Publications by authors named "Alessandro Gori"

58 Publications

A bi-functional polymeric coating for the co-immobilization of proteins and peptides on microarray substrates.

Anal Chim Acta 2021 Dec 9;1187:339138. Epub 2021 Oct 9.

Istituto di Scienze e Tecnologie Chimiche "G.Natta", National Research Council of Italy, Via Mario Bianco 9, 20131, Milan, Italy.

The analytical performance of the microarray technique in screening the affinity and reactivity of molecules towards a specific target, is highly affected by the coupling chemistry adopted to bind probes to the surface. However, the surface functionality limits the biomolecules that can be attached to the surface to a single type of molecule, thus forcing the execution of separate analyses to compare the performance of different species in recognizing their targets. Here we introduce a new N, N-dimethylacrylamide-based polymeric coating, bearing simultaneously different functionalities (N-acryloyloxysuccinimide and azide groups) to allow an easy and straightforward method to co-immobilize proteins and oriented peptides on the same substrate. The bi-functional copolymer has been obtained by partial post polymerization modification of the functional groups of a common precursor. A NMR characterization of the copolymer was conducted to quantify the percentage of NAS that has been transformed into azido groups. The polymer was used to coat surfaces onto which both native antibodies and alkyne modified peptides were immobilized, to perform the phenotype characterization of extracellular vesicles (EVs). This strategy represents a convenient method to reduce the number of analysis, thus possible systematic or random errors, besides offering a drastic shortage in time, reagents and costs.
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http://dx.doi.org/10.1016/j.aca.2021.339138DOI Listing
December 2021

The tumor suppressor folliculin inhibits lactate dehydrogenase A and regulates the Warburg effect.

Nat Struct Mol Biol 2021 08 11;28(8):662-670. Epub 2021 Aug 11.

Department of Urology, SUNY Upstate Medical University, Syracuse, NY, USA.

Aerobic glycolysis in cancer cells, also known as the 'Warburg effect', is driven by hyperactivity of lactate dehydrogenase A (LDHA). LDHA is thought to be a substrate-regulated enzyme, but it is unclear whether a dedicated intracellular protein also regulates its activity. Here, we identify the human tumor suppressor folliculin (FLCN) as a binding partner and uncompetitive inhibitor of LDHA. A flexible loop within the amino terminus of FLCN controls movement of the LDHA active-site loop, tightly regulating its enzyme activity and, consequently, metabolic homeostasis in normal cells. Cancer cells that experience the Warburg effect show FLCN dissociation from LDHA. Treatment of these cells with a decapeptide derived from the FLCN loop region causes cell death. Our data suggest that the glycolytic shift of cancer cells is the result of FLCN inactivation or dissociation from LDHA. Together, FLCN-mediated inhibition of LDHA provides a new paradigm for the regulation of glycolysis.
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http://dx.doi.org/10.1038/s41594-021-00633-2DOI Listing
August 2021

Nanogold Functionalized With Lipoamide-DGR: A Simple, Robust and Versatile Nanosystem for αvβ3-Integrin Targeting.

Front Chem 2021 28;9:690357. Epub 2021 May 28.

Tumor Biology and Vascular Targeting Unit, Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Gold nanoparticles functionalized with DGR, a tripeptide motif that recognizes αvβ3 integrin overexpressed in tumor vessels, have been used as nano-vectors for the delivery of cytokines to tumors. Functionalization of nanogold with this peptide has been achieved by coating nanoparticles with a peptide-albumin conjugate consisting of heterogeneous molecules with a variable number of linkers and peptides. To reduce nanodrug heterogeneity we have designed, produced and preclinically evaluated a homogeneous and well-defined reagent for nanogold functionalization, consisting of a head-to-tail cyclized CGDGRG peptide () coupled its thiol group to maleimide-PEG-lipoamide (LPA). The resulting -PEG-LPA compound can react with nanogold lipoamide to form a stable bond. studies have shown that , after coupling to nanogold, maintains its capability to bind purified αvβ3 and αvβ3-expressing cells. Nanogold functionalized with this peptide can also be loaded with bioactive tumor necrosis factor-α (TNF) to form a bi-functional nanodrug that can be stored for three days at 37°C or >1 year at low temperatures with no loss αvβ3-binding properties and TNF-cytolytic activity. Nanoparticles functionalized with both and TNF induced tumor eradication in WEHI-164 fibrosarcoma-bearing mice more efficiently than nanoparticles lacking the targeting moiety. These results suggest that -PEG-LPA is an efficient and well-defined reagent that can be used to produce robust and more homogeneous nano-vectors for the delivery of TNF and other cytokines to αvβ3 positive cells.
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http://dx.doi.org/10.3389/fchem.2021.690357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8194705PMC
May 2021

Enhancement of doxorubicin anti-cancer activity by vascular targeting using IsoDGR/cytokine-coated nanogold.

