Publications by authors named "Laura Belvisi"

58 Publications

A trifunctional self-immolative spacer enables drug release with two non-sequential enzymatic cleavages.

Chem Commun (Camb) 2021 Jul 15. Epub 2021 Jul 15.

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

Cyclative cleavage of an amine-carbamate self-immolative spacer to deliver a hydroxyl cargo was inhibited by spacer derivatisation with a phosphate monoester handle. This trifunctional spacer was installed in a model anticancer prodrug that showed fast drug release only when incubated with both a protease and a phosphatase enzyme.
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http://dx.doi.org/10.1039/d1cc02895bDOI Listing
July 2021

Prediction and Validation of a Druggable Site on Virulence Factor of Drug Resistant Burkholderia cenocepacia*.

Chemistry 2021 Jul 1;27(40):10341-10348. Epub 2021 May 1.

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

Burkholderia cenocepacia is an opportunistic Gram-negative bacterium that causes infections in patients suffering from chronic granulomatous diseases and cystic fibrosis. It displays significant morbidity and mortality due to extreme resistance to almost all clinically useful antibiotics. The bacterial lectin BC2L-C expressed in B. cenocepacia is an interesting drug target involved in bacterial adhesion and subsequent deadly infection to the host. We solved the first high resolution crystal structure of the apo form of the lectin N-terminal domain (BC2L-C-nt) and compared it with the ones complexed with carbohydrate ligands. Virtual screening of a small fragment library identified potential hits predicted to bind in the vicinity of the fucose binding site. A series of biophysical techniques and X-ray crystallographic screening were employed to validate the interaction of the hits with the protein domain. The X-ray structure of BC2L-C-nt complexed with one of the identified active fragments confirmed the ability of the site computationally identified to host drug-like fragments. The fragment affinity could be determined by titration microcalorimetry. These structure-based strategies further provide an opportunity to elaborate the fragments into high affinity anti-adhesive glycomimetics, as therapeutic agents against B. cenocepacia.
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http://dx.doi.org/10.1002/chem.202100252DOI Listing
July 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

Side chain effect in the modulation of αβ/αβ integrin activity via clickable isoxazoline-RGD-mimetics: development of molecular delivery systems.

Sci Rep 2020 05 4;10(1):7410. Epub 2020 May 4.

Department of Chemistry "G.Ciamician", University of Bologna, Via Selmi 2, 40126, Bologna, Italy.

Construction of small molecule ligand (SML) based delivery systems has been performed starting from a polyfunctionalized isoxazoline scaffold, whose αβ and αβ integrins' potency has been already established. The synthesis of this novel class of ligands was obtained by conjugation of linkers to the heterocyclic core via Huisgen-click reaction, with the aim to use them as "shuttles" for selective delivery of diagnostic agents to cancer cells, exploring the effects of the side chains in the interaction with the target. Compounds 17b and 24 showed excellent potency towards αβ integrin acting as selective antagonist and agonist respectively. Further investigations confirmed their effects on target receptor through the analysis of fibronectin-induced ERK1/2 phosphorylation. In addition, confocal microscopy analysis allowed us to follow the fate of EGFP conjugated αβ integrin and 17b FITC-conjugated (compound 31) inside the cells. Moreover, the stability in water solution at different values of pH and in bovine serum confirmed the possible exploitation of these peptidomimetic molecules for pharmaceutical application.
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http://dx.doi.org/10.1038/s41598-020-64396-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198601PMC
May 2020

Fast Cyclization of a Proline-Derived Self-Immolative Spacer Improves the Efficacy of Carbamate Prodrugs.

Angew Chem Int Ed Engl 2020 03 22;59(10):4176-4181. Epub 2020 Jan 22.

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

Self-immolative (SI) spacers are sophisticated chemical constructs designed for molecular delivery or material degradation. We describe herein a (S)-2-(aminomethyl)pyrrolidine SI spacer that is able to release different types of anticancer drugs (possessing either a phenolic or secondary and tertiary hydroxyl groups) through a fast cyclization mechanism involving carbamate cleavage. The high efficiency of drug release obtained with this spacer was found to be beneficial for the in vitro cytotoxic activity of protease-sensitive prodrugs, compared with a commonly used spacer of the same class. These findings expand the repertoire of degradation machineries and are instrumental for the future development of highly efficient delivery platforms.
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http://dx.doi.org/10.1002/anie.201916394DOI Listing
March 2020

Stem-Like Cancer Cells in a Dynamic 3D Culture System: A Model to Study Metastatic Cell Adhesion and Anti-Cancer Drugs.

Cells 2019 11 13;8(11). Epub 2019 Nov 13.

Dipartmento di Scienze del Farmaco, Università degli Studi di Pavia, 27100 Pavia, Italy.

