Synthesis and Biological Characterization of Monomeric and Tetrameric RGD-Cryptophycin Conjugates

The effective delivery of cytotoxic agents to tumor cells is a key challenge in anticancer therapy. Multivalent integrinspecific ligands are considered a promising tool to increase the binding affinity, selectivity, and internalization efficiency of small-molecule drug conjugates. Herein, we report the synthesis and biological evaluation of a multimeric conjugate containing the high-affinity integrin α_vβ₃ binding ligand RAFT-c(RGDfK)₄, a lysosomally cleavable Val-Cit linker, and cryptophycin-55 glycinate, a potent inhibitor of tubulin polymerization. In vitro cytotoxicity assays verified that the multimeric RGD-cryptophycin conjugate displays improved potency compared to the monomeric analogue in integrin α_vβ₃ overexpressing tumor cell lines, while significantly reduced activity was observed in the integrin-negative cell line.     

Synthesis and Biological Evaluation of RGD Peptidomimetic–Paclitaxel Conjugates Bearing Lysosomally Cleavable Linkers

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 α_vβ₃‐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 α_Vβ₃‐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 (α_Vβ₃?) and its subclone CCRF‐CEM α_Vβ₃ (α_Vβ₃+). 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 α_Vβ₃ 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.     

Cyclic isoDGR and RGD Peptidomimetics Containing Bifunctional Diketopiperazine Scaffolds are Integrin Antagonists

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 α_vβ₃ and α_vβ₅ integrin receptors. IC₅₀ values ranged from low nanomolar (ligand 3 ) to submicromolar with α_vβ₃ integrin. The biological activities of ligands cyclo[DKP3‐RGD] 1 and cyclo[DKP3‐isoDGR] 3 , bearing the same bifunctional DKP scaffold and showing similar α_Vβ₃ 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.     

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

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_vβ₃ 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_vβ₃ 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.     

A Combined NMR and Computational Approach to Determine the RGDechi‐hCit‐αvβ3 Integrin Recognition Mode in Isolated Cell Membranes

The critical role of integrins in tumor progression and metastasis has stimulated intense efforts to identify pharmacological agents that can modulate integrin function. In recent years, α_vβ₃ and α_vβ₅ integrin antagonists were demonstrated to be effective in blocking tumor progression. RGDechi‐hCit, a chimeric peptide containing a cyclic RGD motif linked to an echistatin C‐terminal fragment, is able to recognize selectively α_vβ₃ integrin both in vitro and in vivo. High‐resolution molecular details of the selective α_vβ₃ recognition of the peptide are certainly required, nonetheless RGDechi‐hCit internalization limited the use of classical in cell NMR experiments. To overcome such limitations, we used WM266 isolated cellular membranes to accomplish a detailed NMR interaction study that, combined with a computational analysis, provides significant structural insights into α_vβ₃ molecular recognition by RGDechi‐hCit. Remarkably, on the basis of the identified molecular determinants, we design a RGDechi‐hCit mutant that is selective for α_vβ₅ integrin.     

The Importance of Detail: How Differences in Ligand Structures Determine Distinct Functional Responses in Integrin αvβ3

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_vβ₃ 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.     

Radiosynthesis and evaluation of 188Re-c(RGDyK)-His as a novel radiotherapeutic agent for integrin αvβ3 targeting tumour

The successes of noninvasive methods to visualize and quantify integrin α_vβ₃ expression in vivo have paved the way for radiolabeling anti-integrin therapy in clinic. Arginine-glycine-aspartice (RGD) peptide and related derivatives labeled with radionuclides for radio-therapy, which specifically targeting integrin α_vβ₃-positive tumors, could be used to treat these tumors. We have labeled c(RGDyK)-His, a RGD derivative, with ¹⁸⁸Re and the radio-therapy efficiency has been evaluated in model nude mice. c(RGDyK)-His was labeled with ¹⁸⁸Re by chelating with [¹⁸⁸Re(CO)₃(H₂O)₃]⁺ under a slightly basic condition. The in vitro specific binding affinity to U87 MG cell lines and the biodistribution of ¹⁸⁸Re-c(RGDyK)-His in the animal tumor models was measured. The inhibitory effects of ¹⁸⁸Re-c(RGDyK)-His were observed more than 1 month, and evaluated by microPET/CT imaging with ¹⁸F-FDG. Results of in vivo, cell uptake demonstrated ¹⁸⁸Re-c(RGDyK)-His had a high specific binding affinity to receptor integrin α_vβ₃. In biodistribution experiment, ¹⁸⁸Re-c(RGDyK)-His was accumulated in the tumor and cleared fast from the normal tissues. In radiotherapy study, tumor growth inhibition was significantly higher in the treatment groups than in the control groups. These studies showed that ¹⁸⁸Re-c(RGDyK)-His could be effectively used for integrin α_vβ₃ targeting therapy. This may offer a potential therapeutic strategy for the treatment of integrin-positive tumors in clinic.     