J Nanobiotechnology 2021 May 5;19(1):128. Epub 2021 May 5.

Tumor Biology and Vascular Targeting Unit, Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, via Olgettina 58, 20132, Milan, Italy.

Background: Gold nanospheres tagged with peptides containing isoDGR (isoAsp-Gly-Arg), an αvβ3 integrin binding motif, represent efficient carriers for delivering pro-inflammatory cytokines to the tumor vasculature. We prepared bi- or trifunctional nanoparticles bearing tumor necrosis factor-α (TNF) and/or interleukin-12 (IL12) plus a peptide containing isoDGR, and we tested their anti-cancer effects, alone or in combination with doxorubicin, in tumor-bearing mice.

Results: In vitro biochemical studies showed that both nanodrugs were monodispersed and functional in terms of binding to TNF and IL12 receptors and to αvβ3. In vivo studies performed in a murine model of fibrosarcoma showed that low doses of bifunctional nanoparticles bearing isoDGR and TNF (corresponding to few nanoparticles per cell) delayed tumor growth and increased the efficacy of doxorubicin without worsening its toxicity. Similar effects were obtained using trifunctional nanoparticles loaded with isoDGR, TNF and IL12. Mechanistic studies showed that nanoparticles bearing isoDGR and TNF could increase doxorubicin penetration in tumors a few hours after injection and caused vascular damage at later time points.

Conclusion: IsoDGR-coated gold nanospheres can be exploited as a versatile platform for single- or multi-cytokine delivery to cells of the tumor vasculature. Extremely low doses of isoDGR-coated nanodrugs functionalized with TNF or TNF plus IL12 can enhance doxorubicin anti-tumor activity.
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http://dx.doi.org/10.1186/s12951-021-00871-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097910PMC
May 2021

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis.

Cells 2021 03 4;10(3). Epub 2021 Mar 4.

Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" (SCITEC), Consiglio Nazionale delle Ricerche, Via Alfonso Corti 12, 20133 Milano, Italy.

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators' safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.
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http://dx.doi.org/10.3390/cells10030544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001372PMC
March 2021

SARS-CoV-2 Epitope Mapping on Microarrays Highlights Strong Immune-Response to N Protein Region.

Vaccines (Basel) 2021 Jan 11;9(1). Epub 2021 Jan 11.

National Research Council of Italy, Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" (SCITEC-CNR), Via Mario Bianco 9, 20131 Milano, Italy.

A workflow for rapid SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 spike (S), nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155-71) providing good diagnostic performance in discriminating Covid-19 positive vs. healthy individuals. Using this epitope, 92% sensitivity and 100% specificity were reached for IgG detection in Covid-19 samples, and no cross-reactivity with common cold coronaviruses was detected. Likewise, IgM immunoreactivity in samples collected within the first month after symptoms onset showed discrimination ability. Overall, epitope 155-171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.
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http://dx.doi.org/10.3390/vaccines9010035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827214PMC
January 2021

Self-Assembling Peptide Hydrogels for 3D Microarrays.

Methods Mol Biol 2021 ;2237:179-189

National Research Council of Italy-Istituto di Scienze e Tecnologie Chimiche (SCITEC-CNR), Milan, Italy.

Recent advances in biosensing analytical platforms have brought relevant outcomes for novel diagnostic and therapy-oriented applications. In this context, hydrogels have emerged as appealing matrices to locally confine biomolecules onto sensing surfaces under solution mimetic conditions, preserving their structural integrity and function. Here, we describe the application of a self-assembling peptide hydrogel as a suitable matrix for 3D microarray bioassays. The hydrogel is printable and self-adhesive and allows for fast analyte diffusion. As a showcase example, we describe its application in a diagnostic immunoassay for the detection of arbovirus infection.
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http://dx.doi.org/10.1007/978-1-0716-1064-0_14DOI Listing
March 2021

Multi-biomarker approach and IBR index to evaluate the effects of different contaminants on the ecotoxicological status of Apis mellifera.

Ecotoxicol Environ Saf 2021 Jan 30;208:111486. Epub 2020 Oct 30.

Department of Physical, Earth and Environmental Sciences, University of Siena, via Mattioli, 4, 53100 Siena, Italy.

