Controlled Delivery of Slit3 Proteins from Alginate Microbeads Inhibits In Vitro Angiogenesis.

BACKGROUND: The Slit-Robo pathway is a key regulator of angiogenesis and cellular function in experimental models. Slit3 proteins exhibit both proangiogenic and antiangiogenic properties, but the exact mechanism remains unclear. It is theorized that Slit3 may be a potential treatment for vascular diseases and cancer. METHODS: Slit3 labeled with I-125 was encapsulated in microbeads composed of low-viscosity alginate of high-glucuronic acid content, first coated with poly-L-ornithine for various durations and finally with low-viscosity high mannuronic acid. Gamma counter was used to measure microbead encapsulation efficiency and Slit3 release. Markers of angiogenesis were assessed with Boyden chamber, scratch wound, and Matrigel tube formation assays using human umbilical vein and mouse endothelial cells. RESULTS: On incubation of Slit3-loaded microbeads, there was an initial burst phase release of Slit3 for the first 24 h followed by sustained release for 6 to 12 d. Microbead composition determined encapsulation efficiency and rate of release; Slit3 encapsulation was most efficient in microbeads with lower low-viscosity alginate of high-glucuronic acid content concentrations (1.5%) and no poly-L-ornithine coating. Compared with controls (media alone), Slit3 microbeads significantly inhibited in vitro cellular migration, endothelial cell migration for wound closure at 24 and 48 h and endothelial tube formation (P < 0.001, respectively). CONCLUSIONS: Slit3 can be effectively encapsulated and delivered via a controlled release pattern using alginate microbeads. Microbead encapsulation reduces in vitro endothelial tube formation and inhibits cellular migration to impair angiogenesis. Thus, Slit3 microparticles may be explored as a therapeutic option to mitigate tumor proliferation.

Pharmacokinetic modeling reveals parameters that govern tumor targeting and delivery by a pH-Low Insertion Peptide (pHLIP).

A pH-Low Insertion Peptide (pHLIP) is a pH-sensitive peptide that undergoes membrane insertion, resulting in transmembrane helix formation, on exposure to acidity at a tumor cell surface. As a result, pHLIPs preferentially accumulate within tumors and can be used for tumor-targeted imaging and drug delivery. Here we explore the determinants of pHLIP insertion, targeting, and delivery through a computational modeling approach. We generate a simple mathematical model to describe the transmembrane insertion process and then integrate it into a pharmacokinetic model, which predicts the tumor vs. normal tissue biodistribution of the most studied pHLIP, "wild-type pHLIP," over time after a single intravenous injection. From these models, we gain insight into the various mechanisms behind pHLIP tumor targeting and delivery, as well as the various biological parameters that influence it. Furthermore, we analyze how changing the properties of pHLIP can influence the efficacy of tumor targeting and delivery, and we predict the properties for optimal pHLIP phenotypes that have superior tumor targeting and delivery capabilities compared with wild-type pHLIP.

Digestion and Transport across the Intestinal Epithelium Affects the Allergenicity of Ara h 1 and 3 but Not of Ara h 2 and 6.

SCOPE: No accepted and validated methods are currently available which can accurately predict protein allergenicity. In this study, the role of digestion and transport on protein allergenicity is investigated. METHODS AND RESULTS: Peanut allergens (Ara h 1, 2, 3, and 6) and a milk allergen (ß-lactoglobulin) are transported across pig intestinal epithelium using the InTESTine model and afterward basophil activation is measured to assess the (remaining) functional properties. Additionally, allergens are digested by pepsin prior to epithelial transport and their allergenicity is assessed in a human mast cell activation assay. Remarkably, transported Ara h 1 and 3 are not able to activate basophils, in contrast to Ara h 2 and 6. Digestion prior to transport results in a significant increase in mast cell activation of Ara h 1 and 3 dependent on the length of digestion time. Activation of mast cells by Ara h 2 and 6 is unaffected by digestion prior to transport. CONCLUSIONS: Digestion and transport influences the allergenicity of Ara h 1 and 3, but not of Ara h 2 and 6. The influence of digestion and transport on protein allergenicity may explain why current in vitro assays are not predictive for allergenicity.

Characterization of PROPPIN-Phosphoinositide Binding by Stopped-Flow Fluorescence Spectroscopy.

