Adhesion of Crithidia fasciculata promotes a rapid change in developmental fate driven by cAMP signaling.

Trypanosomatids are single-celled parasites responsible for human and animal disease. Typically, colonization of an insect host is required for transmission. Stable attachment of parasites to insect tissues via their single flagellum coincides with differentiation and morphological changes. Although attachment is a conserved stage in trypanosomatid life cycles, the molecular mechanisms are not well understood. To study this process, we elaborate upon an in vitro model in which the swimming form of the trypanosomatid Crithidia fasciculata rapidly differentiates following adhesion to artificial substrates. Live imaging of cells transitioning from swimming to attached shows parasites undergoing a defined sequence of events, including an initial adhesion near the base of the flagellum immediately followed by flagellar shortening, cell rounding, and the formation of a hemidesmosome-like attachment plaque between the tip of the shortened flagellum and the substrate. Quantitative proteomics of swimming versus attached parasites suggests differential regulation of cyclic adenosine monophosphate (cAMP)-based signaling proteins. We have localized two of these proteins to the flagellum of swimming C. fasciculata; however, both are absent from the shortened flagellum of attached cells. Pharmacological inhibition of cAMP phosphodiesterases increased cAMP levels in the cell and prevented attachment. Further, treatment with inhibitor did not affect the growth rate of either swimming or established attached cells, indicating that its effect is limited to a critical window during the early stages of adhesion. These data suggest that cAMP signaling is required for attachment of C. fasciculata and that flagellar signaling domains may be reorganized during differentiation and attachment.IMPORTANCETrypanosomatid parasites cause significant disease burden worldwide and require insect vectors for transmission. In the insect, parasites attach to tissues, sometimes dividing as attached cells or producing motile, infectious forms. The significance and cellular mechanisms of attachment are relatively unexplored. Here, we exploit a model trypanosomatid that attaches robustly to artificial surfaces to better understand this process. This attachment recapitulates that observed in vivo and can be used to define the stages and morphological features of attachment as well as conditions that impact attachment efficiency. We have identified proteins that are enriched in either swimming or attached parasites, supporting a role for the cyclic AMP signaling pathway in the transition from swimming to attached. As this pathway has already been implicated in environmental sensing and developmental transitions in trypanosomatids, our data provide new insights into activities required for parasite survival in their insect hosts.

Multiplex Detection of Fluorescent Chemokine Binding to CXC Chemokine Receptors by NanoBRET.

NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.

Pharmacological Characterization and Radiolabeling of VUF15485, a High-Affinity Small-Molecule Agonist for the Atypical Chemokine Receptor ACKR3.

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this ß-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based ß-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (K d = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.

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.

Aquaporin-4 and GPRC5B: old and new players in controlling brain oedema.

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.

Optimization of Peptide Linker-Based Fluorescent Ligands for the Histamine H1 Receptor.

The histamine H1 receptor (H1R) has recently been implicated in mediating cell proliferation and cancer progression; therefore, high-affinity H1R-selective fluorescent ligands are desirable tools for further investigation of this behavior in vitro and in vivo. We previously reported a H1R fluorescent ligand, bearing a peptide-linker, based on antagonist VUF13816 and sought to further explore structure-activity relationships (SARs) around the linker, orthostere, and fluorescent moieties. Here, we report a series of high-affinity H1R fluorescent ligands varying in peptide linker composition, orthosteric targeting moiety, and fluorophore. Incorporation of a boron-dipyrromethene (BODIPY) 630/650-based fluorophore conferred high binding affinity to our H1R fluorescent ligands, remarkably overriding the linker SAR observed in corresponding unlabeled congeners. Compound 31a, both potent and subtype-selective, enabled H1R visualization using confocal microscopy at a concentration of 10 nM. Molecular docking of 31a with the human H1R predicts that the optimized peptide linker makes interactions with key residues in the receptor.

Discovery of Diaryl Ether Substituted Tetrahydrophthalazinones as TbrPDEB1 Inhibitors Following Structure-Based Virtual Screening.

