A time-resolved Förster resonance energy transfer assay to investigate drug and inhibitor binding to ABCG2.

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.

ThermoBRET: A Ligand-Engagement Nanoscale Thermostability Assay Applied to GPCRs.

Measurements of membrane protein thermostability reflect ligand binding. Current thermostability assays often require protein purification or rely on pre-existing radiolabelled or fluorescent ligands, limiting their application to established targets. Alternative methods, such as fluorescence-detection size exclusion chromatography thermal shift, detect protein aggregation but are not amenable to high-throughput screening. Here, we present a ThermoBRET method to quantify the relative thermostability of G protein coupled receptors (GPCRs), using cannabinoid receptors (CB1 and CB2 ) and the ß2 -adrenoceptor (ß2 AR) as model systems. ThermoBRET reports receptor unfolding, does not need labelled ligands and can be used with non-purified proteins. It uses Bioluminescence Resonance Energy Transfer (BRET) between Nanoluciferase (Nluc) and a thiol-reactive fluorescent dye that binds cysteines exposed by unfolding. We demonstrate that the melting point (Tm ) of Nluc-fused GPCRs can be determined in non-purified detergent solubilised membrane preparations or solubilised whole cells, revealing differences in thermostability for different solubilising conditions and in the presence of stabilising ligands. We extended the range of the assay by developing the thermostable tsNLuc by incorporating mutations from the fragments of split-Nluc (Tm of 87 °C versus 59 °C). ThermoBRET allows the determination of GPCR thermostability, which is useful for protein purification optimisation and drug discovery screening.

Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2.

The inhibition of CXC chemokine receptor 2 (CXCR2), a key inflammatory mediator, is a potential strategy in the treatment of several pulmonary diseases and cancers. The complexity of endogenous chemokine interaction with the orthosteric binding site has led to the development of CXCR2 negative allosteric modulators (NAMs) targeting an intracellular pocket near the G protein binding site. Our understanding of NAM binding and mode of action has been limited by the availability of suitable tracer ligands for competition studies, allowing direct ligand binding measurements. Here, we report the rational design, synthesis, and pharmacological evaluation of a series of fluorescent NAMs, based on navarixin (2), which display high affinity and preferential binding for CXCR2 over CXCR1. We demonstrate their application in fluorescence imaging and NanoBRET binding assays, in whole cells or membranes, capable of kinetic and equilibrium analysis of NAM binding, providing a platform to screen for alternative chemophores targeting these receptors.

Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta.

Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.

Development of fluorescent peptide G protein-coupled receptor activation biosensors for NanoBRET characterization of intracellular allosteric modulators.

G protein-coupled receptors (GPCRs) are widely therapeutically targeted, and recent advances in allosteric modulator development at these receptors offer further potential for exploitation. Intracellular allosteric modulators (IAM) represent a class of ligands that bind to the receptor-effector interface (e.g., G protein) and inhibit agonist responses noncompetitively. This potentially offers greater selectivity between receptor subtypes compared to classical orthosteric ligands. However, while examples of IAM ligands are well described, a more general methodology for assessing compound interactions at the IAM site is lacking. Here, fluorescent labeled peptides based on the Gα peptide C terminus are developed as novel binding and activation biosensors for the GPCR-IAM site. In TR-FRET binding studies, unlabeled peptides derived from the Gαs subunit were first characterized for their ability to positively modulate agonist affinity at the ß2 -adrenoceptor. On this basis, a tetramethylrhodamine (TMR) labeled tracer was synthesized based on the 19 amino acid Gαs peptide (TMR-Gαs19cha18, where cha = cyclohexylalanine). Using NanoBRET technology to detect binding, TMR-Gαs19cha18 was recruited to Gs coupled ß2 -adrenoceptor and EP2 receptors in an agonist-dependent manner, but not the Gi-coupled CXCR2 receptor. Moreover, NanoBRET competition binding assays using TMR-Gαs19cha18 enabled direct assessment of the affinity of unlabeled ligands for ß2 -adrenoceptor IAM site. Thus, the NanoBRET platform using fluorescent-labeled G protein peptide mimetics offers novel potential for medium-throughput screens to identify IAMs, applicable across GPCRs coupled to a G protein class. Using the same platform, Gs peptide biosensors also represent useful tools to probe orthosteric agonist efficacy and the dynamics of receptor activation.

