Structural basis for selectivity and antagonism in extracellular GPCR-nanobodies.

G protein-coupled receptors (GPCRs) are pivotal therapeutic targets, but their complex structure poses challenges for effective drug design. Nanobodies, or single-domain antibodies, have emerged as a promising therapeutic strategy to target GPCRs, offering advantages over traditional small molecules and antibodies. However, an incomplete understanding of the structural features enabling GPCR-nanobody interactions has limited their development. In this study, we investigate VUN701, a nanobody antagonist targeting the atypical chemokine receptor 3 (ACKR3). We determine that an extended CDR3 loop is required for ACKR3 binding. Uncommon in most nanobodies, an extended CDR3 is prevalent in GPCR-targeting nanobodies. Combining experimental and computational approaches, we map an inhibitory ACKR3-VUN701 interface and define a distinct conformational mechanism for GPCR inactivation. Our results provide insights into class A GPCR-nanobody selectivity and suggest a strategy for the development of these new therapeutic tools.

C­X­C receptor 7 agonist acts as a C­X­C motif chemokine ligand 12 inhibitor to ameliorate osteoclastogenesis and bone resorption.

The C­X­C receptor (CXCR) 7 agonist, VUF11207, is a chemical compound that binds specifically to CXCR7, and negatively regulates C­X­C motif chemokine ligand 12 (CXCL12) and CXCR4­induced cellular events. Lipopolysaccharide (LPS) can induce inflammatory cytokines and pathological bone loss. LPS also induces expression of CXCL12, enhancing sensitivity to receptor activator of NF­κB ligand (RANKL) and tumor necrosis factor­α (TNF­α) in vivo. RANKL and TNF­α induce the differentiation of osteoclasts into osteoclast precursors and bone resorption. The current study was performed to examine the effects of a CXCR7 agonist on osteoclastogenesis and bone resorption induced by LPS in vivo. In addition, the mechanisms underlying these in vivo effects were investigated by in vitro experiments. Eight­week­old male C57BL/6J mice were subcutaneously injected over the calvariae with LPS alone or LPS and CXCR7 agonist. After sacrifice, the number of osteoclasts and the bone resorption area were measured. In vitro experiments were performed to investigate the effects of CXCL12 and CXCR7 agonist on osteoclastogenesis induced by RANKL and TNF­α. Mice injected with LPS and CXCR7 agonist showed significantly reduced osteoclastogenesis and bone resorption compared with mice injected with LPS alone. Moreover, the CXCR7 agonist inhibited CXCL12 enhancement of RANKL­ and TNF­α­induced osteoclastogenesis in vitro. Thus, CXCR7 agonist inhibited LPS­induced osteoclast­associated cytokines, such as RANKL and TNF­α, as well as RANKL­ and TNF­α­induced osteoclastogenesis in vitro by modulating CXCL12­mediated enhancement of osteoclastogenesis. In conclusion, CXCR7 agonist reduced CXCL12­mediated osteoclastogenesis and bone resorption.

CXCR4 and CXCR7 Inhibition Ameliorates the Formation of Platelet-Neutrophil Complexes and Neutrophil Extracellular Traps through Adora2b Signaling.