Several members of the 3',5'-cyclic nucleotide phosphodiesterase (PDE) family play an essential role in cellular processes, which has labeled them as interesting targets for various diseases. The parasitic protozoan Trypanosoma brucei, causative agent of human African trypanosomiasis, contains several cyclic AMP specific PDEs from which TbrPDEB1 is validated as a drug target. The recent discovery of selective TbrPDEB1 inhibitors has increased their potential for a novel treatment for this disease. Compounds characterized by a rigid biphenyl tetrahydrophthalazinone core structure were used as starting point for the exploration of novel TbrPDEB1 inhibitors. Using a virtual screening campaign and structure-guided design, diaryl ether substituted phthalazinones were identified as novel TbrPDEB1 inhibitors with IC50 values around 1 µM against T. brucei. This study provides important structure-activity relationship (SAR) information for the future design of effective parasite-specific PDE inhibitors.

Covalent Inhibition of the Histamine H3 Receptor.

Covalent binding of G protein-coupled receptors by small molecules is a useful approach for better understanding of the structure and function of these proteins. We designed, synthesized and characterized a series of 6 potential covalent ligands for the histamine H3 receptor (H3R). Starting from a 2-amino-pyrimidine scaffold, optimization of anchor moiety and warhead followed by fine-tuning of the required reactivity via scaffold hopping resulted in the isothiocyanate H3R ligand 44. It shows high reactivity toward glutathione combined with appropriate stability in water and reacts selectively with the cysteine sidechain in a model nonapeptide equipped with nucleophilic residues. The covalent interaction of 44 with H3R was validated with washout experiments and leads to inverse agonism on H3R. Irreversible binder 44 (VUF15662) may serve as a useful tool compound to stabilize the inactive H3R conformation and to study the consequences of prolonged inhibition of the H3R.

4-(3-Aminoazetidin-1-yl)pyrimidin-2-amines as High-Affinity Non-imidazole Histamine H3 Receptor Agonists with in Vivo Central Nervous System Activity.

Despite the high diversity of histamine H3 receptor (H3R) antagonist/inverse agonist structures, partial or full H3R agonists have typically been imidazole derivatives. An in-house screening campaign intriguingly afforded the non-imidazole 4-(3-azetidin-1-yl)pyrimidin-2-amine 11b as a partial H3R agonist. Here, the design, synthesis, and structure-activity relationships of 11b analogues are described. This series yields several non-imidazole full agonists with potencies varying with the alkyl substitution pattern on the basic amine following the in vitro evaluation of H3R agonism using a cyclic adenosine monophosphate response element-luciferase reporter gene assay. The key compound VUF16839 (14d) combines nanomolar on-target activity (pKi = 8.5, pEC50 = 9.5) with weak activity on cytochrome P450 enzymes and good metabolic stability. The proposed H3R binding mode of 14d indicates key interactions similar to those attained by histamine. In vivo evaluation of 14d in a social recognition test in mice revealed an amnesic effect at 5 mg/kg intraperitoneally. The excellent in vitro and in vivo pharmacological profiles and the non-imidazole structure of 14d make it a promising tool compound in H3R research.

The Landscape of Atypical and Eukaryotic Protein Kinases.

Kinases are attractive anticancer targets due to their central role in the growth, survival, and therapy resistance of tumor cells. This review explores the two primary kinase classes, the eukaryotic protein kinases (ePKs) and the atypical protein kinases (aPKs), and provides a structure-centered comparison of their sequences, structures, hydrophobic spines, mutation and SNP hotspots, and inhibitor interaction patterns. Despite the limited sequence similarity between these two classes, atypical kinases commonly share the archetypical kinase fold but lack conserved eukaryotic kinase motifs and possess altered hydrophobic spines. Furthermore, atypical kinase inhibitors explore only a limited number of binding modes both inside and outside the orthosteric binding site. The distribution of genetic variations in both classes shows multiple ways they can interfere with kinase inhibitor binding. This multilayered review provides a research framework bridging the eukaryotic and atypical kinase classes.

The human cytomegalovirus-encoded G protein-coupled receptor UL33 exhibits oncomodulatory properties.

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein-coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies.

Modulators of CXCR4 and CXCR7/ACKR3 Function.