The honeybee, Apis mellifera L. (Hymenoptera: Apidae), a keystone pollinator of wild plant species and agricultural crops, is disappearing globally due to parasites and diseases, habitat loss, genetic constraints, beekeeper management issues and to the widespread use of pesticides. Besides insecticides, widely studied in this species, honeybees are also exposed to herbicides and fungicides and heavy metals whose lethal and sublethal effects need to be investigated. In this context, our study aimed to evaluate the effects of fungicides and of heavy metals on honeybees and to develop and apply a multi-biomarker approach that include an Integrated Biological Index (IBRv2) to assess the toxicological status of this species. Biomarkers of neurotoxicity (AChE and CaE), metabolic alteration (ALP, and GST) and immune system (LYS, granulocytes) were measured, following honeybees' exposure to cadmium or to a crop fungicide, using the genotoxic compound EMS as positive control. A biomarker of genotoxicity (NA assay) was developed and applied for the first time in honeybees. At the doses tested, all the contaminants showed sublethal toxicity to the bees, highlighting in particular genotoxic effects. The data collected were analyzed by an IBRv2 index, which integrated the seven biomarkers used in this study. IBRv2 index increased with increasing cadmium or fungicide concentrations. The IBRv2 represents a simple tool for a general description of honeybees ecotoxicological health status. Results highlight the need for more in-depth investigations on the effects of fungicides on non-target organisms, such as honeybees, using sensitive methods for the determination of sublethal effects. This study contributes to the development of a multi-biomarker approach to be used for a more accurate ecotoxicological environmental monitoring of these animals.
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http://dx.doi.org/10.1016/j.ecoenv.2020.111486DOI Listing
January 2021

Hexokinase 2 displacement from mitochondria-associated membranes prompts Ca -dependent death of cancer cells.

EMBO Rep 2020 07 8;21(7):e49117. Epub 2020 May 8.

Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy.

Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca overload caused by Ca release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca entry through plasma membrane. This results in Ca -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.
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http://dx.doi.org/10.15252/embr.201949117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332982PMC
July 2020

Membrane-binding peptides for extracellular vesicles on-chip analysis.

J Extracell Vesicles 2020 17;9(1):1751428. Epub 2020 Apr 17.

Consiglio Nazionale delle Ricerche, Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" (SCITEC), Milan, Italy.

Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a "universal" marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis.
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http://dx.doi.org/10.1080/20013078.2020.1751428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178839PMC
April 2020

Chemical Perturbation of Oncogenic Protein Folding: from the Prediction of Locally Unstable Structures to the Design of Disruptors of Hsp90-Client Interactions.

Chemistry 2020 Aug 8;26(43):9459-9465. Epub 2020 Jul 8.

SCITEC-CNR, via Mario Bianco 9, 20131, Milano, Italy.

Protein folding quality control in cells requires the activity of a class of proteins known as molecular chaperones. Heat shock protein-90 (Hsp90), a multidomain ATP driven molecular machine, is a prime representative of this family of proteins. Interactions between Hsp90, its co-chaperones, and client proteins have been shown to be important in facilitating the correct folding and activation of clients. Hsp90 levels and functions are elevated in tumor cells. Here, we computationally predict the regions on the native structures of clients c-Abl, c-Src, Cdk4, B-Raf and Glucocorticoid Receptor, that have the highest probability of undergoing local unfolding, despite being ordered in their native structures. Such regions represent potential ideal interaction points with the Hsp90-system. We synthesize mimics spanning these regions and confirm their interaction with partners of the Hsp90 complex (Hsp90, Cdc37 and Aha1) by Nuclear Magnetic Resonance (NMR). Designed mimics selectively disrupt the association of their respective clients with the Hsp90 machinery, leaving unrelated clients unperturbed and causing apoptosis in cancer cells. Overall, selective targeting of Hsp90 protein-protein interactions is achieved without causing indiscriminate degradation of all clients, setting the stage for the development of therapeutics based on specific chaperone:client perturbation.
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http://dx.doi.org/10.1002/chem.202000615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415569PMC
August 2020

Halogenation of the -Terminus Tyrosine 10 Promotes Supramolecular Stabilization of the Amyloid-β Sequence 7-12.

ChemistryOpen 2020 02 25;9(2):253-260. Epub 2020 Feb 25.

Dept. Chem., Mater., and Chem. Eng. "Giulio Natta" Politecnico di Milano Via L. Mancinelli 7 20131 Milano Italy.