Peritonitis and peritonitis-associated sepsis are characterized by an increased formation of platelet-neutrophil complexes (PNCs), which contribute to an excessive migration of polymorphonuclear neutrophils (PMN) into the inflamed tissue. An important neutrophilic mechanism to capture and kill invading pathogens is the formation of neutrophil extracellular traps (NETs). Formation of PNCs and NETs are essential to eliminate pathogens, but also lead to aggravated tissue damage. The chemokine receptors CXCR4 and CXCR7 on platelets and PMNs have been shown to play a pivotal role in inflammation. Thereby, CXCR4 and CXCR7 were linked with functional adenosine A2B receptor (Adora2b) signaling. We evaluated the effects of selective CXCR4 and CXCR7 inhibition on PNCs and NETs in zymosan- and fecal-induced sepsis. We determined the formation of PNCs in the blood and, in addition, their infiltration into various organs in wild-type and Adora2b-/- mice by flow cytometry and histological methods. Further, we evaluated NET formation in both mouse lines and the impact of Adora2b signaling on it. We hypothesized that the protective effects of CXCR4 and CXCR7 antagonism on PNC and NET formation are linked with Adora2b signaling. We observed an elevated CXCR4 and CXCR7 expression in circulating platelets and PMNs during acute inflammation. Specific CXCR4 and CXCR7 inhibition reduced PNC formation in the blood, respectively, in the peritoneal, lung, and liver tissue in wild-type mice, while no protective anti-inflammatory effects were observed in Adora2b-/- animals. In vitro, CXCR4 and CXCR7 antagonism dampened PNC and NET formation with human platelets and PMNs, confirming our in vivo data. In conclusion, our study reveals new protective aspects of the pharmacological modulation of CXCR4 and CXCR7 on PNC and NET formation during acute inflammation.

Target engagement of the first-in-class CXCR7 antagonist ACT-1004-1239 following multiple-dose administration in mice and humans.

Antagonism of the chemokine receptor CXCR7 has shown promising effects in diverse disease areas through modulation of its ligands, CXCL11 and CXCL12. Preclinical data of the first-in-class CXCR7 antagonist, ACT-1004-1239, showed efficacy in animal models of multiple sclerosis and acute lung injury. In healthy humans, single-dose administration of ACT-1004-1239 revealed a favorable clinical profile. Here, we report the target engagement of ACT-1004-1239 in healthy mice and humans after multiple doses using CXCL11 and CXCL12 as biomarkers. In addition, safety/tolerability, concentration-QTc relationship, and pharmacokinetics (PK) were assessed in a randomized, double-blind, placebo-controlled Phase 1 clinical study. Multiple-dose ACT-1004-1239 dose-dependently increased CXCL12 plasma concentration across the investigated dose range in mice and humans (mice: 1-100 mg/kg b.i.d.; humans: 30-200 mg o.d.) when compared to vehicle/placebo demonstrating target engagement. Mouse and human PK/PD models predicted that CXCL12 concentration approached a plateau within these dose ranges. In humans, ACT-1004-1239 was rapidly absorbed (tmax: 1.75-3.01 h) and the terminal t1/2 was approximately 19 h. Steady-state conditions were reached by Day 3 with an accumulation index of 1.2. Female subjects had overall higher exposure compared to males. Multiple-dose ACT-1004-1239 was well tolerated up to 200 mg once daily in humans. There was no evidence of ACT-1004-1239-mediated QTc interval prolongation. Overall, multiple oral doses of ACT-1004-1239 showed target engagement with CXCR7 in healthy mice and humans, therefore, assessment of CXCL12 as translational tool for further investigations in patients is warranted. Favorable safety/tolerability and PK profiles allow for further clinical development.

Discovery and evaluation of non-basic small molecule modulators of the atypical chemokine receptor CXCR7.

The atypical chemokine receptor C-X-C chemokine receptor type 7 (CXCR7) is an attractive therapeutic target for a variety of cardiac and immunological diseases. As a strategy to mitigate known risks associated with the development of higher molecular weight, basic compounds, a series of pyrrolidinyl-azolopyrazines were identified as promising small-molecule CXCR7 modulators. Using a highly enabled parallel medicinal chemistry strategy, structure-activity relationship studies geared towards a reduction in lipophilicity and incorporation of saturated heterocycles led to the identification of representative tool compound 20. Notably, compound 20 maintained good potency against CXCR7 with a suitable balance of physicochemical properties to support in vivo pharmacokinetic studies.

CXCL12-CXCR4/CXCR7 Axis in Colorectal Cancer: Therapeutic Target in Preclinical and Clinical Studies.

