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isoDGR-Peptides for Integrin Targeting: Is the Time Up for RGD? Carsten Höltke*
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Institut für Klinische Radiologie, Westfälische Wilhelms-Universität Münster, Albert-Schweitzer Campus 1, D-48149 Münster, Germany ABSTRACT: Integrins are the main cell adhesion mediators on the cell surface. Especially integrin αvβ3 has gained a lot of attention as a target in cancer therapy because it mediates diverse angiogenic processes during tumor development. The peptide sequence Arg-Gly-Asp (RGD), which is present in a number of endogenous integrin ligands like fibronectin, vitronectin, and related proteins of the extracellular matrix (ECM), has been extensively used as a targeting vector for therapeutic as well as diagnostic purposes, and cilengitide, a cyclic RGD peptide, has entered clinical trials for the treatment of various cancers. However, recent advancements utilizing isoDGR, a sequence that was found in aged fibronectin, already show that RGD-based targeting is not the end of the line. Novel developments and a closer investigation of the binding mode of these peptides now show promising results for the future use of such compounds.
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he family of integrins comprises heterodimeric transmembrane proteins mediating cell−cell and cell− extracellular matrix (ECM) interactions. Integrins are composed of an α and a β subunit, and in mammals 18 different α-subunits and 8 β-subunits combine to form 24 different known integrin subtypes, including fibronectin receptor α5β1, vitronectin receptor αvβ3, and lymphocyte Peyer patch adhesion molecule α4β7. They regulate a number of cellular functions like cell adhesion, proliferation, and migration, and they are unique in their capability of bidirectional signaling.1 Intracellular signals can lead to the binding of activator proteins to the cytoplasmic tail of the β subunit and in turn to an activation of the integrin receptor, represented by a change in conformation, whereas in response to binding of ECM components integrins initiate complex internal signaling cascades. The pathophysiological activation of integrins, e.g., in cancer diseases, where they are expressed on the surfaces of both tumor cells and virtually all other cell types present in the tumor microenvironment, contributes to processes like vascular sprouting and tumor cell invasion and dissemination.2 Several integrins have been demonstrated to bind the Arg-Gly-Asp (RGD) motif, a peptide sequence that is found in a number of ECM proteins. Especially, integrins known to be highly active in tumor neoangiogenesis, such as α5β1 and αvβ3, display a high affinity to RGD.3 One problem with exogenous integrin inhibitors, including cyclo-RGDf(NMe)V (cilengitide), is their capability to induce conformational changes, therefore inducing agonist-like activities resulting in adverse effects (Figure 1).4 In this context, the interrogation of receptor allosteric events induced by ligand binding could help in the development of new integrin blockers. The peptide sequence isoDGR, which has been found in aged fibronectin, where it is formed by deamidation of Asn in an asparagine-glycine-arginine (NGR) site, has been described to also bind αvβ3 integrin with high affinity. The orientation of isoDGR at the integrin binding site, however, is inverted compared to RGD-based peptides, albeit occupying the same binding cleft at the junction between αv and β3, and © 2018 American Chemical Society
Figure 1. Dormant αvβ3 can be activated by multiple intracellular signals, promoting the recruitment of activator proteins to the β chain cytoplasmic tail. Alternatively, a transformation to the active conformation may be induced by allosteric activation via binding of extracellular agonists. Such activated integrins may then bind to their endogenous ligands, inducing a variety of further intracellular signals.
importantly, the isoDGR-based cyclopeptide CisoDGRC (cyclized through disulfide bridge formation) has been found to inhibit receptor allosteric activation, therefore behaving as a pure receptor antagonists.5 In this issue of the Journal of Medicinal Chemistry Nardelli and colleagues describe their investigation of isoDGR-based cyclopeptides concerning αvβ3 binding characteristics, generating novel antagonists with enhanced affinity toward αvβ3.6 In their studies they used the head-to-tail-cyclized hexapeptide cyclo(CGisoDGRG) and the Received: July 17, 2018 Published: August 28, 2018 7471
DOI: 10.1021/acs.jmedchem.8b01123 J. Med. Chem. 2018, 61, 7471−7473
Journal of Medicinal Chemistry
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Figure 2. Chemical structures of (from left to right) cilengitide, the original cyclic hexapeptide cycloCGisoDGRG, and conjugate 2 with improved affinity.