Combination of click chemistry and sulfonamides to develop three-armed triazole compounds

Fragment-based drug discovery is a valuable tool in hit identification, as well as the combination of different small fragments showing a minimal binding activity against biological receptors or enzymes to give merged hits. A high number of fragments on the same scaffold improve the probability to find a candidate showing single- or multi-target affinities. A rapid and versatile approach for synthesizing libraries of densely fragment-functionalized scaffolds is reported. Many fragments were assembled in few steps around a triazole ring starting from amino alcohols and other readily available building blocks. A binding assay against integrin α_vβ₃ was used as a test-bed in order to demonstrate the potential of such an approach in hit discovery strategies.     

New cyclic RGD peptides: synthesis,characterization, and theoretical activity towards αvβ3 integrin

Two new cyclic RGD peptides were prepared using a click chemistry approach. The linear RGDfV peptide was synthesized by solid-phase peptide synthesis using a 9-fluorenylmetoxicarbonyl (Fmoc) strategy and a 2-chlorotrityl chloride resin. After coupling 5-hexynoic acid the peptide was cleaved from the resin and linked to propargylamine. The bis-alkynyl RGDfV peptide was then reacted with two different bis-azides by treatment with copper iodide and triethylamine. These two cyclic RGD peptides were characterized by NMR and HRMS. In order to evaluate the interaction of these new compounds with integrin α_vβ₃ docking experiments were carried out and the results compared with those obtained with cyclo(RGDf[N–Me]V) (Cilengitide). The two new cyclic RGD peptides showed a higher affinity to the α_vβ₃ integrin when compared with Cilengitide thus representing two new potential integrin α_vβ₃ antagonists.     

Multifunctional Glycoconjugates for Recruiting Natural Antibodies against Cancer Cells

We have developed a fully synthetic and multifunctional antibody-recruiting molecule (ARM) to guide natural antibodies already present in the blood stream against cancer cells without pre-immunization. Our ARM is composed of antibody and tumor binding modules (i.e., ABM and TBM) displaying clustered rhamnose and cyclo-RGD, respectively. By using a stepwise approach, we have first demonstrated the importance of multivalency for efficient recognition with naturel IgM and α_vβ₃ integrin expressing M21 tumor cell line. Once covalently conjugated by click chemistry, we confirmed by flow cytometry and confocal microscopy that the recognition properties of both the ABM and TBM are conserved, and more importantly, that the resulting ARM promotes the formation of a ternary complex between natural IgM and cancer cells, which is required for the stimulation of the cytotoxic immune response in vivo. Due to the efficiency of the synthetic process, a larger diversity of heterovalent ligands could be easily explored by using the same multivalent approach and could open new perspectives in this field.     

Synthesis of enantiomerically pure αvβ3 integrin ligands based on a 5,6-dihydropyridin-2-one scaffold

The fundamental importance of α_vβ₃ integrin in a diverse range of biological processes, makes the search for new ligands of this receptors a significant therapeutic goal. We herein report our initial results on the synthesis of 5,6-dihydropyridin-2-one based ligands, containing a rigid heterocyclic core and two appendages mimicking arginine and aspartic acid moieties. In particular, we explored the influence of the scaffold stereochemistry on bioactivity, performing SK-MEL-24 cell-fibronectin adhesion tests on diastereoisomers, that differ in the configuration at C6.     

