Barrie Kellam

Genetic tagging of G protein-coupled receptors (GPCRs) with bioluminescent or fluorescent proteins is a well-established method for the study of ligand-binding and protein-protein interactions using resonance energy transfer approaches. Here we present a two-step, ligand-directed covalent labeling (LDCL) method that allows attachment of different fluorescent labels to an untagged adenosine A₁ receptor using click chemistry. We also describe a range of biophysical approaches to confirm that the orthosteric binding site remains available to interact with endogenous ligands, agonists and antagonists, and access to the orthosteric binding site is not sterically hindered by the transferred cargo (fluorophore or click-reactive group).

The study of protein function and dynamics in their native cellular environment is essential for progressing fundamental science. To overcome the requirement of genetic modification of the protein or the limitations of dissociable fluorescent ligands, ligand-directed (LD) chemistry has most recently emerged as a complementary, bioorthogonal approach for labeling native proteins. Here, we describe the rational design, development, and application of the first ligand-directed chemistry approach for labeling the A₁AR in living cells. We pharmacologically demonstrate covalent labeling of A₁AR expressed in living cells while the orthosteric binding site remains available. The probes were imaged using confocal microscopy and fluorescence correlation spectroscopy to study A₁AR localization and dynamics in living cells. Additionally, the probes allowed visualization of the specific localization of A₁ARs endogenously expressed in dorsal root ganglion (DRG) neurons. LD probes developed here hold promise for illuminating ligand-binding, receptor signaling, and trafficking of the A₁AR in more physiologically relevant environments.

Fluorescent ligands have proved to be powerful tools in the study of G protein‐coupled receptors in living cells. Here we have characterized a new fluorescent ligand PSB603‐BY630 that has high selectivity for the human adenosine A_2B receptor (A_2BR). The A_2BR appears to play an important role in regulating immune responses in the tumor microenvironment. Here we have used PSB603‐BY630 to monitor specific binding to A_2BRs in M1‐ and M2‐like macrophages derived from CD14+ human monocytes. PSB603‐BY630 bound with high affinity (18.3 nM) to nanoluciferase‐tagged A_2BRs stably expressed in HEK293G cells. The ligand exhibited very high selectivity for the A_2BR with negligible specific‐binding detected at NLuc‐A_2AR, NLuc‐A₁R, or NLuc‐A₃R receptors at concentrations up to 500 nM. Competition binding studies showed the expected pharmacology at A_2BR with the A_2BR‐selective ligands PSB603 and MRS‐1706 demonstrating potent inhibition of the specific binding of 50 nM PSB603‐BY630 to A_2BR. Functional studies in HEK293G cells using Glosensor to monitor G_s‐coupled cyclic AMP responses indicated that PSB603‐BY630 acted as a negative allosteric regular of the agonist responses to BAY 60–6583. Furthermore, flow cytometry analysis confirmed that PSB603‐BY630 could be used to selectively label endogenous A_2BRs expressed on human macrophages. This ligand should be an important addition to the library of fluorescent ligands which are selective for the different adenosine receptor subtypes, and will enable study of the role of A_2BRs on immune cells in the tumor microenvironment.

CXCL17 is a chemokine principally expressed by mucosal tissues, where it facilitates chemotaxis of monocytes, dendritic cells, and macrophages and has antimicrobial properties. CXCL17 is also implicated in the pathology of inflammatory disorders and progression of several cancers, and its expression is increased during viral infections of the lung. However, the exact role of CXCL17 in health and disease requires further investigation, and there is a need for confirmed molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET)–based assays, here we demonstrated that CXCL17 inhibited CXCR4-mediated signaling and ligand binding. Moreover, CXCL17 interacted with neuropillin-1, a VEGFR2 coreceptor. In addition, we found that CXCL17 only inhibited CXCR4 ligand binding in intact cells and demonstrated that this effect was mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. Disruption of putative GAG binding domains in CXCL17 prevented CXCR4 binding. This indicated that CXCL17 inhibited CXCR4 by a mechanism of action that potentially required the presence of a glycosaminoglycan-containing accessory protein. Together, our results revealed that CXCL17 is an endogenous inhibitor of CXCR4 and represents the next step in our understanding of the function of CXCL17 and regulation of CXCR4 signaling.

Pseudomonas aeruginosa is one of the top priority pathogens that requires immediate attention according to the World Health Organisation (WHO). Due to the alarming shortage of novel antimicrobials, targeting quorum sensing (QS), a bacterial cell to cell signaling system controlling virulence, has emerged as a promising approach as an antibiotic adjuvant therapy. Interference with the pqs system, one of three QS systems in P. aeruginosa, results in reduction of bacterial virulence gene expression and biofilm maturation. Herein, we report a hit to lead process to fine-tune the potency of our previously reported inhibitor 1 (IC₅₀ 3.2 μM in P. aeruginosa PAO1-L), which led to the discovery of 2-(4-(3-((6-chloro-1-isopropyl-1H-benzo[d]imidazol-2-yl)amino)-2-hydroxypropoxy)phenyl)acetonitrile (6f) as a potent PqsR antagonist. Compound 6f inhibited the PqsR-controlled P_pqsA-lux transcriptional reporter fusion in P. aeruginosa at low submicromolar concentrations. Moreover, 6f showed improved efficacy against P. aeruginosa CF isolates with significant inhibition of pyocyanin, 2-alkyl-4(1H)-quinolones production.

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 (pK_d 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.

The human ATP-binding cassette (ABC) transporter, ABCG2 is responsible for multidrug resistance in some tumours. Detailed knowledge of its activity is crucial for understanding drug transport and resistance in cancer, and has implications for wider pharmacokinetics. The binding of substrates and inhibitors is a key stage in the transport cycle of ABCG2. Here, we describe a novel binding assay using a high affinity fluorescent inhibitor based on Ko143 and time-resolved Förster resonance energy transfer (TR-FRET) to measure saturation binding to ABCG2. This binding is displaced by Ko143 and other known ABCG2 ligands, and is sensitive to the addition of AMP-PNP, a non-hydrolysable ATP analogue. This assay complements the arsenal of methods for determining drug:ABCG2 interactions and has the possibility of being adaptable for other multidrug pumps. Highlights: ABCG2 is a multidrug pump which moves between states having low or high affinity for substrates and inhibitors We introduce a time-resolved Förster resonance energy transfer assay to measure interaction of substrates and inhibitors to ABCG2 We confirm that NBD dimerization is associated with a switch from a high to a low affinity site for an ABCG2 inhibitor

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.