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 β-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 β-propeller 3 and 4 of LRP6. The peptides that showed to bind NT4 represented the portion of LRP6 located on the top face of β-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.

BACKGROUND:Near-infrared quantum dots (NIR QDs) are a new class of fluorescent labels with excellent bioimaging features, such as high fluorescence intensity, good fluorescence stability, sufficient electron density, and strong tissue-penetrating ability. For all such features, NIR QDs have great potential for early cancer diagnosis, in vivo tumor imaging and high resolution electron microscopy studies on cancer cells.RESULTS:In the present study we constructed NIR QDs functionalized with the NT4 cancer-selective tetrabranched peptides (NT4-QDs). We observed specific uptake of NT4-QDs in human cancer cells in in vitro experiments and a much higher selective accumulation and retention of targeted QDs at the tumor site, compared to not targeted QDs, in a colon cancer mouse model.CONCLUSIONS:NIR QDs labelled with the tetrabranched NT4 peptide have very promising performance for selective addressing of tumor cells in vitro and in vivo, proving rising features of NT4-QDs as theranostics.

Taxanes are highly effective chemotherapeutic drugs against proliferating cancer and an established option in the standard treatment of ovarian and breast cancer. However, treatment with paclitaxel is associated with severe side effects, including sensory axonal neuropathy and its poor solubility in water complicates its formulation. In this paper we report the in vitro and in vivo activity of a new form of paclitaxel, modified for conjugation with a tumor-selective tetrabranched peptide carrier (NT4). NT4 selectively targets tumor cells by binding to membrane sulfated glycosaminoglycans (GAG) and to endocytic receptors, like LRP1 and LRP6, which are established tumor markers. Biological activity of NT4-paclitaxel was tested in vitro on MDA-MB 231 and SKOV-3 cell lines, representing breast and ovarian cancer, respectively and in vivo in an orthotopic mouse model of human breast cancer. Using in vivo bioluminescence imaging, we found that conjugation of paclitaxel with the NT4 peptide led to increased therapeutic activity of the drug in vivo . NT4-paclitaxel induced tumor regression, whereas treatment with unconjugated paclitaxel only produced a reduction in tumor growth. Moreover, unlike paclitaxel, NT4-paclitaxel is very hydrophilic, which may improve its pharmacokinetic profile and allow the use of less toxic dilution buffers, further decreasing its general chemotherapic toxicity.

M33 is a branched peptide currently under preclinical characterization for the development of a new antibacterial drug against gram-negative bacteria. Here, we report its pegylation at the C-terminus of the three-lysine-branching core and the resulting increase in stability to Pseudomonas aeruginosa elastase. This protease is a virulence factor that acts by destroying peptides of the native immune system. Peptide resistance to this protease is an important feature for M33-Peg activity against Pseudomonas.

In previous papers we demonstrated that tetrabranched peptides containing the sequence of human neurotensin, NT4, are much more selective than native monomeric analogues for binding to different human cancer cells and tissues. We show here that the much higher binding of NT4 peptides, with respect to native neurotensin, to either cancer cell lines or human cancer surgical samples is generated by a switch in selectivity toward additional membrane receptors, which are specifically expressed by different human cancers. We demonstrate that the branched structure provides NT4 with ability to bind heparin and receptors belonging to the low density lipoprotein receptor (LDLR) family, known to be involved in cancer biology. Systematic modification of neurotensin sequence in NT4 peptides led to identification of a multimeric positively charged motif, which mediates interaction with both heparin and endocytic receptors. Our findings provide the molecular basis for construction of cancer theranostics with high cancer selectivity.

Abstract In previous paper we reported on the much higher selectivity toward cancer cells and tissues of tetra-branched neurotensin peptides (NT4) compared to monomeric NT peptide. We also demonstrated that NT4 can be coupled to many different functional units for cancer cell tracing and drug delivery and can induce tumor growth reduction in animal studies. We then proposed NT4 as promising cancer selective theranostics for different human cancers, including CRC, pancreas adenocarcinoma and urinary bladder cancer. Nonetheless, multimeric binding of tetrabranched peptides, together with the chemical modification produced by coupling to the branched core, might have modified receptor selectivity of NT4 with respect to native monomeric NT and actually we had no conclusive indication on which receptor our branched NT4 peptides were binding to. Data reported in the present paper demonstrate that synthesis of neurotensin sequence in a tetra-branched form induce a switching of receptor selectivity, by decreasing affinity to the NT high affinity receptor NTR1 and contemporarily acquiring binding to additional receptors, which produces a much higher cancer cell selectivity of NT4 with respect to monomeric NT peptides. We demonstrate here that NT4 binds sortilin and SorLa and also acquire the ability to bind different receptors belonging to the Low Density Lipoprotein Receptor Related Protein (LRP) family as well as heparin and other Heparan Sulfate Proteoglycans (HSPG). The much higher binding of NT4 in respect to native NT to either cancel cell lines or human cancer surgical samples, as well as the higher selectivity toward human cancer tissues of NT4 is due to binding to different membrane receptors, which are very selectively expressed by many different human cancers. Moreover, our results confirm that sulfated proteoglycan can mimic the ligand binding site of different LRP receptors and indicate that targeting of multiple LRP receptors together with sulfated proteoglycans produce an extremely high selectivity towards many different human cancers. Citation Format: Luisa Bracci, Chiara Falciani, Jlenia Brunetti, Barbara Lelli, Niccolò Ravenni, Luisa Lozzi, Lorenzo Depau, Alessandro Pini. Targeting different LRP receptors and sulfated proteoglycan by branched neurotensin provide high cancer selectivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5625. doi:10.1158/1538-7445.AM2013-5625

