Publications by authors named "Leonardo Manzoni"

32 Publications

Retromer stabilization results in neuroprotection in a model of Amyotrophic Lateral Sclerosis.

Nat Commun 2020 07 31;11(1):3848. Epub 2020 Jul 31.

INSPE-Institute of Experimental Neurology, San Raffaele Scientific Institute, Milano, Italy.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the degeneration of upper and lower motor neurons (MNs). We find a significant reduction of the retromer complex subunit VPS35 in iPSCs-derived MNs from ALS patients, in MNs from ALS post mortem explants and in MNs from SOD1G93A mice. Being the retromer involved in trafficking of hydrolases, a pathological hallmark in ALS, we design, synthesize and characterize an array of retromer stabilizers based on bis-guanylhydrazones connected by a 1,3-phenyl ring linker. We select compound 2a as a potent and bioavailable interactor of VPS35-VPS29. Indeed, while increasing retromer stability in ALS mice, compound 2a attenuates locomotion impairment and increases MNs survival. Moreover, compound 2a increases VPS35 in iPSCs-derived MNs and shows brain bioavailability. Our results clearly suggest the retromer as a valuable druggable target in ALS.
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http://dx.doi.org/10.1038/s41467-020-17524-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395176PMC
July 2020

Interfering with HuR-RNA Interaction: Design, Synthesis and Biological Characterization of Tanshinone Mimics as Novel, Effective HuR Inhibitors.

J Med Chem 2018 02 31;61(4):1483-1498. Epub 2018 Jan 31.

Chemistry Department, University of Milan , Via Golgi 19, 20133 Milan, Italy.

The human antigen R (HuR) is an RNA-binding protein known to modulate the expression of target mRNA coding for proteins involved in inflammation, tumorigenesis, and stress responses and is a valuable drug target. We previously found that dihydrotanshinone-I (DHTS, 1) prevents the association of HuR with its RNA substrate, thus imparing its function. Herein, inspired by DHTS structure, we designed and synthesized an array of ortho-quinones (tanshinone mimics) using a function-oriented synthetic approach. Among others, compound 6a and 6n turned out to be more effective than 1, showing a nanomolar K and disrupting HuR binding to RNA in cells. A combined approach of NMR titration and molecular dynamics (MD) simulations suggests that 6a stabilizes HuR in a peculiar closed conformation, which is incompatible with RNA binding. Alpha screen and RNA-electrophoretic mobility shift assays (REMSA) data on newly synthesized compounds allowed, for the first time, the generation of structure activity relationships (SARs), thus providing a solid background for the generation of highly effective HuR disruptors.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01176DOI Listing
February 2018

Regulation of HuR structure and function by dihydrotanshinone-I.

Nucleic Acids Res 2017 Sep;45(16):9514-9527

Centre for Integrative Biology, CIBIO, University of Trento, Trento 38122, Italy.

The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.
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http://dx.doi.org/10.1093/nar/gkx623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766160PMC
September 2017

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

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

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

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

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

Dimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part II: Structural and biological characterization.

Bioorg Med Chem 2012 Nov 24;20(22):6709-23. Epub 2012 Sep 24.

Dipartimento di Oncologia Sperimentale e Medicina Molecolare, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo 42, Milan, Italy.

Novel pro-apoptotic, homodimeric and heterodimeric Smac mimetics/IAPs inhibitors connected through head-head (8), tail-tail (9) or head-tail linkers (10), were biologically and structurally characterized. In vitro characterization (binding to BIR3 and linker-BIR2-BIR3 domains from XIAP and cIAP1, cytotoxicity assays) identified early leads from each dimer family. Computational models and structural studies (crystallography, NMR, gel filtration) partially rationalized the observed properties for each dimer class. Tail-tail dimer 9a was shown to be active in a breast and in an ovary tumor model, highlighting the potential of dimeric Smac mimetics/IAP inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold as potential antineoplastic agents.
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http://dx.doi.org/10.1016/j.bmc.2012.09.041DOI Listing
November 2012

Homo- and heterodimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part I: Synthesis.

