Publications by authors named "Donatella Diana"

29 Publications

  • Page 1 of 1

Cooperative Binding of the Cationic Porphyrin Tris-T4 Enhances Catalytic Activity of 20S Proteasome Unveiling a Complex Distribution of Functional States.

Int J Mol Sci 2020 Sep 29;21(19). Epub 2020 Sep 29.

Dipartimento di Farmacia, Università di Napoli "Federico II", Via D. Montesano 49, 80131 Napoli, Italy.

The present study provides new evidence that cationic porphyrins may be considered as tunable platforms to interfere with the structural "key code" present on the 20S proteasome α-rings and, by consequence, with its catalytic activity. Here, we describe the functional and conformational effects on the 20S proteasome induced by the cooperative binding of the tri-cationic 5-(phenyl)-10,15,20-(tri -methyl-4-pyridyl) porphyrin (Tris-T4). Our integrated kinetic, NMR, and in silico analysis allowed us to disclose a complex effect on the 20S catalytic activity depending on substrate/porphyrin concentration. The analysis of the kinetic data shows that Tris-T4 shifts the relative populations of the multiple interconverting 20S proteasome conformations leading to an increase in substrate hydrolysis by an allosteric pathway. Based on our Tris-T4/h20S interaction model, Tris-T4 is able to affect gating dynamics and substrate hydrolysis by binding to an array of negatively charged and hydrophobic residues present on the protein surface involved in the 20S molecular activation by the regulatory proteins (RPs). Accordingly, despite the fact that Tris-T4 also binds to the α3ΔN mutant, allosteric modulation is not observed since the molecular mechanism connecting gate dynamics with substrate hydrolysis is impaired. We envisage that the dynamic view of the 20S conformational equilibria, activated through cooperative Tris-T4 binding, may work as a simplified model for a better understanding of the intricate network of 20S conformational/functional states that may be mobilized by exogenous ligands, paving the way for the development of a new generation of proteasome allosteric modulators.
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http://dx.doi.org/10.3390/ijms21197190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582714PMC
September 2020

SPR and NMR characterization of the molecular interaction between A9 peptide and a model system of HER2 receptor: A fragment approach for selecting peptide structures specific for their target.

J Pept Sci 2020 Feb 20;26(2):e3231. Epub 2019 Nov 20.

Institute of Biostructures and Bioimaging, National Research Council, 80134, Naples, Italy.

The binding process of A9 peptide toward HER2-DIVMP, a synthetic model of the receptor domain IV, was studied by using the surface plasmon resonance (SPR) technique, with the aim of validating it as a fast and reliable screening method for selecting peptide ligands specifically targeting a domain of their target. To investigate the structural basis of A9 binding to the model of HER2-DIVMP, multiple ligand-based nuclear magnetic resonance (NMR) methods were applied. The use of saturation transfer difference (STD) and WaterLOGSY NMR experiments identified key residues in the peptide for the receptor binding. Moreover, the bound conformation of the A9 peptide was obtained using transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments. The NMR data revealed an extended binding surface that confirms an in silico model previously reported. These structural findings could provide good starting points for future lead structures optimization specific for the receptor target.
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http://dx.doi.org/10.1002/psc.3231DOI Listing
February 2020

Biochemical and Conformational Characterization of Recombinant VEGFR2 Domain 7.

Mol Biotechnol 2019 Nov;61(11):860-872

Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134, Naples, Italy.

Angiogenesis is a biological process finely tuned by a plethora of pro- and anti-angiogenic molecules, among which vascular endothelial growth factors (VEGFs). Their biological activity is expressed through the interaction with three cognate receptor tyrosine kinases, VEGFR1, 2, and 3. VEGFR2 is the primary regulator of angiogenesis. Ligand-induced VEGFR2 dimerization and activation depend on direct ligand binding to extracellular domains 2 and 3 of receptor and in the establishment of interactions between proximal membrane domains. VEGFR2 domain 7 has been shown to play a crucial role in receptor dimerization and regulation, therefore, representing a convenient target for the allosteric modulation of VEGFR2 activity. The ability to prepare a functional VEGFR2D7 domain represents the starting point to the development of novel VEGFR2 binders acting as allosteric inhibitors of receptor activity. Here, we describe a robust and efficient procedure for the preparation in E. coli of the VEGFR2 domain 7. The protein was obtained with a good yield and was properly folded. It was investigated in a biochemical and structural study, providing information on its conformational arrangement and in solution properties.
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http://dx.doi.org/10.1007/s12033-019-00211-4DOI Listing
November 2019

Human Recombinant VEGFR2D4 Biochemical Characterization to Investigate Novel Anti-VEGFR2D4 Antibodies for Allosteric Targeting of VEGFR2.

