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.

T he atypical chemokine receptor 3 (ACKR3), previously known as CXC-chemokine receptor 7 (CXCR7), is an atypical chemokine receptor belonging to the class A G protein-coupled receptor (GPCR) family.Although the Received: October 19, 2023 Revised: November 24, 2023 Accepted: November 29, 2023 Published: December 8, 2023 Figure 1.Examples of reported small molecule ACKR3 ligands: 1, 10 2, 11 3, 12 4, 13 5, 14 6, 15 7, 16 and 8. 17 Details of the affinity or potency of the compounds are shown in the Supporting Information (Table S1).  10 suggests the highlighted 3-methoxy group present in VUF11207 (1) is not essential for ACKR3 binding and can be targeted for linker and fluorophore attachment (b) Docking of VUF11207 (R)-1 into ACKR3 (pdb 7SK9) suggests substitution on the 3-position of the aryl ring would be an appropriate choice for linker and fluorophore attachment.Docking experiments were performed using OEDOCKING Hybrid docking. 24,25iological role of ACKR3 is not entirely understood, it is reported to function as a scavenger of CXCL12 (C-X-C chemokine 12, also known as SDF-1, stromal cell-derived factor 1) establishing CXCL12 gradients, thereby modulating CXCR4 signaling. 1,2It has been postulated to regulate a range of biological functions that occur after binding of the endogenous ligand CXCL12 and subsequent recruitment of the multifunctional intracellular protein β-arrestin, resulting in phosphorylation-dependent receptor internalization without detectable activation of G-proteins. 3 Expression of ACKR3 on the surface of platelets has been shown to be up-regulated in patients suffering with acute myocardial infarction and subsequent elevation of ACKR3 expression leads to an improvement in recovery. 4,5Additionally, increased infarct size and subsequent patient mortality have been observed, where ACKR3 expression has been decreased, signifying the importance of ACKR3 in promoting proliferation and angiogenesis. 6ACKR3 is known to be overexpressed in numerous cancer types, indicating its involvement in the modulation of tumor cell proliferation and migration and tumor angiogenesis, contributing to cancer progression and metastasis. 7Due to the increasing literature for the role of ACKR3 in disease, several structurally diverse small molecule ACKR3 ligands have been reported (Figure 1). 8,9rrently, the most widely used compound to study ACKR3 function is the endogenous ligand CXCL12.Although human CXCL12 and its radiolabeled and fluorescently labeled    versions are available through commercial sources, their arduous synthesis makes them very expensive to employ in both in vitro and in vivo imaging.Antibodies and nanobodies have also emerged as highly selective tools to study ACKR3 18,19 but similar to CXCL12, the development of ACKR3-specific antibodies and nanobodies is difficult and time-consuming, making them also very expensive for the medicinal chemist to routinely employ.Small molecule ligands that selectively target ACKR3 can offer several advantages over chemokines and antibodies as tool compounds to probe receptor function.−23 We report the synthesis of the first fluorescent ACKR3 probes, based on the receptor agonist VUF11207 (1). 10 An evaluation of the reported structure−activity relationship (SAR) of the small molecule inhibitor, combined with in silico docking experiments utilizing the recently disclosed Cryo-EM structure of ACKR3 complexed with the partial agonist 8 CCX662, 17 informed the synthetic strategy for linker design and fluorophore attachment (Figure 2).
The resulting fluorescent compounds were characterized in a BRET-based assay, enabled by a NanoLuciferase (NLuc)-ACKR3 construct.The recently developed NanoBRET methodology has allowed characterization of various (fluorescent) probes targeting GPCRs, even when under endogenous promotion. 20,21,26he synthesis of fluorescent derivatives of VUF11207 was based on procedures that were described by Wijtmans in the development of VUF11207. 10Zarca et al. recently reported on the pharmacological evaluation of the synthesized single enantiomers of VUF11207 (1) showing that (R)-1 had a pEC 50 of 8.3 ± 0.1 compared to (S)-1, which has a corresponding pEC 50 of 7.7 ± 0.1 in a [ 125 I] CXCL12 displacement assay. 27ynthesis started with an aldol reaction between 2fluorobenzaldehyde and propionaldehyde, which under basic conditions provided (E)-3-(2-fluorophenyl)-2-methylacrylaldehyde 10 in excellent yield.A reductive amination with a picoline borane complex and (R)-2-(1-methylpyrrolidin-2yl)ethanamine gave the homochiral precursor 11 in good yield.With this key fragment in hand, we set out to synthesize the various linkers.Here, we chose to develop linkers of three different lengths, with PEG chains ranging from 0 to 2. Commercially available alcohol-carbamates 12a−c were first converted into tosylates using tosyl chloride 13a−c.O-Alkylation using methyl 3-hydroxy-4,5-dimethoxybenzoate efficiently installed the linkers on the 3′-position.Hydrolysis of the methyl ester to the benzoic acids 15a−c using lithium hydroxide proceeded with quantitative yields, allowing subsequent peptide coupling with key intermediate 11 to give 16a−c and after N-Boc deprotection, the congeners 17a− c were ready for conjugation to commercially available fluorescent dyes (Scheme 1).
The congeners 17a−c were reacted with the commercial BODIPY FL-X succinimidyl ester to give the corresponding fluorescent ligands 18a−c, after purification by reverse phase HPLC.The fluorescent ligands were prepared in >95% purity as defined through analytical HPLC (Scheme 2).
Pharmacological Evaluation of Fluorescent ACKR3 Antagonists.The fluorescent conjugates (18a−c) were evaluated by using a range of pharmacological assays.Initially, saturation binding experiments were used to determine the affinity of the fluorescent conjugates toward the ACKR3 receptor.The fluorescent properties of the compounds allowed detection of the proximity of the fluorescent ligands to an Nterminal NanoLuciferase-tagged receptor (NLuc-ACKR3) by means of bioluminescence resonance energy transfer (Nano-BRET). 20The three fluorescent conjugates produced clear saturable specific binding to the NLuc-ACKR3 receptor that was associated with low levels of nonspecific binding (determined in the presence of unlabeled (R)-1) resulting in pK d values ranging from 6.8 to 7.9 (Figure 3 and Table 1).
To further evaluate the use of 18a in the NanoBRET-ligand binding assay, affinities of ACKR3 ligands 4, 9, 19, and 20 were determined in competition binding experiments (Table 2).
The availability of high affinity green fluorescent ACKR3 receptor ligands suggested utility for live cell imaging.Confocal microscopy images of fluorescent ligand 18a incubated with HEK293 cells transiently expressing N-terminal SNAPTag-ACKR3 (referred to as SNAP-ACKR3) for 30 min at 37 °C were captured.Under these conditions, SNAP-ACKR3 labeled with the cell impermeable SNAP-AF647 showed a predominantly vesicular intracellular location, with a small amount on the cell membrane (Figure 4, second column).This is consistent with its known high levels of constitutive ACKR3 cycling.Ligand 18a (100 nM) showed a very similar distribution of mainly intracellular fluorescence, which was colocalized with that of the SNAP-ACKR3 receptor (Figure 4) and may also therefore indicate some ligand induced internalization.Images collected at various time points during incubation of 50 nM 18a (Supporting Information, Figure S1) indicated that 18a was initially bound to the cell surface at early time points and then internalized with SNAP-ACKR3.When cells were pretreated with (R)-1, its level of binding was significantly reduced, suggesting that the majority of observed fluorescence was specific binding of 18a to the SNAP-ACKR3 receptor.
We have reported the characterization of the first new small molecule-based fluorescent probes for ACKR3.Compounds (18a−c) retained good affinity toward the ACKR3 receptor, as shown by NanoBRET saturation experiments.We further demonstrated that 18a is a useful screening tool for discovering new ACKR3 agonists.Compound 18a displayed good signalto-noise in NanoBRET competition-binding experiments and was displaced by the established small molecule agonist (R)-1, close analogues, and a structurally diverse agonist 4. The fluorescent ACKR3 ligands (18a−c) can be used in live cell confocal microscopy experiments and in combination with the NanoBRET approach may shed further light on ACKR3 function and its participation in pathophysiological conditions.