The two G protein-coupled receptors (GPCRs) C-X-C chemokine receptor type 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are part of the class A chemokine GPCR family and represent important drug targets for human immunodeficiency virus (HIV) infection, cancer, and inflammation diseases. CXCR4 is one of only three chemokine receptors with a US Food and Drug Administration approved therapeutic agent, the small-molecule modulator AMD3100. In this review, known modulators of the two receptors are discussed in detail. Initially, the structural relationship between receptors and ligands is reviewed on the basis of common structural motifs and available crystal structures. To date, no atypical chemokine receptor has been crystallized, which makes ligand design and predictions for these receptors more difficult. Next, the selectivity, receptor activation, and the resulting ligand-induced signaling output of chemokines and other peptide ligands are reviewed. Binding of pepducins, a class of lipid-peptides whose basis is the internal loop of a GPCR, to CXCR4 is also discussed. Finally, small-molecule modulators of CXCR4 and ACKR3 are reviewed. These modulators have led to the development of radio- and fluorescently labeled tool compounds, enabling the visualization of ligand binding and receptor characterization both in vitro and in vivo. SIGNIFICANCE STATEMENT: To investigate the pharmacological modulation of CXCR4 and ACKR3, significant effort has been focused on the discovery and development of a range of ligands, including small-molecule modulators, pepducins, and synthetic peptides. Imaging tools, such as fluorescent probes, also play a pivotal role in the field of drug discovery. This review aims to provide an overview of the aforementioned modulators that facilitate the study of CXCR4 and ACKR3 receptors.

Chemokine Receptor Crystal Structures: What Can Be Learned from Them?

Chemokine receptors belong to the class A of G protein-coupled receptors (GPCRs) and are implicated in a wide variety of physiologic functions, mostly related to the homeostasis of the immune system. Chemokine receptors are also involved in multiple pathologic processes, including immune and autoimmune diseases, as well as cancer. Hence, several members of this GPCR subfamily are considered to be very relevant therapeutic targets. Since drug discovery efforts can be significantly reinforced by the availability of crystal structures, substantial efforts in the area of chemokine receptor structural biology could dramatically increase the outcome of drug discovery campaigns. This short review summarizes the available data on chemokine receptor crystal structures, discusses the numerous applications from chemokine receptor structures that can enhance the daily work of molecular pharmacologists, and describes the challenges and pitfalls to consider when relying on crystal structures for further research applications. SIGNIFICANCE STATEMENT: This short review summarizes the available data on chemokine receptor crystal structures, discusses the numerous applications from chemokine receptor structures that can enhance the daily work of molecular pharmacologists, and describes the challenges and pitfalls to consider when relying on crystal structures for further research applications.

Screening of a PDE-focused library identifies imidazoles with in vitro and in vivo antischistosomal activity.

We report the evaluation of 265 compounds from a PDE-focused library for their antischistosomal activity, assessed in vitro using Schistosoma mansoni. Of the tested compounds, 171 (64%) displayed selective in vitro activity, with 16 causing worm hypermotility/spastic contractions and 41 inducing various degrees of worm killing at 100 µM, with the surviving worms displaying sluggish movement, worm unpairing and complete absence of eggs. The compounds that did not affect worm viability (n = 72) induced a complete cessation of ovipositing. 82% of the compounds had an impact on male worms whereas female worms were barely affected. In vivo evaluation in S. mansoni-infected mice with the in vitro 'hit' NPD-0274 at 20 mg/kg/day orally for 5 days resulted in worm burden reductions of 29% and intestinal tissue egg load reduction of 35% at 10 days post-treatment. Combination of praziquantel (PZQ) at 10 mg/kg/day for 5 days with NPD-0274 or NPD-0298 resulted in significantly higher worm killing than PZQ alone, as well as a reduction in intestinal tissue egg load, disappearance of immature eggs and an increase in the number of dead eggs.

The constitutive activity of the virally encoded chemokine receptor US28 accelerates glioblastoma growth.

Glioblastoma (GBM) is the most aggressive and an incurable type of brain cancer. Human cytomegalovirus (HCMV) DNA and encoded proteins, including the chemokine receptor US28, have been detected in GBM tumors. US28 displays constitutive activity and is able to bind several human chemokines, leading to the activation of various proliferative and inflammatory signaling pathways. Here we show that HCMV, through the expression of US28, significantly enhanced the growth of 3D spheroids of U251- and neurospheres of primary glioblastoma cells. Moreover, US28 expression accelerated the growth of glioblastoma cells in an orthotopic intracranial GBM-model in mice. We developed highly potent and selective US28-targeting nanobodies, which bind to the extracellular domain of US28 and detect US28 in GBM tissue. The nanobodies inhibited chemokine binding and reduced the constitutive US28-mediated signaling with nanomolar potencies and significantly impaired HCMV/US28-mediated tumor growth in vitro and in vivo. This study emphasizes the oncomodulatory role of HCMV-encoded US28 and provides a potential therapeutic approach for HCMV-positive tumors using the nanobody technology.

