The atypical chemokine receptor 3 (ACKR3) is an attractive drug target for a new generation of chemotherapies. The inhibition of chemokine binding to ACKR3 by small-molecule agonists has shown reduced cancer activity in vivo and in vitro. However, despite the potential, a relatively low number of ligands has been disclosed in peer-reviewed scientific literature. The start of this study was the investigation of the peer-reviewed literature but also the patent literature to retrieve the entire arsenal of ACKR3 ligands. We have consulted four databases, namely Reaxys®, PubChem, ChEMBL, and BindingDB, to identify 3915 small molecule ligands for ACKR3. Most ligands were retrieved from patent literature, and these were analysed to map the explored chemical space. The physical-chemical properties of the ligands described in nine patent applications were analysed. We selected the nine patents based on the number of ligands present and if only ACKR3 was targeted by the disclosed ligands. Retrieved patent WO20180199929 from Idorsia showed the first ACKR3 antagonist. The compounds in this patent were selected as starting points to obtain tool compounds and discover new ACKR3 ligands (Chapter 4). In Chapter 4, high-affinity ligands from patent WO201819929, with a clinical candidate later known as ACT-1004-1239, were taken as starting points. The first aim was to reduce the molecular complexity (i.e., reducing the number of chiral centers). Exploration of the structure-activity relationships for these series of compounds resulted amongst others in replacement of the piperidine core using a scaffold hopping approach. The affinity was reduced 25-fold; however, substituting the piperidine nitrogen atom with various groups to replace the aliphatic ring provided ample opportunities to optimize this scaffold. The best performing compound was compound VUF-0025663 with a pIC50 value of 8.2. In chapter 5, the structure-activity studies focused on optimizing the linker that connects the piperidine nitrogen atom with a terminal aromatic ring. Having optimized this part of the ligand, the aromatic ring system was further explored. This study shows that the binding affinity is highly dependent on the placement of the heteroatoms in the 5-membered ring. At best, an analog with almost the same activity was found. Overall compound VUF-0025661 emerged as an additional promising compound which was selected together with VUF-0025663 for initial in vivo evaluation. In Chapter 6, we aimed to find a hit molecule that could be used in more elaborated drug discovery programs. We used a custom-designed small-molecule radioligand as a tracer in a radioligand displacement assay that allows for a fragment library screen. The screening campaign resulted in a hit rate of 1.5%, which is lower than for many other GPCR targets that were probed with this library but still resulted in several interesting hits. The small-molecule radioligand displacement assay was also used to guide hit exploration studies and to develop ACKR3 structure-activity relationships. Quinazoline-containing fragment hits were optimized in a fragment-growing campaign leading to VUF-0025449 as the most potent ACKR3 ligand (pKi =6.7). The best-performing compounds were tested for their functionality in NanoLuc-based β-arrestin2 recruitment assays, showing the agonistic effect of VUF-0025449