PROPPINs (ß-propellers that bind polyphosphoinositides) are a protein family that binds preferentially phosphatidylinositol 3-phosphate (PtdIns(3)P) and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) via its FRRG motif. PROPPINs are involved in autophagic functions, but their molecular mechanism is still elusive. To unravel the molecular mechanism of PROPPINs, it is essential to understand the PROPPIN-phosphoinositide binding. Here, we describe a protocol to study the kinetics of the PROPPIN-phosphoinositide binding using a fluorescence resonance energy transfer (FRET) stopped-flow approach. We use FRET between fluorophore-labeled protein and fluorophore-labeled liposomes, monitoring the increase of the acceptor emission in labeled liposomes after the protein-membrane binding. Through this approach, we studied the kinetics of the PROPPIN Atg18 (Autophagy-related protein 18) from Pichia angusta (PaAtg18) and a mutant of its FRRG motif, called FTTG mutant. Stopped-flow experiments demonstrated that the main function of the FRRG motif is to retain, instead of to drive, Atg18 to the membrane, decreasing the Atg18 dissociation rate. Furthermore, this method is suitable for the study of other PI-binding proteins.

Protein Delivery by Peptide-Based Stealth Liposomes: A Biomolecular Insight into Enzyme Replacement Therapy.

Infantile neural ceroid lipofuscinosis (INCL) is a lysosomal storage disorder characterized by mutations in the CLN1 gene that leads to lack of the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1), which causes the progressive death of cortical neurons. Enzyme replacement therapy (ERT) is one of the most promising treatments, but its translation toward a clinical use is hampered by the need to deliver the enzyme to the central nervous system and a more detailed understanding of its capability to restore physiologic conditions at the biochemical and protein level, beyond the simple regulation of enzymatic activity. Targeted nanoparticles can promote protein delivery to the central nervous system and affect biological pathways inside cells. Here, we describe an innovative peptide-based stealth nanoparticle that inhibits serum protein adsorption exploiting transferrin-driven internalization to convey the PPT1 enzyme to transferrin receptor-mediated pathways (endocytosis in this work, or transcytosis, in perspective, in vivo). These enzyme-loaded nanoparticles were able to restore stable levels of enzymatic activity in CLN1 patient's fibroblasts, comparable with the free enzyme, demonstrating that delivery after encapsulation in the nanocarrier does not alter uptake or intracellular trafficking. We also investigate, for the first time, dysregulated pathways of proteome and palmitoylome and their alteration upon enzyme delivery. Our nanoparticles were able of halving palmitoylated protein levels restoring conditions similar to the normal cells. From proteomic analysis, we also highlighted the reduction of the different groups of proteins after treatments with the free or encapsulated enzyme. In conclusion, our system is able to deliver the enzyme to a model of CLN1 disease restoring normal conditions in cells. Investigation of molecular details of pathologic state and enzyme-based correction reveals dysregulated pathways with unprecedented details for CLN1. Finally, we unveil for the first time the dysregulation landscape of palmitoylome and proteome in primary patient-derived fibroblasts and their modifications in response to enzyme administration. These findings will provide a guideline for the validation of future therapeutic strategies based on enzyme replacement therapy or acting at different metabolic levels.

pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery.

Fluorescence imaging has seen enduring use in blood flow visualization and is now finding a new range of applications in image-guided surgery. In this paper, we report a translational study of a new fluorescent agent for use in surgery, pHLIP ICG, where ICG (indocyanine green) is a surgical fluorescent dye used widely for imaging blood flow. We studied pHLIP ICG interaction with the cell membrane lipid bilayer, the pharmacology and toxicology in vitro and in vivo (mice and dogs), and the biodistribution and clearance of pHLIP ICG in mice. The pHLIP ICG tumor targeting and imaging efficacy studies were carried out in several murine and human mouse tumor models. Blood vessels were imaged in mice and pigs. Clinical Stryker imaging instruments for endoscopy and open surgery were used in the study. Intravenously administered pHLIP ICG exhibits a multi-hour circulation half-life, offering protracted delineation of vasculature. As it clears from the blood, pHLIP ICG targets tumors and tumor stroma, marking them for surgical removal. pHLIP ICG is non-toxic, marks blood flow for hours after injection, and effectively delineates tumors for improved resection on the day after administration.

