Delta-Opioid Receptor (δOR) Targeted Near-Infrared Fluorescent Agent for Imaging of Lung Cancer: Synthesis and Evaluation In Vitro and In Vivo.

In the United States, lung cancer is the leading cause of cancer death and ranks second in the number of new cases annually among all types of cancers. Better methods or tools for diagnosing and treating this disease are needed to improve patient outcomes. The delta-opioid receptor (δOR) is reported to be overexpressed in lung cancers and not expressed in normal lung. Thus, we decided to develop a lung cancer-specific imaging agent targeting this receptor. We have previously developed a δOR-targeted fluorescent imaging agent based on a synthetic peptide antagonist (Dmt-Tic) conjugated to a Cy5 fluorescent dye. In this work, we describe the synthesis of Dmt-Tic conjugated to a longer wavelength near-infrared fluorescent (NIRF) dye, Li-cor IR800CW. Binding affinity of Dmt-Tic-IR800 for the δOR was studied using lanthanide time-resolved fluorescence (LTRF) competitive binding assays in cells engineered to overexpress the δOR. In addition, we identified lung cancer cell lines with high and low endogenous expression of the δOR. We confirmed protein expression in these cell lines using confocal fluorescence microscopy imaging and used this technique to estimate the cell-surface receptor number in the endogenously expressing lung cancer cell lines. The selectivity of Dmt-Tic-IR800 for imaging of the δOR in vivo was shown using both engineered cell lines and endogenously expressing lung cancer cells in subcutaneous xenograft models in mice. In conclusion, the δOR-specific fluorescent probe developed in this study displays excellent potential for imaging of lung cancer.

Hetero-bivalent GLP-1/glibenclamide for targeting pancreatic ß-cells.

G protein-coupled receptor (GPCR) cell signalling cascades are initiated upon binding of a specific agonist ligand to its cell surface receptor. Linking multiple heterologous ligands that simultaneously bind and potentially link different receptors on the cell surface is a unique approach to modulate cell responses. Moreover, if the target receptors are selected based on analysis of cell-specific expression of a receptor combination, then the linked binding elements might provide enhanced specificity of targeting the cell type of interest, that is, only to cells that express the complementary receptors. Two receptors whose expression is relatively specific (in combination) to insulin-secreting pancreatic ß-cells are the sulfonylurea-1 (SUR1) and the glucagon-like peptide-1 (GLP-1) receptors. A heterobivalent ligand was assembled from the active fragment of GLP-1 (7-36 GLP-1) and glibenclamide, a small organic ligand for SUR1. The synthetic construct was labelled with Cy5 or europium chelated in DTPA to evaluate binding to ß-cells, by using fluorescence microscopy or time-resolved saturation and competition binding assays, respectively. Once the ligand binds to ß-cells, it is rapidly capped and presumably removed from the cell surface by endocytosis. The bivalent ligand had an affinity approximately fivefold higher than monomeric europium-labelled GLP-1, likely a result of cooperative binding to the complementary receptors on the ßTC3 cells. The high-affinity binding was lost in the presence of either unlabelled monomer, thus demonstrating that interaction with both receptors is required for the enhanced binding at low concentrations. Importantly, bivalent enhancement was accomplished in a cell system with physiological levels of expression of the complementary receptors, thus indicating that this approach might be applicable for ß-cell targeting in vivo.

Spectrophotometric determination and removal of unchelated europium ions from solutions containing Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates.

Europium chelates conjugated with peptide ligands are routinely used as probes for conducting in vitro binding experiments. The presence of unchelated Eu ions in these formulations gives high background luminescence and can lead to poor results in binding assays. In our experience, the reported methods for purification of these probes do not achieve adequate removal of unchelated metal ions in a reliable manner. In this work, a xylenol orange-based assay for the quantification of unchelated metal ions was streamlined and used to determine levels of metal ion contamination as well as the success of metal ion removal on attempted purification. We compared the use of Empore chelating disks and Chelex 100 resin for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Both purification methods gave complete and selective removal of the contaminant metal ions. However, Empore chelating disks were found to give much higher recoveries of the probes under the conditions used. Related to the issue of probe recovery, we also describe a significantly more efficient method for the synthesis of one such probe using Rink amide AM resin in place of Tentagel S resin.

Development of highly potent protease-activated receptor 2 agonists via synthetic lipid tethering.

