Publications by authors named "Daniela Arosio"

48 Publications

Trehalose-based neuroprotective autophagy inducers.

Bioorg Med Chem Lett 2021 May 9;40:127929. Epub 2021 Mar 9.

Istituto di Scienze e Tecnologie Chimiche (SCITEC) "Giulio Natta", Consiglio Nazionale delle Ricerche (CNR), Via C. Golgi 19, I-20133 Milan, Italy and Via G. Fantoli 16/15, I-20138 Milan, Italy. Electronic address:

A small set of trehalose-centered putative autophagy inducers was rationally designed and synthesized, with the aim to identify more potent and bioavailable autophagy inducers than free trehalose, and to acquire information about their molecular mechanism of action. Several robust, high yield routes to key trehalose intermediates and small molecule prodrugs (2-5), putative probes (6-10) and inorganic nanovectors (12a - thiol-PEG-triazole-trehalose constructs 11) were successfully executed, and compounds were tested for their autophagy-inducing properties. While small molecules 2-11 showed no pro-autophagic behavior at sub-millimolar concentrations, trehalose-bearing PEG-AuNPs 12a caused measurable autophagy induction at an estimated 40 μM trehalose concentration without any significant toxicity at the same concentration.
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http://dx.doi.org/10.1016/j.bmcl.2021.127929DOI Listing
May 2021

Cyclic RGD and DGR Integrin Ligands Containing -2-amino-1-cyclopentanecarboxylic (-β-ACPC) Scaffolds.

Molecules 2020 Dec 16;25(24). Epub 2020 Dec 16.

Dipartimento di Scienza e Alta Tecnologia, Università degli Studi dell'Insubria, Via Valleggio 11, 22100 Como, Italy.

Integrin ligands containing the tripeptide sequences Arg-Gly-Asp (RGD) and -Asp-Gly- Arg (DGR) were actively investigated as inhibitors of tumor angiogenesis and directing unit in tumor-targeting drug conjugates. Reported herein is the synthesis, of two RGD and one DGR cyclic peptidomimetics containing (1,2) and (1,2) -2-amino-1-cyclopentanecarboxylic acid (-β-ACPC), using a mixed solid phase/solution phase synthetic protocol. The three ligands were examined in vitro in competitive binding assays to the purified αβ and αβ receptors using biotinylated vitronectin (αβ) and fibronectin (αβ) as natural displaced ligands. The IC50 values of the ligands ranged from nanomolar (the two RGD ligands) to micromolar (the isoDGR ligand) with a pronounced selectivity for αβ over αβ. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin αβ). The two RGD ligands showed IC values in the same micromolar range as the reference compound ([RGDfV]), while for the DGR derivative an IC value could not be measured for the cell adhesion assay. A conformational analysis of the free RGD and DGR ligands by NMR (VT-NMR and NOESY experiments) and computational studies (MC/EM and MD), followed by docking simulations performed in the αβ integrin active site, provided a rationale for the behavior of these ligands toward the receptor.
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http://dx.doi.org/10.3390/molecules25245966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766232PMC
December 2020

Shifting Towards α β Integrin Ligands Using Novel Aminoproline-Based Cyclic Peptidomimetics.

Chemistry 2020 Oct 21;26(59):13468-13475. Epub 2020 Sep 21.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università di Parma, Parco Area delle Scienze 27A, 43124, Parma, Italy.

In recognition of the key role played by integrins in several life-threatening dysfunctions, the search for novel small-molecule probes that selectively recognize these surface receptors is still open and widely pursued. Inspired by previously established aminoproline (Amp)-RGD based cyclopeptidomimetics with attracting α β integrin affinity and selectivity, the design and straightforward synthesis of 18 new AmpRGD chemotypes bearing additional structural variants were herein implemented, to shift toward peptide-like α β integrin targeted binders. The ligand competence of the synthesized products toward α β was evaluated in competitive binding assays on isolated receptors, and α β /α β selectivity was determined for a subgroup of compounds, resulting in the identification of four very promising candidates. SAR considerations and docking simulations allowed us to appreciate the key structural features responsible for the observed activity.
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http://dx.doi.org/10.1002/chem.202002554DOI Listing
October 2020

Multimeric Presentation of RGD Peptidomimetics Enhances Integrin Binding and Tumor Cell Uptake.

Chemistry 2020 Jun 19;26(33):7492-7496. Epub 2020 May 19.

Department of Molecular Chemistry, University Grenoble Alpes, CNRS, 570, rue de la chimie, CS 40700, 38041, GRENOBLE Cedex 9, France.

The use of multimeric ligands is considered as a promising strategy to improve tumor targeting for diagnosis and therapy. Herein, tetrameric RGD (Arg-Gly-Asp) peptidomimetics were designed to target α β integrin-expressing tumor cells. These compounds were prepared by an oxime chemoselective assembly of cyclo(DKP-RGD) ligands and a cyclodecapeptide scaffold, which allows a tetrameric presentation. The resulting tetrameric RGD peptidomimetics were shown to improve α β integrin binding compared with the monomeric form. Interestingly, these compounds were also able to enhance tumor cell endocytosis in the same way as tetrameric RGD peptides. Altogether, the results show the potential of the tetrameric cyclo(DKP-RGD) ligands for in vivo imaging and drug delivery.
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http://dx.doi.org/10.1002/chem.202001115DOI Listing
June 2020

A dimeric bicyclic RGD ligand displays enhanced integrin binding affinity and strong biological effects on U-373 MG glioblastoma cells.