Mol Biotechnol 2019 Jul;61(7):513-520

Istituto di Biostrutture e Bioimmagini, CNR, Via Nizza 52, 10126, Torino, Italy.

VEGF-A/VEGFR2 complex is the major signaling pathway involved in angiogenesis and the inhibition of this axis retards tumor growth and inflammatory disorders progression, reducing vessel sprouting. Signaling by VEGFR2 requires receptor dimerization and a well-defined orientation of monomers in the active dimer. The extracellular portion of receptor is composed of seven Ig-like domains, of which D2-3 are the ligand binding domains, while D4 and D7, establishing homotypic contacts, allosterically regulate receptor activity. The allosteric targeting of VEGFR2 represents a promising alternative to study neovascular disorders overcoming drawbacks related to competition with VEGF. In this work, we expressed in bacterial host domain 4 of VEGFR2 (VEGFR2D4). After protein refolding, we characterized the purified domain and administered it in mice for monoclonal antibodies production. One of them, mAbD4, was tested in ELISA assays, showing a nanomolar affinity for VEGFR2D4. Finally, the methodology here described could contribute to the development of antibodies which can allosterically bind VEGFR2 and therefore to be used for imaging purposes or to modulate receptor signaling.
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http://dx.doi.org/10.1007/s12033-019-00181-7DOI Listing
July 2019

VEGFR Recognition Interface of a Proangiogenic VEGF-Mimetic Peptide Determined In Vitro and in the Presence of Endothelial Cells by NMR Spectroscopy.

Chemistry 2018 Aug 9;24(44):11461-11466. Epub 2018 Jul 9.

Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134, Naples, Italy.

QK peptide is a vascular endothelial growth factor (VEGF)-mimetic molecule with significant proangiogenic activity. In particular, QK is able to bind and activate VEGF receptors (VEGFRs) to stimulate a functional response in endothelial cells. To characterize the peptide bioactivity and its molecular recognition properties, a detailed picture of the interaction between peptide QK and VEGF receptors is reported. By combining NMR spectroscopy studies in solution on the purified receptor and in the presence of intact endothelial cells, a molecular description of the binding interaction between peptide QK and VEGFR2 in the cellular context is obtained. These results reveal useful insights into the peptide biological mechanism, which opens the way to further optimization of this class of VEGF-mimicking peptides.
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http://dx.doi.org/10.1002/chem.201802117DOI Listing
August 2018

Ligand-Based NMR Study of C-X-C Chemokine Receptor Type 4 (CXCR4)-Ligand Interactions on Living Cancer Cells.

J Med Chem 2018 04 19;61(7):2910-2923. Epub 2018 Mar 19.

Dipartimento di Farmacia, Università di Napoli Federico II , 80131 Naples , Italy.

Peptide-binding G protein-coupled receptors (GPCRs) are key effectors in numerous pathological and physiological pathways. The assessment of the receptor-bound conformation of a peptidic ligand within a membrane receptor such as a GPCR is of great impact for a rational drug design of more potent analogues. In this work, we applied multiple ligand-based nuclear magnetic resonance (NMR) methods to study the interaction of peptide heptamers, derived from the C-X-C Motif Chemokine 12 (CXCL12), and the C-X-C Chemokine Receptor Type 4 (CXCR4) on membranes of human T-Leukemia cells (CCRF-CEM cells). This study represents the first structural investigation reporting the receptor-bound conformation of a peptide to a GPCR directly on a living cell. The results obtained in the field of CXCL12/CXCR4 are proofs of concept, although important information for researchers dealing with the CXCR4 field arises. General application of the presented NMR methodologies is possible and surely may help to boost the development of new therapeutic agents targeting GPCRs.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01830DOI Listing
April 2018

Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-β-OTX2-SNAIL via PTEN inhibition.

