Publications by authors named "Cesare Gennari"

45 Publications

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

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

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

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

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

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

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

Riding the Wave of Monodentate Ligand Revival: From the A/B Concept to Noncovalent Interactions.

Chem Rec 2016 12 18;16(6):2544-2560. Epub 2016 Jul 18.

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

The rediscovery of chiral monodentate ligands made in the period 1999-2003 had important consequences in enantioselective transition-metal catalysis, such as the introduction of the A/B concept (i.e., use of monodentate ligand mixtures) and, later, a renewed interest in supramolecular ligands capable of ligand-ligand and ligand-substrate interactions. This Personal Account summarizes the contributions made by our research group in this area in the period 2004-2015, which reflect the abovementioned developments. Within this area, we introduced some original concepts, such as 1) the use of chiral tropos ligand mixtures; 2) the development of new strategies to maximize heterocomplex formation from combinations of simple monodentate ligands; 3) the investigation of new ligand-ligand interactions to achieve selective heterocomplex formation; and 4) the development of highly efficient and synthetically accessible supramolecular ligands.
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http://dx.doi.org/10.1002/tcr.201600087DOI Listing
December 2016

Asymmetric Hydrogenation of 3-Substituted Pyridinium Salts.

Chemistry 2016 Jul 3;22(28):9528-32. Epub 2016 Jun 3.

DSM Ahead R&D B.V.-Innovative Synthesis, P. O. Box 18, 6160 MD, Geleen, The Netherlands.

The use of an equivalent amount of an organic base leads to high enantiomeric excess in the asymmetric hydrogenation of N-benzylated 3-substituted pyridinium salts into the corresponding piperidines. Indeed, in the presence of Et3 N, a Rh-JosiPhos catalyst reduced a range of pyridinium salts with ee values up to 90 %. The role of the base was elucidated with a mechanistic study involving the isolation of the various reaction intermediates and isotopic labeling experiments. Additionally, this study provided some evidence for an enantiodetermining step involving a dihydropyridine intermediate.
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http://dx.doi.org/10.1002/chem.201601501DOI Listing
July 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

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

Determination of the binding epitope of RGD-peptidomimetics to αvβ3 and α(IIb)β3 integrin-rich intact cells by NMR and computational studies.

Org Biomol Chem 2013 Jun;11(23):3886-93

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

NMR experiments (transferred NOE and Saturation Transfer Difference) were used to shed light on the binding epitope of RGD peptidomimetics 1-3 with integrins αvβ3 and α(IIb)β3, expressed on the membrane of ECV304 bladder cancer cells and human platelets, respectively. The NMR results were supported by docking calculations of 1-3 in the active sites of αvβ3 and α(IIb)β3 integrin receptors and were compared to the results of competitive αvβ3 receptor binding assays and competitive ECV304 cell adhesion experiments. While cis RGD ligand 1 interacts mainly with the α integrin subunit through its basic guanidine group, trans RGD ligands 2 and 3 are able to interact with both the α and β integrin subunits via an electrostatic clamp.
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http://dx.doi.org/10.1039/c3ob40540kDOI Listing
June 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

Synthesis and biological evaluation (in vitro and in vivo) of cyclic arginine-glycine-aspartate (RGD) peptidomimetic-paclitaxel conjugates targeting integrin αVβ3.

J Med Chem 2012 Dec 19;55(23):10460-74. Epub 2012 Nov 19.

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

A small library of integrin ligand-paclitaxel conjugates 10-13 was synthesized with the aim of using the tumor-homing cyclo[DKP-RGD] peptidomimetics for site-directed delivery of the cytotoxic drug. All the paclitaxel-RGD constructs 10-13 inhibited biotinylated vitronectin binding to the purified αVβ3 integrin receptor at low nanomolar concentration and showed in vitro cytotoxic activity against a panel of human tumor cell lines similar to that of paclitaxel. Among the cell lines, the cisplatin-resistant IGROV-1/Pt1 cells expressed high levels of integrin αVβ3, making them attractive to be tested in in vivo models. cyclo[DKP-f3-RGD]-PTX 11 displayed sufficient stability in physiological solution and in both human and murine plasma to be a good candidate for in vivo testing. In tumor-targeting experiments against the IGROV-1/Pt1 human ovarian carcinoma xenotransplanted in nude mice, compound 11 exhibited a superior activity compared with paclitaxel, despite the lower (about half) molar dosage used.
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http://dx.doi.org/10.1021/jm301058fDOI Listing
December 2012

A library approach to the development of BenzaPhos: highly efficient chiral supramolecular ligands for asymmetric hydrogenation.

Chemistry 2012 Aug 19;18(33):10368-81. Epub 2012 Jun 19.

Università degli Studi di Milano, Dipartimento di Chimica Organica e Industriale, Istituto di Scienze e Tecnologie Molecolari (ISTM) del CNR, Via G. Venezian, 21, 20133, Milano, Italy.

A library of chiral supramolecular ligands, named BenzaPhos, of straightforward preparation (two steps from commercially or readily available starting materials) and modular structure, was designed and synthesized. The ligands were screened in the search for new rhodium catalysts for the enantioselective hydrogenation of several benchmark and industrially relevant substrates. Once a series of hits were identified, structural modifications were introduced on three of the best ligands and a small second-generation library was created. Members of the latter library showed outstanding levels of activity and enantioselectivity in the hydrogenation of challenging olefins, such as enamide S4 and β-dehydroamino ester S5 (>99% ee: best value ever reported in both cases). A series of control experiments were undertaken to clarify the role of hydrogen bonding in determining the catalytic properties of the new ligands. The results of these experiments, together with those of computational studies carried out on four dihydride complexes involved in the catalytic hydrogenation of substrate S4, strongly suggest that a substrate orientation takes place in the catalytic cycle by formation of a hydrogen bond between the ligand amide oxygen atom and the substrate amide NH atom.
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http://dx.doi.org/10.1002/chem.201201032DOI Listing
August 2012

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

Chemistry 2012 May 19;18(20):6195-207. Epub 2012 Apr 19.

