Molecular definition of the interaction between a tumor-specific tetrabranched peptide and LRP6 receptor.

The tumor-specific tetrabranched peptide NT4 binds membrane sulfate glycosaminoglycans and receptors belonging to the low density lipoprotein receptor-related protein (LRP) family, like LRP6, which are overexpressed in cancer. The binding occurs through a multimeric positively-charged motif of NT4 that interacts with negatively charged motives in both glycosaminoglycans and LRP receptors. LRP6 has an essential function in canonical Wnt signaling, acting together with receptors of the Frizzled family as coreceptor for Wnt ligands. The extracellular domain of LRP6 contains four YWTD ß-propellers, which are fundamental for interactions with ligands, such as Wnt and Wnt inhibitors. To investigate the molecular interactions between the NT4 peptide and LRP6 receptor, we synthesized a library of epitope mapping peptides reproducing the YWTD ß-propeller 3 and 4 of LRP6. The peptides that showed to bind NT4 represented the portion of LRP6 located on the top face of ß-propeller 3 and contained negatively charged residues, including glutamic acid-708 which is known to be involved in Wnt3a interaction. The results pave the way for a possible development of peptide inhibitors of Wnt3a pathway to be used as drugs in oncology.

NTS2-selective neurotensin mimetics with tetrahydrofuran amino acids.

Stimulation of the NTS2 neurotensin receptor causes antipsychotic effects and leads to a promotion of the µ-opioid-independent antinociception, which is important in the modulation of tonic pain sensitivity. We report the synthesis and properties of a small library of peptidic agonists based on the active neurotensin fragment NT(8-13). Two tetrahydrofuran amino acid derivatives were synthesized to replace Tyr11 in NT(8-13). Additionally, Arg8, Arg9, and Ile12 of the lead peptide were exchanged by Lys, Lys, and Gly, respectively. The new compounds showed substantial NTS2 binding affinity and up to 1000-fold selectivity over NTS1. The highest selectivity (Ki(NTS2): 29nM, Ki(NTS1): 35,000nM) was observed for the peptide analog 17Rtrans.

Synthesis and binding characteristics of [(3)H]neuromedin N, a NTS2 receptor ligand.

Neurotensin (NT) and its analog neuromedin N (NN) are formed by the processing of a common precursor in mammalian brain tissue and intestines. The biological effects mediated by NT and NN (e.g. analgesia, hypothermia) result from the interaction with G protein-coupled receptors. The goal of this study consisted of the synthesis and radiolabeling of NN, as well as the determination of the binding characteristics of [(3)H]NN and G protein activation by the cold ligand. In homologous displacement studies a weak affinity was determined for NN, with IC50 values of 454nM in rat brain and 425nM in rat spinal cord membranes. In saturation binding experiments the Kd value proved to be 264.8±30.18nM, while the Bmax value corresponded to 3.8±0.2pmol/mg protein in rat brain membranes. The specific binding of [(3)H]NN was saturable, interacting with a single set of homogenous binding sites. In sodium sensitivity experiments, a very weak inhibitory effect of Na(+) ions was observed on the binding of [(3)H]NN, resulting in an IC50 of 150.6mM. In [(35)S]GTPγS binding experiments the Emax value was 112.3±1.4% in rat brain and 112.9±2.4% in rat spinal cord membranes and EC50 values of 0.7nM and 0.79nM were determined, respectively. NN showed moderate agonist activities in stimulating G proteins. The stimulatory effect of NN could be maximally inhibited via use of the NTS2 receptor antagonist levocabastine, but not by the opioid receptor specific antagonist naloxone, nor by the NTS1 antagonist SR48692. These observations allow us to conclude that [(3)H]NN labels NTS2 receptors in rat brain membranes.

Peptide Bioconjugates of Electron-Poor Metallocenes: Synthesis, Characterization, and Anti-Proliferative Activity.