Brain 2018 05;141(5):1300-1319

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Naples, Italy.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-β signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-β activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-β/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.
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http://dx.doi.org/10.1093/brain/awy039DOI Listing
May 2018

Conformational stabilization of a β-hairpin through a triazole-tryptophan interaction.

Org Biomol Chem 2018 01;16(5):787-795

Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy.

Molecular tools to stabilize the β-hairpin conformation are needed as β-hairpin peptides are useful molecules for pharmaceutical, biological and materials applications. We explored the use of a "triazole bridge", a covalent link between two β-hairpin strands obtained through Cu-catalyzed alkyne-azide cycloaddition, combined with an aromatic-aromatic interaction. Highly conformationally stable peptides were identified by NMR screening of a small collection of cyclic peptides based on the Trpzip2 scaffold. The characteristic Trp-Trp interaction of Trpzip2 was replaced by a diagonal triazole bridge of variable length. NMR and CD analyses showed that triazole and indole rings could favorably interact to stabilize a β-hairpin conformation. The conformational stabilization depends on the length of the triazole bridge and the reciprocal position between the aromatic rings. Combining aromatic interactions and the covalent inter-strand triazole bridge is a useful strategy to obtain peptides with a high β-hairpin content.
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http://dx.doi.org/10.1039/c7ob02815fDOI Listing
January 2018

Unveiling a VEGF-mimetic peptide sequence in the IQGAP1 protein.

Mol Biosyst 2017 Jul;13(8):1619-1629

Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, Napoli, 80134, Italy.

The ability to modulate angiogenesis by chemical tools has several important applications in different scientific fields. With the perspective of finding novel proangiogenic molecules, we searched peptide sequences with a chemical profile similar to that of the QK peptide, a well described VEGF mimetic peptide. We found that residues 1617-1627 of the IQGAP1 protein show molecular features similar to those of the QK peptide sequence. The IQGAP1-derived synthetic peptide was analyzed by NMR spectroscopy and its biological activity was characterized in endothelial cells. These studies showed that this IQGAP1-derived peptide has a biological activity similar to that of VEGF and could be considered as a novel tool for reparative angiogenesis.
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http://dx.doi.org/10.1039/c7mb00190hDOI Listing
July 2017

Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response.

Sci Rep 2016 08 8;6:31295. Epub 2016 Aug 8.

Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134, Napoli, Italy.

The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor.
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http://dx.doi.org/10.1038/srep31295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976335PMC
August 2016

Cationic porphyrins are tunable gatekeepers of the 20S proteasome.

Chem Sci 2016 Feb 9;7(2):1286-1297. Epub 2015 Nov 9.

Dipartimento di Scienze Chimiche , Università di Catania , Viale Andrea Doria 6 , 95125 Catania , Italy . Email:

The 20S proteasome is a barrel-shaped enzymatic assembly playing a critical role in proteome maintenance. Access of proteasome substrates to the catalytic chamber is finely regulated through gating mechanisms which involve aromatic and negatively charged residues located at the N-terminal tails of α subunits. However, despite the importance of gates in regulating proteasome function, up to now very few molecules have been shown to interfere with the equilibrium by which the catalytic channel exchanges between the open and closed states. In this light, and inspired by previous results evidencing the antiproteasome potential of cationic porphyrins, here we combine experimental (enzyme kinetics, UV stopped flow and NMR) and computational (bioinformatic analysis and docking studies) approaches to inspect proteasome inhibition by -tetrakis(4--methylpyridyl)-porphyrin (HT4) and its two - and -isomers. We show that in a first, fast binding event HT4 accommodates in a pocket made of negatively charged and aromatic residues present in α1 (Asp10, Phe9), α3 (Tyr5), α5 (Asp9, Tyr8), α6 (Asp7, Tyr6) and α7 (Asp9, Tyr8) subunits thereby stabilizing the closed conformation. A second, slower binding mode involves interaction with the grooves which separate the α- from the β-rings. Of note, the proteasome inhibition by - and -HT4 decreases significantly if compared to the parent compound, thus underscoring the role played by spatial distribution of the four peripheral positive charges in regulating proteasome-ligand interactions. We think that our results may pave the way to further studies aimed at rationalizing the molecular basis of novel, and more sophisticated, proteasome regulatory mechanisms.
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http://dx.doi.org/10.1039/c5sc03312hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5975898PMC
February 2016

Long range Trp-Trp interaction initiates the folding pathway of a pro-angiogenic β-hairpin peptide.