amide derivative of the bifunctional cross-linking agent sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfoSMCC). The maleimide moiety of this crosslinker reacts with the sulfide of the cysteine residue and the succinimidyl group is hydrolyzed and amidated, resulting in conjugate 2 (Figure 2). In earlier work, the same group found a higher affinity of albumin-conjugates of cyclo(CGisoDGRG), synthesized with the same linker strategy, toward αvβ3, compared to the pure peptide.7 Further investigations then led to the perception that additional polar contacts by the linker molecular scaffold close to the RGD binding site caused the higher affinity. In retrospect, this finding is comprehensible; however, to prove it, it required elaborate NMR, computational, and biochemical investigations. Nardelli and co-workers first investigated the effect of the truncated linker on the macrocycle conformation in solution by extensive NMR experiments. After evaluating the acquired homonuclear 2D (NOESY, ROESY, TOCSY) and heteronuclear experiments (1H−13C HSQC, HMBC), the authors could conclude that the highly flexible linker does not interact with the cyclopeptide and does not influence its overall conformation. The most probable macrocycle equilibrium conformations were then computed by bias-exchange metadynamics simulations on the original hexapeptide cycloCGisoDGRG. These molecular dynamics calculations led to four conformations with high population numbers (14−27%) containing stabilizing intramolecular H-bonds. Further NMR experiments were necessary to depict the putative peptide− receptor interaction sites. Saturation transfer difference (STD) experiments are a popular ligand-based NMR method that is widely used to probe ligand−protein interactions to offer binding information at atomic resolution. The method allows the identification of the protons that are closest to the receptor. The cyclopeptides were investigated in the presence of membrane preparations of cells containing and devoid of αvβ3. Furthermore, a cyclopeptide not binding αvβ3 (cycloCDGRC) was used as control. STD effects were observed for a number of protons of both the cyclopeptide and the linker, and the control experiments showed that the interaction is mainly due to the presence of αvβ3 in the preparation. Molecular docking calculations, using the coordinates of the Xray crystal structure of αvβ3 headpiece in complex with cilengitide (1L5G) and a total of 800 conformers as input for the constrained docking calculations, then yielded further insight into possible binding modes. Four top-scored clusters were extracted, in which the linker moiety adopted a wide range of conformations, one of them showing the succinimide oxygen of the linker forming a hydrogen bond with the hydroxyl group of Tyr122β3 (Figure 3). Intriguingly, the
Figure 3. Representative binding mode of conjugate 2 at the RGD binding site of αvβ3. αv and β3 integrin subunits are represented in pale brown and green surfaces. The cyclopeptide is represented as yellow sticks. Receptor amino acids forming direct electrostatic interactions with the ligand (solid lines) are shown in sticks and labeled with the one-letter code.