Protease-activated receptor-2 (PAR2) is a G-protein coupled receptor (GPCR) associated with a variety of pathologies. However, the therapeutic potential of PAR2 is limited by a lack of potent and specific ligands. Following proteolytic cleavage, PAR2 is activated through a tethered ligand. Hence, we reasoned that lipidation of peptidomimetic ligands could promote membrane targeting and thus significantly improve potency and constructed a series of synthetic tethered ligands (STLs). STLs contained a peptidomimetic PAR2 agonist (2-aminothiazol-4-yl-LIGRL-NH2) bound to a palmitoyl group (Pam) via polyethylene glycol (PEG) linkers. In a high-throughput physiological assay, these STL agonists displayed EC50 values as low as 1.47 nM, representing a ∼200 fold improvement over the untethered parent ligand. Similarly, these STL agonists were potent activators of signaling pathways associated with PAR2: EC50 for Ca(2+) response as low as 3.95 nM; EC50 for MAPK response as low as 9.49 nM. Moreover, STLs demonstrated significant improvement in potency in vivo, evoking mechanical allodynia with an EC50 of 14.4 pmol. STLs failed to elicit responses in PAR2(-/-) cells at agonist concentrations of >300-fold their EC50 values. Our results demonstrate that the STL approach is a powerful tool for increasing ligand potency at PAR2 and represent opportunities for drug development at other protease activated receptors and across GPCRs.

Lanthanide labeling of a potent protease activated receptor-2 agonist for time-resolved fluorescence analysis.

Protease activated receptor-2 (PAR(2)) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR(2) can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR(2) to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR(2) peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH(2) (2-f-LIGRLO-dtpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO-dtpa is a potent and specific PAR(2) agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO-dtpa can be utilized in an affinity binding assay to evaluate the ligand-receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH(2), 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH(2), 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH(2), 6-an-LIGRL) and PAR(2). A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH(2), 3-ia-LIGRL) that does not activate PAR(2) signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR(2) binding. In summary, we have developed a europium-containing PAR(2) agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR(2) ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR(2) ligands.

A Synthetic Heterobivalent Ligand Composed of Glucagon-Like Peptide 1 and Yohimbine Specifically Targets ß Cells Within the Pancreas.

PURPOSE: ß Cell specificity for a heterobivalent ligand composed of glucagon-like peptide-1 (GLP-1) linked to yohimbine (GLP-1/Yhb) was evaluated to determine its utility as a noninvasive imaging agent. PROCEDURES: Competition binding assays were performed on ßTC3 cells and isolated rat islets. Immunostaining for insulin was used to co-localized intravenously injected Cy5-labeled GLP-1/Yhb in ß cells of Sprague-Dawley rats. Rats were intravenously injected with In-111-labeled GLP-1/Yhb to determine clearance rates and tissue biodistribution. Tissue-specific binding was confirmed by competition with pre-administration of unlabeled GLP-1/Yhb and in Streptozotocin-induced diabetic rats. RESULTS: In ßTC3 cells, high affinity binding of GLP-1/Yhb required interactions with both receptors because monovalent competition or receptor knockdown with RNAi lowered specificity and avidity of the heterobivalent ligand. Binding specificity for isolated islets was 2.6-fold greater than that of acinar tissue or islets pre-incubated with excess unlabeled GLP-1/Yhb. Immunofluorescent localization of Cy5-labeled GLP-1/Yhb was restricted to pancreatic islets. Within 30 min, ~90% of the In-111-labeled GLP-1/Yhb was cleared from blood. Tissue-specific accumulation of radiolabeled ligand was apparent in the pancreas, but not in other tissues within the abdominal imaging field. Pancreas specificity was lost in Streptozotocin-induced diabetic rats. CONCLUSIONS: The GLP-1/Yhb exhibits high specificity for ß cells, rapid blood clearance rates, and low non-specific uptake by other tissues within the abdominal imaging field. These characteristics of GLP-1/Yhb are desirable for application to ß cell imaging in vivo and provide a basis for developing additional multivalent ß cell-specific targeting agents to aid in the management of type 1 diabetes.

Sensitive and rapid isocratic liquid chromatography method for the quantitation of curcumin in plasma.

An HPLC assay was developed using three methods of plasma sample preparation in order to quantitate curcumin, the main constituent in the herbal dietary supplement turmeric. Each method involves simple and rapid processing of samples (either an ethyl acetate or chloroform extraction) with resulting different quantitation limits for curcumin. The assay was developed in an effort to quantify extremely low curcumin plasma concentrations observed in preliminary in vivo studies. The most sensitive assay can reliably detect concentrations down to 2.5 ng/ml. Plasma quantitation was precise and accurate based on both intra- and inter-day validations as indicated by low values for coefficients of variation and bias, respectively (< or =15%). The analytical validation was reproducible between different analysts. The resulting analytical method couples desired sensitivity with the ease of an isocratic system.