Sci Rep 2015 Nov 25;5:16651. Epub 2015 Nov 25.

Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Seconda Università degli Studi Napoli, via Vivaldi 43, 81100, Caserta (Italy).

HPLW, a designed VEGF (Vascular Endothelium Growth Factor) receptor-binding peptide, assumes a well folded β-hairpin conformation in water and is able to induce angiogenesis in vivo. In this study, we investigated at atomic resolution the thermal folding/unfolding pathway of HPLW by means of an original multi-technique approach combining DSC, NMR, MD and mutagenesis analyses. In particular, careful NMR investigation of the single proton melting temperatures together with DSC analysis accurately delineate the peptide folding mechanism, which is corroborated by computational folding/unfolding simulations. The HPLW folding process consists of two main events, which are successive but do not superimpose. The first folding step initiates at 320 K upon the hydrophobic collapse of the Trp5 and Trp13 side-chains which stabilizes the concurrent β-turn formation, whose COi-HNi + 3 hydrogen bond (Asp10 → Arg7) appears particularly stable. At 316 K, once the β-turn is completely formed, the two β-strands pair, very likely starting by Trp5 and Trp13, which thus play a key role also in the final step of the β-hairpin folding. Overall, here we describe a multi-state hierarchical folding pathway of a highly structured β-hairpin, which can be classified as a broken-zipper mechanism.
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http://dx.doi.org/10.1038/srep16651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4658480PMC
November 2015

Insight into conformational modification of alpha-synuclein in the presence of neuronal whole cells and of their isolated membranes.

FEBS Lett 2015 Mar 18;589(7):798-804. Epub 2015 Feb 18.

Istituto di Biostrutture e Bioimmagini, C.N.R. via Mezzocannone, 16, 80134 Napoli, Italy. Electronic address:

A change in the conformational plasticity of α-Synuclein (α-Syn) is hypothesised to be a key step in the pathogenic mechanism of Parkinson's disease (PD). Here, we report the study of extracellular α-Syn interaction with whole cells and membranes isolated from the neuronal SH-SY5Y cells, exploiting NMR and CD spectroscopies. In addition, the crosslinking agent DSG was used to freeze the conformational and oligomeric state of α-Syn in the presence of cells. These data, in a quasi-physiological environment, confirm the protein monomeric state with a propensity to adopt a transient alpha helical following interaction with biological membranes.
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http://dx.doi.org/10.1016/j.febslet.2015.02.012DOI Listing
March 2015

Functional binding surface of a β-hairpin VEGF receptor targeting peptide determined by NMR spectroscopy in living cells.

Chemistry 2015 Jan 6;21(1):91-5. Epub 2014 Nov 6.

Istituto di Biostrutture e Bioimmagini, C.N.R., via Mezzocannone 16, 80134, Napoli (Italy).

In this study, the functional interaction of HPLW peptide with VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) was determined by using fast (15)N-edited NMR spectroscopic experiments. To this aim, (15)N uniformly labelled HPLW has been added to Porcine Aortic Endothelial Cells. The acquisition of isotope-edited NMR spectroscopic experiments, including (15)N relaxation measurements, allowed a precise characterization of the in-cell HPLW epitope recognized by VEGFR2.
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http://dx.doi.org/10.1002/chem.201403335DOI Listing
January 2015

Towards understanding the molecular recognition process in prokaryotic zinc-finger domain.

Eur J Med Chem 2015 Feb 16;91:100-8. Epub 2014 Sep 16.

