Luca Bagnasco

A retro-inverso peptide homologous to helix 1 of c-Myc is a potent and specific inhibitor of proliferation in different cellular systems

In 1998 we reported that an L‐peptide derived from H1 of c‐Myc (Int‐H1‐S6A,F8A), linked to an internalization sequence from the third α‐helix of Antennapedia, was endowed with an antiproliferative and proapoptotic activity toward a human mammary cancer cell line: The activity apparently depends upon the presence of the Myc motif. In the present work we have added new dimensions to our original findings. It is known that short retro‐inverso (RI‐) peptides can assume a 3D conformation very close to their corresponding L‐forms and can be recognized by the same monoclonal antibody. We synthesized a RI‐peptide form of our original L‐peptide: It was much more resistant to serum peptidases than the original molecule (a half life of days rather than hours); in addition, the RI‐form of the original Antennapedia internalization sequence was perfectly capable of carrying a D‐peptide into human cells. We have studied three different potentially active peptides. L‐peptides: Int‐H1wt, Int‐H1‐S6A,F8A. D‐peptides: RI‐Int‐H1‐S6A,F8A. We have also studied three presumed control peptides: Int and RI‐Int (no H1 motif), H1‐S6A,F8A (no internalization sequence). Both ‘active’ and ‘control’ peptides have essentially confirmed our expectations, however, in cells treated with the higher concentration (10 μM) of the control peptide RI‐Int, non‐Myc related side effects could be detected. In order to investigate whether the antiproliferative activities displayed by some of our molecules were indeed related to an interference with the role of c‐Myc (and molecules of the family), we chose an iso‐amphipathic modified peptide of the H1 motif, with a proximity coefficient >50% and where the major change was at position 7 (F→A). From a family of 73 H1 motifs belonging to (H1‐Loop‐H2) human sequences, the smallest evolutionary distance from our reference peptide was observed for the H1 of N‐Myc, L‐Myc, c‐Myc, H1‐S6A,F8A of c‐Myc, and Max, in that order. Our reference peptide was therefore appropriate as a check of whether we were indeed observing activities related to Myc functions. Both Int‐H1isoamph and the corresponding RI‐Int‐H1isoamph peptide were synthesized and studied. In terms of biological targets, we added to the human mammary cancer line of our previous work (MCF‐7 cells) a colon cancer line (HCT‐116 cells) and also a system of normal cells: human peripheral blood lymphocytes (PBLs) stimulated with phytohemoagglutinin (PHA). Peptides carrying an iso‐amphipathic‐modified H1 sequence were always very clearly (3‐10 times) less active than the corresponding peptides carrying a conserved “H1 of Myc” motif. This finding was noted in five independent situations (all the cellular models considered at the present time): MCF‐7 cells treated with L‐peptides; MCF‐7 cells treated with RI‐peptides; HCT‐116 cells treated with L‐peptides; PBLs treated with L‐peptides; PBLs treated with RI‐peptides. Modulation of transcription levels of ornithine decarboxylase (ODC), p53, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), in PBLs treated with our different molecules, was well compatible with an interference by our active peptides at the level of Myc transcriptional activity. We had already reported a similar observation in MCF‐7 cells. On a molar basis, RI‐peptides were about 5–10 times more potent and 30–35 times more stable in complete culture medium, than their corresponding L‐forms. RI‐Int can probably internalize longer peptido‐mimetic molecules (for instance molecules mimetic of (H1‐Loop‐H2), or even more. These possibilities open the way to rodent studies and to more potent/selective Myc inhibitors—two steps closer to a potential drug.

Binding of abscisic acid to human LANCL2

The phytohormone abscisic acid (ABA) is the central regulator of abiotic stress in plants and plays important roles during plant growth and development. In animal cells, ABA was shown to be an endogenous hormone, acting as a stress signal and stimulating cell functions involved in inflammatory responses and in insulin release. Recently, we demonstrated that Lanthionine synthetase component C-like protein 2 (LANCL2) is required for ABA binding to the plasmamembrane of granulocytes and for the activation of the signaling pathway triggered by ABA in human granulocytes and in rat insulinoma cells. In order to investigate whether ABA activates LANCL2 via direct interaction, we performed specific binding studies on human LANCL2 recombinant protein using different experimental approaches (saturation binding, scintillation proximity assays, dot blot experiments and affinity chromatography). Altogether, results indicate that human recombinant LANCL2 binds ABA directly and provide the first demonstration of ABA binding to a mammalian ABA receptor.