Shifting Towards αVβ6 Integrin Ligands Using Novel Aminoproline-Based Cyclic Peptidomimetics

In recognition of the key role played by integrins in several life-threatening dysfunctions, the search for novel small-molecule probes that selectively recognize these surface receptors is still open and widely pursued. Inspired by previously established aminoproline (Amp)-RGD based cyclopeptidomimetics with attracting α_Vβ₃ integrin affinity and selectivity, the design and straightforward synthesis of 18 new AmpRGD chemotypes bearing additional structural variants were herein implemented, to shift toward peptide-like α_Vβ₆ integrin targeted binders. The ligand competence of the synthesized products toward α_Vβ₆ was evaluated in competitive binding assays on isolated receptors, and α_Vβ₆/α_Vβ₃ selectivity was determined for a subgroup of compounds, resulting in the identification of four very promising candidates. SAR considerations and docking simulations allowed us to appreciate the key structural features responsible for the observed activity.     

Phenothiazine-Biaryl-Containing Fluorescent RGD Peptides

Cyclic RGD peptides are well-known ligands of integrins. The integrins α_Vβ₃ and α₅β₁ are involved in angiogenesis, and integrin α_Vβ₃ is abundantly present on cancer cells, thus representing a therapeutic target. Hence, synthetic and biophysical studies continuously are being directed towards the understanding of ligand-integrin interaction. In this context, the development of versatile synthetic strategies to obtain fluorescent building blocks that can add molecular diversity and modular spectral characteristics while not compromising binding affinity or selectivity is a relevant task. An on-resin intramolecular Suzuki–Miyaura cross-coupling (SMC) between l - or d -7-bromotryptophan (7BrTrp) and a phenothiazine (Ptz) boronic acid affords fluorescent cyclic RGD pseudopeptides, c(RGD(W/w)Ptz). Ring closure by SMC establishes a phenothiazine–indole moiety with axial chirality. An array of eight novel compounds has been synthesized, among them one fluorescent compound with good affinity to integrin α_Vβ₃. The fluorescence properties of the analogues can be efficiently tuned depending on the substituents in Ptz moiety even for fluorescence emission in the visible (red) spectral range.     

TERS Detection of αVβ3 Integrins in Intact Cell Membranes

Integrins are important membrane receptors that form focal adhesions with the extracellular matrix and are transmembrane signaling proteins. We demonstrate that nanoparticles functionalized with c‐RGDfC ligands bind to intact cell membranes and selectively enhance the amino acid signals of the integrin receptor when coupled with tip‐enhanced Raman scattering (TERS) detection. Controlling the plasmonic interaction between the functionalized nanoparticle and the TERS tip provides a clear Raman signal from α_Vβ₃ integrins in the cell membrane that matches the signal of the purified integrin receptor. Random aggregation of nanoparticles on the cell does not provide the same spectral information. Chemical characterization of membrane receptors in intact cellular membranes is important for understanding membrane signaling and drug targeting. These results provide a new method to investigate the chemical interactions associated with ligand binding to membrane receptors in cells.     

Synthesis of [64Cu]DOTA-ADIBON3-Ala-PEG28-A20FMDV2 via copper-free click chemistry for PET imaging of integrin αvβ6

Catalyst-free click reactions are effective chemical tools for synthesis of radiometal-based radiopharmaceuticals offering advantages towards preparation of non-toxic agents with high specific activity. In the present study the radiotracer [⁶⁴Cu]DOTA-ADIBON₃-Ala-PEG₂₈-A20FMDV2, [⁶⁴Cu]3, was synthesized for positron emission tomography imaging of integrin α_vβ₆ expressing tumors via a strain-promoted click reaction using both a “pre-click” and “post-click” approaches. The radiotracer, prepared in >99 % radiochemical yield, was evaluated in vitro (64.6 ± 2.8 % binding to α_vβ₆-positive cells vs. <5 % to α_vβ₆-negative cells) and in vivo (α_vβ₆-positive tumor uptake: 1.52 ± 0.16 % ID/g, 24 h p.i.). While the high initial renal uptake (76.2 ± 10.7 % ID/g at 1 h p.i.) was comparable to a previously reported radiotracer, [⁶⁴Cu]DOTA-PEG₂₈-A20FMDV2, [⁶⁴Cu]3 showed notably improved renal clearance (11.3 ± 2.5 % ID/g at 24 h p.i.). Thus, the introduction of a chelator-strained alkyne system resulted in improved pharmacokinetics for the present radiotracer, highlighting the attractive prospects of strain-promoted click-based preparations in the construction of radiometalated bioconjugates for targeted molecular imaging and therapy.     

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

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_vβ₃ 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.     