The tetra-branched peptide M33 (Pini et al. in FASEB J 24:1015–1022, 2010) is under evaluation in animal models for its activity as antimicrobial agent in lung infections and sepsis. The preclinical development of a new drug requires medium-scale manufacture for tests of efficacy, biodistribution, pharmacokinetics and toxicity. In order to produce the most suitable peptide form for these purposes, we evaluated the behaviour of the peptide M33 obtained with different counter-ions. We compared activity and toxicity in vitro and in vivo of the peptide M33 produced as trifluoroacetate salt (TFacetate) and as acetate salt. The two forms did not differ substantially in terms of efficacy in vitro or in vivo but showed different toxicities for human cells and in animals. M33-TFacetate proved to be 5–30% more toxic than M33-acetate for cells derived from normal bronchi and cells carrying ΔF508 mutation in the CFTR gene, the most frequent variant in cystic fibrosis. M33-TFacetate produced manifest signs of in vivo toxicity immediately after administration, whereas M33-acetate only generated mild signs, which disappeared within a few hours. The peptide M33-acetate proved more suitable for the development of a new drug, and was therefore chosen for further characterization.

The branched M33 antimicrobial peptide was previously shown to be very active against Gram-negative bacterial pathogens, including multidrug-resistant strains. In an attempt to produce back-up molecules, we synthesized an M33 peptide isomer consisting of D-aminoacids (M33-D). This isomeric version showed 4 to 16-fold higher activity against Gram-positive pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, than the original peptide, while retaining strong activity against Gram-negative bacteria. The antimicrobial activity of both peptides was influenced by their differential sensitivity to bacterial proteases. The better activity shown by M33-D against S. aureus compared to M33-L was confirmed in biofilm eradication experiments where M33-L showed 12% activity with respect to M33-D, and in vivo models where Balb-c mice infected with S. aureus showed 100% and 0% survival when treated with M33-D and M33-L, respectively. M33-D appears to be an interesting candidate for the development of novel broad-spectrum antimicrobials active against bacterial pathogens of clinical importance.

This chapter discusses the cell cycle inhibitors in cancer in the current status and future directions. The current clinical experience with the first generation of the new targeted anticancer agents against specific tumor kinases suggests that the majority of targeted cancer drugs will probably provide dramatic responses, but only against tumors where the function of the target kinase is aberrant, and thus in fewer patients than hoped. Although genetic and proteomic profiling of individual patients' tumor biopsies can predict responsiveness towards such agents, the difficulties in implementing personalized medicine strategies are of course substantial. For this reason, multi-targeted drugs, especially kinase inhibitors, are now being advocated on the basis that they may be more effective against cancers – most of which are inherently heterogeneous – than mono-specific drugs, but that they are still not so promiscuous as to be toxic like traditional chemotherapeutics. Although the clinical development of some cell cycle agents, especially CDK inhibitors, has been under way for some time and has shown some promise, most targeted cell cycle therapies are only now starting to enter clinical trials in cancer patients. Considering the current focus on addressing mechanistically validated targets and advancing targeted agents that should be able to be developed rationally using biomarker-based methods, there is considerable hope that cell cycle agents will benefit cancer patients in the not too distant future.