Figure 2 .
Figure 2. (a) Reported SAR10 suggests the highlighted 3-methoxy group present in VUF11207 (1) is not essential for ACKR3 binding and can be targeted for linker and fluorophore attachment (b) Docking of VUF11207 (R)-1 into ACKR3 (pdb 7SK9) suggests substitution on the 3-position of the aryl ring would be an appropriate choice for linker and fluorophore attachment.Docking experiments were performed using OEDOCKING Hybrid docking.24,25

Figure 3 .
Figure 3. Saturation binding of (R)-1 using 18a−c in HEK293G_N-Luc-ACKR3 cells.HEK293G_NLuc-ACKR3 cells were treated with 18a−c in the presence and absence of 10 μM unlabeled (R)-1 fulllength N terminal NanoLuciferase-ACKR3 stably expressing HEK293 cells.Compounds were added simultaneously and incubated for 60 min at 37 °C in HBSS containing 0.2% BSA.Furimazine (1:400 final dilution) was added and plates incubated for 5 min.Fluorescence and luminescence emissions were measured using a BMG Pherastar FS.The raw BRET ratio was calculated by dividing the fluorescent signal by the bioluminescent signal and specific binding was calculated by deducting nonspecific binding from the total binding values.

Table 1
a pK d values were calculated from the negative logarithm of the equilibrium dissociation constant (K d ) determined from saturationbinding experiments using increasing concentrations of labeled ligand in the presence or absence of (R)-1 (10 μM).Data are expressed as mean ± SEM, where each experiment was performed in triplicate.

Table 2 .
Binding Affinities of a Series of Known ACKR3 Ligands a Data are combined mean ± SEM, where each experiment was performed in triplicate.