Università degli Studi di Milano, Dipartimento di Chimica Organica e Industriale, Via Venezian, 21, I-20133, Milan, Italy.

The synthesis of eight bifunctional diketopiperazine (DKP) scaffolds is described; these were formally derived from 2,3-diaminopropionic acid and aspartic acid (DKP-1-DKP-7) or glutamic acid (DKP-8) and feature an amine and a carboxylic acid functional group. The scaffolds differ in the configuration at the two stereocenters and the substitution at the diketopiperazinic nitrogen atoms. The bifunctional diketopiperazines were introduced into eight cyclic peptidomimetics containing the Arg-Gly-Asp (RGD) sequence. The resulting RGD peptidomimetics were screened for their ability to inhibit biotinylated vitronectin binding to the purified integrins α(v)β(3) and α(v)β(5), which are involved in tumor angiogenesis. Nanomolar IC(50) values were obtained for the RGD peptidomimetics derived from trans DKP scaffolds (DKP-2-DKP-8). Conformational studies of the cyclic RGD peptidomimetics by (1)H NMR spectroscopy experiments (VT-NMR and NOESY spectroscopy) in aqueous solution and Monte Carlo/Stochastic Dynamics (MC/SD) simulations revealed that the highest affinity ligands display well-defined preferred conformations featuring intramolecular hydrogen-bonded turn motifs and an extended arrangement of the RGD sequence [Cβ(Arg)-Cβ(Asp) average distance ≥8.8 Å]. Docking studies were performed, starting from the representative conformations obtained from the MC/SD simulations and taking as a reference model the crystal structure of the extracellular segment of integrin α(v)β(3) complexed with the cyclic pentapeptide, Cilengitide. The highest affinity ligands produced top-ranked poses conserving all the important interactions of the X-ray complex.
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http://dx.doi.org/10.1002/chem.201200457DOI Listing
May 2012

Rhodium-catalyzed asymmetric hydrogenation of olefins with PhthalaPhos, a new class of chiral supramolecular ligands.

Chemistry 2012 Jan 28;18(5):1383-400. Epub 2011 Dec 28.

Università degli Studi di Milano, Dipartimento di Chimica Organica e Industriale, Centro Interdipartimentale CISI, Istituto di Scienze e Tecnologie Molecolari del CNR, Milano, Italy.

A library of 19 binol-derived chiral monophosphites that contain a phthalic acid diamide group (PhthalaPhos) has been designed and synthesized in four steps. These new ligands were screened in the rhodium-catalyzed enantioselective hydrogenation of prochiral dehydroamino esters and enamides. Several members of the library showed excellent enantioselectivity with methyl 2-acetamido acrylate (6 ligands gave >97% ee), methyl (Z)-2-acetamido cinnamate (6 ligands gave >94% ee), and N-(1-phenylvinyl)acetamide (9 ligands gave >95% ee), whilst only a few representatives afforded high enantioselectivities for challenging and industrially relevant substrates N-(3,4-dihydronaphthalen-1-yl)-acetamide (96% ee in one case) and methyl (E)-2-(acetamidomethyl)-3-phenylacrylate (99% ee in one case). In most cases, the new ligands were more active and more stereoselective than their structurally related monodentate phosphites (which are devoid of functional groups that are capable of hydrogen-bonding interactions). Control experiments and kinetic studies were carried out that allowed us to demonstrate that hydrogen-bonding interactions involving the diamide group of the PhthalaPhos ligands strongly contribute to their outstanding catalytic properties. Computational studies carried out on a rhodium precatalyst and on a conceivable intermediate in the hydrogenation catalytic cycle shed some light on the role played by hydrogen bonding, which is likely to act in a substrate-orientation effect.
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http://dx.doi.org/10.1002/chem.201102018DOI Listing
January 2012

Supramolecular ligand-ligand and ligand-substrate interactions for highly selective transition metal catalysis.

Dalton Trans 2011 May 17;40(17):4355-73. Epub 2011 Feb 17.

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

The use of non covalent supramolecular ligand-ligand and ligand-substrate interactions in transition metal-catalysed transformations is a new, rapidly emerging area of research. Non-covalent interactions between monodentate ligands such as hydrogen bonding, coordinative bonding, ion pairing, π-π interactions and the formation of inclusion compounds, have been shown to impart higher activity and chemo-, regio-, and stereoselectivity to the corresponding transition metal complexes in a number of catalytic applications. Analogously, supramolecular ligand-substrate interactions, and particularly hydrogen bonding, have been used to direct the regio- and stereochemistry of several metal-catalysed reactions. The catalytic systems relying on supramolecular interactions are generally capable of self-assembling from simpler components in the environment where catalysis is to take place, and are therefore very well-suited for combinatorial catalyst discovery strategies and high-throughput screening.
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http://dx.doi.org/10.1039/c0dt01517bDOI Listing
May 2011

PhthalaPhos: chiral supramolecular ligands for enantioselective rhodium-catalyzed hydrogenation reactions.

Angew Chem Int Ed Engl 2010 Sep;49(37):6633-7

Università degli Studi di Milano, Dipartimento di Chimica Organica e Industriale, Centro Interdipartimentale CISI, Istituto di Scienze e Tecnologie Molecolari (ISTM) del CNR, Via G. Venezian, 21, 20133 Milano, Italy.

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http://dx.doi.org/10.1002/anie.201002958DOI Listing
September 2010

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