Bioorg Med Chem 2012 Nov 21;20(22):6687-708. Epub 2012 Sep 21.

Istituto di Scienze e Tecnologie Molecolari (ISTM), Consiglio Nazionale delle Ricerche (CNR), Via Golgi 19, Milan, Italy.

Novel pro-apoptotic, homo- and heterodimeric Smac mimetics/IAPs inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold were prepared from monomeric structures connected through a head-head (8), tail-tail (9) or head-tail (10) linker. The selection of appropriate decorating functions for the scaffolds, and of rigid and flexible linkers connecting them, is described. The synthesis, purification and analytical characterization of each prepared dimer 8-10 is thoroughly described.
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http://dx.doi.org/10.1016/j.bmc.2012.09.020DOI Listing
November 2012

Design, synthesis, and biological evaluation of novel cRGD-paclitaxel conjugates for integrin-assisted drug delivery.

Bioconjug Chem 2012 Aug 20;23(8):1610-22. Epub 2012 Jul 20.

Centro Interdipartimentale Studi Biomolecolari e Applicazioni Industriali, Università degli Studi di Milano, Via Fantoli 16/15, I-20138 Milano, Italy.

The efficacy of taxane-based antitumor therapy is limited by several drawbacks which result in a poor therapeutic index. Thus, the development of approaches that favor selective delivery of taxane drugs (e.g., paclitaxel, PTX) to the disease area represents a truly challenging goal. On the basis of the strategic role of integrins in tumor cell survival and tumor progression, as well as on integrin expression in tumors, novel molecular conjugates were prepared where PTX is covalently attached to either cyclic AbaRGD (Azabicycloalkane-RGD) or AmproRGD (Aminoproline-RGD) integrin-recognizing matrices via structurally diverse connections. Receptor-binding assays indicated satisfactory-to-excellent α(V)β(3) binding capabilities for most conjugates, while in vitro growth inhibition assays on a panel of human tumor cell lines revealed outstanding cell sensitivity values. Among the nine conjugate ensemble, derivative 21, bearing a robust triazole ring connected to ethylene glycol units by an amide function and showing excellent cell sensitivity properties, was selected for in vivo studies in an ovarian carcinoma model xenografted in immunodeficient mice. Remarkable antitumor activity was attained, superior to that of PTX itself, which was associated with a marked induction of aberrant mitoses, consistent with the mechanism of action of spindle poisons. Overall, the novel cRGD-PTX conjugates disclosed here represent promising candidates for further advancement in the domain of targeted antitumor therapy.
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http://dx.doi.org/10.1021/bc300164tDOI Listing
August 2012

Synthesis of Gd and (68)Ga complexes in conjugation with a conformationally optimized RGD sequence as potential MRI and PET tumor-imaging probes.

ChemMedChem 2012 Jun 5;7(6):1084-93. Epub 2012 Apr 5.

Consiglio Nazionale delle Ricerche, Istituto di Scienze e Tecnologie Molecolari, Via Fantoli 16/15, 20138, Milano, Italy.

We report the synthesis of novel chelates of Gd and (68)Ga with DPTA, DOTA, HP-DOA3, as well as with AAZTA, a novel chelating agent developed by our research group. These chelating agents were appropriately conjugated, prior to metal complexation, with DB58, an RGD peptidomimetic, conformationally constrained on an azabicycloalkane scaffold and endowed with high affinity for integrin α(ν)β(3) . Because α(ν)β(3) is involved in neo-angiogenesis in solid tumors and is also directly expressed in cancer cells (e.g. glioblastomas, melanomas) and ovarian, breast, and prostate cancers, these constructs could prove useful as molecular imaging probes in cancer diagnosis by MRI or PET techniques. Molecular modeling, integrin binding assays, and relaxivity assessments allowed the selection of compounds suitable for multiple expression on dendrimeric or nanoparticulate structures. These results also led us to an exploratory investigation of (68)Ga complexation for the promising (68)Ga-PET technique; the AAZTA complex 15((68)Ga) exhibited uptake in a xenograft model of glioblastoma, suggesting potentially useful developments with new probes with improved affinity.
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http://dx.doi.org/10.1002/cmdc.201200043DOI Listing
June 2012

A new optical imaging probe targeting αVβ3 integrin in glioblastoma xenografts.