Metastatic spread is mainly sustained by cancer stem cells (CSC), a subpopulation of cancer cells that displays stemness features. CSC are thought to be derived from cancer cells that undergo epithelial to mesenchymal transition (EMT), thus acquiring resistance to anoikis and anti-cancer drugs. After detachment from the primary tumor mass, CSC reach the blood and lymphatic flow, and disseminate to the target tissue. This process is by nature dynamic and in vitro models are quite far from the in vivo situation. In this study, we have tried to reproduce the adhesion process of CSC to a target tissue by using a 3D dynamic cell culture system. We isolated two populations of 3D tumor spheroids displaying CSC-like features from breast carcinoma (MCF-7) and lung carcinoma (A549) cell lines. Human fibroblasts were layered on a polystyrene scaffold placed in a dynamically perfused millifluidic system and then the adhesion of tumor cell derived from spheroids to fibroblasts was investigated under continuous perfusion. After 24 h of perfusion, we found that spheroid cells tightly adhered to fibroblasts layered on the scaffold, as assessed by a scanning electron microscope (SEM). To further investigate mechanisms involved in spheroid cell adhesion to fibroblasts, we tested the effect of three RGD integrin antagonists with different molecular structures on cell adhesion; when injected into the circuit, only cilengitide was able to inhibit cell adhesion to fibroblasts. Although our model needs further refinements and improvements, we do believe this study could represent a promising approach in improving current models to study metastatic infiltration in vitro and a new tool to screen new potential anti-metastatic molecules.
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http://dx.doi.org/10.3390/cells8111434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912649PMC
November 2019

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

Innovative Linker Strategies for Tumor-Targeted Drug Conjugates.

Chemistry 2019 Nov 20;25(65):14740-14757. Epub 2019 Sep 20.

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

The covalent conjugation of potent cytotoxic agents to either macromolecular carriers or small molecules represents a well-known approach to increase the therapeutic index of these drugs, thus improving treatment efficacy and minimizing side effects. In general, cytotoxic activity is displayed only upon cleavage of a specific chemical bond (linker) that connects the drug to the carrier. The perfect balance between the linker stability and its selective cleavage represents the key for success in these therapeutic approaches and the chemical toolbox to reach this goal is continuously expanding. In this Review article, we highlight recent advances on the different modalities to promote the selective release of cytotoxic agents, either by exploiting specific hallmarks of the tumor microenvironment (e.g. pH, enzyme expression) or by the application of external triggers (e.g. light and bioorthogonal reactions).
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http://dx.doi.org/10.1002/chem.201903127DOI Listing
November 2019

Bromine-Promoted Glycosidation of Conformationally Superarmed Thioglycosides.

Chemistry 2019 Sep 20;25(51):11831-11836. Epub 2019 Aug 20.

Department of Chemistry and Biochemistry, University of Missouri-St. Louis, One University Boulevard, St. Louis, Missouri, 63121, USA.

Presented herein is a study of the conformation and reactivity of highly reactive thioglycoside donors. The structural studies have been conducted using NMR spectroscopy and computational methods. The reactivity of these donors has been investigated in bromine-promoted glycosylations of aliphatic and sugar alcohols. Swift reaction times, high yields, and respectable 1,2-cis stereoselectivity were observed in a majority of these glycosylations.
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http://dx.doi.org/10.1002/chem.201901969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742554PMC
September 2019

Exploring E-cadherin-peptidomimetics interaction using NMR and computational studies.

PLoS Comput Biol 2019 06 3;15(6):e1007041. Epub 2019 Jun 3.

Dipartimento di Chimica, Università degli Studi di Milano, Milan, Italy.

Cadherins are homophilic cell-cell adhesion molecules whose aberrant expression has often been shown to correlate with different stages of tumor progression. In this work, we investigate the interaction of two peptidomimetic ligands with the extracellular portion of human E-cadherin using a combination of NMR and computational techniques. Both ligands have been previously developed as mimics of the tetrapeptide sequence Asp1-Trp2-Val3-Ile4 of the cadherin adhesion arm, and have been shown to inhibit E-cadherin-mediated adhesion in epithelial ovarian cancer cells with millimolar potency. To sample a set of possible interactions of these ligands with the E-cadherin extracellular portion, STD-NMR experiments in the presence of two slightly different constructs, the wild type E-cadherin-EC1-EC2 fragment and the truncated E-cadherin-(Val3)-EC1-EC2 fragment, were carried out at three temperatures. Depending on the protein construct, a different binding epitope of the ligand and also a different temperature effect on STD signals were observed, both suggesting an involvement of the Asp1-Trp2 protein sequence among all the possible binding events. To interpret the experimental results at the atomic level and to probe the role of the cadherin adhesion arm in the dynamic interaction with the peptidomimetic ligand, a computational protocol based on docking calculations and molecular dynamics simulations was applied. In agreement with NMR data, the simulations at different temperatures unveil high variability/dynamism in ligand-cadherin binding, thus explaining the differences in ligand binding epitopes. In particular, the modulation of the signals seems to be dependent on the protein flexibility, especially at the level of the adhesive arm, which appears to participate in the interaction with the ligand. Overall, these results will help the design of novel cadherin inhibitors that might prevent the swap dimer formation by targeting both the Trp2 binding pocket and the adhesive arm residues.
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http://dx.doi.org/10.1371/journal.pcbi.1007041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6564044PMC
June 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

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

The Importance of Detail: How Differences in Ligand Structures Determine Distinct Functional Responses in Integrin α β.