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex.

A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and inflammation. ADAM10 is a "molecular scissor" that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.

The Peptide PnPP-19, a Spider Toxin Derivative, Activates µ-Opioid Receptors and Modulates Calcium Channels.

The synthetic peptide PnPP-19 comprehends 19 amino acid residues and it represents part of the primary structure of the toxin δ-CNTX-Pn1c (PnTx2-6), isolated from the venom of the spider Phoneutria nigriventer. Behavioural tests suggest that PnPP-19 induces antinociception by activation of CB1, µ and δ opioid receptors. Since the peripheral and central antinociception induced by PnPP-19 involves opioid activation, the aim of this work was to identify whether this synthetic peptide could directly activate opioid receptors and investigate the subtype selectivity for µ-, δ- and/or κ-opioid receptors. Furthermore, we also studied the modulation of calcium influx driven by PnPP-19 in dorsal root ganglion neurons, and analyzed whether this modulation was opioid-mediated. PnPP-19 selectively activates µ-opioid receptors inducing indirectly inhibition of calcium channels and hereby impairing calcium influx in dorsal root ganglion (DRG) neurons. Interestingly, notwithstanding the activation of opioid receptors, PnPP-19 does not induce ß-arrestin2 recruitment. PnPP-19 is the first spider toxin derivative that, among opioid receptors, selectively activates µ-opioid receptors. The lack of ß-arrestin2 recruitment highlights its potential for the design of new improved opioid agonists.

Optically Pure, Structural, and Fluorescent Analogues of a Dimeric Y4 Receptor Agonist Derived by an Olefin Metathesis Approach.

The dimeric peptide 1 (BVD-74D, as a diastereomeric mixture) is a potent and selective neuropeptide Y Y4 receptor agonist. It represents a valuable candidate in developing traceable ligands for pharmacological studies of Y4 receptors and as a lead compound for antiobesity drugs. Its optically pure stereoisomers along with analogues and fluorescently labeled variants were prepared by exploiting alkene metathesis reactions. The (2R,7R)-diaminosuberoyl containing peptide, (R,R)-1, had markedly higher affinity and agonist efficacy than its (S,S)-counterpart. Furthermore, the sulfo-Cy5 labeled (R,R)-14 retained high agonist potency as a novel fluorescent ligand for imaging Y4 receptors.

Plasma membrane dynamics and tetrameric organisation of ABCG2 transporters in mammalian cells revealed by single particle imaging techniques.

ABCG2 is one of three human ATP binding cassette (ABC) transporters involved in the export from cells of a chemically and structurally diverse range of compounds. This multidrug efflux capability, together with a broad tissue distribution in the body, means that ABCG2 exerts a range of effects on normal physiology such as kidney urate transport, as well as contributing towards the pharmacokinetic profiles of many exogenous drugs. The primary sequence of ABCG2 contains only half the number of domains required for a functioning ABC transporter and so it must oligomerise in order to function, yet its oligomeric state in intact cell membranes remains uncharacterized. We have analysed ABCG2 in living cell membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, and stepwise photobleaching to demonstrate a predominantly tetrameric structure for ABCG2 in the presence or absence of transport substrates. These results provide the essential basis for exploring pharmacological manipulation of oligomeric state as a strategy to modulate ABCG2 activity in future selective therapeutics.

Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis.

The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

Dimerization of ABCG2 analysed by bimolecular fluorescence complementation.