Here, we demonstrate that introduction of halogen atoms at the tyrosine 10 phenol ring of the DSGYEV sequence derived from the flexible amyloid-β -terminus, promotes its self-assembly in the solid state. In particular, we report the crystal structures of two halogen-modified sequences, which we found to be stabilized in the solid state by halogen-mediated interactions. The structural study is corroborated by Non-Covalent Interaction (NCI) analysis. Our results prove that selective halogenation of an amino acid enhances the supramolecular organization of otherwise unstructured biologically-relevant sequences. This method may develop as a general strategy for stabilizing highly polymorphic peptide regions.
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http://dx.doi.org/10.1002/open.201900350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041548PMC
February 2020

A nicotinamide phosphoribosyltransferase-GAPDH interaction sustains the stress-induced NMN/NAD salvage pathway in the nucleus.

J Biol Chem 2020 03 27;295(11):3635-3651. Epub 2020 Jan 27.

Department of Pharmaceutical Sciences, University of Piemonte Orientale, Largo Donegani 2, 28100 Novara, Italy. Electronic address:

All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD, its phosphorylated variant NAD(P), and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes ( poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD pool is maintained and how it is replenished upon NAD consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to HO or -nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD salvage pathway.
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http://dx.doi.org/10.1074/jbc.RA119.010571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076215PMC
March 2020

A stapled chromogranin A-derived peptide is a potent dual ligand for integrins αvβ6 and αvβ8.

Chem Commun (Camb) 2019 Dec;55(98):14777-14780

IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.

Combining 2D STD-NMR, computation, biochemical assays and click-chemistry, we have identified a chromogranin-A derived compound (5) that has high affinity and bi-selectivity for αvβ6 and αvβ8 integrins and is stable in microsomal preparations. 5 is suitable for nanoparticle functionalization and delivery to cancer cells, holding promise for diagnostic and/or therapeutic applications.
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http://dx.doi.org/10.1039/c9cc08518aDOI Listing
December 2019

Peptides for Infectious Diseases: From Probe Design to Diagnostic Microarrays.

Antibodies (Basel) 2019 Mar 12;8(1). Epub 2019 Mar 12.

Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Via Mario Bianco 9, 20131 Milano, Italy.

Peptides and peptidomimetics have attracted revived interest regarding their applications in chemical biology over the last few years. Their chemical versatility, synthetic accessibility and the ease of storage and management compared to full proteins have made peptides particularly interesting in diagnostic applications, where they proved to efficiently recapitulate the molecular recognition properties of larger protein antigens, and were proven to be able to capture antibodies circulating in the plasma and serum of patients previously exposed to bacterial or viral infections. Here, we describe the development, integration and application of strategies for computational prediction and design, advanced chemical synthesis, and diagnostic deployment in multiplexed assays of peptide-based materials which are able to bind antibodies of diagnostic as well as therapeutic interest. By presenting successful applications of such an integrated strategy, we argue that they will have an ever-increasing role in both basic and clinical realms of research, where important advances can be expected in the next few years.
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http://dx.doi.org/10.3390/antib8010023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640701PMC
March 2019

Clickable cellulosic surfaces for peptide-based bioassays.

Talanta 2019 Dec 12;205:120152. Epub 2019 Jul 12.

Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Via Mario Bianco, 9, 20131, Milano, Italy. Electronic address:

The use of peptides in paper-based analytics is a highly appealing field, yet it suffers from severe limitations. This is mostly due to the loss of effective target recognition properties of this relatively small probes upon nonspecific adsorption onto cellulose substrates. Here we address this issue by introducing a simple polymer-based strategy to obtain clickable cellulose surfaces, that we exploited for the chemoselective bioconjugation of peptide bioprobes. Our method largely outperformed standard adsorption-based immobilization strategy in a challenging, real case immunoassay, namely the diagnostic discrimination of Zika + individuals from healthy controls. Of note, the clickable polymeric coating not only allows efficient peptides bioconjugation, but it provides favorable anti-fouling properties to the cellulosic support. We envisage our strategy to broaden the repertoire of cellulosic materials manipulation and promote a renewed interest in peptide-based paper bioassays.
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http://dx.doi.org/10.1016/j.talanta.2019.120152DOI Listing
December 2019

Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment.

EMBO Rep 2019 10 14;20(10):e47788. Epub 2019 Aug 14.

Biomolecular NMR Laboratory, Division of Genetics and Cell Biology, IRCCS Ospedale San Raffaele, Milan, Italy.

