Site-specific conjugation of the quencher on peptide's N-terminal for the synthesis of a targeted non-spreading activatable optical probe.

Optical imaging offers high sensitivity and portability at low cost. The design of 'smart' or 'activatable' probes can decrease the background noise and increase the specificity of the signal. By conjugating a fluorescent dye and a compatible quencher on each side of an enzyme's substrate, the signal remains in its 'off ' state until it reaches the area where a specific enzyme is expressed. However, the signal can leak from that area unless the dye is attached to a molecule able to bind to a specific target also presented in that area. The aim of this study was to (i) specifically conjugate the quencher on the α-amino group of the peptide's N-terminus, (ii) conjugate the dye on the ε-amino group of a lysine in C-terminus, and (iii) conjugate the carboxyl group of the peptide's C-terminus to an amino group present on an antibody, using carbodiimide chemistry. The use of protecting groups, such as Boc or Fmoc, to allow site-specific conjugation, presents several drawbacks including 'on beads labeling', additional steps required for deprotection and removal from the resin, decreased yield, and dye degradation. A method of preferential labeling of α-amino N-terminal group in slightly acidic solution, proposed by Selo et al. (1996) has partially solved the problem. The present study reports improvements of the method allowing to (i) avoid the homo-bilabeling, (ii) increase the yield of the N-terminal labeling by two folds, and (iii) decrease the cost by 44-fold. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Optimal dye-quencher pairs for the design of an "activatable" nanoprobe for optical imaging.

Optical imaging offers high sensitivity and portability at low cost. The design of an optimal "activatable" imaging agent could greatly decrease the background noise and increase specificity of the signal. Five different molecules have been used to quench basal fluorescence of an enzyme substrate labeled with Cy5, Cy5.5 or IR800 at a distance of 8 amino acids (32 Å): a 6 nm gold nanoparticle (NP), a 20 nm and a 30 nm iron oxide (FeO) NP, the black hole quencher BHQ-3 and the IRdye quencher QC-1. The quenching efficiencies were 99% for QC1-IR800, 98% for QC1-Cy5.5, 96% for 30 nm FeO NP-Cy5.5, 89% for BHQ3-Cy5, 84% for BHQ3-Cy5.5, 77-90% for 6 nm gold NP-Cy5.5, depending on the number of dyes around the NP, 79% for 20 nm FeO NP-Cy5.5 and 77% for Cy5.5-Cy5. Signal activation upon cleavage by the matrix metalloproteinase MMP9 was proportional to the quenching efficiencies, ranging from 3-fold with Cy5.5-Cy5 to 67-fold with QC1-IR800. This independent work reports on the properties of the dyes and quenchers explaining the superior performance of QC-1 and 30 nm FeO NPs.

Shark cartilage extract induces cytokines expression and release in endothelial cells and induces E-selectin, plasminogen and t-PA genes expression through an antioxidant-sensitive mechanism.

Neovastat® is a standardized extract of marine cartilage, an avascular tissue, which contains many biologically active molecules and has multiple antiangiogenic properties. In addition to VEGFR2 and MMPs inhibition, shark cartilage extract (SCE) has recently been shown to induce tissue plasminogen activator gene (PLAT) expression in bovine endothelial cells in a TNF like manner, by inducing the typical mediators NF-κB and JNK. There is now compelling evidences that the NF-κB and JNK pathways are activated by cytokines induced generation of reactive oxygen species (ROS). We used macroarray genes expression analysis on human umbilical vein endothelial cells, to investigate if that mechanism could mediate the effect of SCE. Transcriptomic results showed that SCE induced expression of several cytokines. Their impact must be important, given that treatment of endothelial cells with the cytokine TNF-α was able to reproduce most of the effects of cartilage extract on genes expression. In addition, most of the genes, known to be inducible by NF-κB or JNK following cytokines stimulation, were less induced by SCE when endothelial cells were pretreated with the antioxidant N-Acetylcysteine (NAC), suggesting a role of ROS in endothelial cell activation by SCE. Finally, the possible effects of PLAT, PLG, SELE, IL8 and PRDX2 (those validated by q-PCR) on angiogenesis, will also be discussed.

Induction of the fibrinolytic system by cartilage extract mediates its antiangiogenic effect in mouse glioma.