The Effect of the Food Matrix on the In Vitro Bio-Accessibility and IgE Reactivity of Peanut Allergens.

SCOPE: Factors such as food processing, the food matrix, and antacid medication may affect the bio-accessibility of proteins in the gastrointestinal tract and hence their allergenic activity. However, at present they are poorly understood. METHODS AND RESULTS: Roasted peanut flour was incorporated into either a chocolate dessert or cookie matrix and bio-accessibility were assessed using an in vitro digestion system comprising a model chew and simulated gastric and duodenal digestion. Protein digestion was monitored by SDS-PAGE and immunoreactivity analyzed by immunoblotting and immunoassay. IgE reactivity was assessed by immunoassay using serum panels from peanut-allergic subjects. Roasted peanut flour proteins proved highly digestible following gastro-duodenal digestion even when incurred into a food matrix, with only low molecular weight polypeptides of Mr < 8 kDa remaining. When gastric digestion was performed at pH 6.5 (simulating the effect of antacid medication), peanut proteins are not digested; subsequent duodenal digestion is also limited. IgE reactivity of the major peanut allergens Ara h 1, Ara h 2, and Ara h 6, although reduced, was retained after oral-gastro-duodenal digestion irrespective of digestion conditions employed. CONCLUSION: Peanut allergen bio-accessibility is unaffected by the dessert or cookie matrices whilst high intra-gastric pH conditions render allergens more resistant to digestion.

Inhibition of allogeneic islet graft rejection by VISTA-conjugated liposome.

The Ig superfamily member V-domain Ig-containing suppressor of T-cell activation (VISTA) is a negative regulator with broad-spectrum activities and has reported that blockade of VISTA or combination with other negative checkpoint receptors sufficiently break tumor tolerance. However, it remains unclear whether VISTA could induce allogeneic T-cell hyporesponsiveness and inhibit allograft rejection. Here we found VISTA treatment significantly inhibited lymphocyte proliferation and activation in allogeneic MLR assay through impairing SYK-VAV pathway. Interestingly, though neither VISTA protein nor VISTA-Fc fusion protein administration exerted satisfactory immunosuppressive effect on allograft survival due to their short half-life in circulation, this problem was solved by conjugating VISTA protein on liposome by biotin-streptavidin system, which markedly prolonged its circulating half-life to 60 h. With islet transplant model, administration of VISTA-conjugated liposome could markedly prolong allograft survival by inhibition of SYK-VAV pathway, thus maintained the normal blood glucose level of recipients during treatment period. The results indicate VISTA is a promising therapeutic target to treat allograft rejection of islet transplantation.

Efficient Delivery of Tyrosinase Related Protein-2 (TRP2) Peptides to Lymph Nodes using Serum-Derived Exosomes.

Exosomes (EXO) are considered to be versatile carriers for biomolecules; however, the delivery of therapeutic peptides using EXOs poses several challenges. In this study, the efficiency of serum-derived EXOs in delivering tyrosinase-related protein-2 (TRP2) peptides to lymph nodes is determined. TRP2 peptides are successfully incorporated into EXOs, which show a uniform and narrow size distribution of around 45 nm. The TRP2-incorporated exosomes (EXO-TRP2) are efficiently internalized into macrophages and dendritic cells, and are seen to display a punctate distribution. EXOs loaded with TRP2 together with MPLA, (EXO-MPLA-TRP2) result in a strong release of proinflammatory cytokines (TNF-α and IL-6) from both RAW264.7 and DC2.4 cells. Finally, subcutaneous injection of fluorescently labeled EXO-TRP2 followed by ex vivo imaging using in vivo imaging system (IVIS) show a strong fluorescent signal in the lymph nodes after only 1 h, which is maintained until at least 4 h after injection. Taken together, the findings suggest that serum-derived EXOs can serve as promising carriers to deliver therapeutic peptides to lymph nodes for immunotherapy.

Diagnostic Utility of Prostein, Uroplakin II and SATB2 for Diagnosing Carcinoma of Unknown Primary Origin: A Systematic Immunohistochemical Profiling.