Contrast Media Mol Imaging 2011 Nov-Dec;6(6):449-58

Molecular Biotechnology Center, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

α(V)β(3) Integrins are a widely recognized target for in vivo molecular imaging of pathological conditions such as inflammation, cancer and rheumatoid arthritis. We have evaluated the sensitivity of a new, near-infrared fluorescence (NIRF), RGD cyclic probe (DA364) in noninvasive detection of α(V) β(3) integrin-overexpressing tumors. DA364's binding affinity for α(V)β(3) integrin was first evaluated in vitro. Human α(V)β(3) integrin-positive, U-87 MG glioblastoma cells were then xenografted in nude mice, and DA364 was injected intravenously (i.v.) to evaluate its in vivo distribution, specificity and sensitivity in comparison with a commercially available probe. DA364 bound α(V)β(3) integrin on U-87 MG cells with high affinity and specificity, both in vitro and in vivo. This binding specificity was corroborated by the strong inhibition of its tumor uptake induced by nonfluorescent, cyclic-RGD peptides. Ex vivo analysis showed that DA364 accumulated at the tumor site, whereas very low levels were detected in liver and spleen. In conclusion, DA364 allows sensitive and specific detection of transplantable glioblastoma by NIRF imaging, and is thus a promising candidate for the elaboration of imaging and therapeutic probes for α(V)β(3) integrin-overexpressing tumors.
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http://dx.doi.org/10.1002/cmmi.444DOI Listing
April 2012

Cyclic RGD functionalized gold nanoparticles for tumor targeting.

Bioconjug Chem 2011 Apr 24;22(4):664-72. Epub 2011 Mar 24.

CNR-Istituto di Scienze e Tecnologie Molecolari ( ISTM ), Via Fantoli 16/15, I-20138, Milan, Italy.

Integrin α(v)β(3) is an adhesion molecule involved in physiological and pathological angiogenesis as well as tumor invasion and metastasis. Therefore, it is considered an important target for molecular imaging and delivery of therapeutics for cancer, and there is a strong interest in developing novel agents interacting with this protein. Nevertheless, the interaction of individual ligands is often still weak for efficient tumor targeting, and many research groups have synthesized multivalent displays in order to overcome this problem. Gold nanoparticles can be considered a smart platform for polyvalent presentation on account of their globular shape, tunable size, facile surface chemistry, and biocompatibility. Moreover, their unique physical properties render gold nanoparticles ideal candidates for tumor diagnosis and therapy. Here, we report the synthesis and characterization of gold nanoparticles functionalized with cRGD integrin ligand and their employment for targeting human cancer cells expressing α(v)β(3) integrin.
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http://dx.doi.org/10.1021/bc100448rDOI Listing
April 2011

Novel second mitochondria-derived activator of caspases (Smac) mimetic compounds sensitize human leukemic cell lines to conventional chemotherapeutic drug-induced and death receptor-mediated apoptosis.

Invest New Drugs 2011 Dec 8;29(6):1264-75. Epub 2010 Jul 8.

Fondazione Matarelli, Dipartimento di Farmacologia Chemioterapia e Tossicologia Medica, Università degli Studi di Milano, Via Vanvitelli 32, 20129, Milan, Italy.

The Inhibitor of Apoptosis Proteins (IAPs) are important regulators of programmed cell death. XIAP is the most potent among them and is over-expressed in several hematological malignancies. Its activity is endogenously antagonized by SMAC/DIABLO, and also by small molecules mimicking Smac that can induce apoptosis in tumor cells. Here we describe the activity of 56 newly synthesized Smac-mimetics in human leukemic cell lines and normal CD34(+) progenitor cells. Our compounds bind to XIAP with high affinity, reduce the levels of cIAP1 and are cytotoxic at nanomolar or low micromolar concentrations. Furthermore, the Smac-mimetics synergize with Cytarabine, Etoposide and especially with TRAIL in combination treatments. Apoptosis activation was clearly detectable by the occurrence of sub G(1) apoptotic peak and the accumulation of cleaved PARP, caspase 8 and caspase 3. Interestingly, the down-regulation of XIAP sensitized Jurkat cells to drugs too, confirming the role of this protein in drug-resistance. In conclusion, while being very active in leukemic cells, our Smac-mimetics have modest effects on normal hematopoietic progenitors, suggesting their promising therapeutic potential as a new class of anticancer drugs in onco-hematology, particularly when combined with TRAIL, to overcome the resistance of cancer cells.
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http://dx.doi.org/10.1007/s10637-010-9475-6DOI Listing
December 2011