Department of Environmental, Biological and Pharmaceutical Sciences and Technology, Via Vivaldi 43, 81100 Caserta, Italy; Interuniversity Centre for Research on Bioactive Peptides (CIRPEB), University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy. Electronic address:

Eukaryotic Cys2His2 zinc finger domain is one of the most common and important structural motifs involved in protein-DNA interaction. The recognition motif is characterized by the tetrahedral coordination of a zinc ion by conserved cysteine and histidine residues. We have characterized the prokaryotic Cys2His2 zinc finger motif, included in the DNA binding region (Ros87) of Ros protein from Agrobacterium tumefaciens, demonstrating that, although possessing a similar zinc coordination sphere, this domain presents significant differences from its eukaryotic counterpart. Furthermore, basic residues flanking the zinc binding region on either side have been demonstrated, by Electrophoretic Mobility Shift Assay (EMSA) experiments, to be essential for Ros DNA binding. In spite of this wealth of knowledge, the structural details of the mechanism through which the prokaryotic zinc fingers recognize their target genes are still unclear. Here, to gain insights into the molecular DNA recognition process of prokaryotic zinc finger domains we applied a strategy in which we performed molecular docking studies using a combination of Nuclear Magnetic Resonance (NMR) and Molecular Dynamics (MD) simulations data. The results demonstrate that the MD ensemble provides a reasonable picture of Ros87 backbone dynamics in solution. The Ros87-DNA model indicates that the interaction involves the first two residue of the first α-helix, and several residues located in the basic regions flanking the zinc finger domain. Interestingly, the prokaryotic zinc finger domain, mainly with the C-terminal tail that is wrapped around the DNA, binds a more extended recognition site than the eukaryotic counterpart. Our analysis demonstrates that the introduction of the protein flexibility in docking studies can improve, in terms of accuracy, the quality of the obtained models and could be particularly useful for protein showing high conformational heterogeneity as well as for computational drug design applications.
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http://dx.doi.org/10.1016/j.ejmech.2014.09.040DOI Listing
February 2015

Design, structural and biological characterization of a VEGF inhibitor β-hairpin-constrained peptide.

Eur J Med Chem 2014 Feb 24;73:210-6. Epub 2013 Dec 24.

Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134 Napoli, Italy. Electronic address:

The design, structural and biological characterization of a novel VEGF inhibitor peptide is described. The peptide was designed on the β5-β6 hairpin region of Placenta Growth Factor. NMR studies showed that the peptide assumes in solution a β-hairpin conformation similarly to the corresponding region of the natural ligand. In vitro experiments on endothelial cells demonstrated that the peptide is able to inhibit VEGF biological activity. This molecule represents a novel molecular entity to modulate VEGF activity and with potential application in therapy and diagnosis of angiogenesis-dependent diseases.
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http://dx.doi.org/10.1016/j.ejmech.2013.12.016DOI Listing
February 2014

Zinc to cadmium replacement in the prokaryotic zinc-finger domain.

Metallomics 2014 Jan;6(1):96-104

Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Via Vivaldi 43, 81100 Caserta, Italy.

Given the similar chemical properties of zinc and cadmium, zinc finger domains have been often proposed as mediators of the toxic and carcinogenic effects exerted by this xenobiotic metal. The effects of zinc replacement by cadmium in different eukaryotic zinc fingers have been reported. In the present work, to evaluate the effects of such substitution in the prokaryotic zinc finger, we report a detailed study of its functional and structural consequences on the Ros DNA binding domain (Ros87). We show that this protein, which bears important structural differences with respect to the eukaryotic domains, appears to structurally tolerate the zinc to cadmium substitution and the presence of cadmium does not affect the DNA binding activity of the protein. Moreover, we show for the first time how zinc to cadmium replacement can also take place in a cellular context. Our findings both complement and extend previous results obtained for different eukaryotic zinc fingers, suggesting that metal substitution in zinc fingers may be of relevance to the toxicity and/or carcinogenicity mechanisms of this metal.
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http://dx.doi.org/10.1039/c3mt00208jDOI Listing
January 2014

Mapping functional interaction sites of human prune C-terminal domain by NMR spectroscopy in human cell lysates.

Chemistry 2013 Sep 12;19(37):12217-20. Epub 2013 Aug 12.

Istituto di Biostrutture e Bioimmagini, CNR via Mezzocannone 16, 80134, Napoli (Italy).

