ACKR3 agonism induces heterodimerization with chemokine receptor CXCR4 and attenuates platelet function.

BACKGROUND: Platelet receptors ACKR3 and CXCR4 play a crucial role in a variety of cardiovascular diseases. Like most chemokine receptors, CXCR4 is a G protein coupled receptor that induces platelet activation. In contrast, the atypical chemokine receptor 3 (ACKR3) lacks the ability to activate heterotrimeric G proteins and its activation leads to platelet inhibition and attenuates thrombus formation. In nucleated cells, heterodimerization of ACKR3 with CXCR4 regulates CXCL12-dependent signalling. The aim of our study was to investigate the formation of ACKR3/CXCR4 heterodimers in platelets and the subsequent consequences for platelet function. METHODS AND RESULTS: Using a proximity ligation assay (PLA, Duolink®) to screen for CXCR4/ACKR3 heterodimerization inducing compounds, we found that ACKR3 agonism but not conventional platelet agonists or endogen ligands lead to heterodimer formation. To further characterize the formation of ACKR3/CXCR4 heterodimers, we studied the CXCL12-dependent platelet activation via CXCR4. Both, CXCL12-dependent platelet aggregation and collagen-dependent ex vivo thrombus formation were significantly downregulated by ACKR3 agonism. Moreover, platelet intracellular calcium and Akt signalling were increased by CXCL12 and again suppressed by ACKR3-specific agonists. Previously, CXCL12 was shown to decrease platelet cAMP levels via CXCR4. Treatment with a specific ACKR3 agonist counteracted this CXCL12/CXCR4-dependent cAMP decrease. CONCLUSION: Our results reveal that the formation of platelet ACKR3/CXCR4 heterodimers is dependent on ACKR3 rather than CXCR4. Furthermore, ACKR3 agonism induced heterodimerization is associated with mitigating CXCL12/CXCR4-dependent platelet activation possibly by modulating CXCR4-dependent G protein signalling. Our results indicate possible ACKR3 agonist functions and reinforce the potential therapeutic applications of ACKR3 agonists.

Hemin-induced platelet activation is regulated by the ACKR3 chemokine surface receptor and has implications for passivation of vulnerable atherosclerotic plaques.

In vulnerable atherosclerotic plaques, intraplaque hemorrhages (IPH) result in hemolysis of red blood cells and release of hemoglobin and free hemin. Hemin activates platelets and leads to thrombosis. Agonism of the inhibitory platelet receptor ACKR3 inhibits hemin-dependent platelet activation and thrombus formation. To characterize the effect of hemin and ACKR3 agonism on isolated human platelets, multi-color flow cytometry and classical experimental setup such as light transmission aggregometry and a flow chamber assay were used. Hemin induces platelet aggregation and ex vivo platelet-dependent thrombus formation on immobilized collagen under a low shear rate of 500 s-1, indicating that free hemin is a strong activator of platelet-dependent thrombosis. Recently, we described that ACKR3 is a prominent inhibitory receptor of platelet activation. Specific ACKR3 agonists but not conventional antiplatelet compounds such as COX-1 inhibitor (indometacin), ADP-receptor blocker (cangrelor), or PAR1 inhibitor (ML161) inhibit both hemin-dependent aggregation and thrombus formation. To further characterize the effect of hemin on platelet subpopulations, we established a multi-color flow cytometry assay. We found that hemin induces procoagulant (CD42bpos/PAC-1neg/AnnexinVpos), aggregatory (CD42bpos/PAC-1pos/AnnexinVneg), and inflammatory (CD42bpos/CXCR4pos/ACKR3pos/AnnexinVpos) platelet subpopulations. Treatment with ACKR3 agonists significantly decreased the formation of procoagulant and ACKR3pos platelets in response to hemin. We conclude that hemin is a strong activator for the formation of procoagulant platelets and thrombus formation which is dependent on the function of ACKR3. Activation of ACKR3 using specific agonists may offer a therapeutic strategy to regulate the vulnerability of atherosclerotic plaques in areas of IPH.

Novel Small-Molecule Atypical Chemokine Receptor 3 Agonists: Design, Synthesis, and Pharmacological Evaluation for Antiplatelet Therapy.

ACKR3, an atypical chemokine receptor, has been associated with prothrombotic events and the development of cardiovascular events. We designed, synthesized, and evaluated a series of novel small molecule ACKR3 agonists. Extensive structure-activity relationship studies resulted in several promising agonists with potencies ranging from the low micromolar to nanomolar range, for example, 23 (EC50 = 111 nM, Emax = 95%) and 27 (EC50 = 69 nM, Emax = 82%) in the ß-arrestin-recruitment assay. These compounds are selective for ACKR3 versus ACKR2, CXCR3, and CXCR4. Several agonists were subjected to investigations of their P-selectin expression reduction in the flow cytometry experiments. In particular, compounds 23 and 27 showed the highest potency for platelet aggregation inhibition, up to 80% and 97%, respectively. The most promising compounds, especially 27, exhibited good solubility, metabolic stability, and no cytotoxicity, suggesting a potential tool compound for the treatment of platelet-mediated thrombosis.

Discovery and Development of First-in-Class ACKR3/CXCR7 Superagonists for Platelet Degranulation Modulation.

The atypical chemokine receptor 3 (ACKR3), formerly known as CXC-chemokine receptor 7 (CXCR7), has been postulated to regulate platelet function and thrombus formation. Herein, we report the discovery and development of first-in-class ACKR3 agonists, which demonstrated superagonistic properties with Emax values of up to 160% compared to the endogenous reference ligand CXCL12 in a ß-arrestin recruitment assay. Initial in silico screening using an ACKR3 homology model identified two hits, C10 (EC50 19.1 µM) and C11 (EC50 = 11.4 µM). Based on these hits, extensive structure-activity relationship studies were conducted by synthesis and testing of derivatives. It resulted in the identification of the novel thiadiazolopyrimidinone-based compounds 26 (LN5972, EC50 = 3.4 µM) and 27 (LN6023, EC50 = 3.5 µM). These compounds are selective for ACKR3 versus CXCR4 and show metabolic stability. In a platelet degranulation assay, these agonists effectively reduced P-selectin expression by up to 97%, suggesting potential candidates for the treatment of platelet-mediated thrombosis.

Super-conserved receptors expressed in the brain: biology and medicinal chemistry efforts.

The super-conserved receptors expressed in the brain (SREB) constitute a family of orphan G protein-coupled receptors that include GPR27 (SREB1), GPR85 (SREB2) and GPR173 (SREB3). Their sequences are highly conserved in vertebrates, and they are almost exclusively expressed in the central nervous system. This family of receptors has attracted much attention due to their putative physiological functions and their potential as novel drug targets. The SREB family has been postulated to play important roles in a wide range of different diseases, including pancreatic ß-cell insulin secretion and regulation, schizophrenia, autism and atherosclerosis. This review intends to provide a comprehensive overview of the SREB family and its recent advances in biology and medicinal chemistry.

In recent years, the super-conserved receptors expressed in the brain called GPR27, GPR85 and GPR173 have attracted much interest in the field of medicinal science. They have one important feature in common: they are all almost entirely found in the brain. Researchers have investigated their functions in the body in various animal models, as well as their utility in future drug development. GPR27 has been found to be involved in insulin and blood sugar processes in the body and therefore may be important for diabetes treatment. GPR85 is thought to be linked to brain diseases such as schizophrenia and autism. GPR173 is linked to many different illnesses, including atherosclerosis (the buildup of fats, cholesterol and other substances in arteries) and Type 2 diabetes.