Org Biomol Chem 2019 10 26;17(39):8913-8917. Epub 2019 Sep 26.

Università degli Studi di Milano, Dipartimento di Chimica, Via C. Golgi, 19, I-20133, Milan, Italy.

A C-symmetric bicyclic peptide bearing two RGD motifs was developed as a dimeric ligand, and it displayed enhanced inhibition of ECM protein binding to purified integrin receptors as compared to monomeric RGD analogues. Moreover, the dimeric bicyclic ligand induced cell detachment and inhibited FAK phosphorylation in U-373 MG glioblastoma cells.
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http://dx.doi.org/10.1039/c9ob01811eDOI Listing
October 2019

Conjugates of Cryptophycin and RGD or DGR Peptidomimetics for Targeted Drug Delivery.

ChemistryOpen 2019 Jun 7;8(6):737-742. Epub 2019 Jun 7.

Organic and Bioorganic Chemistry, Department of Chemistry Bielefeld University Universitätsstraße 25 DE-33615 Bielefeld Germany.

RGD-cryptophycin and DGR-cryptophycin conjugates were synthetized by combining peptidomimetic integrin ligands and cryptophycin, a highly potent tubulin-binding antimitotic agent across lysosomally cleavable Val-Ala or uncleavable linkers. The conjugates were able to effectively inhibit binding of biotinylated vitronectin to integrin αβ, showing a binding affinity in the same range as that of the free ligands. The antiproliferative activity of the novel conjugates was evaluated on human melanoma cells M21 and M21-L with different expression levels of integrin αβ, showing nanomolar potency of all four compounds against both cell lines. Conjugates containing uncleavable linker show reduced activity compared to the corresponding cleavable conjugates, indicating efficient intracellular drug release in the case of cryptophycin-based SMDCs. However, no significant correlation between the in vitro biological activity of the conjugates and the integrin αβ expression level was observed, which is presumably due to a non-integrin-mediated uptake. This reveals the complexity of effective and selective αβ integrin-mediated drug delivery.
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http://dx.doi.org/10.1002/open.201900110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6587324PMC
June 2019

Kiss and Run: Promoting Effective and Targeted Cellular Uptake of a Drug Delivery Vehicle Composed of an Integrin-Targeting Diketopiperazine Peptidomimetic and a Cell-Penetrating Peptide.

Bioconjug Chem 2019 07 19;30(7):2011-2022. Epub 2019 Jun 19.

University of Cologne , Department of Chemistry, Biochemistry , Zülpicher Strasse 47a , D-50674 Cologne , Germany.

Cell-penetrating peptides (CPPs) have emerged as powerful tools in terms of drug delivery. Those short, often cationic peptides are characterized by their usually low toxicity and their ability to transport diverse cargos inside almost any kinds of cells. Still, one major drawback is their nonselective uptake making their application in targeted cancer therapies questionable. In this work, we aimed to combine the power of a CPP (sC18) with an integrin-targeting unit ([DKP-RGD]). The latter is composed of the Arg-Gly-Asp peptide sequence cyclized via a diketopiperazine scaffold and is characterized by its high selectivity toward integrin αβ. The two parts were linked via copper-catalyzed alkyne-azide click reaction (CuAAC), while the CPP was additionally functionalized with either a fluorescent dye or the anticancer drug daunorubicin. Both functionalities allowed a careful biological evaluation of these novel peptide-conjugates regarding their cellular uptake mechanism, as well as cytotoxicity in αβ integrin receptor expressing cells versus cells that do not express αβ. Our results show that the uptake follows a "kiss-and-run"-like model, in which the conjugates first target and recognize the receptor, but translocate mainly by CPP mediation. Thereby, we observed significantly more pronounced toxic effects in αβ expressing U87 cells compared to HT-29 and MCF-7 cells, when the cells were exposed to the substances with only very short contact times (15 min). All in all, we present new concepts for the design of cancer selective peptide-drug conjugates.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00292DOI Listing
July 2019

β-Glucuronidase triggers extracellular MMAE release from an integrin-targeted conjugate.

Org Biomol Chem 2019 05;17(19):4705-4710

Università degli Studi di Milano, Dipartimento di Chimica, Via C. Golgi, 19 I-20133, Milan, Italy.

A non-internalizing αvβ3 integrin ligand was conjugated to the anticancer drug MMAE through a β-glucuronidase-responsive linker. In the presence of β-glucuronidase, only the conjugate bearing a PEG4 spacer inhibited the proliferation of integrin-expressing cancer cells at low nanomolar concentrations, indicating important structural requirements for the efficacy of these therapeutics.
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http://dx.doi.org/10.1039/c9ob00617fDOI Listing
May 2019

Rational Design of Antiangiogenic Helical Oligopeptides Targeting the Vascular Endothelial Growth Factor Receptors.

Front Chem 2019 29;7:170. Epub 2019 Mar 29.