Targeting a Subpocket in Trypanosoma brucei Phosphodiesterase B1 (TbrPDEB1) Enables the Structure-Based Discovery of Selective Inhibitors with Trypanocidal Activity.

Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, Trypanosoma brucei PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, and pharmacological evaluation and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, 8 (NPD-008) and 9 (NPD-039), were potent ( Ki = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC50 = 5.5 and 6.7 µM, respectively). Treatment of parasites with 8 caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of T. brucei cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis.

Molecular interaction fingerprint approaches for GPCR drug discovery.

Protein-ligand interaction fingerprints (IFPs) are binary 1D representations of the 3D structure of protein-ligand complexes encoding the presence or absence of specific interactions between the binding pocket amino acids and the ligand. Various implementations of IFPs have been developed and successfully applied for post-processing molecular docking results for G Protein-Coupled Receptor (GPCR) ligand binding mode prediction and virtual ligand screening. Novel interaction fingerprint methods enable structural chemogenomics and polypharmacology predictions by complementing the increasing amount of GPCR structural data. Machine learning methods are increasingly used to derive relationships between bioactivity data and fingerprint descriptors of chemical and structural information of binding sites, ligands, and protein-ligand interactions. Factors that influence the application of IFPs include structure preparation, binding site definition, fingerprint similarity assessment, and data processing and these factors pose challenges as well possibilities to optimize interaction fingerprint methods for GPCR drug discovery.

The viral G protein-coupled receptor ORF74 unmasks phospholipase C signaling of the receptor tyrosine kinase IGF-1R.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth factor (IGF)-1 receptor, a receptor tyrosine kinase, also plays an essential role in Kaposi's sarcoma growth and survival. In this study we examined the effect of the constitutively active viral receptor ORF74 on human IGF-1R signaling. Constitutive and CXCL1-induced ORF74 signaling did not transactivate IGF-1R. In contrast, IGF-1 stimulated phospholipase C (PLC) activation in an ORF74-dependent manner without affecting chemokine binding to ORF74. Inhibition of constitutive ORF74 activity by mutagenesis or the inverse agonist CXCL10, or neutralizing IGF-1R with an antibody or silencing IGF-1R expression using siRNA inhibited PLC activation by IGF-1. Transactivation of ORF74 in response to IGF-1 occurred independently of Src, PI3K, and secreted ORF74 ligands. Furthermore, tyrosine residues in the carboxyl-terminus and intracellular loop 2 of ORF74 are not essential for IGF-1-induced PLC activation. Interestingly, PLC activation in response to IGF-1 is specific for ORF74 as IGF-1 was unable to activate PLC in cells expressing the constitutively active human cytomegalovirus (HCMV)-encoded GPCR US28. Interestingly, IGF-1 does not induce ß-arrestin recruitment to ORF74. The proximity ligation assay revealed close proximity between ORF74 and IGF-1R on the cell surface, but a physical interaction was not confirmed by co-immunoprecipitation. Unmasking IGF-1R signaling to PLC in response to IGF-1 is a previously unrecognized action of ORF74.

Molecular Pharmacology of Chemokine Receptors.

Chemokine receptors are involved in various pathologies such as inflammatory diseases, cancer, and HIV infection. Small molecule and antibody-based antagonists have been developed to inhibit chemokine-induced receptor activity. Currently two small molecule inhibitors targeting CXCR4 and CCR5 are on the market for stem cell mobilization and the treatment of HIV infection, respectively. Antibody fragments (e.g., nanobodies) targeting chemokine receptors are primarily orthosteric ligands, competing for the chemokine binding site. This is opposed by most small molecules, which act as allosteric modulators and bind to the receptor at a topographically distinct site as compared to chemokines. Allosteric modulators can be distinguished from orthosteric ligands by unique features, such as a saturable effect and probe dependency. For successful drug development, it is essential to determine pharmacological parameters (i.e., affinity, potency, and efficacy) and the mode of action of potential drugs during early stages of research in order to predict the biological effect of chemokine receptor targeting drugs in the clinic. This chapter explains how the pharmacological profile of chemokine receptor targeting ligands can be determined and quantified using binding and functional experiments.