We report the synthesis of metallocene compounds Cp2 M with two different electron-withdrawing substituents on both cyclopentadienyl rings (hexafluoroacetone (HFA) and chlorobenzoyl (1-5); HFA and COOH (6 and 7), M=Fe or Ru). The COOH-containing derivatives were used to synthesize peptide bioconjugates with enkephalin (8 and 9) and neurotensin (10 and 11) as well as fluorescein-labeled neurotensin (12). All the molecules were fully characterized, including X-ray structures for 6 and 7. The physicochemical properties (lipophilicity and electrochemistry) and cytotoxicity on MCF-7, HT-29, and PT-45 cancer cells were evaluated for selected compounds. Electrochemical investigation by cyclic voltammetry revealed that all bis-substituted metallocenes are up to 300 mV harder to oxidize compared to the monosubstituted 2-ferrocenylhexafluoropropan-2-ol (FcHFA: Δ${E{{{\rm f}\hfill \atop 0\hfill}}}$=214 mV; disubstituted derivatives: up to Δ${E{{{\rm f}\hfill \atop 0\hfill}}}$=512 mV; both vs. FcH(0/+) ). For the bis-substituted compounds, log P determinations by RP-HPLC showed increased lipophilicity in comparison to the monosubstituted FcHFA and RcHFA. Cellular uptake was investigated by fluorescence microcopy, and this revealed endosomal entrapment for 12.

Facile Preparation of a Thiol-Reactive (18)F-Labeling Agent and Synthesis of (18)F-DEG-VS-NT for PET Imaging of a Neurotensin Receptor-Positive Tumor.

UNLABELLED: Accumulating evidence suggests that neurotensin receptors (NTRs) play key roles in cancer growth and survival. In this study, we developed a simple and efficient method to radiolabel neurotensin peptide with (18)F for NTR-targeted imaging. METHODS: The thiol-reactive reagent (18)F-(2-(2-(2-fluoroethoxy)ethoxy)ethylsulfonyl)ethane ((18)F-DEG-VS) was facilely prepared through 1-step radiofluorination. After high-pressure liquid chromatography purification, (18)F-DEG-VS was incubated with the c(RGDyC) and c(RGDyK) peptide mixture to evaluate its specificity toward the reactive thiol. Thiolated neurotensin peptide was then labeled with (18)F using this novel synthon, and the resulting imaging probe was subjected to receptor-binding assay and small-animal PET studies in a murine xenograft model. The imaging results and metabolic stability of (18)F-DEG-VS-NT were compared with the thiol-specific maleimide derivative N-[2-(4-(18)F-fluorobenzamido)ethyl]maleimide-neurotensin ((18)F-FBEM-NT). RESULTS: (18)F-DEG-VS was obtained in high labeling yield. The reaction of (19)F-DEG-VS was highly specific for thiols at neutral pH, whereas the lysine of c(RGDyK) reacted at a pH greater than 8.5. (18)F-DEG-VS-c(RGDyC) was the preferred product when both c(RGDyK) and c(RGDyC) were incubated together with (18)F-DEG-VS. Thiolated neurotensin peptide (Cys-NT) efficiently reacted with (18)F-DEG-VS, with a 95% labeling yield (decay-corrected). The radiochemical purity of the (18)F-DEG-VS-NT was greater than 98%, and the specific activity was about 19.2 ± 4.3 TBq/mmol. Noninvasive small-animal PET demonstrated that (18)F-DEG-VS-NT had an NTR-specific tumor uptake in subcutaneous HT-29 xenografts. The tumor-to-muscle, tumor-to-liver, and tumor-to-kidney ratios reached 30.65 ± 22.31, 11.86 ± 1.98, and 1.91 ± 0.43 at 2 h after injection, respectively, based on the biodistribution study. Receptor specificity was demonstrated by blocking experiment. Compared with (18)F-FBEM-NT, (18)F-DEG-VS-NT was synthesized with fewer steps and provided significantly improved imaging quality in vivo. CONCLUSION: We have established a facile (18)F-labeling method for site-specific labeling of the Cys-NT. Using this method, we synthesized an NTR-targeted PET agent, which demonstrated high tumor-to-background contrast.

Relevance of dopamine D(2)/neurotensin NTS1 and NMDA/neurotensin NTS1 receptor interaction in psychiatric and neurodegenerative disorders.