Department of Chemical Science and Technologies, University of Rome Tor Vergata, Rome, Italy.

Tumor angiogenesis, essential for cancer development, is regulated mainly by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), which are overexpressed in cancer cells. Therefore, the VEGF/VEGFR interaction represents a promising pharmaceutical target to fight cancer progression. The VEGF surface interacting with VEGFRs comprises a short α-helix. In this work, helical oligopeptides mimicking the VEGF-C helix were rationally designed based on structural analyses and computational studies. The helical conformation was stabilized by optimizing intramolecular interactions and by introducing helix-inducing C-disubstituted amino acids. The conformational features of the synthetic peptides were characterized by circular dichroism and nuclear magnetic resonance, and their receptor binding properties and antiangiogenic activity were determined. The best hits exhibited antiangiogenic activity at nanomolar concentrations and were resistant to proteolytic degradation.
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http://dx.doi.org/10.3389/fchem.2019.00170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449863PMC
March 2019

Intracisternal delivery of PEG-coated gold nanoparticles results in high brain penetrance and long-lasting stability.

J Nanobiotechnology 2019 Apr 3;17(1):49. Epub 2019 Apr 3.

Experimental Imaging Centre, San Raffaele Scientific Institute, 20132, Milan, Italy.

Background: The increasing use of gold nanoparticles (AuNPs) in the field of neuroscience instilled hope for their rapid translation to the clinical practice. AuNPs can be engineered to carry therapeutics or diagnostics in the diseased brain, possibly providing greater cell specificity and low toxicity. Although there is a general enthusiasm for these tools, we are in early stages of their development. Overall, their brain penetrance, stability and cell specificity are critical issues that must be addressed to drive AuNPs to the clinic.

Results: We studied the kinetic, distribution and stability of PEG-coated AuNPs in mice receiving a single injection into the cisterna magna of the 4th ventricle. AuNPs were conjugated with the fluorescent tag Cy5.5 (Cy5.5-AuNPs) to track their in vivo distribution. Fluorescence levels from such particles were detected in mice for weeks. In situ analysis of brains by immunofluorescence and electron microscopy revealed that Cy5.5-AuNPs penetrated the brain parenchyma, spreading in the CNS parenchyma beneath the 4th ventricle. Cy5.5-AuNPs were preferentially found in neurons, although a subset of resting microglia also entrapped these particles.

Conclusions: Our results suggest that the ICM route for delivering gold particles allows the targeting of neurons. This approach might be pursued to carry therapeutics or diagnostics inside a diseased brain with a surgical procedure that is largely used in gene therapy approaches. Furthermore, this approach could be used for radiotherapy, enhancing the agent's efficacy to kill brain cancer cells.
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http://dx.doi.org/10.1186/s12951-019-0481-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6448280PMC
April 2019

Synthesis and Biological Evaluation of RGD and isoDGR-Monomethyl Auristatin Conjugates Targeting Integrin α β.

ChemMedChem 2019 05 22;14(9):938-942. Epub 2019 Mar 22.

Università degli Studi dell'Insubria, Dipartimento di Scienza e Alta Tecnologia, Via Valleggio, 11, 22100, Como, Italy.

This work reports the synthesis of a series of small-molecule-drug conjugates containing the α β -integrin ligand cyclo[DKP-RGD] or cyclo[DKP-isoDGR], a lysosomally cleavable Val-Ala (VA) linker or an "uncleavable" version devoid of this sequence, and monomethyl auristatin E (MMAE) or F (MMAF) as the cytotoxic agent. The conjugates were obtained via a straightforward synthetic scheme taking advantage of a copper-catalyzed azide-alkyne cycloaddition as the key step. The conjugates were tested for their binding affinity for the isolated α β receptor and were shown to retain nanomolar IC values, in the same range as those of the free ligands. The cytotoxic activity of the conjugates was evaluated in cell viability assays with α β integrin overexpressing human glioblastoma (U87) and human melanoma (M21) cells. The conjugates possess markedly lower cytotoxic activity than the free drugs, which is consistent with inefficient integrin-mediated internalization. In almost all cases the conjugates featuring isoDGR as integrin ligand exhibited higher potency than their RGD counterparts. In particular, the cyclo[DKP-isoDGR]-VA-MMAE conjugate has low nanomolar IC values in cell viability assays with both cancer cell lines tested (U87: 11.50±0.13 nm; M21: 6.94±0.09 nm) and is therefore a promising candidate for in vivo experiments.
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http://dx.doi.org/10.1002/cmdc.201900049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593765PMC
May 2019

Neutrophil Elastase Promotes Linker Cleavage and Paclitaxel Release from an Integrin-Targeted Conjugate.

Chemistry 2019 Feb 27;25(7):1696-1700. Epub 2018 Dec 27.

Università degli Studi di Milano, Dipartimento di Chimica, Via C. Golgi, 19, I-20133, Milan, Italy.