Extracellular HMGB1 triggers inflammation following infection or injury and supports tumorigenesis in inflammation-related malignancies. HMGB1 has several redox states: reduced HMGB1 recruits inflammatory cells to injured tissues forming a heterocomplex with CXCL12 and signaling via its receptor CXCR4; disulfide-containing HMGB1 binds to TLR4 and promotes inflammatory responses. Here we show that diflunisal, an aspirin-like nonsteroidal anti-inflammatory drug (NSAID) that has been in clinical use for decades, specifically inhibits in vitro and in vivo the chemotactic activity of HMGB1 at nanomolar concentrations, at least in part by binding directly to both HMGB1 and CXCL12 and disrupting their heterocomplex. Importantly, diflunisal does not inhibit TLR4-dependent responses. Our findings clarify the mode of action of diflunisal and open the way to the rational design of functionally specific anti-inflammatory drugs.
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http://dx.doi.org/10.15252/embr.201947788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776901PMC
October 2019

Towards precision medicine: the role and potential of protein and peptide microarrays.

Analyst 2019 Sep;144(18):5353-5367

Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Via Mario Bianco, 9, 20131, Milano, Italy.

Although the traditional strategy of developing general medical treatments for heterogeneous patient populations has a well-established track record, the acknowledgment that one-size-does-not-fit-all is pushing health-care to enter a new era of tailored interventions. The advent of precision medicine is fueled by the high-throughput analysis of individual DNA variants and mRNA expression profiles. However, due to the role of proteins in providing a more direct view of disease states than genomics alone, the ability to comprehensively analyze protein alterations and post translational modifications (PTMs) is a necessary step to unravel disease mechanisms, develop novel biomarkers and targeted therapies. Protein and peptide microarrays can play a major role in this frame, due to high-throughput, low sample consumption and wide applicability. Here, their current role and potentialities are discussed through the review of some promising applications in the fields of PTMs analysis, enzyme screening, high-content immune-profiling and the phenotyping of extracellular vesicles.
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http://dx.doi.org/10.1039/c9an01142kDOI Listing
September 2019

Computational Analysis of Dengue Virus Envelope Protein (E) Reveals an Epitope with Flavivirus Immunodiagnostic Potential in Peptide Microarrays.

Int J Mol Sci 2019 Apr 18;20(8). Epub 2019 Apr 18.

Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Via Mario Bianco 9, 20131 Milano, Italy.

The mosquito-borne viral disease caused by the Dengue virus is an expanding global threat. Diagnosis in low-resource-settings and epidemiological surveillance urgently requires new immunoprobes for serological tests. Structure-based epitope prediction is an efficient method to design diagnostic peptidic probes able to reveal specific antibodies elicited in response to infections in patients' sera. In this study, we focused on the Dengue viral envelope protein (E); computational analyses ranging from extensive Molecular Dynamics (MD) simulations and energy-decomposition-based prediction of potentially immunoreactive regions identified putative epitope sequences. Interestingly, one such epitope showed internal dynamic and energetic properties markedly different from those of other predicted sequences. The epitope was thus synthesized as a linear peptide, modified for chemoselective immobilization on microarrays and used in a serological assay to discriminate Dengue-infected individuals from healthy controls. The synthetic epitope probe showed a diagnostic performance comparable to that of the full antigen in terms of specificity and sensitivity. Given the high level of sequence identity among different flaviviruses, the epitope was immune-reactive towards Zika-infected sera as well. The results are discussed in the context of the quest for new possible structure-dynamics-based rules for the prediction of the immunoreactivity of selected antigenic regions with potential pan-flavivirus immunodiagnostic capacity.
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http://dx.doi.org/10.3390/ijms20081921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514720PMC
April 2019

In Situ Generation of Chiroptically-Active Gold-Peptide Superstructures Promoted by Iodination.

ACS Nano 2019 02 16;13(2):2158-2166. Epub 2019 Jan 16.

Hyber Center of Excellence, Department of Applied Physics , Aalto University , Puumiehenkuja 2 , FI-00076 Espoo , Finland.

Peptide-mediated routes to the synthesis of plasmonic nanoparticles have been drawing increasing attention for the development of chiroptically active nanoscale architectures. However, designing a multifunctional peptide able to drive the formation of structurally defined nanomaterials endowed with specific functionalities is still challenging. In this work, iodination has been devised as a strategy to strengthen Au-reduction capability of the amyloidogenic peptide DFNKF and combine it with its distinctive self-assembly features. Thanks to the Au-mediated C-I activation on the phenylalanine iodobenzenes, the peptides yield efficient Au-reduction ability promoting the synthesis of Au nanoparticles, and simultaneously working as templates for their spontaneous self-assembly into spherical superstructures endowed with chiroptical activities. The reaction occurs in situ through a one-pot process in aqueous media. The generality of this approach has been demonstrated using an iodinated derivative of the peptide KLVFF, which also showed reducing and templating abilities forming chiroptically active helical superstructures decorated with Au nanoparticles.
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http://dx.doi.org/10.1021/acsnano.8b08805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396319PMC
February 2019

Enhancement of Tumor Homing by Chemotherapy-Loaded Nanoparticles.