Get well prune: The C-terminal third domain of h-prune is largely unfolded and involved in relevant protein-protein interactions, particularly with Nm23-H1 (see figure), GSK-3β and gelsolin. This study shows that protein functions mediated by protein-protein interactions can be accurately followed in cell lysates by using fast NMR spectroscopy, which could be easily used for a very efficient NMR drug-discovery strategy.
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http://dx.doi.org/10.1002/chem.201302168DOI Listing
September 2013

Neuroblastoma tumorigenesis is regulated through the Nm23-H1/h-Prune C-terminal interaction.

Sci Rep 2013 ;3:1351

Centro di Ingegneria Genetica e Biotecnologie Avanzate-CEINGE, Naples, Italy.

Nm23-H1 is one of the most interesting candidate genes for a relevant role in Neuroblastoma pathogenesis. H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers. Our study focuses on the role of the Nm23-H1/h-Prune protein complex in Neuroblastoma. Using NMR spectroscopy, we performed a conformational analysis of the h-Prune C-terminal to identify the amino acids involved in the interaction with Nm23-H1. We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation. Meta-analysis performed on three Neuroblastoma cohorts showed Nm23-H1 as the gene highly associated to Neuroblastoma aggressiveness. We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP. These data suggest a new avenue for potential clinical application of CPP in Neuroblastoma treatment.
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http://dx.doi.org/10.1038/srep01351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584926PMC
August 2013

Structural investigation of the VEGF receptor interaction with a helical antagonist peptide.

J Pept Sci 2013 Apr 19;19(4):214-9. Epub 2013 Feb 19.

Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy.

Angiogenesis is mainly regulated by the vascular endothelial growth factor (VEGF), a mitogen specific for endothelial cells, which binds two tyrosine kinase receptors, VEGFR1 and VEGFR2, on the surface of endothelial cells. Molecules targeting VEGF receptors are attractive to pharmacologically treat diseases associated with angiogenesis or to be used as probes in angiogenesis imaging. Recently, we reported a designed peptide targeting VEGF receptors and able to inhibit the VEGF-angiogenic response in vitro and in vivo. In this study, we employed NMR and molecular modeling methodology to investigate the molecular determinants of the interaction peptide-receptor. In particular, the peptide binding site on VEGFR1 domain 2 and the residues involved in receptor recognition have been determined. These results provide significant information to develop a new class of molecules able to recognize the VEGF receptors overexpressed in pathological angiogenesis.
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http://dx.doi.org/10.1002/psc.2480DOI Listing
April 2013

C-terminal truncation of Vascular Endothelial Growth Factor mimetic helical peptide preserves structural and receptor binding properties.

Biochem Biophys Res Commun 2012 Jul 27;424(2):290-4. Epub 2012 Jun 27.

Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134 Napoli, Italy.

Vascular Endothelial Growth Factor mimetic peptides have interesting applications in therapeutic angiogenesis. Recently, we described the proangiogenic properties of a 15 mer peptide designed on the N-terminal helix 17-25 of VEGF. The peptide was stabilized introducing well known peptide chemical tools among which N- and C-terminal capping sequence. Here, we show that the C-terminal sequence does not affect the structural and biological properties of the full-length peptide. In fact, a C-terminal truncated analog peptide resulted in a well folded and stable helix retaining the ability to bind to VEGF receptors. This study will allow to develop smaller peptidomimetic analogs able to modulate the VEGF-dependent angiogenesis.
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http://dx.doi.org/10.1016/j.bbrc.2012.06.109DOI Listing
July 2012

β-Hairpin stabilization through an interstrand triazole bridge.

Chem Commun (Camb) 2012 Jan 1;48(5):762-4. Epub 2011 Dec 1.

Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134, Napoli, Italy.

β-Hairpin peptides were conformationally stabilized through a 1,4 disubstituted 1,2,3-triazole interstrand linkage. A NMR conformational analysis revealed that the β-hairpin content depends on the number and position of substituent methylene units of the 1,2,3-triazole ring. These results will allow the design of metabolically stable peptidomimetic analogs of bioactive β-hairpin peptides.
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http://dx.doi.org/10.1039/c1cc16017fDOI Listing
January 2012

β-hairpin peptide that targets vascular endothelial growth factor (VEGF) receptors: design, NMR characterization, and biological activity.