Both the antiangiogenic and antitumoral activity of shark cartilage extracts (SCE) have been demonstrated in animal models and clinical trials. Studies reported that SCE induces the expression of tissue plasminogen activator gene (PLAT) in endothelial cells and increases the activity of the protein (t-PA) in vitro. The aim of this study was to demonstrate the crucial role of t-PA induction in the antiangiogenic and antitumor activity of SCE in experimental glioma. This study showed antiangiogenic and antitumoral effects of SCE in three mice glioma models (C6, HGD and GL26). Histological examination suggested perivascular proteolysis and edema as well as important intratumoral necrosis, which artefactually increased the tumor volume at high doses. Thus, the antiangiogenic effect of SCE correlated with the presence of t-PA and angiostatin in degenerating vessels. Functional in vivo experiments were conducted to modulate the plasminogen pathway. No antiangiogenic effect was observed on tumors overexpressing the plasminogen activator inhibitor-1 (PAI-1). Moreover, therapeutical effects were neutralized in mice that were cotreated with ε-aminocaproic acid (EACA, 120 mg/kg p.o.), an inhibitor that blocks the high-affinity lysine binding sites of both plasminogen and plasmin. In contrast, cotreatment with N-acetylcysteine (NAC, 7,5mg/kg i.p.), a sulfhydril donor that reduces plasmin into angiostatin or other antiangiogenic fragments, increased the benefit of SCE on mice survival. In subcutaneous models, NAC prevented the increase in tumor volume caused by high doses of cartilage extract. In conclusion, this study indicates that induction of t-PA by shark cartilage extract plays an essential role in its antiangiogenic activity, but that control of excessive proteolysis by a plasmin reductor could prevent edema and uncover the full benefit of shark cartilage extract in the treatment of intracranial tumors.

Identification, characterization and potent antitumor activity of ECO-4601, a novel peripheral benzodiazepine receptor ligand.

PURPOSE: ECO-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through DECIPHER technology, Thallion's proprietary drug discovery platform. The compound was shown to have a broad cytotoxic activity in the low micromolar range when tested in the NCI 60 cell line panel. In the work presented here, ECO-4601 was further evaluated against brain tumor cell lines. Preliminary mechanistic studies as well as in vivo antitumor evaluation were performed. METHODS: Since ECO-4601 has a benzodiazepinone moiety, we first investigated if it binds the central and/or peripheral benzodiazepine receptors. ECO-4601 was tested in radioligand binding assays on benzodiazepine receptors obtained from rat hearts. The ability of ECO-4601 to inhibit the growth of CNS cancers was evaluated on a panel of mouse, rat and human glioma cell lines using a standard MTT assay. Antitumor efficacy studies were performed on gliomas (rat and human), human breast and human prostate mouse tumor xenografts. Antitumor activity and pharmacokinetic analysis of ECO-4601 was evaluated following intravenous (i.v.), subcutaneous (s.c.), and intraperitoneal (i.p.) bolus administrations. RESULTS: ECO-4601 was shown to bind the peripheral but not the central benzodiazepine receptor and inhibited the growth of CNS tumor cell lines. Bolus s.c. and i.p. administration gave rise to low but sustained drug exposure, and resulted in moderate to significant antitumor activity at doses that were well tolerated. In a rat glioma (C6) xenograft model, ECO-4601 produced up to 70% tumor growth inhibition (TGI) while in a human glioma (U-87MG) xenograft, TGI was 34%. Antitumor activity was highly significant in both human hormone-independent breast (MDA-MB-231) and prostate (PC-3) xenografts, resulting in TGI of 72 and 100%, respectively. On the other hand, i.v. dosing was followed by rapid elimination of the drug and was ineffective. CONCLUSIONS: Antitumor efficacy of ECO-4601 appears to be associated with the exposure parameter AUC and/or sustained drug levels rather than C (max). These in vivo data constitute a rationale for clinical studies testing prolonged continuous administration of ECO-4601.

Human urotensin-II enhances plasma extravasation in specific vascular districts in Wistar rats.