4-Aminoproline-based arginine-glycine-aspartate integrin binders with exposed ligation points: practical in-solution synthesis, conjugation and binding affinity evaluation.

Org Biomol Chem 2009 Dec 6;7(23):4924-35. Epub 2009 Oct 6.

Dipartimento Farmaceutico, Università degli Studi di Parma, Viale G. P. Usberti 27A, I-43100 Parma, Italy.

An expedient and practical in-solution synthesis of three new 4-aminoproline-based arginine-glycine-aspartate integrin binders--compounds 15, 17 and 19--is presented. Two candidates carrying exposed azide and amine functional points were further advanced to trimeric platform 21 as well as fluorescein- and DOTA-conjugates 23 and 25. The new compounds were assayed for their binding affinity towards human alpha(V)beta3 and alpha(V)beta5 integrin receptors. Both monomeric candidates and covalent conjugates revealed potent ligand competence for the alpha(V)beta3 receptor in the one-digit nanomolar range (IC50 alpha(V)beta3 = 0.2-8.0 nM; IC50 alpha(V)beta5 = 5.0-1621 nM), thus demonstrating that conjugation does not impair the exquisite binding profile of this new generation of integrin ligands.
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http://dx.doi.org/10.1039/b914836aDOI Listing
December 2009

Cyclic RGD-peptidomimetics containing bifunctional diketopiperazine scaffolds as new potent integrin ligands.

Chemistry 2009 Nov;15(45):12184-8

Università degli Studi dell'Insubria, Dipartimento di Scienze Chimiche e Ambientali, Via Valleggio 11, 22100 Como, Italy.

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http://dx.doi.org/10.1002/chem.200902398DOI Listing
November 2009

Functionalized cyclic RGD peptidomimetics: conjugable ligands for αvβ3 receptor imaging.

Bioconjug Chem 2009 Aug 22;20(8):1611-7. Epub 2009 Jul 22.

CNR-Istituto di Scienze e Tecnologie Molecolari (ISTM), Milan, Italy.

The α(v)β(3) integrin is an adhesion molecule involved in physiological and pathological angiogenesis as well as in tumor invasion and metastasis, and therefore, there is a strong interest in developing novel agents interacting with this molecule. We report the synthesis and characterization of fluorescent α(v)β(3) integrin probes and their use to visualize integrin α(v)β(3) expression on human normal and cancer cells. The fluorescent probes we describe here may be of use for noninvasive imaging of α(V)β(3) integrin expression also in vivo.
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http://dx.doi.org/10.1021/bc900155jDOI Listing
August 2009

Rational design, synthesis and characterization of potent, non-peptidic Smac mimics/XIAP inhibitors as proapoptotic agents for cancer therapy.

Bioorg Med Chem 2009 Aug 10;17(16):5834-56. Epub 2009 Jul 10.

Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, Milan, Italy.

Novel proapoptotic Smac mimics/IAPs inhibitors have been designed, synthesized and characterized. Computational models and structural studies (crystallography, NMR) have elucidated the SAR of this class of inhibitors, and have permitted further optimization of their properties. In vitro characterization (XIAP BIR3 and linker-BIR2-BIR3 binding, cytotox assays, early ADMET profiling) of the compounds has been performed, identifying one lead for further in vitro and in vivo evaluation.
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http://dx.doi.org/10.1016/j.bmc.2009.07.009DOI Listing
August 2009

Structural basis for bivalent Smac-mimetics recognition in the IAP protein family.