BACKGROUND/AIM: Immunohistochemistry was used to evaluate 600 carcinomas of major histological types from various organs to determine the tissue distributions of the novel markers prostein, uroplakin II and SATB2. MATERIALS AND METHODS: We retrieved 30 cases from 20 different carcinomas of systemic organs. RESULTS: All prostate adenocarcinomas were immunopositive for prostein, and its reactivity was consistently diffuse. There was faint labeling of prostein in few cases of the 570 non-prostatic carcinomas. Uroplakin II was immunopositive in 53% and 60% of urothelial carcinomas (UC) of the bladder and the ureter, respectively. There was focal and weak positivity of uroplakin II in a few cases of non-urinary tract carcinomas. SATB2 was frequently positive in adenocarcinomas of the digestive organs, and was also expressed in a minority of the non-colorectal adenocarcinomas. CONCLUSION: Prostein and uroplakin II are immunohistochemical biomarkers of prostate adenocarcinomas and UCs of the urinary tract.

Role of Extracellular Vesicle Surface Proteins in the Pharmacokinetics of Extracellular Vesicles.

Extracellular vesicles (EVs) are small membrane vesicles secreted from cells and have great potential as drug delivery carriers. Surface proteins on EV membranes might play roles in pharmacokinetics. One method which can be used to study the role of surface membrane of EV is to modify the inner space of EV. In the present study, we constructed a plasmid DNA expressing a fusion protein of Gag protein derived from Moloney murine leukemia virus (Gag) and Gaussia luciferase (gLuc) (Gag-gLuc) to modify the inner space of EVs. EVs were collected from B16BL6 melanoma cells, transfected with the plasmid, and isolated by a differential ultracentrifugation method. Gag-gLuc EVs were negatively charged globular vesicles with a diameter of approximately 100 nm. gLuc labeling of the Gag-gLuc EVs was stable in serum. gLuc activity of Gag-gLuc EVs was minimally decreased by proteinase K (ProK) treatment, indicating that gLuc was modified in the inner space of EV. Then, to evaluate the effect of the surface proteins of EVs on their pharmacokinetics, Gag-gLuc EVs treated with ProK were intravenously administered to mice. Volume of distribution (Vd) was significantly smaller for treated EVs than untreated EVs. Moreover, integrin α6ß1, an integrin known to be involved in lung targeting, was degraded after ProK treatment. The ProK treatment significantly reduced the lung distribution of EVs after intravenous injection. These results indicate that the surface proteins of EVs such as integrin α6ß1 play some roles in pharmacokinetics in terms of reducing Vd and their distribution to the lung.

PET Imaging of Extracellular pH in Tumors with (64)Cu- and (18)F-Labeled pHLIP Peptides: A Structure-Activity Optimization Study.

pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides ((64)Cu and (18)F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with (64)Cu or [(18)F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either (18)F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or (64)Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on (18)F- or (64)Cu-labeled NO2A-cysVar3.

Cellular Uptake and Ultrastructural Localization Underlie the Pro-apoptotic Activity of a Hydrocarbon-stapled BIM BH3 Peptide.

Hydrocarbon stapling has been applied to restore and stabilize the α-helical structure of bioactive peptides for biochemical, structural, cellular, and in vivo studies. The peptide sequence, in addition to the composition and location of the installed staple, can dramatically influence the properties of stapled peptides. As a result, constructs that appear similar can have distinct functions and utilities. Here, we perform a side-by-side comparison of stapled peptides modeled after the pro-apoptotic BIM BH3 helix to highlight these principles. We confirm that replacing a salt-bridge with an i, i + 4 hydrocarbon staple does not impair target binding affinity and instead can yield a biologically and pharmacologically enhanced α-helical peptide ligand. Importantly, we demonstrate by electron microscopy that the pro-apoptotic activity of a stapled BIM BH3 helix correlates with its capacity to achieve cellular uptake without membrane disruption and accumulate at the organellar site of mechanistic activity.

Mixed nanomicelles as potential carriers for systemic delivery of Z-GP-Dox, an FAPα-based doxorubicin prodrug: formulation and pharmacokinetic evaluation.