J Biol Chem 2011 Dec 3;286(48):41680-41691. Epub 2011 Oct 3.

Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, via Mezzocannone 16, 80134 Napoli, Italy. Electronic address:

VEGF receptors have been the target of intense research aimed to develop molecules able to inhibit or stimulate angiogenesis. Based on the x-ray structure of the complex placental growth factor-VEGF receptor 1(D2), we designed a VEGF receptor-binding peptide reproducing the placental growth factor β-hairpin region Gln(87)-Val(100) that is involved in receptor recognition. A conformational analysis showed that the designed peptide adopts the expected fold in pure water. Moreover, a combination of NMR interaction analysis and cell binding studies were used to demonstrate that the peptide targets VEGF receptors. The VEGF receptor 1(D2)-interacting residues were characterized at the molecular level, and they correspond to the residues recognizing the placental growth factor sequence Gln(87)-Val(100). Finally, the peptide biological activity was characterized in vitro and in vivo, and it showed a VEGF-like behavior. Indeed, the peptide activated VEGF-dependent intracellular pathways, induced endothelial cell proliferation and rescue from apoptosis, and promoted angiogenesis in vivo. This compound is one of the few peptides known with proangiogenic activity, which makes it a candidate for the development of a novel peptide-based drug for medical applications in therapeutic angiogenesis.
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http://dx.doi.org/10.1074/jbc.M111.257402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308877PMC
December 2011

Characterization of a designed vascular endothelial growth factor receptor antagonist helical peptide with antiangiogenic activity in vivo.

J Med Chem 2011 Mar 31;54(5):1391-400. Epub 2011 Jan 31.

Dipartimento di Scienze Farmaceutiche, Università di Salerno and BioUniverSA SRL, Salerno, Italy.

Angiogenesis is a fundamental process underlining physiological and pathological conditions. It is mainly regulated by the vascular endothelial growth factor (VEGF) and its receptors, which are the main targets of molecules able to modulate the angiogenic response. Pharmaceutical therapies based on antiangiogenic drugs represent a promising approach for the treatment of several socially important diseases. We report the biological and structural characterization of a VEGF receptor binder peptide designed on the N-terminal helix of VEGF. The reported experimental evidence shows that the peptide assumes in water a well-defined helical conformation and indicates that this peptide is a VEGF receptor antagonist and possesses antiangiogenic biological activity. In particular, it inhibits VEGF stimulated endothelial cell proliferation, activation, and survival, as well as angiogenesis and tumor progression in vivo. This peptide is a candidate for the development of novel peptide-based drugs for the treatment of diseases associated with excessive VEGF-dependent angiogenesis.
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http://dx.doi.org/10.1021/jm101435rDOI Listing
March 2011

VEGFR1(D2) in drug discovery: Expression and molecular characterization.

Biopolymers 2010 ;94(6):800-9

Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. Its biological activity is mediated by the binding to the extracellular domain of two tyrosine kinase transmembrane receptors: VEGFR1 and VEGFR2. Deletion studies showed that VEGF binding site resides in the first three domains of VEGFR1 and in domains 2 and 3 of VEGFR2. In particular, the second extracellular domain of VEGFR1 (VEGFR1(D2)) contains most of the VEGF binding requirements. Here, we report an efficient expression protocol and the molecular characterization by spectroscopic techniques of VEGFR1(D2). The protein was expressed in E. coli and refolded from inclusion bodies. The recombinant protein assumes the correct fold as assessed by a combination of biochemical and functional assays as well as by NMR characterization. Furthermore, the recombinant VEGFR1(D2) was analyzed by circular dichroism and fluorescence spectroscopy. The protein obtained by this procedure is suitable for the structural characterization of the complexes with receptor binders and to be used in interaction/screening studies.
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http://dx.doi.org/10.1002/bip.21448DOI Listing
March 2011

Biochemical and structural analysis of the binding determinants of a vascular endothelial growth factor receptor peptidic antagonist.

J Med Chem 2010 Jun;53(11):4428-40

Université Paris Descartes, UFR Biomédicale, Laboratoire de Pharmacochimie Moléculaire et Cellulaire, INSERM U648, 75006 Paris, France.

