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. 2015 Sep 11;290(37):22385-97.
doi: 10.1074/jbc.M115.675108. Epub 2015 Jul 27.

Dual targeting of the chemokine receptors CXCR4 and ACKR3 with novel engineered chemokines

Melinda S Hanes  1 , Catherina L Salanga  1 , Arnab B Chowdry  1 , Iain Comerford  2 , Shaun R McColl  2 , Irina Kufareva  1 , Tracy M Handel  3
Affiliations

Dual targeting of the chemokine receptors CXCR4 and ACKR3 with novel engineered chemokines

Melinda S Hanes et al. J Biol Chem. .

Abstract

The chemokine CXCL12 and its G protein-coupled receptors CXCR4 and ACKR3 are implicated in cancer and inflammatory and autoimmune disorders and are targets of numerous antagonist discovery efforts. Here, we describe a series of novel, high affinity CXCL12-based modulators of CXCR4 and ACKR3 generated by selection of N-terminal CXCL12 phage libraries on live cells expressing the receptors. Twelve of 13 characterized CXCL12 variants are full CXCR4 antagonists, and four have Kd values <5 nm. The new variants also showed high affinity for ACKR3. The variant with the highest affinity for CXCR4, LGGG-CXCL12, showed efficacy in a murine model for multiple sclerosis, demonstrating translational potential. Molecular modeling was used to elucidate the structural basis of binding and antagonism of selected variants and to guide future designs. Together, this work represents an important step toward the development of therapeutics targeting CXCR4 and ACKR3.

Keywords: ACKR3; C-X-C chemokine receptor type 4 (CXCR-4); CXCL12; G protein-coupled receptor (GPCR); SDF-1a; chemokine; chemokine receptors; experimental autoimmune encephalomyelitis (EAE); phage display; signaling.