ABCG2 is one of three human ATP binding cassette transporters that are functionally capable of exporting a diverse range of substrates from cells. The physiological consequence of ABCG2 multidrug transport activity in leukaemia, and some solid tumours is the acquisition of cancer multidrug resistance. ABCG2 has a primary structure that infers that a minimal functional transporting unit would be a homodimer. Here we investigated the ability of a bimolecular fluorescence complementation approach to examine ABCG2 dimers, and to probe the role of individual amino acid substitutions in dimer formation. ABCG2 was tagged with fragments of venus fluorescent protein (vYFP), and this tagging did not perturb trafficking or function. Co-expression of two proteins bearing N-terminal and C-terminal fragments of YFP resulted in their association and detection of dimerization by fluorescence microscopy and flow cytometry. Point mutations in ABCG2 which may affect dimer formation were examined for alterations in the magnitude of fluorescence complementation signal. Bimolecular fluorescence complementation (BiFC) demonstrated specific ABCG2 dimer formation, but no changes in dimer formation, resulting from single amino acid substitutions, were detected by BiFC analysis.

Y(4) receptors mediate the inhibitory responses of pancreatic polypeptide in human and mouse colon mucosa.

The antisecretory effects of several Y agonists, including pancreatic polypeptide (PP), indicate the presence of Y(1), Y(2), and Y(4) receptors in mouse and human (h) colon mucosae. Here, we used preparations from human and from wild-type (WT), Y(4), and Y(1) receptor knockout ((-/-)) mice, alongside Y(4) receptor-transfected cells to define the relative functional contribution of the Y(4) receptor. First, rat (r) PP antisecretory responses were lost in murine Y(4)(-/-) preparations, but hPP and Pro(34) peptide YY (PYY) costimulated Y(4) and Y(1) receptors in WT mucosa. The Y(1) antagonist/Y(4) agonist GR231118 [(Ile,Glu,Pro,Dpr,Tyr,Arg,Leu,Arg,Try-NH(2))-2-cyclic(2,4'),(2',4)-diamide] elicited small Y(4)-mediated antisecretory responses in human tissues pretreated with the Y(1) antagonist, BIBO3304 [(R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N(2)-(diphenylacetyl)-argininamide trifluoroacetate)], and attenuated Y(4)-mediated hPP responses in mouse and human mucosa. GR231118 and rPP were also antisecretory in hY(4)-transfected epithelial monolayers but were partial agonists compared with hPP at this receptor. In Y(4)-transfected human embryonic kidney (HEK) 293 cells, Y(4) ligands displaced [(125)I]hPP binding with orders of affinity (pK(i)) at human (hPP = rPP > GR231118 > Pro(34)PYY = PYY) and mouse (rPP = hPP > GR231118 > Pro(34)PYY > PYY) Y(4) receptors. GR231118- and rPP-stimulated guanosine 5'-3-O-(thio)triphosphate binding through hY(4) receptors with significantly lower efficacy than hPP. GR231118 marginally increased basal but abolished further PP-induced hY(4) internalization to recycling (transferrin-labeled) pathways in HEK293 cells. Taken together, these findings show that Y(4) receptors play a definitive role in attenuating colonic anion transport and may be useful targets for novel antidiarrheal agents due to their limited peripheral expression.

Role of the C terminus in neuropeptide Y Y1 receptor desensitization and internalization.

