21 residues and displayed a biased amino acid content with a predominant proportion of Tyr, Gly, Ser and Asp. Repetitive and conserved motifs were observed in the majority of the HCDR3 sequences. The strength and efficacy of the HCDR3 libraries reside in the combination of multiple size peptides and a naturally biased sequence variation. Therefore, HCDR3 libraries represent a powerful and versatile alternative to fully randomized peptide libraries, in particular for difficult targets.

Non-immunized natural human heavy chain CDR3 repertoires allow the isolation of high affinity peptides mimicking a human influenza hemagglutinin epitope.

In this study we constructed two phage libraries displaying non-immunized natural human IgM derived HCDR3 repertoires. One library was structurally constrained by a Gly to Cys substitution at position 104 enabling the formation of a disulfide bridge with the Cys at position 92. Panning of these libraries on an anti-human influenza hemagglutinin (HA) antibody resulted in the selection of 16 different HCDR3 loops displaying different degrees of sequence homology with the HA epitope. The specificity of the HCDR3 loops recovered from the structurally constrained library was confirmed by competition assays using the HA epitope. Only one of these HCDR3 peptides contained Cys104. Structural analysis of these sequences revealed that the loss of Cys104 was associated with an increased preference for the formation of the type I beta-turn required for high affinity binding to the antibody. Affinity studies confirmed that the HCDR3 peptides containing the sequence YDVPDY and Gly104 had affinities in the nanomolar range (K(d)=7.6 nM) comparable to the HA epitope. These findings provided evidence that the recovered HCDR3 sequences may bind to their target in a conformation that is unreachable by the parental antibody from which the HCDR3 was derived. Furthermore, the isolation of target-specific and high affinity binders demonstrates the value of HCDR3 libraries as a source of 'biologically randomized' sequences of human origin for the identification of peptidic lead molecules.