The existence of functional NT/dopamine interactions in the central nervous system has been extensively documented. Among others, a possible molecular mechanism underlying the NT-induced modulation of dopamine release is a direct antagonistic NTS(1)/D(2) receptor interaction. More recently, neurochemical experiments also supported the existence of a possible interaction between NT and N-methyl-d-aspartate (NMDA) receptors. In particular, it has been suggested that NT, by amplifying NMDA receptor signaling, could be involved in neurodegeneration. The present article attempts to provide a summary of current knowledge, mainly emerging from our studies, on the existence of receptor-receptor interactions between NT receptor subtype 1 (NTS1) and dopamine D(2) or NMDA receptors in the brain. Special emphasis is placed on the pre and post-synaptic neurochemical mechanisms possibly underlying the involvement of these interactions in the physiopathology of schizophrenia and acute neurodegenerative disorders.

A novel tetrabranched neurotensin(8-13) cyclam derivative: synthesis, 64Cu-labeling and biological evaluation.

New macrocyclic 1,4,8,11-tetraazacyclotetradecane (cyclam) derivatives with 1, 2 and 4 neurotensin(8-13) units 4, 5 and 7 have been synthesized. Compounds 4 and 5 were prepared by the reaction of non-stabilized neurotensin(8-13) and cyclamtetrapropionic acid 2 using 1-ethyl-3-(3-dimethylaminocarbonyl)carbodiimide-hydrochloride and N-hydroxysulfosuccinimide. The tetrameric compound 7 was synthesized by Michael addition of neurotensin(8-13) acrylamide 6 and cyclam 1. The copper(II) complexation behavior of 4, 5 and 7 was investigated by UV/visible spectrophotometry and shows that the metal center resides inside the N4 chromophore with additional apical interactions established with pendant arms. The novel tetrabranched NT(8-13) cyclam 7 with nanomolar neurotensin receptor 1 binding affinity was efficiently radiolabeled with (64)Cu under mild conditions. (64)Cu⊂7 showed slow transchelation in the presence of a large amount of cyclam as competing ligand, while it completely remains intact in the presence of EDTA. The in vivo behavior of (64)Cu⊂7 was studied in rats and mice. The metabolic stability in rodent models was high with a half-life of intact (64)Cu⊂7 in plasma of 34 min in rats and 60 min in the mice, respectively. The binding affinity was high enough to demonstrate in vivo binding of (64)Cu⊂7 to NTR1 overexpressing HT-29 tumor xenotransplants in nude mice. Regarding elimination, (64)Cu⊂7 showed a substantial renal and reticuloendothelial accumulation. On the other hand, metabolization of the compound in vivo with a resulting metabolite-postulated to be the (64)Cu-cyclam-tetraarginine complex-also showed long retention in the circulating blood, preventing a better contrast of tumor imaging.

Radical arylation of tyrosine and its application in the synthesis of a highly selective neurotensin receptor 2 ligand.

A small library of Fmoc-protected 3-arylated tyrosines was created by radical arylation. The new building blocks were successfully applied in the synthesis of two novel neurotensin receptor ligands. Both isomers showed high affinity for the human NTS2 receptor with K(i) values in the nanomolar range. Interestingly, subtype selectivity strongly depends on the configuration of the peptide in position 11. Isomer (11R)-3 displayed an excellent preference for NTS2 compared to NTS1.

Synthesis and cytotoxicity of a bimetallic ruthenocene dicobalt-hexacarbonyl alkyne peptide bioconjugate.

Organometallic conjugates of receptor-targeting peptides are proposed as interesting candidates for novel cancer therapies since they are capable of targeting a specific kind of cell. Here, we have synthesised a dicobalt hexacarbonyl alkyne compound linked to the neurotensin peptide hormone. In order to circumvent synthetic difficulties encountered when adding a cobalt carbonyl moiety onto the hydrophilic alkyne peptide, and to enhance the cellular uptake we functionalised the alkyne neurotensin(8-13) fragment (NT) first N-terminally by ruthenocene carboxylic acid to form the metallocene-alkyne-NT conjugate 3, before adding Co2(CO)8 to a propargyl glycine residue to form the Co-alkyne derivative 4. Compound 4 represents the first heterobimetallic organometallic peptide conjugate reported to date. It shows moderate cytotoxicity against HeLa, PT45 and HepG2 cell lines.