This work takes advantage of one of the hallmarks of cancer, that is, the presence of tumor infiltrating cells of the immune system and leukocyte-secreted enzymes, to promote the activation of an anticancer drug at the tumor site. The peptidomimetic integrin ligand cyclo(DKP-RGD) was found to accumulate on the surface of α β integrin-expressing human renal cell carcinoma 786-O cells. The ligand was conjugated to the anticancer drug paclitaxel through a Asn-Pro-Val (NPV) tripeptide linker, which is a substrate of neutrophil-secreted elastase. In vitro linker cleavage assays and cell antiproliferative experiments demonstrate the efficacy of this tumor-targeting conjugate, opening the way to potential therapeutic applications.
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http://dx.doi.org/10.1002/chem.201805447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471013PMC
February 2019

Stereodivergent synthesis of 5-aminopipecolic acids and application in the preparation of a cyclic RGD peptidomimetic as a nanomolar αβ integrin ligand.

Org Biomol Chem 2018 05;16(18):3402-3414

Dipartimento di Chimica "U. Schiff", Università degli Studi di Firenze, Via della Lastruccia 13, I-50019 Sesto Fiorentino, Italy.

A stereodivergent strategy was devised to obtain enantiopure cis and trans 5-aminopipecolic acids (5-APAs) in suitably protected forms to be employed in peptide synthesis as conformationally constrained α- and δ-amino acids. The cis isomer was used as a δ-amino acid to construct a cyclic RGD-containing peptidomimetic, the ability of which to compete with biotinylated vitronectin for binding with the isolated αVβ3 integrin was measured (IC50 = 4.2 ± 0.9 nM). A complete 1H NMR and computational conformational analysis was performed to elucidate the reasons for the high affinity of this cyclic peptidomimetic in comparison with cilengitide.
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http://dx.doi.org/10.1039/c8ob00534fDOI Listing
May 2018

Synthesis and biological evaluation of RGD and isoDGR peptidomimetic-α-amanitin conjugates for tumor-targeting.

Beilstein J Org Chem 2018 14;14:407-415. Epub 2018 Feb 14.

Dipartimento di Scienza e Alta Tecnologia, Via Valleggio, 11, 22100, Como, Italy.

RGD-α-amanitin and isoDGR-α-amanitin conjugates were synthesized by joining integrin ligands to α-amanitin via various linkers and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified αβ receptor, retaining good binding affinity, in the same nanomolar range as the free ligands. The antiproliferative activity of the conjugates was evaluated in three cell lines possessing different levels of αβ integrin expression: human glioblastoma U87 (αβ+), human lung carcinoma A549 (αβ-) and breast adenocarcinoma MDA-MB-468 (αβ-). In the U87, in the MDA-MB-468, and partly in the A549 cancer cell lines, the cyclo[DKP-isoDGR]-α-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were found to be slightly more potent than α-amanitin. Apparently, for all these α-amanitin conjugates there is no correlation between the cytotoxicity and the expression of αβ integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-α-amanitin conjugates is governed by an integrin-mediated binding and internalization process, competition experiments were carried out in which the conjugates were tested with U87 (αβ+, αβ+, αβ-, αβ+) and MDA-MB-468 (αβ-, αβ+, αβ+, αβ-) cells in the presence of excess cilengitide, with the aim of blocking integrins on the cell surface. Using the MDA-MB-468 cell line, a fivefold increase of the IC was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only αβ, but also αβ, αβ, and αβ. These data indicate that in this case the cyclo[DKP-isoDGR]-α-amanitin conjugates are possibly internalized by a process mediated by integrins different from αβ (e.g., αβ).
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http://dx.doi.org/10.3762/bjoc.14.29DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827777PMC
February 2018

Investigating the Interaction of Cyclic RGD Peptidomimetics with αβ₆ Integrin by Biochemical and Molecular Docking Studies.

Cancers (Basel) 2017 Sep 21;9(10). Epub 2017 Sep 21.

Dipartimento di Chimica, Università degli Studi di Milano, via Golgi 19, I-20133 Milano, Italy.

The interaction of a small library of cyclic RGD (Arg-Gly-Asp) peptidomimetics with αβ₆ integrin has been investigated by means of competitive solid phase binding assays to the isolated receptor and docking calculations in the crystal structure of the αβ₆ binding site. To this aim, a rigid receptor-flexible ligand docking protocol has been set up and then applied to predict the binding mode of the cyclic RGD peptidomimetics to αβ₆ integrin. Although the RGD interaction with αβ₆ recapitulates the RGD binding mode observed in αβ₃, differences between the integrin binding pockets can strongly affect the ligand binding ability. In general, the peptidomimetics exhibited IC values for integrin αβ₆ (i.e., the concentration of compound required for 50% inhibition of biotinylated fibronectin binding to isolated αβ₆ integrin) in the nanomolar range (77-345 nM), about 10-100 times higher than those for the related αβ₃ receptor, with a single notable ligand displaying a low nanomolar IC value (2.3 nM). Insights from the properties of the binding pocket combined with the analysis of the docking poses provided a rationale for ligand recognition and selectivity.
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http://dx.doi.org/10.3390/cancers9100128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664067PMC
September 2017

Multivalency Increases the Binding Strength of RGD Peptidomimetic-Paclitaxel Conjugates to Integrin α β.

Chemistry 2017 Oct 6;23(58):14410-14415. Epub 2017 Sep 6.

