Silvia Pileri

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. [Mol Cancer Ther 2007;6(9):2441–8]

Effect of ligand binding on human d-amino acid oxidase: Implications for the development of new drugs for schizophrenia treatment

In human brain the flavoprotein D‐amino acid oxidase (hDAAO) is responsible for the degradation of the neuromodulator D‐serine, an important effector of NMDA‐receptor mediated neurotransmission. Experimental evidence supports the concept that D‐serine concentration increase by hDAAO inhibition may represent a valuable therapeutic approach to improve the symptoms in schizophrenia patients. This study investigated the effects on hDAAO conformation and stability of the substrate D‐serine (or of the pseudo‐substrate trifluoro‐D‐alanine), the FAD cofactor, and two inhibitors (benzoate, a classical substrate‐competitive inhibitor and the drug chlorpromazine (CPZ), which competes with the cofactor). We demonstrated that all these compounds do not alter the interaction of hDAAO with its physiological partner pLG72. The ligands used affect the tertiary structure of hDAAO differently: benzoate or trifluoro‐D‐alanine binding increases the amount of the holoenzyme form in solution and stabilizes the flavoprotein, while CPZ binding favors a protein conformation resembling that of the apoprotein, which is more sensitive to degradation. Interestingly, the apoprotein form of hDAAO binds the substrate D‐serine: this interaction increases FAD binding thus increasing the amount of active holoenzyme in solution. Benzoate and CPZ similarly modify the short‐term cellular D‐serine concentration but affect the cellular concentration of hDAAO differently. In conclusion, the different alteration of hDAAO conformation and stability by the ligands used represents a further parameter to take into consideration during the development of new drugs to cope schizophrenia.

Stable peptide inhibitors prevent binding of lethal and oedema factors to protective antigen and neutralize anthrax toxin in vivo

The lethal and oedema toxins produced by Bacillus anthracis, the aetiological agent of anthrax, are made by association of protective antigen with lethal and oedema factors and play a major role in the pathogenesis of anthrax. In the present paper, we describe the production of peptide-based specific inhibitors in branched form which inhibit the interaction of protective antigen with lethal and oedema factors and neutralize anthrax toxins in vitro and in vivo. Anti-protective antigen peptides were selected from a phage library by competitive panning with lethal factor. Selected 12-mer peptides were synthesized in tetra-branched form and were systematically modified to obtain peptides with higher affinity and inhibitory efficiency.

Modular branched neurotensin peptides for tumor target tracing and receptor-mediated therapy: a proof-of-concept.

The aim of this study was to demonstrate that oligo-branched peptides can be effective either for spotlighting tumor cells that overexpress 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 as selective targeting agents for 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 (EC(50) 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 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 tools for tumor selective targeting.

Functional Characterization of a Small-Molecule Inhibitor of the DKK1-LRP6 Interaction

Background. DKK1 antagonizes canonical Wnt signalling through high-affinity binding to LRP5/6, an essential component of the Wnt receptor complex responsible for mediating downstream canonical Wnt signalling. DKK1 overexpression is known for its pathological implications in osteoporosis, cancer, and neurodegeneration, suggesting the interaction with LRP5/6 as a potential therapeutic target. Results. We show that the small-molecule NCI8642 can efficiently displace DKK1 from LRP6 and block DKK1 inhibitory activity on canonical Wnt signalling, as shown in binding and cellular assays, respectively. We further characterize NCI8642 binding activity on LRP6 by Surface Plasmon Resonance (SPR) technology. Conclusions. This study demonstrates that the DKK1-LRP6 interaction can be the target of small molecules and unlocks the possibility of new therapeutic tools for diseases associated with DKK1 dysregulation.

Surface Interactome in Streptococcus pyogenes

Very few studies have so far been dedicated to the systematic analysis of protein interactions occurring between surface and/or secreted proteins in bacteria. Such interactions are expected to play pivotal biological roles that deserve investigation. Taking advantage of the availability of a detailed map of surface and secreted proteins in Streptococcus pyogenes (group A Streptococcus (GAS)), we used protein array technology to define the “surface interactome” in this important human pathogen. Eighty-three proteins were spotted on glass slides in high density format, and each of the spotted proteins was probed for its capacity to interact with any of the immobilized proteins. A total of 146 interactions were identified, 25 of which classified as “reciprocal,” namely, interactions that occur irrespective of which of the two partners was immobilized on the chip or in solution. Several of these interactions were validated by surface plasmon resonance and supported by confocal microscopy analysis of whole bacterial cells. By this approach, a number of interesting interactions have been discovered, including those occurring between OppA, DppA, PrsA, and TlpA, proteins known to be involved in protein folding and transport. These proteins, all localizing at the septum, might be part, together with HtrA, of the recently described ExPortal complex of GAS. Furthermore, SpeI was found to strongly interact with the metal transporters AdcA and Lmb. Because SpeI strictly requires zinc to exert its function, this finding provides evidence on how this superantigen, a major player in GAS pathogenesis, can acquire the metal in the host environment, where it is largely sequestered by carrier proteins. We believe that the approach proposed herein can lead to a deeper knowledge of the mechanisms underlying bacterial invasion, colonization, and pathogenesis.