[Neurotensin-like oligopeptides as potential antipsychotics: effect on dopamine system].

According to published data, peptide neurotensin is considered as endogenous antipsychotic agent. A series of oligopeptides have been synthesized based on the proposed active center of neurotensin. These oligopeptides (called neurotensin-like peptides, NLPs) have been studied on behavioral models, in which the functional state of the dopamine system of animals was modified by apomorphine injections. The results of verticalization, stereotypy, and yawning tests revealed NLPs that behave as antagonists of dopamine receptors. Radioligand analysis showed that these peptides compete for specific binding to these receptors with sulpiride, which is a D2-type selective antagonist of dopamine receptors. The high degree of NLPs efficiency manifested in the behavioral tests and radioligand analysis suggests that the their antipsychotic action can be mediated by dopamine receptors.

Introduction of lipidization-cationization motifs affords systemically bioavailable neuropeptide Y and neurotensin analogs with anticonvulsant activities.

The neuropeptides galanin (GAL), neuropeptide Y (NPY) or neurotensin (NT) exhibit anticonvulsant activities mediated by their respective receptors in the brain. To transform these peptides into potential neurotherapeutics, their systemic bioavailability and metabolic stability must be improved. Our recent studies with GAL analogs suggested that an introduction of lipoamino acids in the context of oligo-Lys residues (lipidization-cationization motif) significantly increases their penetration into the brain, yielding potent antiepileptic compounds. Here, we describe an extension of this strategy to NPY and NT. Rationally designed analogs of NPY and NT containing the lipidization-cationization motif were chemically synthesized and their physicochemical and pharmacological properties were characterized. The analogs NPY-BBB2 and NT-BBB1 exhibited increased serum stability, possessed log D > 1.1, retained high affinities toward their native receptors and produced potent antiseizure activities in animal models of epilepsy following intraperitoneal administration. Our results suggest that the combination of lipidization and cationization may be an effective strategy for improving systemic bioavailability and metabolic stability of various neuroactive peptides.

Identification and functional characterization of a stable, centrally active derivative of the neurotensin (8-13) fragment as a potential first-in-class analgesic.

The neurotensin hexapapetide fragment NT(8-13) is a potent analgesic when administered directly to the central nervous system but does not cross the blood-brain barrier. A total of 43 novel derivatives of NT(8-13) were evaluated, with one, ABS212 (1), being most active in four rat models of pain when administered peripherally. Compound 1 binds to human neurotensin receptors 1 and 2 with IC(50) of 10.6 and 54.2 nM, respectively, and tolerance to the compound in a rat pain model did not develop after 12 days of daily administration. When it was administered peripherally, serum levels and neurotensin receptor binding potency of 1 peaked within 5 min and returned to baseline within 90-120 min; however, analgesic activity remained near maximum for >240 min. This could be due to its metabolism into an active fragment; however, all 4- and 5-mer hydrolysis products were inactive. This pharmacokinetic/pharmacodynamic dichotomy is discussed. Compound 1 is a candidate for development as a first-in-class analgesic.

Design and in vitro evaluation of branched peptide conjugates: turning nonspecific cytotoxic drugs into tumor-selective agents.

The use of peptide receptors as targets for tumor-selective therapies was envisaged years ago with the findings that receptors for different endogenous regulatory peptides are overexpressed in several primary and metastatic human tumors, and can be used as tumor antigens. Branched peptides can retain or even increase, through multivalent binding, the biological activity of a peptide and are very resistant to proteolysis, thus having a markedly higher in vivo activity compared with the corresponding monomeric peptides. Oligo-branched peptides, containing the human regulatory peptide neurotensin (NT) sequence, have been used as tumor-specific targeting agents. These peptides are able to selectively and specifically deliver effector units, for cell imaging or killing, to tumor cells that overexpress NT receptors. Results obtained with branched NT conjugated to different functional units for tumor imaging and therapy indicate that branched peptides are promising novel multifunctional targeting molecules. This study is focused on the role of the releasing pattern of drug-conjugated branched NT peptides. We present results obtained with oligo-branched neurotensin peptides conjugated to 6-mercaptopurin (6-MP), combretastain A-4 (CA4) and monastrol (MON). Drugs were conjugated to oligo-branched neurotensin through different linkers, and the mode-of-release, together with cytotoxicity, was studied in different human cancer cell lines. The results show that branched peptides are very promising pharmacodelivery options. Among our drug-armed branched peptides, NT4-CA4 was identified as a candidate for further development and evaluation in preclinical pharmacokinetic and pharmacodynamic studies. This peptide-drug exhibits significant activity against pancreas and prostate human cancer cells. Consequently, this derivative is of considerable interest due to the high mortality rates of pancreas neuroendocrine tumors and the high incidence of prostate cancer.