Cyclic peptide antagonist c[YYDEGLEE]-NH(2), which disrupts the interaction between vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), represents a promising tool in the fight against cancer and age-related macular degeneration. Furthermore, coupled to a cyclen derivative, this ligand could be used as a medicinal imaging agent. Nevertheless, before generating such molecular probes, some preliminary studies need to be undertaken in order to define the more suitable positions for introduction of the cyclen macrocycle. Through an Ala-scan study on this peptide, we identified its binding motif, and an NMR study highlights its binding sites on the VEGFR-1D2 Ig-like domain. Guided by the structural relationship results deduced from the effect of the peptides on endothelial cells, new peptides were synthesized and grafted on beads. Used in a pull-down assay, these new peptides trap the VEGFRs, thus confirming that the identified amino acid positions are suitable for further derivatization.
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http://dx.doi.org/10.1021/jm1002167DOI Listing
June 2010

Structural analysis of a helical peptide unfolding pathway.

Chemistry 2010 May;16(18):5400-7

Istituto di Biostrutture e Bioimmagini, C.N.R. via Mezzocannone 16, 80134, Napoli, Italy.

The analysis of the folding mechanism in peptides adopting well-defined secondary structure is fundamental to understand protein folding. Herein, we describe the thermal unfolding of a 15-mer vascular endothelial growth factor mimicking alpha-helical peptide (QK(L10A)) through the combination of spectroscopic and computational analyses. In particular, on the basis of the temperature dependencies of QK(L10A) H(alpha) chemical shifts we show that the first phase of the thermal helix unfolding, ending at around 320 K, involves mainly the terminal regions. A second phase of the transition, ending at around 333 K, comprises the central helical region of the peptide. The determination of high-resolution QK(L10A) conformational preferences in water at 313 K allowed us to identify, at atomic resolution, one intermediate of the folding-unfolding pathway. Molecular dynamics simulations corroborate experimental observations detecting a stable central helical turn, which represents the most probable site for the helix nucleation in the folding direction. The data presented herein allows us to draw a folding-unfolding picture for the small peptide QK(L10A) compatible with the nucleation-propagation model. This study, besides contributing to the basic field of peptide helix folding, is useful to gain an insight into the design of stable helical peptides, which could find applications as molecular scaffolds to target protein-protein interactions.
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http://dx.doi.org/10.1002/chem.200903428DOI Listing
May 2010

Structural determinants of the unusual helix stability of a de novo engineered vascular endothelial growth factor (VEGF) mimicking peptide.

Chemistry 2008 ;14(14):4164-6

Dipartimento di Scienze Ambientali, Seconda Università di Napoli via Vivaldi 43, 81100 Caserta, Italy.

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http://dx.doi.org/10.1002/chem.200800180DOI Listing
July 2008

A new nerve growth factor-mimetic peptide active on neuropathic pain in rats.

J Neurosci 2008 Mar;28(11):2698-709

Laboratorio di Neuroscienze "R. Levi-Montalcini" and Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, 20126 Milan, Italy.

Analysis of the structure of nerve growth factor (NGF)-tyrosine kinase receptor A (TrkA) complex, site-directed mutagenesis studies and results from chemical modification of amino acid residues have identified loop 1, loop 4, and the N-terminal region of the NGF molecule as the most relevant for its biological activity. We synthesized several peptides mimicking the two loops (1 and 4) linked together with an appropriate spacer, with or without the N-terminal region. Two peptides named NL1L4 and L1L4 demonstrated good NGF agonist activity at a concentration as low as 3 mum. They induced differentiation of chick dorsal root ganglia and stimulated tyrosine phosphorylation of TrkA, but not TrkB, receptor. In addition L1L4 was able to induce differentiation of PC12 cells. More interestingly, the peptide with the highest "in vitro" activity (L1L4) was shown to reduce neuropathic behavior and restore neuronal function in a rat model of peripheral neuropathic pain, thereby suggesting a potential therapeutic role for this NGF-mimetic peptide.
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http://dx.doi.org/10.1523/JNEUROSCI.5201-07.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6670672PMC
March 2008