Plasma extravasation (PE) was measured in adult Wistar rats by injecting Evans blue dye (EB) (20 mg kg-1) intravenously in the absence or presence of human urotensin II (U-II) (0.1-10 nmol kg-1). A consistent increase of PE was observed in specific organs (e.g., aorta, from 28.1 +/- 2.4 to 74.6 +/- 3.6 micro g EB g-1 dry tissue; P < 0.001) after an administration of 4.0 nmol kg-1 (a preselected optimal dose) of U-II. The effects of U-II (4.0 nmol kg-1) were compared with those of endothelin-1 (ET-1) (1.0 nmol kg-1). In the thoracic aorta and pancreas, U-II was active, while ET-1 was not. The two agents were equivalent in the heart and kidney, whereas, in the duodenum, ET-1 was more active than U-II. Increases of plasma extravasation induced by U-II, but not by ET-1, were reduced after treatment with [Orn8]U-II (0.3 micromol kg-1). This latter antagonist did not show any significant residual agonistic activity in vivo in the rat. Other specific receptor antagonists for ET-1, such as BQ-123 (endothelin type A (ETA) receptor) and BQ-788 (endothelin type B (ETB) receptor), and for the platelet activating factor (PAF), such as BN50730, failed to modify the action of U-II. The present study is the first report describing the modulator roles of U-II on vascular permeability in specific organs. Moreover, the action of U-II appears specific, since it is independent of the ET-1 and PAF signalling pathways.

The antiangiogenic agent neovastat (AE-941) inhibits vascular endothelial growth factor-mediated biological effects.

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis, which exerts direct effects on vascular endothelial cells, including endothelial cell proliferation and survival, tubulogenesis, and vascular permeability. In this study, we examined whether Neovastat, a naturally occurring multifunctional antiangiogenic drug, could inhibit the endothelial cell response to VEGF stimulation. RESULTS: We demonstrated that Neovastat was able to block the VEGF-dependent microvessel sprouting from Matrigel-embedded rat aortic rings, and it also blocked the VEGF-induced endothelial cell tubulogenesis in vitro. In vivo studies showed that Neovastat was able to specifically inhibit VEGF-induced plasma extravasation in numerous tissues, including pancreas and skin. The mechanism of action of Neovastat on VEGF-mediated effects was also evaluated at the molecular level. Neovastat was shown to compete against the binding of VEGF to its receptor in endothelial cells and significantly inhibited the VEGF-dependent tyrosine phosphorylation of VEGF receptor-2, whereas it had no significant effect on VEGF receptor-1 activity. Moreover, the inhibition of receptor phosphorylation was correlated with a marked decrease in the ability of VEGF to induce pERK activation. Neovastat does not compete against the binding of basic fibroblast growth factor, indicating a preferential inhibitory effect on the VEGF receptor. CONCLUSIONS: Because Neovastat was shown previously to inhibit metalloproteinase activities, these results suggest that Neovastat is able to target multiple steps in tumor neovascularization, further emphasizing its use as a pleiotropic, multifunctional antiangiogenic drug.

Inhibitory effect of a novel bradykinin B1 receptor antagonist, R-954, on enhanced vascular permeability in type 1 diabetic mice.

The morbidity and mortality associated with type 1 diabetes are essentially related to the micro- and macrovascular complications that develop over time and lead to several diabetic complications, including hypertension, atherosclerosis, and retinopathy, as well as coronary and renal failure. Normally absent in physiological conditions, the bradykinin B1 receptor (BKB1-R) was recently found to be overexpressed in pathological conditions, including type 1 diabetes. In the present study, we evaluated the effect of the new BKB1-R antagonist, R-954 (Ac-Orn-[Oic2, alpha-MePhe5, D-betaNal7, Ile8]desArg9-bradykinin, on the increase in vascular permeability in streptozotocin (STZ)-diabetic mice. The capillary permeability to albumin was measured by quantifying the extravasation of albumin-bound Evans blue dye in selected target tissues (liver, pancreas, duodenum, ileum, spleen, heart, kidney, stomach, skin, muscle, and thyroid gland). Acute single administration of R-954 (300 microg/kg, i.v.) to type 1 diabetic mice 4 weeks after STZ significantly inhibited the enhanced vascular permeability in most tissues. These data provide further experimental evidence for the implication of BKB1-R in the enhanced vascular permeability associated with type 1 diabetes.