J Mol Biol 2009 Sep 22;392(3):630-44. Epub 2009 Apr 22.

Department of Biomolecular Sciences and Biotechnology, University of Milano, Italy.

XIAP is an apoptotic regulator protein that binds to the effector caspases -3 and -7 through its BIR2 domain, and to initiator caspase-9 through its BIR3 domain. Molecular docking studies suggested that Smac-DIABLO may antagonize XIAP by concurrently targeting both BIR2 and BIR3 domains; on this basis bivalent Smac-mimetic compounds have been proposed and characterized. Here, we report the X-ray crystal structure of XIAP-BIR3 domain in complex with a two-headed compound (compound 3) with improved efficacy relative to its monomeric form. A small-angle X-ray scattering study of XIAP-BIR2BIR3, together with fluorescence polarization binding assays and compound 3 cytotoxicity tests on HL60 leukemia cell line are also reported. The crystal structure analysis reveals a network of interactions supporting XIAP-BIR3/compound 3 recognition; moreover, analytical gel-filtration chromatography shows that compound 3 forms a 1:1 stoichiometric complex with a XIAP protein construct containing both BIR2 and BIR3 domains. On the basis of the crystal structure and small-angle X-ray scattering, a model of the same BIR2-BIR3 construct bound to compound 3 is proposed, shedding light on the ability of compound 3 to relieve XIAP inhibitory effects on caspase-9 as well as caspases -3 and -7. A molecular modeling/docking analysis of compound 3 bound to cIAP1-BIR3 domain is presented, considering that Smac-mimetics have been shown to kill tumor cells by inducing cIAP1 and cIAP2 ubiquitination and degradation. Taken together, the results reported here provide a rationale for further development of compound 3 as a lead in the design of dimeric Smac mimetics for cancer treatment.
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http://dx.doi.org/10.1016/j.jmb.2009.04.033DOI Listing
September 2009

Cyclic RGD-containing functionalized azabicycloalkane peptides as potent integrin antagonists for tumor targeting.

ChemMedChem 2009 Apr;4(4):615-32

Istituto di Scienze e Tecnologie Molecolari, Consiglio Nazionale delle Ricerche, Milano, Italy.

Cyclic RGD-containing functionalized azabicycloalkane peptides were synthesized with the aim of developing high-affinity selective integrin ligands as carriers for therapeutic and diagnostic purposes. Herein we describe the synthesis and in vitro screening of these RGD derivatives, as well as the determination of their conformational properties in solution by spectroscopic and computational methods. Docking studies with the X-ray crystal structure of the extracellular domain of integrin alpha(v)beta(3) were also performed to elucidate the structural binding requirements and to rationalize the biological results. One compound in particular was found to be the best alpha(v)beta(3) integrin binder (IC(50)=53.7 nM) among the new functionalized RGD cyclic peptides, thus emerging as a promising candidate for covalent bonding and selective homing of useful functional units.
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http://dx.doi.org/10.1002/cmdc.200800422DOI Listing
April 2009

Designing Smac-mimetics as antagonists of XIAP, cIAP1, and cIAP2.

Biochem Biophys Res Commun 2009 Jan 4;378(2):162-7. Epub 2008 Nov 4.

Department of Biomolecular Sciences and Biotechnology, University of Milano, Via Celoria 26, I-20133 Milano, Italy.

Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. Several compounds targeting IAPs and inducing cell death in cancer cells have been developed. Some of these are synthesized mimicking the N-terminal tetrapeptide sequence of Smac/DIABLO, the natural endogenous IAPs inhibitor. Starting from such conceptual design, we generated a library of 4-substituted azabicyclo[5.3.0]alkane Smac-mimetics. Here we report the crystal structure of the BIR3 domain from XIAP in complex with Smac037, a compound designed according to structural principles emerging from our previously analyzed XIAP BIR3/Smac-mimetic complexes. In parallel, we present an in silico docking analysis of three Smac-mimetics to the BIR3 domain of cIAP1, providing general considerations for the development of high affinity lead compounds targeting three members of the IAP family.
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http://dx.doi.org/10.1016/j.bbrc.2008.10.139DOI Listing
January 2009

Targeting the X-linked inhibitor of apoptosis protein through 4-substituted azabicyclo[5.3.0]alkane smac mimetics. Structure, activity, and recognition principles.