Comparison of N-terminal modifications on neurotensin(8-13) analogues correlates peptide stability but not binding affinity with in vivo efficacy.

Neurotensin(8-13) and two related analogues were used as model systems to directly compare various N-terminal peptide modifications representing both commonly used and novel capping groups. Each N-terminal modification prevented aminopeptidase cleavage but surprisingly differed in its ability to inhibit cleavage at other sites, a phenomenon attributed to long-range conformational effects. None of the capping groups were inherently detrimental to human neurotensin receptor 1 (hNTR1) binding affinity or receptor agonism. Although the most stable peptides exhibited the lowest binding affinities and were the least potent receptor agonists, they produced the largest in vivo effects. Of the parameters studied only stability significantly correlated with in vivo efficacy, demonstrating that a reduction in binding affinity at NTR1 can be countered by increased in vivo stability.

Glycosylated neurotensin analogues exhibit sub-picomolar anticonvulsant potency in a pharmacoresistant model of epilepsy.

Neurotensin (NT) is an endogenous neuropeptide involved in a variety of central and peripheral neuromodulatory effects. Herein we show the effects of site-specific glycosylation on the in vitro and in vivo pharmacological properties of this neuropeptide. NT analogues containing O-linked disaccharides (beta-melibiose and alpha-TF antigen) or beta-lactose units linked by a PEG(3) spacer were designed and chemically synthesized using Fmoc chemistry. For the latter analogue, Fmoc-Glu-(beta-Lac-PEG(3)-amide) was prepared. Our results indicate that the addition of the disaccharides does not negatively affect the sub-nanomolar affinity or the low-nanomolar agonist potency for the neurotensin receptor subtype 1 (NTS1). Interestingly, three glycosylated analogues exhibited sub-picomolar potency in the 6 Hz limbic seizure mouse model of pharmacoresistant epilepsy following intracerebroventricular administration. Our results suggest for the first time that chemically modified NT analogues may lead to novel antiepileptic therapies.

Synthesis and biological effects of c(Lys-Lys-Pro-Tyr-Ile-Leu-Lys-Lys-Pro-Tyr-Ile-Leu) (JMV2012), a new analogue of neurotensin that crosses the blood-brain barrier.

The central administration of neurotensin (NT) or of its C-terminal hexapeptide fragment NT(8-13), produces strong analgesic effects in tests evaluating acute pain. The use of NT-derived peptides as pharmaceutical agents to relief severe pain in patients could be of great interest. Unfortunately, peptides do not readily penetrate the blood-brain barrier. We have observed that the cyclic NT(8-13) analogue, c(Lys-Lys-Pro-Tyr-Ile-Leu-Lys-Lys-Pro-Tyr-Ile-Leu) (JMV2012, compound 1), when peripherally administered to mice produced analgesic and hypothermic effects, suggesting the peptide penetrates the blood-brain barrier and functions effectively like a drug. Moreover, dimeric compounds show increased potency compared to their corresponding monomer. We present the synthesis of the cyclic dimer compound 1 (JMV2012). In mice, compound 1 induced a profound hypothermia and a potent analgesia, even when peripherally administered. Compound 1 appears to be an ideal lead compound for the development of bioactive NT analogues as novel analgesics drugs.

Synthesis and biological activity of stable branched neurotensin peptides for tumor targeting.