Chemokines are chemotactic cytokines that promote cancer growth, metastasis, and regulate resistance to chemotherapy. Stromal cell-derived factor 1 (SDF1) also known as C-X-C motif chemokine 12 (CXCL12), a prognostic factor, is an extracellular homeostatic chemokine that is the natural ligand for chemokine receptors C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or cluster of differentiation 184 (CD184) and chemokine receptor type 7 (CXCR7). CXCR4 is the most widely expressed rhodopsin-like G protein coupled chemokine receptor (GPCR). The CXCL12-CXCR4 axis is involved in tumor growth, invasion, angiogenesis, and metastasis in colorectal cancer (CRC). CXCR7, recently termed as atypical chemokine receptor 3 (ACKR3), is amongst the G protein coupled cell surface receptor family that is also commonly expressed in a large variety of cancer cells. CXCR7, like CXCR4, regulates immunity, angiogenesis, stem cell trafficking, cell growth and organ-specific metastases. CXCR4 and CXCR7 are expressed individually or together, depending on the tumor type. When expressed together, CXCR4 and CXCR7 can form homo- or hetero-dimers. Homo- and hetero-dimerization of CXCL12 and its receptors CXCR4 and CXCR7 alter their signaling activity. Only few drugs have been approved for clinical use targeting CXCL12-CXCR4/CXCR7 axis. Several CXCR4 inhibitors are in clinical trials for solid tumor treatment with limited success whereas CXCR7-specific inhibitors are still in preclinical studies for CRC. This review focuses on current knowledge of chemokine CXCL12 and its receptors CXCR4 and CXCR7, with emphasis on targeting the CXCL12-CXCR4/CXCR7 axis as a treatment strategy for CRC.

The CXCL12/CXCR7 signalling axis promotes proliferation and metastasis in cervical cancer.

C-X-C chemokine receptor 7 (CXCR7), a novel receptor of C-X-C motif chemokine ligand 12 (CXCL12), is associated with the occurrence and metastasis of various malignant tumours. However, the role, function and underlying mechanisms of CXCR7 expression in cervical cancer remain undefined. The expression level of CXCR7 was evaluated in cervical cancer samples by immunohistochemistry and real-time PCR analyses. Western blot analysis was used to examine the expression level of CXCR7 in cervical cancer cell lines. HeLa cells were genetically silenced or pharmacologically inhibited for CXCR7 or CXCR4. Transwell and CCK-8 assays were used to examine cell migration and proliferation. The expression levels of MMP2, MMP9, TIMP-1 and TIMP-2 in HeLa cells were assessed by western blot or real-time PCR. HeLa cells silenced for CXCR7 were subcutaneously injected into nude mice to form tumours. The expression of CXCR7 in nude mice was investigated by immunohistochemical staining. Tumour volumes and weights were measured. The in vivo expression levels of MMP2, MMP9, TIMP-1 and TIMP-2 were determined by western blot analysis and real-time PCR. CXCR7 was overexpressed in cervical cancer tissues and cell lines. CXCL12 was highly expressed in cervical cancer lines. CXCR7 silencing or CCX733 treatment rather than CXCR4 silencing or AMD3100 treatment suppressed the proliferation, migration and invasion of cervical cancer cells stimulated by CXCL12. In a xenograft tumour model, CXCR7 silencing or CCX733 treatment inhibited the volumes and weights of xenograft tumours. In addition, downregulation of CXCR7 decreased the expression levels of MMP2 and MMP9 but increased the expression levels of TIMP-1 and TIMP-2 in vivo. These data support the finding that the downregulation of CXCR7 suppresses the proliferation and metastasis of cervical cancer cells. Inhibition of CXCR7 may be a potential targeted therapy for cervical cancer.

ACT-1004-1239, a first-in-class CXCR7 antagonist with both immunomodulatory and promyelinating effects for the treatment of inflammatory demyelinating diseases.