We have studied truncation mutants of the rat neuropeptide Y (NPY) Y1 receptor lacking four (Thr361stop, Y1T361*) or eight (Ser352stop, Y1S352*) potential serine/threonine C-terminal phosphorylation sites. NPY-stimulated hemagglutinin-tagged Y1, Y1T361*, and Y1S352* receptors all efficiently activated G proteins in Chinese hamster ovary (CHO) cell membranes, but desensitization after NPY pretreatment was only prevented in the HAY1S352* clone. In transfected colonic carcinoma epithelial layers, functional Y1 and Y1T361* peptide YY responses became more transient as the agonist concentration increased, whereas those mediated by the Y1S352* receptor remained sustained. NPY-stimulated HAY1 receptor phosphorylation was increased by transient overexpression of G protein-coupled receptor kinase 2, and only Ser352stop truncation abolished this response in CHO or human embryonic kidney (HEK) 293 cells. Rapid internalization of cell-surface HAY1 receptors in HEK293 cells was observed in response to agonist, resulting in partial colocalization with transferrin, a marker for clathrin-mediated endocytosis and recycling. It is surprising that both truncated receptors were constitutively internalized, predominantly in transferrin-positive compartments. NPY increased cell-surface localization of HAY1S352* receptors, whereas the distribution of both mutants was unaltered by BIBO3304. Recruitment of green fluorescent protein-tagged beta-arrestin2 to punctate endosomes was observed only for HAY1 and HAY1T361* receptors and solely under NPY-stimulated conditions. Thus, the key C-terminal sequence between Ser352 and Lys360 is a major site for Y1 receptor phosphorylation, is critical for its desensitization, and contributes to the association between the receptor and beta-arrestin proteins. However, additional beta-arrestin-independent mechanisms control Y1 receptor trafficking under basal conditions.

Common structural basis for constitutive activity of the ghrelin receptor family.

Three members of the ghrelin receptor family were characterized in parallel: the ghrelin receptor, the neurotensin receptor 2 and the orphan receptor GPR39. In transiently transfected COS-7 and human embryonic kidney 293 cells, all three receptors displayed a high degree of ligand-independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G(q), phospholipase C pathway was approximately 50% of their maximal capacity as determined through inositol phosphate accumulation. These two receptors also showed very high constitutive activity in activation of cAMP response element-driven transcription. GPR39 displayed a clear but lower degree of constitutive activity through the inositol phosphate and cAMP response element pathways. In contrast, GPR39 signaled with the highest constitutive activity in respect of activation of serum response element-dependent transcription, in part, possibly, through G(12/13) and Rho kinase. Antibody feeding experiments demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically be tuned up and down depending on the size and hydrophobicity of the side chain in position VI:16 in the context of an aromatic residue at VII:09 and a large hydrophobic residue at VII:06. It is concluded that the three ghrelin-like receptors display an unusually high degree of constitutive activity, the structural basis for which is determined by an aromatic cluster on the inner face of the extracellular ends of TMs VI and VII.

Control of signalling efficacy by palmitoylation of the rat Y1 receptor.

(1) We have investigated the properties of native and haemagglutinin (HA)-tagged neuropeptide Y (NPY) Y(1) receptors after mutation of the palmitoylation site Cys(337) to Ser or Ala. (2) In Chinese hamster ovary cells expressing similar receptor levels, the C337A mutation abolished incorporation of [(3)H]palmitic acid into the HA-Y(1) receptor. (3) Cys(337) substitution did not alter the affinities of Y(1) receptor agonists or antagonists, but it eliminated the ability of guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) to displace [(125)I]PYY-specific binding (compared to approximately 50% inhibition in Y(1) or HA-Y(1) clones). (4) Stimulation of GTPgamma[(35)S] binding by native and HA-Y(1) receptors in standard incubation buffer (100 mM NaCl, 10 micro M GDP) was prevented by Cys(337) mutation. In this assay, the function of Y(1)(C337S) receptors could be partially rescued by reducing the Na(+) concentration, and when overexpressed (B(max): approximately 10 pmol mg(-1)), both HA-Y(1) and HA-Y(1)(C337A) receptors displayed similar responses to NPY and peptide YY (PYY). (5) In stably transfected adenocarcinoma cells expressing Y(1) or Y(1)(C337S) receptors, PYY inhibited anion secretion stimulated by vasoactive intestinal peptide (VIP; measured as short-circuit current, I(SC)) with similar potency (EC(50): 26-53 nM). In contrast to the transient Y(1) receptor-mediated responses observed at maximal PYY concentrations, I(SC) reductions in both Y(1)(C337S) clones were sustained. (6) We conclude that nonpalmitoylation of the Y(1) receptor reduces its coupling efficiency to G proteins, and may also indirectly influence desensitisation processes that depend on the formation of an active agonist-receptor conformation.