Efficient, enantioselective synthesis of a β,β-disubstituted carboxylic acid by Ru-XylPhanePhos-catalyzed asymmetric hydrogenation

Enantioselective preparation of a key α_vβ₃ integrin antagonist intermediate was accomplished via catalytic asymmetric hydrogenation of the corresponding β,β-disubstituted α,β-unsaturated carboxylic acid bearing a 3-quinolinyl moiety. The successful application of a Ru-(R)-XylPhanePhos catalyst to this type of substrate is unprecedented. In situ NMR experiments of pre-catalyst formation/activation by CH₃CO₂H, and reaction parameter modification, revealed that [Ru(COD)(CF₃CO₂)₂]₂/(R)-XylPhanePhos is a highly active and efficient catalytic system.     

Cyclic RGD–Polyethylene Glycol–Polyethylenimine for Intracranial Glioblastoma‐Targeted Gene Delivery

Even though the blood–brain barrier (BBB) is compromised for angiogenesis, therapeutic agents for glioblastoma multiforme (GBM) are particularly inefficient due to the existence of a blood–tumor barrier (BTB), which hampers tumor accumulation and uptake. Integrin α_vβ₃ is overexpressed on glioblastoma U87 cells and neovasculture, thus making its ligands such as the RGD motif target glioblastoma in vitro and in vivo. In the present work, we have designed a modified polyethylene glycol–polyethylenimine (PEG–PEI) gene carrier by conjugating it with a cyclic RGD sequence, c(RGDyK) (cyclic arginine‐glycine‐aspartic acid‐D ‐tyrosine‐lysine). When complexed with plasmid DNA, this gene carrier, termed RGD–PEG–PEI, formed homogenous nanoparticles with a mean diameter of 73 nm. These nanoparticles had a high binding affinity with U87 cells and facilitated targeted gene delivery against intracranial glioblastoma in vivo, thereby leading to a higher gene transfer efficiency compared to the PEG–PEI gene carrier without RGD decoration. This intracranial glioblastoma‐targeted gene carrier also enhanced the therapeutic efficacy of pORF‐hTRAIL, as evidenced by a significantly prolonged survival of intracranial glioblastoma‐bearing nude mice. Considering the contribution of glioblastoma neovasculature to the BBB under angiogenic conditions, our results demonstrated the therapeutic feasibility of treating a brain tumor through mediation of integrin α_vβ₃, as well as the potential of using RGD–PEG–PEI as a targeted gene carrier in the treatment of intracranial glioblastoma.     

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

Integrin ligands containing the tripeptide sequences Arg-Gly-Asp (RGD) and iso-Asp-Gly- Arg (isoDGR) were actively investigated as inhibitors of tumor angiogenesis and directing unit in tumor-targeting drug conjugates. Reported herein is the synthesis, of two RGD and one isoDGR cyclic peptidomimetics containing (1S,2R) and (1R,2S) cis-2-amino-1-cyclopentanecarboxylic acid (cis-β-ACPC), using a mixed solid phase/solution phase synthetic protocol. The three ligands were examined in vitro in competitive binding assays to the purified α_vβ₃ and α₅β₁ receptors using biotinylated vitronectin (α_vβ₃) and fibronectin (α₅β₁) as natural displaced ligands. The IC50 values of the ligands ranged from nanomolar (the two RGD ligands) to micromolar (the isoDGR ligand) with a pronounced selectivity for α_vβ₃ over α₅β₁. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin α_vβ₃). The two RGD ligands showed IC₅₀ values in the same micromolar range as the reference compound (cyclo[RGDfV]), while for the isoDGR derivative an IC₅₀ value could not be measured for the cell adhesion assay. A conformational analysis of the free RGD and isoDGR ligands by NMR (VT-NMR and NOESY experiments) and computational studies (MC/EM and MD), followed by docking simulations performed in the α_Vβ₃ integrin active site, provided a rationale for the behavior of these ligands toward the receptor.     

Molecular dynamics reveal that isoDGR-containing cyclopeptides are true αvβ3 antagonists unable to promote integrin allostery and activation

Ain't got that swing(-out): The cyclopeptide isoDGR is emerging as a new αvβ3 integrin binding motif. Agreement between the results of computational and biochemical studies reveals that isoDGR-containing cyclopeptides are true αvβ3 integrin antagonists that block αvβ3 in its inactive conformation (see scheme). isoDGR-based ligands may give αvβ3 antagonists without paradoxical effects.