Università degli Studi di Milano, Dipartimento di Chimica, Via C. Golgi 19, 20133, Milan, Italy), Fax: (+39) 02-5031-4072.

This work reports the synthesis of three multimeric RGD peptidomimetic-paclitaxel conjugates featuring a number of α β integrin ligands ranging from 2 to 4. These constructs were assembled by conjugation of the integrin α β ligand cyclo[DKP-RGD]-CH NH with paclitaxel via a 2'-carbamate with a self-immolative spacer, the lysosomally cleavable Val-Ala dipeptide linker, a multimeric scaffold, a triazole linkage, and finally a PEG spacer. Two monomeric conjugates were also synthesized as reference compounds. Remarkably, the new multimeric conjugates showed a binding affinity for the purified integrin α β receptor that increased with the number of integrin ligands (reaching a minimum IC value of 1.2 nm for the trimeric), thus demonstrating that multivalency is an effective strategy to strengthen the ligand-target interactions.
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http://dx.doi.org/10.1002/chem.201703093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656903PMC
October 2017

Tumor Targeting with an isoDGR-Drug Conjugate.

Chemistry 2017 Jun 26;23(33):7910-7914. Epub 2017 May 26.

Dipartimento di Chimica, Università degli Studi di Milano, Via C. Golgi 19, 20133, Milano, Italy.

Herein we report the first example of an isoDGR-drug conjugate (2), designed to release paclitaxel selectively within cancer cells expressing integrin α β . Conjugate 2 was synthesized by connecting the isoDGR peptidomimetic 5 with paclitaxel via the lysosomally cleavable Val-Ala dipeptide linker. Conjugate 2 displayed a low nanomolar affinity for the purified integrin α β receptor (IC =11.0 nm). The tumor targeting ability of conjugate 2 was assessed in vitro in anti-proliferative assays on two isogenic cancer cell lines characterized by different integrin α β expression: human glioblastoma U87 (α β +) and U87 β -KO (α β -). The isoDGR-PTX conjugate 2 displayed a remarkable targeting index (TI=9.9), especially when compared to the strictly related RGD-PTX conjugate 4 (TI=2.4).
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http://dx.doi.org/10.1002/chem.201701844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488297PMC
June 2017

4-Connected azabicyclo[5.3.0]decane Smac mimetics-Zn chelators as dual action antitumoral agents.

Bioorg Med Chem Lett 2017 06 13;27(11):2336-2344. Epub 2017 Apr 13.

Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, I-20133 Milan, Italy. Electronic address:

Putative dual action compounds (DACs 3a-d) based on azabicyclo[5.3.0]decane (ABD) Smac mimetic scaffolds linked to Zn-chelating 2,2'-dipicolylamine (DPA) through their 4 position are reported and characterized. Their synthesis, their target affinity (cIAP1 BIR3, Zn) in cell-free assays, their pro-apoptotic effects, and their cytotoxicity in tumor cells with varying sensitivity to Smac mimetics are described. A limited influence of Zn chelation on in vitro activity of DPA-substituted DACs 3a-d was sometimes perceivable, but did not lead to strong cellular synergistic effects. In particular, the linker connecting DPA with the ABD scaffold seems to influence cellular Zn-chelation, with longer lipophilic linkers/DAC 3c being the optimal choice.
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http://dx.doi.org/10.1016/j.bmcl.2017.04.032DOI Listing
June 2017

Insights into the Binding of Cyclic RGD Peptidomimetics to αβ Integrin by using Live-Cell NMR And Computational Studies.

ChemistryOpen 2017 Feb 9;6(1):128-136. Epub 2016 Dec 9.

Dipartimento di Chimica Università degli Studi di Milano Via Golgi, 19 20133 Milano Italy.

The interaction of a small library of cyclic DKP-RGD peptidomimetics with αβ integrin has been investigated by means of an integrated experimental and computational approach. Bioaffinity NMR techniques, including saturation transfer difference (STD) and transferred NOESY, were applied to the ligands in a suspension of intact MDA-MB-231 breast cancer cells, in which integrin αβ is highly expressed. The NMR data were compared with the docking calculations of the RGD ligands in the crystal structure of the αβ binding site, and were integrated with competitive binding assays to the purified αβ integrin. Ligand binding epitopes involve protons of both the RGD moiety and the DKP scaffold, although the stereochemistry and the functionalization of the DKP scaffold as well as the macrocycle conformation determine a great variability in the interaction. The ligand showing the highest number of STD signals is also the most potent αβ ligand of the series, displaying a nanomolar value.
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http://dx.doi.org/10.1002/open.201600112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5288746PMC
February 2017

Synthesis of Novel c(AmpRGD)-Sunitinib Dual Conjugates as Molecular Tools Targeting the αβ Integrin/VEGFR2 Couple and Impairing Tumor-Associated Angiogenesis.

J Med Chem 2017 01 20;60(1):248-262. Epub 2016 Dec 20.

Dipartimento di Farmacia, Università degli Studi di Parma , Parco Area delle Scienze 27A, 43124 Parma, Italy.