Small 2018 11 7;14(45):e1802886. Epub 2018 Oct 7.

Laboratory of Experimental Therapy in Oncology, IRCCS Istituto Giannina Gaslini, Via G. Gaslini 5, 16147, Genoa, Italy.

Targeted delivery of anticancer drugs with nanocarriers can reduce side effects and ameliorate therapeutic efficacy. However, poorly perfused and dysfunctional tumor vessels limit the transport of the payload into solid tumors. The use of tumor-penetrating nanocarriers might enhance tumor uptake and antitumor effects. A peptide containing a tissue-penetrating (TP) consensus motif, capable of recognizing neuropilin-1, is here fused to a neuroblastoma-targeting peptide (pep) previously developed. Neuroblastoma cell lines and cells derived from both xenografts and high-risk neuroblastoma patients show overexpression of neuropilin-1. In vitro studies reveal that TP-pep binds cell lines and cells derived from neuroblastoma patients more efficiently than pep. TP-pep, after coupling to doxorubicin-containing stealth liposomes (TP-pep-SL[doxorubicin]), enhances their uptake by cells and cytotoxic effects in vitro, while increasing tumor-binding capability and homing in vivo. TP-pep-SL[doxorubicin] treatment enhances the Evans Blue dye accumulation in tumors but not in nontumor tissues, pointing to selective increase of vascular permeability in tumor tissues. Compared to pep-SL[doxorubicin], TP-pep-SL[doxorubicin] shows an increased antineuroblastoma activity in three neuroblastoma animal models mimicking the growth of neuroblastoma in humans. The enhancement of drug penetration in tumors by TP-pep-targeted nanoparticles may represent an innovative strategy for neuroblastoma.
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http://dx.doi.org/10.1002/smll.201802886DOI Listing
November 2018

Dynamics of Structural Elements of GB1 β-Hairpin Revealed by Tryptophan-Cysteine Contact Formation Experiments.

J Phys Chem B 2018 12 1;122(49):11468-11477. Epub 2018 Oct 1.

Department of Medical Biotechnology and Translational Medicine , Università degli Studi di Milano , 20122 Milano , Italy.

Quenching of the triplet state of tryptophan by close contact with cysteine provides a tool for measuring the rate of intramolecular contact formation, one of the most elementary events in the folding process, in peptides and proteins using only natural probes. Here we present a study performed on a stabilized mutant of the second β-hairpin of the GB1 domain, where we combine steady-state fluorescence, laser-induced temperature-jump, and contact formation measurements to unveil the role of elementary structural components on hairpin dynamics and overall stability. In particular, our methodology provides access to the conformational dynamics of both the folded and unfolded state of the hairpin under native conditions, revealing the presence of extremely slow dynamics on the microsecond time scale in the unfolded state and coexistence of structures with partial pairing of the tails in the folded state. Comparing model peptides that mimic the turn sequence, we found that both ion pairing and hydrogen bonding due to the threonine side chain contribute to the propensity of turn formation but not to the much slower dynamics of the hydrophobic core formation. Interestingly, the dynamics of the turn region in isolation are significantly faster than the dynamics measured for the unfolded state of the complete hairpin, suggesting that non-native hydrophobic contacts slow down the reconfiguration dynamics of the unfolded state. Overall, the information extracted from these experiments provides kinetic limits on interconversions among conformational populations, hence enabling a simplified multistate free-energy landscape for the GB1 hairpin to be drawn.
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http://dx.doi.org/10.1021/acs.jpcb.8b07399DOI Listing
December 2018

A halogen bond-donor amino acid for organocatalysis in water.

Chem Commun (Camb) 2018 Sep;54(76):10718-10721

Department of Chemistry, Materials, and Chemical Engineering "Giulio Natta", Politecnico di Milano, Via L. Mancinelli 7, 20131 Milan, Italy.

Herein we report a halogen bond-donor amino acid, 4-iodotetrafluorophenylalanine, which behaves as a catalyst for the aqueous synthesis of bis-(heterocyclic)methanes. We also provide experimental evidence that halogen bonding is a plausible explanation for the observed catalytic effect.
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http://dx.doi.org/10.1039/c8cc06010jDOI Listing
September 2018

The calcium-binding type III repeats domain of thrombospondin-2 binds to fibroblast growth factor 2 (FGF2).

Angiogenesis 2019 02 30;22(1):133-144. Epub 2018 Aug 30.

Tumor Angiogenesis Unit, Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Via Stezzano, 87, Bergamo, 24126, Italy.