Current strategies for the treatment of demyelinating diseases such as multiple sclerosis (MS) are based on anti-inflammatory or immunomodulatory drugs. Those drugs have the potential to reduce the frequency of new lesions but do not directly promote remyelination in the damaged central nervous system (CNS). Targeting CXCR7 (ACKR3) has been postulated as a potential therapeutic approach in demyelinating diseases, leading to both immunomodulation by reducing leukocyte infiltrates and promyelination by enhancing myelin repair. ACT-1004-1239 is a potent, selective, insurmountable, and orally available first-in-class CXCR7 receptor antagonist. The effect of ACT-1004-1239 was evaluated in the myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) and the cuprizone-induced demyelination mouse models. In addition, ACT-1004-1239 was assessed in a rat oligodendrocyte precursor cell (OPC) differentiation assay in vitro. In the MOG-induced EAE model, ACT-1004-1239 treatment (10-100 mg/kg, twice daily, orally) showed a significant dose-dependent reduction in disease clinical scores, resulting in increased survival. At the highest dose tested (100 mg/kg, twice daily), ACT-1004-1239 delayed disease onset and significantly reduced immune cell infiltrates into the CNS and plasma neurofilament light chain concentration. Treatment with ACT-1004-1239 dose-dependently increased plasma CXCL12 concentration, which correlated with a reduction of the cumulative disease score. Furthermore, in the cuprizone model, ACT-1004-1239 treatment significantly increased the number of mature myelinating oligodendrocytes and enhanced myelination in vivo. In vitro, ACT-1004-1239 promoted the maturation of OPCs into myelinating oligodendrocytes. These results provide evidence that ACT-1004-1239 both reduces neuroinflammation and enhances myelin repair substantiating the rationale to explore its therapeutic potential in a clinical setting.

A Multipurpose First-in-Human Study With the Novel CXCR7 Antagonist ACT-1004-1239 Using CXCL12 Plasma Concentrations as Target Engagement Biomarker.

The C-X-C chemokine receptor 7 (CXCR7) has evolved as a promising, druggable target mainly in the immunology and oncology fields modulating plasma concentrations of its ligands CXCL11 and CXCL12 through receptor-mediated internalization. This "scavenging" activity creates concentration gradients of these ligands between blood vessels and tissues that drive directional cell migration. This randomized, double-blind, placebo-controlled first-in-human study assessed the safety, tolerability, pharmacokinetics, and pharmacodynamics of ACT-1004-1239, a first-in-class drug candidate small-molecule CXCR7 antagonist. Food effect and absolute bioavailability assessments were also integrated in this multipurpose study. Healthy male subjects received single ascending oral doses of ACT-1004-1239 (n = 36) or placebo (n = 12). At each of six dose levels (1-200 mg), repeated blood sampling was done over 144 hours for pharmacokinetic/pharmacodynamic assessments using CXCL11 and CXCL12 as biomarkers of target engagement. ACT-1004-1239 was safe and well tolerated up to the highest tested dose of 200 mg. CXCL12 plasma concentrations dose-dependently increased and more than doubled compared with baseline, indicating target engagement, whereas CXCL11 concentrations remained unchanged. An indirect-response pharmacokinetic/pharmacodynamic model well described the relationship between ACT-1004-1239 and CXCL12 concentrations across the full dose range, supporting once-daily dosing for future clinical studies. At doses ≥ 10 mg, time to reach maximum plasma concentration ranged from 1.3 to 3.0 hours and terminal elimination half-life from 17.8 to 23.6 hours. The exposure increase across the dose range was essentially dose-proportional and no relevant food effect on pharmacokinetics was determined. The absolute bioavailability was 53.0% based on radioactivity data after oral vs. intravenous 14 C-radiolabeled microtracer administration of ACT-1004-1239. Overall, these comprehensive data support further clinical development of ACT-1004-1239.