On the basis of a previously discovered anti-αβ integrin peptidomimetic (c(AmpRGD)) and the clinically approved antiangiogenic kinase inhibitor sunitinib, three novel dual conjugates were synthesized (compounds 1-3), featuring the covalent and robust linkage between these two active modules. In all conjugates, the ligand binding competence toward αβ (using both isolated receptors and αβ-overexpressing endothelial progenitor EP cells) and the kinase inhibitory activity (toward both isolated kinases and EPCs) remained almost untouched and comparable to the activity of the single active units. Compounds 1-3 showed interesting antiangiogenesis properties in an in vitro tubulogenic assay; furthermore, dimeric-RGD conjugate 3 strongly inhibited in vivo angiogenesis in Matrigel plug assays in FVB mice. These results offer proof-of-concept of how the covalent conjugation of two angiogenesis-related small modules may result in novel and stable molecules, which impair tumor-related angiogenesis with equal or even superior ability as compared to the single modules or their simple combinations.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01266DOI Listing
January 2017

Dual action Smac mimetics-zinc chelators as pro-apoptotic antitumoral agents.

Bioorg Med Chem Lett 2016 10 22;26(19):4613-4619. Epub 2016 Aug 22.

Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, I-20133 Milan, Italy. Electronic address:

Dual action compounds (DACs) based on 4-substituted aza-bicyclo[5.3.0]decane Smac mimetic scaffolds (ABDs) linked to a Zn(2+)-chelating moiety (DPA, o-hydroxy, m-allyl, N-acyl (E)-phenylhydrazone) through their 10 position are reported and characterized. Their synthesis, their target affinity (XIAP BIR3, Zn(2+)) in cell-free assays, their pro-apoptotic effects and cytotoxicity in tumor cells with varying sensitivity to Smac mimetics are described. The results are interpreted to evaluate the influence of Zn(2+) chelators on cell-free potency and on cellular permeability of DACs, and to propose novel avenues towards more potent antitumoral DACs based on Smac mimetics and Zn(2+) chelation.
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http://dx.doi.org/10.1016/j.bmcl.2016.08.065DOI Listing
October 2016

Advancement in integrin facilitated drug delivery.

Adv Drug Deliv Rev 2016 Feb 10;97:111-43. Epub 2015 Dec 10.

Università degli Studi di Milano, Dipartimento di Chimica, Via C. Golgi 19, I-20133 Milan, Italy. Electronic address:

The research of integrin-targeted anticancer agents has recorded important advancements in ingenious design of delivery systems, based either on the prodrug approach, or on nanoparticle carriers, but for now, none of these has reached a clinical stage of development. Past work in this area has been extensively reviewed by us and others. Thus, the purpose and scope of the present review is to survey the advancement reported in the last 3years, with focus on innovative delivery systems that appear to afford openings for future developments. These systems exploit the labelling with conventional and novel integrin ligands for targeting the interface of cancer cells and of endothelial cells involved in cancer angiogenesis, with the proteins of the extracellular matrix, in the circulation, in tissues, and in tumour stroma, as the site of progression and metastatic evolution of the disease. Furthermore, these systems implement the expertise in the development of nanomedicines to the purpose of achieving preferential biodistribution and uptake in cancer tissues, internalisation in cancer cells, and release of the transported drugs at intracellular sites. The assessment of the value of controlling these factors, and their combination, for future developments requires support of biological testing in appropriate mechanistic models, but also imperatively demand confirmation in therapeutically relevant in vivo models for biodistribution, efficacy, and lack of off-target effects. Thus, among many studies, we have tried to point out the results supported by relevant in vivo studies, and we have emphasised in specific sections those addressing the medical needs of drug delivery to brain tumours, as well as the delivery of oligonucleotides modulating gene-dependent pathological mechanism. The latter could constitute the basis of a promising third branch in the therapeutic armamentarium against cancer, in addition to antibody-based agents and to cytotoxic agents.
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http://dx.doi.org/10.1016/j.addr.2015.12.001DOI Listing
February 2016

Synthesis, Characterization, and Biological Evaluation of a Dual-Action Ligand Targeting αvβ3 Integrin and VEGF Receptors.

ChemistryOpen 2015 Oct 2;4(5):633-41. Epub 2015 Jul 2.

Dipartimento di Chimica, Università degli Studi di Milano Via C. Golgi 19, 20133, Milan, Italy.

A dual-action ligand targeting both integrin αVβ3 and vascular endothelial growth factor receptors (VEGFRs), was synthesized via conjugation of a cyclic peptidomimetic αVβ3 Arg-Gly-Asp (RGD) ligand with a decapentapeptide. The latter was obtained from a known VEGFR antagonist by acetylation at the Lys13 side chain. Functionalization of the precursor ligands was carried out in solution and in the solid phase, affording two fragments: an alkyne VEGFR ligand and the azide integrin αVβ3 ligand, which were conjugated by click chemistry. Circular dichroism studies confirmed that both the RGD and VEGFR ligand portions of the dual-action compound substantially adopt the biologically active conformation. In vitro binding assays on isolated integrin αVβ3 and VEGFR-1 showed that the dual-action conjugate retains a good level of affinity for both its target receptors, although with one order of magnitude (10/20 times) decrease in potency. The dual-action ligand strongly inhibited the VEGF-induced morphogenesis in Human Umbilical Vein Endothelial Cells (HUVECs). Remarkably, its efficiency in preventing the formation of new blood vessels was similar to that of the original individual ligands, despite the worse affinity towards integrin αVβ3 and VEGFR-1.
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http://dx.doi.org/10.1002/open.201500062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608532PMC
October 2015

Synthesis and biological evaluation of RGD peptidomimetic-paclitaxel conjugates bearing lysosomally cleavable linkers.