involvement of Tyr122β3 has also been described by Van Agthoven et al. to be responsible for the pure antagonism of a mutant, high-affinity domain of fibronectin (hFN10), albeit carrying an RGD binding motif.8 Van Agthoven and colleagues found a π−π interaction of Tyr122β3 with a Trp side chain of hFN10 and observed that the interaction blocked the inward movement of the α1 helix toward the metal-ion-dependent adhesion site (MIDAS), sufficient to inhibit the associated conformational changes in the βA domain that are mandatory for outside-in signaling. Nardelli and colleagues, however, attribute the absence of allosteric activation solely to the isoDGR sequence because they found that binding of cycloCGisoDGRG to αvβ3 alone inhibited binding of the monoclonal antibody AP5. This antibody attaches to the ligand induce binding site (LIBS) of αvβ3, which is only exposed after activation and which has been used by other groups as well to identify integrin activation states.8,9 In this context, the involvement of Tyr122β3, an amino acid that is conserved in α5β1 and β2 integrins, which, like αvβ3, are drug targets, should be even more emphasized and further investigated. The failure of current integrin-targeted drugs, including RGD-based cilengitide, to show real benefits in clinical trials makes it necessary to have a closer look at the target. With affinities close to that of RGD-based ligands, the novel isoDGR-based compounds are promising leads for integrintargeted drug discovery, especially if allosteric activation of the receptors can be circumvented. The in-depth NMR, computational, and biochemical investigations of Nardelli et al. concerning binding interactions at αvβ3 give a deeper insight 7472
DOI: 10.1021/acs.jmedchem.8b01123 J. Med. Chem. 2018, 61, 7471−7473
Journal of Medicinal Chemistry
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into the structural basis of integrin−ligand interaction and pinpoint possible routes toward novel therapeutic drugs as well as diagnostic agents.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The author declares no competing financial interest.
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REFERENCES
(1) Avraamides, C. J.; Garmy-Susini, B.; Varner, J. A. Integrins in Angiogenesis and Lymphangiogenesis. Nat. Rev. Cancer 2008, 8 (8), 604−617. (2) Brooks, P. C.; Clark, R. A.; Cheresh, D. A. Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science 1994, 264 (5158), 569−571. (3) Garmy-Susini, B.; Varner, J. A. Roles of integrins in tumor angiogenesis and lymphangiogenesis. Lymphatic Res. Biol. 2008, 6 (3− 4), 155−163. (4) Cox, D.; Brennan, M.; Moran, N. Integrins as therapeutic targets: lessons and opportunities. Nat. Rev. Drug Discovery 2010, 9 (10), 804−820. (5) Ghitti, M.; Spitaleri, A.; Valentinis, B.; Mari, S.; Asperti, C.; Traversari, C.; Rizzardi, G. P.; Musco, G. Molecular Dynamics Reveal That IsoDGR-Containing Cyclopeptides Are True αvβ3 Antagonists Unable to Promote Integrin Allostery and Activation. Angew. Chem., Int. Ed. 2012, 51 (31), 7702−7705. (6) Nardelli, F.; Paissoni, C.; Quilici, G.; Gori, A.; Traversari, C.; Valentinis, B.; Sacchi, A.; Corti, A.; Curnis, F.; Ghitti, M.; Musco, G. Succinimide-Based Conjugates Improve IsoDGR Cyclopeptide Affinity to αvβ3 without Promoting Integrin Allosteric Activation. J. Med. Chem. 2018, DOI: 10.1021/acs.jmedchem.8b00745 (7) Curnis, F.; Sacchi, A.; Longhi, R.; Colombo, B.; Gasparri, A.; Corti, A. IsoDGR-Tagged Albumin: A New αvβ3 Selective Carrier for Nanodrug Delivery to Tumors. Small 2013, 9 (5), 673−678. (8) Van Agthoven, J. F.; Xiong, J.-P.; Alonso, J. L.; Rui, X.; Adair, B. D.; Goodman, S. L.; Arnaout, M. A. Structural Basis for Pure Antagonism of Integrin αvβ3 by a High-Affinity Form of Fibronectin. Nat. Struct. Mol. Biol. 2014, 21 (4), 383−388. (9) Takagi, J.; Petre, B. M.; Walz, T.; Springer, T. A. Global Conformational Rearrangements in Integrin Extracellular Domains in Outside-in and inside-out Signaling. Cell 2002, 110 (5), 599−611.
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DOI: 10.1021/acs.jmedchem.8b01123 J. Med. Chem. 2018, 61, 7471−7473