Thrombospondin (TSP)-1 and TSP-2 share similar structures and functions, including a remarkable antiangiogenic activity. We have previously demonstrated that a mechanism of the antiangiogenic activity of TSP-1 is the interaction of its type III repeats domain with fibroblast growth factor-2 (FGF2), affecting the growth factor bioavailability and angiogenic activity. Since the type III repeats domain is conserved in TSP-2, this study aimed at investigating whether also TSP-2 retained the ability to interact with FGF2. The FGF2 binding properties of TSP-1 and TSP-2 and their recombinant domains were analyzed by solid-phase binding and surface plasmon resonance assays. TSP-2 bound FGF2 with high affinity (Kd = 1.3 nM). TSP-2/FGF2 binding was inhibited by calcium and heparin. The FGF2-binding domain of TSP-2 was located in the type III repeats and the minimal interacting sequence was identified as the GVTDEKD peptide in repeat 3C, corresponding to KIPDDRD, the active sequence of TSP-1. A second putative FGF2 binding sequence was also identified in repeat 11C of both TSPs. Computational docking analysis predicted that both the TSP-2 and TSP-1-derived heptapeptides interacted with FGF2 with comparable binding properties. Accordingly, small molecules based on the TSP-1 active sequence blocked TSP-2/FGF2 interaction. Binding of TSP-2 to FGF2 impaired the growth factor ability to interact with its cellular receptors, since TSP-2-derived fragments prevented the binding of FGF2 to both heparin (used as a structural analog of heparan sulfate proteoglycans) and FGFR-1. These findings identify TSP-2 as a new FGF2 ligand that shares with TSP-1 the same molecular requirements for interaction with the growth factor and a comparable capacity to block FGF2 interaction with proangiogenic receptors. These features likely contribute to TSP-2 antiangiogenic and antineoplastic activity, providing the rationale for future therapeutic applications.
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http://dx.doi.org/10.1007/s10456-018-9644-3DOI Listing
February 2019

Succinimide-Based Conjugates Improve IsoDGR Cyclopeptide Affinity to αβ without Promoting Integrin Allosteric Activation.

J Med Chem 2018 09 26;61(17):7474-7485. Epub 2018 Jun 26.

IRCCS Ospedale San Raffaele , Via Olgettina 60 , 20132 Milan , Italy.

The isoDGR sequence is an integrin-binding motif that has been successfully employed as a tumor-vasculature-homing molecule or for the targeted delivery of drugs and diagnostic agents to tumors. In this context, we previously demonstrated that cyclopeptide 2, the product of the conjugation of c(CGisoDGRG) (1) to 4-( N-maleimidomethyl)cyclohexane-1-carboxamide, can be successfully used as a tumor-homing ligand for nanodrug delivery to neoplastic tissues. Here, combining NMR, computational, and biochemical methods, we show that the succinimide ring contained in 2 contributes to stabilizing interactions with αβ, an integrin overexpressed in the tumor vasculature. Furthermore, we demonstrate that various cyclopeptides containing the isoDGR sequence embedded in different molecular scaffolds do not induce αβ allosteric activation and work as pure integrin antagonists. These results could be profitably exploited for the rational design of novel isoDGR-based ligands and tumor-targeting molecules with improved αβ-binding properties and devoid of adverse integrin-activating effects.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00745DOI Listing
September 2018

Mosquito-Derived Anophelin Sulfoproteins Are Potent Antithrombotics.

ACS Cent Sci 2018 Apr 28;4(4):468-476. Epub 2018 Mar 28.

School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia.

The anophelins are small protein thrombin inhibitors that are produced in the salivary glands of the mosquito to fulfill a vital role in blood feeding. A bioinformatic analysis of anophelin sequences revealed the presence of conserved tyrosine residues in an acidic environment that were predicted to be post-translationally sulfated . To test this prediction, insect cell expression of two anophelin proteins, from and , was performed, followed by analysis by mass spectrometry, which showed heterogeneous sulfation at the predicted sites. Homogeneously sulfated variants of the two proteins were subsequently generated by chemical synthesis via a one-pot ligation-desulfurization strategy. Tyrosine sulfation of the anophelins was shown to significantly enhance the thrombin inhibitory activity, with a doubly sulfated variant of the anophelin from exhibiting a 100-fold increase in potency compared with the unmodified homologue. Sulfated anophelins were also shown to exhibit potent anticoagulant and antithrombotic activity.
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http://dx.doi.org/10.1021/acscentsci.7b00612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920608PMC
April 2018

Enhancing Antibody Serodiagnosis Using a Controlled Peptide Coimmobilization Strategy.

ACS Infect Dis 2018 06 29;4(6):998-1006. Epub 2018 Mar 29.

Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM) , Via Mario Bianco, 9 , 20131 Milano , Italy.

Antigen immunoreactivity is often determined by surface regions defined by the 3D juxtapositions of amino acids stretches that are not continuous in the linear sequence. As such, mimicking an antigen immunoreactivity by means of putative linear peptide epitopes for diagnostic purposes is not trivial. Here we present a straightforward and robust method to extend the reach of immune-diagnostic probes design by copresenting peptides belonging to the same antigenic surface. In this case study focused on a computationally predicted Zika virus NS1 protein putative antigenic region, we reached a diagnostic confidence by the oriented and spatially controlled coimmobilization of peptide sequences found adjacent within the protein fold, that cooperatively interacted to provide enhanced immunoreactivity with respect to single linear epitopes. Through our method, we were able to differentiate Zika infected individuals from healthy controls. Remarkably, our strategy fits well with the requirements to build high-throughput screening platforms of linear and mixed peptide libraries, and it could possibly facilitate the rapid identification of conformational immunoreactive regions.
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http://dx.doi.org/10.1021/acsinfecdis.8b00014DOI Listing
June 2018

The enzymatic processing of α-dystroglycan by MMP-2 is controlled by two anchoring sites distinct from the active site.

PLoS One 2018 15;13(2):e0192651. Epub 2018 Feb 15.

CNR Institute for Molecular Recognition, Roma Italy.

Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and β-dystroglycan (β -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of β -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483-628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621-628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613-651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613-651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192651PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813964PMC
April 2018

Glycine -methylation in NGR-Tagged Nanocarriers Prevents Isoaspartate formation and Integrin Binding without Impairing CD13 Recognition and Tumor Homing.

Adv Funct Mater 2017 Sep;27(36)

IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132, Milan, Italy.

NGR (asparagine-glycine-arginine) is a tumor vasculature-homing peptide motif widely used for the functionalization of drugs, nanomaterials and imaging compounds for cancer treatment and diagnosis. Unfortunately, this motif has a strong propensity to undergo rapid deamidation. This reaction, which converts NGR into DGR, is associated with receptor switching from CD13 to integrins, with potentially important manufacturing, pharmacological and toxicological implications. It is found that glycine -methylation of NGR-tagged nanocarriers completely prevents asparagine deamidation without impairing CD13 recognition. Studies in animal models have shown that the methylated NGR motif can be exploited for delivering radiolabeled compounds and nanocarriers, such as tumor necrosis factor-α (TNF)-bearing nanogold and liposomal doxorubicin, to tumors with improved selectivity. These findings suggest that this NGR derivative is a stable and efficient tumor-homing ligand that can be used for delivering functional nanomaterials to tumor vasculature.
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http://dx.doi.org/10.1002/adfm.201701245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624507PMC
September 2017

Multiple epitope presentation and surface density control enabled by chemoselective immobilization lead to enhanced performance in IgE-binding fingerprinting on peptide microarrays.

Anal Chim Acta 2017 Aug 20;983:189-197. Epub 2017 Jun 20.

Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Milano, Italy.

Multiple ligand presentation is a powerful strategy to enhance the affinity of a probe for its corresponding target. A promising application of this concept lies in the analytical field, where surface immobilized probes interact with their corresponding targets in the context of complex biological samples. Here we investigate the effect of multiple epitope presentation (MEP) in the challenging context of IgE-detection in serum samples using peptide microarrays, and evaluate the influence of probes surface density on the assay results. Using the milk allergen alpha-lactalbumin as a model, we have synthesized three immunoreactive epitope sequences in a linear, branched and tandem form and exploited a chemoselective click strategy (CuAAC) for their immobilization on the surface of two biosensors, a microarray and an SPR chip both modified with the same clickable polymeric coating. We first demonstrated that a fine tuning of the surface peptide density plays a crucial role to fully exploit the potential of oriented and multiple peptide display. We then compared the three multiple epitope presentations in a microarray assay using sera samples from milk allergic patients, confirming that a multiple presentation, in particular that of the tandem construct, allows for a more efficient characterization of IgE-binding fingerprints at a statistically significant level. To gain insights on the binding parameters that characterize antibody/epitopes affinity, we selected the most reactive epitope of the series (LAC1) and performed a Surface Plasmon Resonance Imaging (SPRi) analysis comparing different epitope architectures (linear versus branched versus tandem). We demonstrated that the tandem peptide provides an approximately twofold increased binding capacity with respect to the linear and branched peptides, that could be attributed to a lower rate of dissociation (K).
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http://dx.doi.org/10.1016/j.aca.2017.06.027DOI Listing
August 2017
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