Chemistry 2015 Apr 17;21(18):6921-9. Epub 2015 Mar 17.

Università degli Studi di Milano, Dipartimento di Chimica, Via C. Golgi, 19, 20133, Milan (Italy), Fax: (+39) 02-5031-4072.

Two small-molecule-drug conjugates (SMDCs, 6 and 7) featuring lysosomally cleavable linkers (namely the Val-Ala and Phe-Lys peptide sequences) were synthesized by conjugation of the αvβ3-integrin ligand cyclo[DKP-RGD]-CH2NH2 (2) to the anticancer drug paclitaxel (PTX). A third cyclo[DKP-RGD]-PTX conjugate with a nonpeptide "uncleavable" linker (8) was also synthesized to be tested as a negative control. These three SMDCs were able to inhibit biotinylated vitronectin binding to the purified αVβ3-integrin receptor at nanomolar concentrations and showed good stability at pH 7.4 and pH 5.5. Cleavage of the two peptide linkers was observed in the presence of lysosomal enzymes, whereas conjugate 8, which possesses a nonpeptide "uncleavable" linker, remained intact under these conditions. The antiproliferative activities of the conjugates were evaluated against two isogenic cell lines expressing the integrin receptor at different levels: the acute lymphoblastic leukemia cell line CCRF-CEM (αVβ3-) and its subclone CCRF-CEM αVβ3 (αVβ3+). Fairly effective integrin targeting was displayed by the cyclo[DKP-RGD]-Val-Ala-PTX conjugate (6), which was found to differentially inhibit proliferation in antigen-positive CCRF-CEM αVβ3 versus antigen-negative isogenic CCRF-CEM cells. The total lack of activity displayed by the "uncleavable" cyclo[DKP-RGD]-PTX conjugate (8) clearly demonstrates the importance of the peptide linker for achieving the selective release of the cytotoxic payload.
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http://dx.doi.org/10.1002/chem.201500158DOI Listing
April 2015

Cyclic isoDGR and RGD peptidomimetics containing bifunctional diketopiperazine scaffolds are integrin antagonists.

Chemistry 2015 Apr 11;21(16):6265-71. Epub 2015 Mar 11.

Università degli Studi dell'Insubria, Dipartimento di Scienza e Alta Tecnologia, Via Valleggio 11, 22100 Como (Italy).

The cyclo[DKP-isoDGR] peptidomimetics 2-5, containing bifunctional diketopiperazine (DKP) scaffolds that differ in the configuration of the two DKP stereocenters and in the substitution at the DKP nitrogen atoms, were prepared and examined in vitro in competitive binding assays with purified αv β3 and αv β5 integrin receptors. IC50 values ranged from low nanomolar (ligand 3) to submicromolar with αv β3 integrin. The biological activities of ligands cyclo[DKP3-RGD] 1 and cyclo[DKP3-isoDGR] 3, bearing the same bifunctional DKP scaffold and showing similar αV β3 integrin binding values, were compared in terms of their cellular effects in human U373 glioblastoma cells. Compounds 1 and 3 displayed overlapping inhibitory effects on the FAK/Akt integrin activated transduction pathway and on integrin-mediated cell infiltration processes, and qualify therefore, despite the different RGD and isoDGR sequences, as integrin antagonists. Both compounds induced apoptosis in glioma cells after 72 hour treatment.
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http://dx.doi.org/10.1002/chem.201406567DOI Listing
April 2015

Computational design of novel peptidomimetic inhibitors of cadherin homophilic interactions.

Org Biomol Chem 2015 Mar;13(9):2570-3

Dipartimento di Chimica, Università degli Studi di Milano, Via C. Golgi 19, I-20133, Milan, Italy.

We report a first set of peptidomimetic ligands mimicking the adhesive interface identified by recent crystallographic structures of E- and N-cadherin. Compounds 2 and 3 inhibit adhesion of epithelial ovarian cancer (EOC) cells with improved efficacy compared to the ADH-1 peptide, a N-cadherin antagonist that is in early clinical trials in EOC patients.
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http://dx.doi.org/10.1039/c4ob02538eDOI Listing
March 2015

Effective targeting of DC-SIGN by α-fucosylamide functionalized gold nanoparticles.

Bioconjug Chem 2014 Dec 24;25(12):2244-51. Epub 2014 Nov 24.

CNR-Institute of Molecular Science and Technologies (ISTM) , via Golgi 19, I-20133 Milan, Italy.