J Mol Biol 2008 Dec 7;384(3):673-89. Epub 2008 Oct 7.

Department of Biomolecular Sciences and Biotechnology, CNR-INFM and CIMAINA, University of Milano, Via Celoria 26, I-20133 Milan, Italy.

The X-linked inhibitor of apoptosis protein (XIAP) is overexpressed in several malignant cells where it prevents apoptosis by binding to, and blocking, the activation of caspase-3, -7, and -9. Human XIAP (479 residues) is composed of three tandem-repeated baculoviral IAP repeat (BIR) domains (BIR1-3), and by a C-terminal RING domain. Smac-DIABLO [second mitochondria-derived activator of caspases (Smac)-direct IAP binding protein with low pI (DIABLO)], the natural antagonist of XIAP, binds through its N-terminal sequence AVPI to the same surface groove, in the BIR domains, that binds caspases. Synthetic compounds mimicking such tetrapeptide motif effectively block the interaction between IAP and active caspases, thus triggering apoptosis. Peptidomimetics based on an azabicyclo[x.y.0]alkane scaffolds, have been shown to bind the BIR3 domain of XIAP with micromolar to nanomolar affinities, thus presenting attractive features for drug lead optimization. Here we report a study on three newly synthesized Smac mimetics, which have been characterized in their complexes with XIAP BIR3 domain through X-ray crystallography and molecular modelling/docking simulations. Based on analysis of the crystal structures, we show that specific substitutions at the 4-position of the azabicyclo[5.3.0]alkane scaffold results in sizeable effects on the peptidomimetic-BIR3 domain affinity. By means of functional, biophysical and simulative approaches we also propose that the same Smac mimetics can bind XIAP BIR2 domain at a location structurally related to the BIR3 domain AVPI binding groove. Details of the XIAP-Smac mimetic recognition principles highlighted by this study are discussed in light of the drug-like profile of the three (potentially proapoptotic) compounds developed that show improved performance in ADMET (adsorption, distribution, metabolism, excretion and toxicity) tests.
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http://dx.doi.org/10.1016/j.jmb.2008.09.064DOI Listing
December 2008

A potent integrin antagonist from a small library of cyclic RGD pentapeptide mimics including benzyl-substituted azabicycloalkane amino acids.

ChemMedChem 2008 Oct;3(10):1589-603

Istituto di Scienze e Tecnologie Molecolari, Consiglio Nazionale delle Ricerche, Via Fantoli 16/15, 20138 Milano, Italy.

A small library of cyclic RGD pentapeptide mimics, including benzyl-substituted azabicycloalkane amino acids, was synthesized with the aim of developing active and selective integrin antagonists. In vitro binding assays established one particular compound with affinity for both the alpha(v)beta(3) and the alpha(v)beta(5) integrins. The synthesis in solution and the in vitro screening of these RGD derivatives, as well as the determination of the conformational properties of the integrin ligands by spectroscopic and computational methods are described.
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http://dx.doi.org/10.1002/cmdc.200800143DOI Listing
October 2008

Froc: a new fluorous protective group for peptide and oligosaccharide synthesis.

Org Lett 2006 Mar;8(5):955-7

C.N.R. - Istituto di Scienze e Tecnologie Molecolari (ISTM) and Centro Interdisciplinare Studi Biomolecolari e applicazioni Industriali (CISI), Via Venezian 21, I-20133 Milano, Italy.

The synthesis of a new fluorous protecting group, Froc, is described. This new fluorous tag has been used in peptide and carbohydrate synthesis by our group and readily allows us to fully characterize each product (NMR, MS) and monitor each synthetic step by TLC. Purification of the products is generally performed by standard fluorous solid-phase extraction techniques (e.g., F-SPE), but standard chromatographic purifications are also possible if required.
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http://dx.doi.org/10.1021/ol060006eDOI Listing
March 2006

Biological and molecular properties of a new alpha(v)beta3/alpha(v)beta5 integrin antagonist.