Receptors for endogenous regulatory peptides, like the neuropeptide neurotensin, are overexpressed in several human cancers and can be targets for peptide-mediated tumor-selective therapy. Peptides, however, have the main drawback of an extremely short half-life in vivo. We showed that neurotensin and other endogenous peptides, when synthesized as dendrimers, retain biological activity and become resistant to proteolysis. Here, we synthesized the neurotensin functional fragment NT(8-13) in a tetrabranched form linked to different units for tumor therapy or diagnosis. Fluorescent molecules were used to monitor receptor binding and internalization in HT29 human adenocarcinoma cells and receptor binding in HT29 tumor xenografts in nude mice. Linking of chemotherapic molecules like chlorin e6 and methotrexate to dendrimers resulted in a dramatic increase in drug selectivity, uptake of which by target cells became dependent on peptide receptor binding. When nude mice carrying human tumor xenografts were treated with branched NT(8-13)-methotrexate, a 60% reduction in tumor growth was observed with respect to mice treated with the free drug.

Toward stable N4-modified neurotensins for NTS1-receptor-targeted tumor imaging with 99mTc.

A series of Gly-neurotensin(8-13) analogues modified at the N-terminus by acyclic tetraamines (Demotensin 1-4) were obtained by solid-phase peptide synthesis techniques. Strategic replacement of amino acids and/or reduction of sensitive peptide bonds were performed to enhance conjugate resistance against proteolytic enzymes. During 99mTc-labeling, single species radiopeptides, [99mTc]Demotensin 1-4, were easily obtained in high yields and typical specific activities of 1 Ci/micromol. Peptide conjugates displayed a high affinity binding to the human neurotensin subtype 1 receptor (NTS1-R) expressed in colon adenocarcinoma HT-29 or WiDr cells and/or in human tumor sections. [99mTc]Demotensin 1-4 internalized very rapidly in HT-29 or WiDr cells by a NTS1-R-mediated process. [99mTc]Demotensin 3 and 4, which remained stable during 1 h incubation in murine plasma, were selectively studied in nude mice bearing human HT-29 and WiDr xenografts. After injection, [99mTc]Demotensin 3 and 4 effectively and specifically localized in the experimental tumors and were rapidly excreted via the kidneys into the urine, exhibiting overall biodistribution patterns favorable for NTS1-R-targeted tumor imaging in man.

Synthesis and evaluation of novel multimeric neurotensin(8-13) analogs.

Neurotensin(8-13) is a hexapeptide with subnanomolar affinity to the neurotensin receptor 1 which is expressed with high incidence in several human tumor entities. Thus, radiolabeled neurotensin(8-13) might be used for tumor targeting. However, its application is limited by insufficient metabolic stability. The present study aims at improving metabolic stability by the synthesis of multimeric neurotensin(8-13) derivatives rather than commonly employed chemical modifications of the peptide itself. Thus, different dimeric and tetrameric peptides carrying C- or N-terminal attached neurotensin(8-13) moieties have been synthesized and their binding affinity toward the neurotensin receptor has been determined. The results demonstrate that branched compounds containing neurotensin(8-13) attached via its C-terminus only show low receptor affinities, whilst derivatives with neurotensin(8-13) attached via the N-terminus show IC50 values in the nanomolar range. Moreover, within the multimeric neurotensin(8-13) derivatives with neurotensin(8-13) attached via the N-terminus an increasing number of branching units lead to higher binding affinities toward the neurotensin receptor.

Novel 99mTc-labeled neurotensin analogues with optimized biodistribution properties.

Two new 99mTc-labeled neurotensin(8-13) analogues containing the retro-N(alpha)-carboxymethyl-histidine ((N(alpha)His)Ac) chelator were synthesized as potential radiopharmaceuticals for visualization of pancreatic carcinoma. To improve the pharmacokinetic properties, (N(alpha)His)Ac-Arg-NMeArg-Pro-Tyr-Tle-Leu (NT-XII), which is metabolically stabilized at two positions, was further modified. Shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) was introduced to obtain a more hydrophilic peptide (NT-XVIII), or Tyr11 was replaced by 2,6-dimethyltyrosine (Dmt) resulting in a triple-stabilized NT(8-13) analogue (NT-XIX). The latter has the best biodistribution profile.