Z-GP-Dox, the FAPα (fibroblast activation protein-α)-based doxorubicin prodrug, demonstrates excellent tumor targeting effects and a favorable toxicokinetic profile. However, the insoluble nature of Z-GP-Dox becomes a significant barrier to drug administration, particularly when it comes to the clinical stage. Here we developed a nanomicelle system to facilitate the systemic delivery of Z-GP-Dox, and evaluated its disposition in rats following administration of the micelles using a physiologically-based pharmacokinetic model. Z-GP-Dox-loaded mixed nanomicelles (ZGD-MNs) were prepared by dispersion of an ethanol solution of Z-GP-Dox, lecithin, and sodium oleate in water. The obtained ZGD-MNs were 86.6 nm in size with a drug loading of 14.03%. ZGD-MNs were fairly stable in phosphate-buffered saline and showed satisfactory physical and chemical stability over a 2-week observation period. Accumulative drug release was more than 56% within 24 hours. Further, the physiologically-based pharmacokinetic rat model consisting of various organs (ie, heart, liver, spleen, lung, kidney, and intestine) was fitted to the experimental data following administration of ZGD-loaded cosolvent (control) or micelles. Derived partition coefficient values revealed that the nanomicelles significantly altered the biodistribution of Z-GP-Dox. Of note, drug distribution to the lung, liver, and spleen was greatly enhanced and the fold change ranged from 2.4 to 33. In conclusion, this is the first report of a mixed micelle system being a viable carrier for delivery of Z-GP-Dox. Also, the pharmacokinetic behavior of Z-GP-Dox was satisfactorily described by the physiologically-based pharmacokinetic model.

Pharmacokinetics and toxicology of a fibroblast activation protein (FAP)-activated prodrug in murine xenograft models of human cancer.

BACKGROUND: As carcinoma progresses, the stroma undergoes a variety of phenotypic changes, including the presence of carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). FAP is a post-prolyl endopeptidase whose expression in a healthy adult is largely restricted to the cancer-associated stroma. FAP-targeted prodrugs with a 100-fold greater therapeutic window over the parent compound were previously generated. METHODS: Prodrugs and non-cleavable controls were incubated in the presence of FAP. Plasma and tumor half-lives (t1/2) of the full-length and active forms of the prodrugs were determined using LCMS. Biodistribution studies of prodrug activation were performed. Histopathological analysis of tissues from treated animals were compared to vehicle-treated controls. Toxicity and efficacy studies were performed in human breast (MDA-MB-231 and MCF-7) and prostate (LNCaP) cancer xenografts models. RESULTS: These FAP-activated prodrugs have a significantly slower clearance from tumor tissue than the circulation (∼12 vs. ∼4.5 hr). Micromolar concentrations of active drug persist in the tumor. Active drug is detected in non-target tissues; however, histopathologic evaluation reveals no evidence of drug-induced toxicity. A FAP-activated prodrug (ERGETGP-S12ADT) inhibits tumor growth in multiple human breast and prostate cancer xenograft models. The anti-tumor effect is comparable to that observed with docetaxel, but results in significantly less toxicity. CONCLUSION: FAP-activated prodrugs are a viable strategy for the management of prostate and other cancers. These prodrugs exhibit less toxicity than a commonly used chemotherapeutic agent. Further refinement of the FAP cleavage site for greater specificity may reduce prodrug activation in non-target tissues and enhance clinical benefit.

Kidins220/ARMS is dynamically expressed during Xenopus laevis development.

Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin Repeat-rich Membrane Spanning) is a conserved scaffold protein that acts as a downstream substrate for protein kinase D and mediates multiple receptor signalling pathways. Despite the dissecting of the function of this protein in mammals, using both in vitro and in vivo studies, a detailed characterization of its gene expression during early phases of embryogenesis has not been described yet. Here, we have used Xenopus laevis as a vertebrate model system to analyze the gene expression and the protein localization of Kidins220/ARMS. We found its expression was dynamically regulated during development. Kidins220/ARMS mRNA was expressed from neurula to larval stage in different embryonic regions including the nervous system, eye, branchial arches, heart and somites. Similar to the transcript, the protein was present in multiple embryonic domains including the central nervous system, cranial nerves, motor nerves, intersomitic junctions, retinal ganglion cells, lens, otic vesicle, heart and branchial arches. In particular, in some regions such as the retina and somites, the protein displayed a differential localization pattern in stage 42 embryos when compared to the earlier examined stages. Taken together our results suggest that this multidomain protein is involved in distinct spatio-temporal differentiative events.

Peptide targeting and imaging of damaged lung tissue in influenza-infected mice.