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Figures

FIGURE 1.
FIGURE 1.
The steps in the phage display experimental design are shown in the middle panel. Multiple CXCL12 phagemid constructs were tested when optimizing the CXCL12 phage display system (left, magenta box), and the experimental conditions for generating recombinant CXCL12 phage are given (left, green box). Selections were performed on live cells at 37 °C, which is permissive of internalization, to bias the output toward receptor antagonists (right, blue box). MBP, maltose-binding protein; Ubq, ubiquitin; IPTG, isopropyl β-d-thiogalactopyranoside.
FIGURE 2.
FIGURE 2.
CXCL12 recombinant phage are detected in a sandwich ELISA experiment over control phage. Recombinant CXCL12 phage or control WT phage were captured with an anti-CXCL12 antibody and detected with an anti-phage antibody. Error bars represent S.D.
FIGURE 3.
FIGURE 3.
Characterization of CXCL12 variants. Competition binding curves for CXCL12 proteins against CXCR4 (A) or ACKR3 (B) in a scintillation proximity assay binding assay with 40 pm 125I-CXCL12 as a tracer are shown. A control experiment was performed between untransfected HEK293S cells and wild type CXCL12 (inset). The observed background signal is not competed off by increasing concentrations of CXCL12; thus the endogenous levels of CXCR4 are not sufficient to produce a binding signal. All binding assays were performed in quadruplicate and plotted as mean ± S.D. (error bars). C, the affinity of each CXCL12 variant for ACKR3 (IC50 value in nm) is plotted versus its affinity for CXCR4 (Kd value in nm). D, calcium flux dose-response curve for CXCL12 proteins, including WT CXCL12, LKQV, P2G, LGGG, LRHQ, LRSQ, MLGI, VPGA, QWVA, QFNI, SQCS, SQLA, and SQSQ. Data for all nonfunctional mutants are shown as overlapping cross marks (×), and the line appears as a flat baseline. Data shown are mean ± S.D. (error bars) of triplicates. E, cell migration of Jurkat cells in response to a chemokine gradient for CXCL12 proteins, including WT CXCL12, LKQV, P2G, LGGG, LRHQ, LRSQ, MLGI, VPGA, QWVA, QFNI, SQCS, SQLA, and SQSQ. Data for all nonfunctional mutants are shown as overlapping cross marks (×), and the line appears as a flat baseline. Data shown are mean ± S.D. of triplicates. F, CXCR4 internalization assay for WT CXCL12 and two of the highest affinity variants, LGGG-CXCL12 and LRHQ-CXCL12. For these experiments, MDA-MD-231 cells were transfected with HA epitope-tagged CXCR4, and result shown are the means ± S.E. (error bars) from three independent experiments performed in duplicate.
FIGURE 4.
FIGURE 4.
LGGG-CXCL12 slows onset of EAE and reduces peak and cumulative disease. EAE was induced in SJL/J mice as described, and mice were treated with 100 μg of LGGG-CXCL12, CXCL12, or CCL2Ala on even days following immunization up to day 17. A, the mice were scored daily for clinical signs of the disease. Statistically significant differences were observed between the CCL2Ala and LGGG-CXCL12 groups at p < 0.05 on days 9–12 and 19. B, cumulative disease over the disease course. Statistically significant differences were observed between the CCL2Ala and LGGG-CXCL12 groups at p < 0.05 on days 12–26. The data are presented as mean ± S.E. (error bars) (n = 7). C, post-test comparison of the day of disease onset (left) and peak disease score (right) where LGGG-CXCL12 and CCL2Ala were statistically different at p < 0.05 (analysis of variance). In all three cases (CCL2Ala, LGGG-CXCL12, and CXCL12), the incidence of the disease was seven of seven.
FIGURE 5.
FIGURE 5.
Predicted binding modes of CXCL12 and its antagonist variants to CXCR4. A, overall architecture of the complexes with the conserved CRS1 interaction shown between the globular core of the chemokine (skin) and the N terminus of the receptor (black ribbon and sticks). Acidic and basic residues in the chemokine are colored red and blue, respectively; they form favorable contacts with the basic (Lys-25) and acidic (Asp-22 and Glu-26) residues in the CXCR4 N terminus. The sulfated N-terminal tyrosine of CXCR4 (sY21) interacts with the backbone amines of Arg-20 and Ala-21 as well as the side chain of Arg-20 in a way that is observed for sulfate groups and free sulfate ions in several x-ray structures of CXCL12 (71–74). The predicted positions of the N termini of four CXCL12 variants are shown in ribbon: orange, WT; green, P2G; purple, LGGG; cyan, LRHQ. B–E, close-up views and important residue contacts for the four CXCL12 variants in the CRS2. The transmembrane helices of CXCR4 are numbered (roman numerals).
FIGURE 6.
FIGURE 6.
A, a Venn diagram for 71 sequences selected by biopanning of N-addition CXCL12 library against cells expressing CXCR4 and ACKR3. Sequences are shown as dots; each sequence is shown as many times as it appeared in the selected set. Sequences for which Kd values against CXCR4 were determined are colored black (pKd > 9), dark gray (8 < pKd < 9), gray (7 < pKd < 8), and light gray (6 < pKd < 7). 63 of 71 sequences had at least one of glutamine (Q; cyan), leucine (L; black), arginine (R; blue), or histidine (H; gray) and are pictured inside the corresponding Venn diagram regions; the remaining eight sequences are pictured outside and labeled “other.” The diagram describes the amino acid composition of the selected variants regardless of the relative positions of the amino acids within the sequences. B, a sequence profile for the 71 sequences where the relative conservation of amino acids at each of the four N-terminal positions of the chemokine is indicated by height. The graphic was prepared with WebLogo.
FIGURE 7.
FIGURE 7.
Maps of polar residues in the binding pockets of CXCR4 (A) and ACKR3 (B) built from an x-ray structure and a homology model, respectively. Each pocket is split approximately in half by a plane perpendicular to the membrane, and two sides of each pocket are shown: one comprising the N terminus; TM helices 1, 6, 7; and ECL3 (left), and the other is formed by TMs 2, 3, 4, and 5 and ECLs 1 and 2 (right). The surfaces are colored according to the character of the amino acid side chains forming them: acidic, red; basic blue; polar neutral, cyan. The maps illustrate that the distribution of pocket charged residues is very different between CXCR4 and ACKR3.
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