Abstract The aim of this study was to validate oligo-branched peptides as selective targeting agents that might be effective either for spotlighting tumor cells that over-express peptide receptors, or for killing them, simply by exchanging the functional moiety coupled to the conserved receptor-targeting core. Tetra-branched peptides containing neurotensin (NT) sequence are described here for selective targeting of human colon, pancreas and prostate cancer. Fluorophore-conjugated peptides were used to measure tumor versus healthy tissue binding in human surgical samples, resulting in validation of neurotensin receptors as highly promising tumor-biomarkers. Drug-armed branched peptides were synthesized with different conjugation methods, resulting in uncleavable adducts or drug-releasing molecules. Cytotoxicity on human cell lines from colon (HT-29), pancreas (PANC-1) or prostate (PC-3) carcinoma indicated branched NT conjugated with MTX and 5-FdU as the most active agents on PANC-1 (EC50 4.4e-007 M) and HT-29 (1.1e-007 M), respectively. Tetra-branched NT armed with 5-FdU was used for in vivo experiments in HT-29-xenografted mice and produced a 50% reduction in tumor growth with respect to animals treated with the same amount of free drug. An unrelated branched peptide carrying the same drug was completely ineffective. In vitro and in vivo results indicated that branched peptides are valuable new tools for tumor selective targeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2582.

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.

3982 The finding that receptors for different endogenous regulatory peptides, like the neuropeptide neurotensin (NT), are over-expressed in different human cancers, has opened new perspectives on the use of synthetic peptides for tumor-selective targeting (1). Peptide ligands carrying cytotoxic moieties or radiotracers might act as drugs or diagnostics. Nonetheless, the use of peptides as drugs has largely been limited by their short half-life. Peptides are physiologically hydrolysed by proteases and peptidases and the chemical modifications necessary to stabilize endogenous sequence can deeply modify peptide affinity and specificity. We demonstrated that NT, like several other endogenous peptides, retains its biological activity and becomes resistant to proteolysis when synthesized in a branched form (2). NT receptors are over-expressed in different human tumors with high incidence and mortality rate, like small cell lung cancer, colon, prostate and pancreas carcinomas. For this reason, high affinity and stable NT peptides can have important applications as specific tumor-targeting agents. We synthesized the short functional fragment NT(8-13) in a tetra-branched form (NT4), which was coupled during the synthesis to different units designed for tumor tracing or therapy. Cell internalization of branched NT4 molecules together with NT receptor regulation and trafficking, were followed by confocal microscopy. NT4 conjugated to fluorescent probes was also used for NT receptor tracing in human specimens from colon cancer surgical resections. Comparison with normal tissues from the same patients showed a clear over-expression of NT receptor in a high percentage of tumors even at early differentiation stages. Cytotoxicity of NT4 conjugated to methotrexate or to the photosensitizer chlorine e6, was tested on human colon adenocarcinoma cell lines. In both cases, conjugation to branched NT dramatically decreased non-specific toxicity of the drugs by inducing receptor-specific uptake of the toxic moiety by target cells. NT4 conjugated to methotrexate was also tested in in vivo experiments using HT29 human colon adenocarcinoma xenografts in nude mice. Animals treated with NT4 conjugated to methotrexate showed a clear decrease in tumor growth with respect to untreated mice or mice treated with equimolar amount of the unconjugated drug. Branched NT molecules are good candidates for the development of peptide tumor-targeting agents that might have the dual use of specific NT receptor tracing followed by targeted therapy of NT receptor-expressing tumors. Reubi JC. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev. 2003; 24(4): 389-427. Bracci L. et al. Synthetic peptides in the form of dendrimers become resistant to protease activity. J Biol Chem. 2003; 278(47): 46590-5.

We found that synthetic peptides in the form of dendrimers become resistant to proteolysis. To determine the molecular basis of this resistance, different bioactive peptides were synthesized in monomeric, two‐branched and tetra‐branched form and incubated with human plasma and serum. Proteolytic resistance of branched multimeric sequences was compared to that of the same peptides synthesized as multimeric linear molecules. Unmodified peptides and cleaved sequences were detected by high pressure liquid chromatography and mass spectrometry. An increase in peptide copies did not increase peptide resistance in linear multimeric sequences, whereas multimericity progressively enhanced proteolytic stability of branched multimeric peptides. A structure‐based hypothesis of branched peptide resistance to proteolysis by metallopeptidases is presented.

A structural genomics approach is proposed for the development of new diagnostic kits. It combines molecular modelling, peptide synthesis and immunological tests. The preliminary step is the development of a reliable three-dimensional structure of an immunodominant protein of the target pathogenic organism using the various bioinformatic strategies that are now available to structural biologists. Once the protein structure is obtained, the most surface-exposed fragments with minimal sequence variability among the different strains reported in the genomic data bank are reproduced synthetically as linear peptides. These peptides are then tested for immunoreactivity with the plasma of infected patients to determine whether the synthetic molecules have antigenic activity and can therefore be used to detect infecting agents. This structurally driven selection of mimotopes was successfully performed for the human hepatitis C virus, as five peptides that specifically interact with the plasma of HCV-infected patients were identified solely on the basis of the three-dimensional structure predicted for the E2 homodimer of the 1a viral subtype. A similar approach could easily be extended to a large variety of immunogenic proteins from other pathogenic organisms.