Dendritic Cells (DCs), the most potent antigen-presenting cells, play a critical role in the detection of invading pathogens, which are recognized also by multiple carbohydrate-specific receptors. Among them, DC-SIGN is one of the best characterized, with high-mannose and Lewis-type glycan specificity. In this study, we present a potent DC-SIGN targeting device developed using gold nanoparticles functionalized with α-fucosyl-β-alanyl amide. The nanoparticles bound to cellular DC-SIGN and induced internalization as effectively as similar particles coated with comparable amounts of Lewis(X) oligosaccharide. They were found to be neutral toward dendritic cell maturation and IL-10 production, thus envisaging a possible use as targeted imaging tools and antigen delivery devices.
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http://dx.doi.org/10.1021/bc500467uDOI Listing
December 2014

Enhancement of the uptake and cytotoxic activity of doxorubicin in cancer cells by novel cRGD-semipeptide-anchoring liposomes.

Mol Pharm 2014 Jul 22;11(7):2280-93. Epub 2014 May 22.

Dipartimento di Farmacia, Università degli Studi di Parma , Parma 43124, Italy.

Novel liposemipeptides hanging cyclic azabicycloalkane-RGD or aminoproline-RGD terminals were synthesized and incorporated into liposomal nanoparticles cAba/cAmpRGD-LNP5 3C/3D. Liposomes with similar composition and lacking semipeptide conjugates were constructed for comparison (LNP, 3A), and physical encapsulation of the anticancer doxorubicin drug in both targeted and untargeted liposomes was accomplished. Microstructural analysis performed by dynamic light scattering (DLS), small-angle neutron scattering (SANS), and electron paramagnetic resonance (EPR) revealed that the conjugated nanoparticles presented an average size of 80 nm and were constituted by 5 nm thick unilamellar liposome bilayer. Flow cytometry and fluorescent microscopy studies showed that 3C-DOXO and 3D-DOXO efficiently delivered the drug into the nuclei of both quiescent and proliferating cells even in a high serum concentration environment. The uptake of doxorubicin when carried by liposomes was faster than that of the free drug, and 30 min incubation was sufficient to load cell nuclei with doxorubicin. Targeted liposomes significantly induced cell death of human breast adenocarcinoma MCF7 cells (IC50 = 144 nM, 3C-DOXO; IC50 = 274 nM, 3D-DOXO), about 2- to 6-fold more potent than free doxorubicin or 3A-DOXO controls (IC50 = 527 and 854 nM, respectively). These results suggest that cAba/cAmpRGD liposomal nanoparticles hold promise for the rapid and efficient delivery of chemotherapeutic agents to αVβ3-expressing tumor cells.
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http://dx.doi.org/10.1021/mp400718jDOI Listing
July 2014

MicroPET/CT imaging of αvβ₃ integrin via a novel ⁶⁸Ga-NOTA-RGD peptidomimetic conjugate in rat myocardial infarction.

Eur J Nucl Med Mol Imaging 2013 Aug 15;40(8):1265-74. Epub 2013 May 15.

CNR-Institute of Clinical Physiology (IFC), Via Moruzzi 1, 56124 Pisa, Italy.

Purpose: The αvβ3 integrin is expressed in angiogenic vessels and is a potential target for molecular imaging of evolving pathological processes. Its expression is upregulated in cancer lesions and metastases as well as in acute myocardial infarction (MI) as part of the infarct healing process. The purpose of our study was to determine the feasibility of a new imaging approach with a novel (68)Ga-2,2',2″-(1,4,7-triazonane-1,4,7-triyl)triacetic acid (NOTA)-arginine-glycine-aspartic acid (RGD) construct to assess integrin expression in the evolving MI.

Methods: A straightforward labelling chemistry to attach the radionuclide (68)Ga to a NOTA-based chelating agent conjugated with a cyclic RGD peptidomimetic is described. Affinity for αvβ₃ integrin was assessed by in vitro receptor binding assay. The proof-of-concept in vivo studies combined the (68)Ga-NOTA-RGD with the flow tracer (13)N-NH₃ imaging in order to obtain positron emission tomography (PET)/CT imaging of both integrin expression and perfusion defect at 4 weeks after infarction. Hearts were then processed for immunostaining of integrin β₃.

Results: NOTA-RGD conjugate displayed a binding affinity for αvβ₃ integrin of 27.9 ± 6.8 nM. (68)Ga-NOTA-RGD showed stability without detectable degradation or formation of by-products in urine up to 2 h following injection in the rat. MI hearts exhibited (68)Ga-NOTA-RGD uptake in correspondence to infarcted and border zone regions. The tracer signal drew a parallel with vascular remodelling due to ischaemia-induced angiogenesis as assessed by immunohistochemistry.

Conclusion: As compared to similar imaging approaches using the (18)F-galacto-derivative, we documented for the first time with microPET/CT imaging the (68)Ga-NOTA-RGD derivative that appears eligible for PET imaging in animal models of vascular remodelling during evolving MI. The simple chemistry employed to synthesize the (68)Ga-based radiotracer may greatly facilitate its translation to a clinical setting.
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http://dx.doi.org/10.1007/s00259-013-2432-9DOI Listing
August 2013

Cyclic isoDGR peptidomimetics as low-nanomolar αvβ3 integrin ligands.

Chemistry 2013 Mar 19;19(11):3563-7. Epub 2013 Feb 19.

Università degli Studi di Milano, Dipartimento di Chimica, Via C. Golgi, 19, 20133, Milan, Italy.

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http://dx.doi.org/10.1002/chem.201204639DOI Listing
March 2013