A novel Bim-BH3-derived Bcl-XL inhibitor: biochemical characterization, in vitro, in vivo and ex-vivo anti-leukemic activity.

BH3-only members of the Bcl-2 family exert a fundamental role in apoptosis induction. This work focuses on the development of a novel peptidic molecule based on the BH3 domain of Bim. The antiapoptotic molecule Bcl-XL, involved in cancer development/progression and tumour resistance to cytotoxic drugs, is a target for Bim. According to a rational study of the structural interactions between wt Bim-BH3 and Bcl-XL, we replaced specific residues of Bim-BH3 with natural and non-natural aminoacids and added an internalizing sequence, thus increasing dramatically the inhibitory activity of our modified Bim-BH3 peptide, called 072RB. Confocal microscopy and flow cytometry demonstrated cellular uptake and internalization of 072RB, followed by co-localization with mitochondria. Multiparameter flow cytometry demonstrated that the 072RB dose-dependent growth inhibition of leukaemia cell lines was due to apoptotic cell death. No effect was observed when cells were treated with the internalizing vector alone or a mutated control peptide (single aminoacid substitution L94A). Ex-vivo derived leukemic cells from acute myeloid leukaemia (AML) patients underwent cell death when cultured in vitro in the presence of 072RB. Conversely, no significant cytotoxic effect was observed when 072RB was administered to cultures of peripheral blood mononuclear cells, either resting or PHA-stimulated, and bone marrow cells of normal donors. Xenografts of human AML cells in NOD/SCID mice displayed a significant delay of leukemic cell growth upon treatment with 072RB administered intravenously (15 mg/Kg three times, 48 hours after tumour cell injection). Altogether, these observations support the therapeutic potentials of this novel BH3 mimetic.

Internalization via Antennapedia protein transduction domain of an scFv antibody toward c-Myc protein

We constructed a single‐chain variable fragment miniantibody (G11‐scFv) directed toward the transactivation domain of c‐Myc, which is fused with the internalization domain Int of Antennapedia at its carboxyl terminus (a cargo‐carrier construct). In ELISA experiments, an EC50 for binding saturation was achieved at concentrations of G11‐scFv‐Int(—)of ~10−8 M. Internalization of a fluoresceinated R‐G11‐scFv‐Int(+) construct was observed in intact human cultured cells with confocal microscopy. After 5 h of incubation in medium containing 1 μM Fl‐G11‐scFv‐Int(+) or Fl‐G11‐scFv‐Int(—), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of Fl‐G11‐scFv‐Int(+), relative to the extracellular culture medium concentration, were 4–5 times higher in the cytoplasm, 7–8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the Fl‐G11‐scFv‐Int(—) construct was 3–4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 μM G11‐scFv‐Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets. Avignolo, C., Bagnasco, L., Biasotti, B., Melchiori, A., Tomati, V., Bauer, I., Salis, A., Chiossone, L., Mingari, M. C., Orecchia, P., Carnemolla, B., Neri, D., Zardi, L., Parodi, S. Internalization via Antennapedia protein trans‐duction domain of an scFv antibody toward c‐Myc protein. FASEB J. 22, 1237‐1245 (2008)

Inhibition of a protein-protein interaction between INI1 and c-Myc by small peptidomimetic molecules inspired by Helix-1 of c-Myc: identification of a new target of potential antineoplastic interest