Chemistry 2019 Apr 28;25(23):5959-5970. Epub 2019 Mar 28.

Dipartimento di Chimica, Università degli Studi di Pavia, Viale Taramelli 12, 27100, Pavia, Italy.

Ligand-based control of protein functional motions can provide novel opportunities in the study of fundamental biological mechanisms and in the development of novel therapeutics. In this work we addressed the ligand-based modulation of integrin functions. Inhibitors of integrin α β are interesting anticancer agents but their molecular mechanisms are still unclear: Peptides and peptidomimetics characterized by the Arg-Gly-Asp (RGD) or isoAsp-Gly-Arg (isoDGR) binding motifs have shown controversial agonist/antagonist effects. We have investigated the differential mechanisms of integrin activation/deactivation by three distinct ligands (cyclo-RGDf(NMe)V (Cilengitide), cyclo[DKP3-RGD], cyclo[DKP3-isoDGR]; DKP=diketopiperazine) through a comparative analysis of ligand-controlled protein internal dynamics: Although RGD facilitates the onset of dynamic states leading to activation, isoDGR induces a diffuse rigidification of the complex consistent with antagonist activities. Computational predictions have been experimentally probed by showing that the antibody AP5, which is capable of recognizing the active form of integrin, binds specifically to the RGD complexes and not to the isoDGR complex, which supports opposite functional roles of the two motifs targeting the same binding site.
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http://dx.doi.org/10.1002/chem.201900169DOI Listing
April 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

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

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

High Affinity vs. Native Fibronectin in the Modulation of αvβ3 Integrin Conformational Dynamics: Insights from Computational Analyses and Implications for Molecular Design.

PLoS Comput Biol 2017 01 23;13(1):e1005334. Epub 2017 Jan 23.

Istituto di Chimica del Riconoscimento Molecolare CNR, Milan, Italy.

Understanding how binding events modulate functional motions of multidomain proteins is a major issue in chemical biology. We address several aspects of this problem by analyzing the differential dynamics of αvβ3 integrin bound to wild type (wtFN10, agonist) or high affinity (hFN10, antagonist) mutants of fibronectin. We compare the dynamics of complexes from large-scale domain motions to inter-residue coordinated fluctuations to characterize the distinctive traits of conformational evolution and shed light on the determinants of differential αvβ3 activation induced by different FN sequences. We propose an allosteric model for ligand-based integrin modulation: the conserved integrin binding pocket anchors the ligand, while different residues on the two FN10's act as the drivers that reorganize relevant interaction networks, guiding the shift towards inactive (hFN10-bound) or active states (wtFN10-bound). We discuss the implications of results for the design of integrin inhibitors.
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http://dx.doi.org/10.1371/journal.pcbi.1005334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293283PMC
January 2017

New β-Lactam Derivatives Modulate Cell Adhesion and Signaling Mediated by RGD-Binding and Leukocyte Integrins.

J Med Chem 2016 11 21;59(21):9721-9742. Epub 2016 Oct 21.

Department of Chemistry "G. Ciamician", University of Bologna , Via Selmi 2, 40126 Bologna, Italy.

A novel series of β-lactam derivatives that was designed and synthesized to target RGD-binding and leukocyte integrins is reported. The compound library was evaluated by investigating the effects on integrin-mediated cell adhesion and cell signaling in cell lines expressing αβ, αβ, αβ, αβ, αβ, αβ, and αβ integrins. SAR analysis of the new series of azetidinones enabled the recognition of structural elements associated with integrin selectivity. We obtained selective and potent agonists that could induce cell adhesion and promote cell signaling mediated by αβ, αβ, αβ, or αβ integrin, and antagonists for the integrins αβ and αβ as well as αβ and αβ, preventing the effects elicited by the respective endogenous agonists.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00576DOI Listing
November 2016

Crystal Structure of Human E-Cadherin-EC1EC2 in Complex with a Peptidomimetic Competitive Inhibitor of Cadherin Homophilic Interaction.

J Med Chem 2016 05 3;59(10):5089-94. Epub 2016 May 3.

Center for Nano Science and Technology @PoliMi, Istituto Italiano di Tecnologia , Via G. Pascoli 70/3, 20133 Milano, Italy.