Mol Cancer Ther 2005 Nov;4(11):1670-80

Organic and Industrial Chemistry Department, Centre for Biomolecular Interdisciplinary Studies and Industrial Applications, University of Milan, Italy.

The aim of the present study was to identify specific alpha(v)beta3/alpha(v)beta5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp-containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for alpha(v)beta3 and alpha(v)beta5 integrins with negligible interacting with alpha5beta1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of alpha(v)beta3/alpha(v)beta5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of alpha(v)beta3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-alpha(v)beta3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-alpha(v)beta3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual alpha(v)beta3/alpha(v)beta5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.
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http://dx.doi.org/10.1158/1535-7163.MCT-05-0120DOI Listing
November 2005

Targeting integrins: insights into structure and activity of cyclic RGD pentapeptide mimics containing azabicycloalkane amino acids.

Bioorg Med Chem 2006 Jan 7;14(1):169-80. Epub 2005 Oct 7.

Dipartimento di Chimica Organica e Industriale and Centro Interdisciplinare Studi bio-molecolari e applicazioni Industriali, (CISI), Università degli Studi di Milano, via G. Venezian 21, I-20133 Milan, Italy.

A small library of cyclic RGD pentapeptide mimics incorporating stereoisomeric 5,6- and 5,7-fused bicyclic lactams was synthesized. This library was found to contain high-affinity ligands for the alpha(v)beta3 integrin. The aim of this study was to investigate activity, selectivity, and structure of these ligands in order to identify new specific alpha(v)-integrin antagonists that could be evaluated as tumor angiogenesis inhibitors. In vitro screening, including receptor-binding assays to purified alpha(v)beta3, alpha(v)beta5, and alpha5beta1 integrins, and platelet aggregation assay, revealed ST1646 as a potent, highly selective alpha(v)beta3/alpha(v)beta5 integrin antagonist. Structure determination of the cyclic RGD pentapeptide mimics performed by a combination of NMR spectroscopy, and molecular mechanics and dynamics calculations showed a strong dependence of the RGD cyclopeptide conformation on lactam ring size and stereochemistry. ST1646 revealed the highest ability within the library to adopt the proper RGD orientation required for binding to the alpha(v)beta3 integrin, as deduced from the recently solved crystal structure of the extracellular segment of integrin alpha(v)beta3 in complex with a cyclic pentapeptide ligand.
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http://dx.doi.org/10.1016/j.bmc.2005.08.048DOI Listing
January 2006

Functionalized azabicycloalkane amino acids by nitrone 1,3-dipolar intramolecular cycloaddition.

J Org Chem 2005 May;70(10):4124-32

C.N.R.-Istituto di Scienze e Tecnologie Molecolari, and Centro Interdisciplinare Studi bio-molecolari e applicazioni Industriali, Via Venezian 21, I-20133, Milano, Italy.

[reaction: see text] An efficient and operationally simple method for the synthesis of functionalized azaoxobicyclo[X.3.0]alkane amino acids has been devised. The key step is an intramolecular nitrone cycloaddition on 5-allyl- or 5-homoallylproline that was found to be completely regio- and stereoselective.
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http://dx.doi.org/10.1021/jo0500683DOI Listing
May 2005

Synthesis of the Lewis a trisaccharide based on an anomeric silyl fluorous tag.

Org Lett 2004 Nov;6(23):4195-8

C.N.R.-Istituto di Scienze e Tecnologie Molecolari, Dipartimento di Chimica Organica e Industriale, Milano, Italy.

The synthesis of the trisaccharide Lewis a was performed using an anomeric fluorous silyl protective group. This methodology allowed us to fully characterize each product (NMR, MS) and monitor each synthetic step (TLC). Although the product purifications could be performed by fluorous-solid-phase extraction (F-SPE) technology, standard chromatography could be used to effect purification if necessary. Trichloroethoxy carbonyl (Troc) protection of the amino group of the glucosamine moiety was found essential to allow protecting group manipulation of the fluorous protected sugar.
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http://dx.doi.org/10.1021/ol048474gDOI Listing
November 2004