c‐Myc is a transcription modulator proto‐oncogene. When overexpressed, it becomes an important contributor to the multi‐hit process of malignant transformation. In two earlier papers in this journal (see refs. 19, 20) we reported that retro‐inverso pep‐tidomimetic molecules inspired by the Helix‐1 of c‐Myc motif could be sequence‐specific antiproliferative agents active in the low micromolar range. We also found that our peptides were not opening the four‐alpha‐helix Myc:Max bundle. Their antiproliferative activity in cancer cell lines needs the presence of side chains projecting outside of the bundle in the corresponding native H1 motif. This observation suggested interference with an external partner. In this study we investigated the INI1:Myc interaction. INI1 is a subunit of the SWI/SNF complex (component of the enhanceo‐some surrounding Myc:Max heterodimer). The INI1: Myc interaction was confirmed via pull down, ELISA, and fluorescence anisotropy assays. According to the length of INI1 fragments used, we calculated Kds ranging between 1.3×10−6 and 4.8×10−7 M. The three different techniques applied showed that the INI1:Myc interaction was also the target of our retro‐inverso peptidomimetic molecules, which seem to bind specifically at INI1. A Myc binding, 21aa INI1 fragment (minimum interacting sequence), could inspire the synthesis of a new class of more selective c‐Myc inhibitors.—Bagnasco, L., Tortolina, L., Biasotti, B., Castagnino, N., Ponassi, R., Tomati, V., Nieddu, E., Stier, G., Malacarne, D., and Parodi, S. Inhibition of a protein‐protein interaction between INI1 and c‐Myc by small peptidomimetic molecules inspired by Helix‐1 of c‐Myc: identification of a new target of potential antineoplastic interest. FASEB J. 21, 1256–1263 (2007)

Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: Identification of differentially expressed and MAR-binding proteins

Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.

Selection and Characterization of Single Stranded DNA Aptamers for the Hormone Abscisic Acid

The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions in higher plants. Recently, ABA has been also identified as an endogenous hormone in mammals, regulating different cell functions including inflammatory processes, stem cell expansion, insulin release, and glucose uptake. Aptamers are short, single-stranded (ss) oligonucleotidesable to recognize target molecules with high affinity. The small size of the ABA molecule represented a challenge for aptamer development and the aim of this study was to develop specific anti-ABA DNA aptamers. Biotinylated abscisic acid (bio-ABA) was immobilized on streptavidin-coated magnetic beads. DNA aptamers against bio-ABA were selected with 7 iterative rounds of the systematic evolution of ligands by exponential enrichment method (SELEX), each round comprising incubation of the ABA-binding beads with the ssDNA sequences, DNA elution, electrophoresis, and polymerase chain reaction (PCR) amplification. The PCR product was cloned and sequenced. The binding affinity of several clones was determined using bio-ABA immobilized on streptavidin-coated plates. Aptamer 2 and aptamer 9 showed the highest binding affinity, with dissociation constants values of 0.98 ± 0.14 μM and 0.80 ± 0.07 μM, respectively. Aptamers 2 and 9 were also able to bind free, unmodified ABA and to discriminate between different ABA enantiomers and isomers. Our findings indicate that ssDNA aptamers can selectively bind ABA and could be used for the development of ABA quantitation assays.

Functional characterization of a synthetic abscisic acid analog with anti-inflammatory activity on human granulocytes and monocytes.

The phytohormone abscisic acid (ABA), in addition to regulating several important physiological functions in plants, is also produced and released by human granulocytes and monocytes where it stimulates cell activities involved in the innate immune response. Here we describe the properties of an ABA synthetic analog that competes with the hormone for binding to human granulocyte membranes and to purified recombinant LANCL2 (the human ABA receptor) and inhibits several ABA-triggered inflammatory functions of granulocytes and monocytes in vitro: chemotaxis, phagocytosis, reactive oxygen species production and release of prostaglandin E(2) (PGE(2)) by human granulocytes, release of PGE(2) and of monocyte chemoattractant protein-1 by human monocytes. This observation provides a proof of principle that ABA antagonists may represent a new class of anti-inflammatory agents.

Dynamic Simulations of Pathways Downstream of TGFβ, Wnt and EGF-Family Growth Factors, in Colorectal Cancer, including Mutations and Treatments with Onco-Protein Inhibitors

With reference to colorectal cancer, we have reconstructed a Molecular Interaction Map downstream of TGFβ, Wnt and EGF-family. Based on an extensive and systematic direct/indirect data extrapolation from several dozens of published experimental papers, and some data interpolation that could fit with the general behavior of this signaling-network region, we were able to obtain an operative mathematical simulation model. We could simulate normal conditions of the network, behavior in the presence of important colorectal cancer mutations, behavior in the presence of virtual drug inhibitors of different specifically altered onco-proteins affected by excess of function. The dynamic behavior of the simulation seems quite reasonable, in terms of what is known about the physiology and the pathology of this signaling-network region. Preliminary experimental verification experiments look encouraging.

Innovative leads for antineoplastic drugs suggested by a more intimate familiarity with structural motifs of oncoproteins

Innovative leads for antineoplastic drugs suggested by a more intimate familiarity with structural motifs of oncoproteins