Cadherins are transmembrane cell adhesion proteins whose aberrant expression often correlates with cancer development and proliferation. We report the crystal structure of an E-cadherin extracellular fragment in complex with a peptidomimetic compound that was previously shown to partially inhibit cadherin homophilic adhesion. The structure reveals an unexpected binding mode and allows the identification of a druggable cadherin interface, thus paving the way to a future structure-guided design of cell adhesion inhibitors against cadherin-expressing solid tumors.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01487DOI Listing
May 2016

New Insights into the Molecular Mechanism of E-Cadherin-Mediated Cell Adhesion by Free Energy Calculations.

J Chem Theory Comput 2015 Apr;11(4):1354-9

Department of Chemistry and Institute of Structural and Molecular Biology, University College London , 20 Gordon Street, London WC1H 0AJ, United Kingdom.

Three-dimensional domain swapping is an important mode of protein association leading to the formation of stable dimers. Monomers associating via this mechanism mutually exchange a domain to form a homodimer. Classical cadherins, an increasingly important target for anticancer therapy, use domain swapping to mediate cell adhesion. However, despite its importance, the molecular mechanism of domain swapping is still debated. Here, we study the conformational changes that lead to activation and dimerization via domain swapping of E-cadherin. Using state-of-the-art enhanced sampling atomistic simulations, we reconstruct its conformational free energy landscape, obtaining the free energy profile connecting the inactive and active form. Our simulations predict that the E-cadherin monomer populates the open and closed forms almost equally, which is in agreement with the proposed "selected fit" mechanism in which monomers in an active conformational state bind to form a homodimer, analogous to the conformational selection mechanism often observed in ligand-target binding. Moreover, we find that the open state population is increased in the presence of calcium ions at the extracellular boundary, suggesting their possible role as allosteric activators of the conformational change.
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http://dx.doi.org/10.1021/ct5010164DOI Listing
April 2015

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

Metadynamics Simulations Rationalise the Conformational Effects Induced by N-Methylation of RGD Cyclic Hexapeptides.

Chemistry 2015 Sep 6;21(40):14165-70. Epub 2015 Aug 6.

Biomolecular NMR Unit, Ospedale S. Raffaele, Via Olgettina 58, 20132 Milan (Italy).

We combined metadynamics, docking and molecular mechanics/generalised born surface area (MM/GBSA) re-scoring methods to investigate the impact of single and multiple N-methylation on a set of RGD cyclopeptides displaying different affinity for integrin αIIbβ3. We rationalised the conformational effects induced by N-methylation and its interplay with receptor affinity, obtaining good agreement with experimental data. This approach can be exploited before entering time-consuming and expensive synthesis and binding experiments.
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http://dx.doi.org/10.1002/chem.201501196DOI Listing
September 2015

αvβ3 Integrin-Targeted Peptide/Peptidomimetic-Drug Conjugates: In-Depth Analysis of the Linker Technology.

Curr Top Med Chem 2016 ;16(3):314-29

Universita degli Studi di Milano, Dipartimento di Chimica, via C. Golgi, 19, I-20133, Milan (Italy).

Covalent conjugation of anticancer drugs to targeting carriers (e.g., antibodies or small molecules) capable of selectively binding to tumor-specific antigens, is emerging as a successful strategy to overcome the drawbacks of traditional chemotherapy. Due to its overexpression on blood vessels of human tumors, αvβ3 integrin is one of the most studied receptors of tumor-targeted therapeutics: several peptides and peptidomimetics, bearing the RGD (Arg-Gly-Asp) recognition sequence, have been developed as integrin ligands and linked to different anticancer drugs. The resulting integrin- targeted small molecule-drug conjugates (SMDCs) are able to release the cytotoxic agents upon cleavage of a linker under specific conditions (i.e., hydrolysis, enzymatic action or reduction). Despite the significant efforts made in this field, αvβ3 integrin-targeted SMDCs are still far from the clinic. In this review, we survey this approach with a special focus on the different linkers employed and the reported biological activities in vitro and in vivo.
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http://dx.doi.org/10.2174/1568026615666150701114343DOI Listing
July 2016

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

Designing nanomolar antagonists of DC-SIGN-mediated HIV infection: ligand presentation using molecular rods.

Chem Commun (Camb) 2015 Mar;51(18):3816-9

Università degli Studi di Milano (UniMI), Dip. Chimica, via Golgi 19, 20133, Milano, Italy.

DC-SIGN antagonists were designed combining one selective monovalent glycomimetic ligand with trivalent dendrons separated by a rigid core of controlled length. The design combines multiple multivalency effects to achieve inhibitors of HIV infection, which are active in nanomolar concentration.
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http://dx.doi.org/10.1039/c4cc09709bDOI Listing
March 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
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