AIM: In this study, we investigate whether pH (low) insertion peptide (pHLIP) can target regions of lung injury associated with influenza infection. MATERIALS & METHODS: Fluorophore-conjugated pHLIP was injected intraperitoneally into mice infected with a sublethal dose of H1N1 influenza and visualized histologically. RESULTS: pHLIP specifically targeted inflamed lung tissues of infected mice in the later stages of disease and at sites where alveolar type I and type II cells were depleted. Regions of pHLIP-targeted lung tissue were devoid of peroxiredoxin 6, the lung-abundant antioxidant enzyme, and were deficient in pneumocytes. Interestingly, a pHLIP variant possessing mutations that render it insensitive to pH changes was also able to target damaged lung tissue. CONCLUSION: pHLIP holds potential for delivering therapeutics for lung injury during influenza infection. Furthermore, there may be more than one mechanism that enables pHLIP variants to target inflamed lung tissue.

Polymeric delivery of therapeutic RAE-1 plasmid to the pancreatic islets for the prevention of type 1 diabetes.

The activating receptor NKG2D plays an important role in the development of type-1 diabetes. Exploiting a natural phenomenon observed in tumors, plasmid DNA encoding for a soluble ligand to NKG2D (sRAE-1γ) was isolated and engineered into a plasmid expression system. A polymeric gene delivery system was developed to deliver the soluble RAE-1 plasmid to the pancreatic islets. The bioreducible cationic polymer poly(cystamine bisacrylamide-diamino hexane) (p(CBA-DAH)) was modified with poly(ethylene glycol) (PEG) and the targeting peptide CHVLWSTRC, known to target the EphA2 and EphA4 receptors. We observed a higher uptake of the targeting polymer Eph-PEG-p(CBA-DAH) in the pancreas of NOD mice compared to non-targeting controls. To evaluate the efficacy of preventing diabetes, the Eph-PEG-p(CBA-DAH)/RAE-1 complex (polyplex) was intravenously injected into 6-week-old female NOD mice. Within 17 weeks blood glucose levels were stabilized in animals injected with polyplex, while those treated without therapeutic plasmid developed progressive hyperglycemia. Additionally, the degree of insulitis and the infiltration of CD8⁺ T-cells in the polyplex treated group were improved over the targeting polymer only treated group. The current study suggests that the therapy of the Eph-PEG-p (CBA-DAH) delivering therapeutic sRAE-1 gene may be used to protect ß-cells from autoimmune destruction and prevent type-1 diabetes.

Efficient (18)F-labeling of large 37-amino-acid pHLIP peptide analogues and their biological evaluation.

Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pH(e) < 7). Labeling of peptides with [(18)F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known "click" methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC "click chemistry" for the simple and efficient (18)F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and an L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[(18)F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥98%. The subsequent Cu(I)-catalyzed "click" reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [(18)F]-D-WT-pHLIP and [(18)F]-L-K-pHLIP were obtained with total radiochemical yields of 5-20% after HPLC purification. The total reaction time was 85 min including formulation. In vitro stability tests revealed high stability of the [(18)F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65% and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [(18)F]-D-WT-pHLIP and the negative control [(18)F]-L-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be useful for the (18)F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [(18)F]-pHLIP analogues as potential PET tracers.

FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development.

Latrophilins (LPHNs) are a small family of G protein-coupled receptors known to mediate the massive synaptic exocytosis caused by the black widow spider venom α-latrotoxin, but their endogenous ligands and function remain unclear. Mutations in LPHN3 are strongly associated with attention deficit hyperactivity disorder, suggesting a role for latrophilins in human cognitive function. Using affinity chromatography and mass spectrometry, we identify the FLRT family of leucine-rich repeat transmembrane proteins as endogenous postsynaptic ligands for latrophilins. We demonstrate that the FLRT3 and LPHN3 ectodomains interact with high affinity in trans and that interference with this interaction using soluble recombinant LPHN3, LPHN3 shRNA, or FLRT3 shRNA reduces excitatory synapse density in cultured neurons. In addition, reducing FLRT3 levels with shRNA in vivo decreases afferent input strength and dendritic spine number in dentate granule cells. These observations indicate that LPHN3 and its ligand FLRT3 play an important role in glutamatergic synapse development.