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Salicylaldehyde (SA) derivatives are emerging as useful fragments to obtain reversible-covalent inhibitors interacting with the lysine residues of the target protein. Here the SA installation at the C terminus of an integrin-binding cyclopeptide, leading to enhanced ligand affinity for the receptor as well as stronger biological activity in cultured glioblastoma cells is reported., Chemistry - A European Journal, 29 (19), ISSN:0947-6539, ISSN:1521-3765

Amine-carbamate self-immolative (SI) spacers represent practical and versatile tools in targeted prodrugs, but their slow degradation mechanism limits drug activation at the site of disease. We engineered a pyrrolidine-carbamate SI spacer with a tertiary amine handle which strongly accelerates the spacer cyclization to give a bicyclic urea and the free hydroxy groups of either cytotoxic (Camptothecin) or immunostimulatory (Resiquimod) drugs. In silico conformational analysis and pK

2 pags

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

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).

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 v β 3 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.

Burkholderia cenocepacia is an opportunistic Gram‐negative bacterium that causes infections in patients suffering from chronic granulomatous diseases and cystic fibrosis. It displays significant morbidity and mortality due to extreme resistance to almost all clinically useful antibiotics. The bacterial lectin BC2L‐C expressed in B. cenocepacia is an interesting drug target involved in bacterial adhesion and subsequent deadly infection to the host. We solved the first high resolution crystal structure of the apo form of the lectin N‐terminal domain (BC2L‐C‐nt) and compared it with the ones complexed with carbohydrate ligands. Virtual screening of a small fragment library identified potential hits predicted to bind in the vicinity of the fucose binding site. A series of biophysical techniques and X‐ray crystallographic screening were employed to validate the interaction of the hits with the protein domain. The X‐ray structure of BC2L‐C‐nt complexed with one of the identified active fragments confirmed the ability of the site computationally identified to host drug‐like fragments. The fragment affinity could be determined by titration microcalorimetry. These structure‐based strategies further provide an opportunity to elaborate the fragments into high affinity anti‐adhesive glycomimetics, as therapeutic agents against B. cenocepacia., A new druggable site for anti‐adhesive therapy. We used fragment‐based virtual screening to explore the druggability of a region in the vicinity of the fucose binding site in a lectin from an opportunistic and highly drug‐resistant pathogen. The crystal structure of the target protein complexed with the lead fragment validated the existence of a secondary binding site, paving the way for the design of potent ligands to be employed in anti‐adhesive therapy.

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-&beta, ACPC), using a mixed solid phase/solution phase synthetic protocol. The three ligands were examined in vitro in competitive binding assays to the purified &alpha, v&beta, 3 and &alpha, 5&beta, 1 receptors using biotinylated vitronectin (&alpha, 3) and fibronectin (&alpha, 1) 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 &alpha, 3 over &alpha, 1. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin &alpha, 3). 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 &alpha, V&beta, 3 integrin active site, provided a rationale for the behavior of these ligands toward the receptor.

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 αvβ3 and α5β1 receptors using biotinylated vitronectin (αvβ3) 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 αvβ3 over α5β1. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin αvβ3). 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 αVβ3 integrin active site, provided a rationale for the behavior of these ligands toward the receptor.

Construction of small molecule ligand (SML) based delivery systems has been performed starting from a polyfunctionalized isoxazoline scaffold, whose αvβ3 and α5β1 integrins’ potency has been already established. The synthesis of this novel class of ligands was obtained by conjugation of linkers to the heterocyclic core via Huisgen-click reaction, with the aim to use them as “shuttles” for selective delivery of diagnostic agents to cancer cells, exploring the effects of the side chains in the interaction with the target. Compounds 17b and 24 showed excellent potency towards α5β1 integrin acting as selective antagonist and agonist respectively. Further investigations confirmed their effects on target receptor through the analysis of fibronectin-induced ERK1/2 phosphorylation. In addition, confocal microscopy analysis allowed us to follow the fate of EGFP conjugated α5β1 integrin and 17b FITC-conjugated (compound 31) inside the cells. Moreover, the stability in water solution at different values of pH and in bovine serum confirmed the possible exploitation of these peptidomimetic molecules for pharmaceutical application.

Theranostic RGD-camptothecin conjugates, possessing a disulfide linker and a fluorescent naphthalimide moiety, were synthesized and biologically evaluated. The conjugates showed nanomolar affinity for the purified ?V?3-integrin receptor. For antiproliferative assays, the U87 human glioblastoma were chosen as ?V?3-expressing cells, whereas a non ?V?3-expressing clone (U87 ?3-KO) was generated as negative control. Although the U87 ?3-KO cells treated with the conjugates showed a statistically significant reduced fluorescence intensity (in the range 7-12 %) compared to the parental U87, internalization of the conjugates was clearly observed in both cell lines. Stability studies showed premature cleavage of the disulfide linker in the cell media, with consequent release of free camptothecin. Consistent with the results of the internalization and stability studies, the conjugates did not show significant selectivity against the U87 cells compared to the U87 ?3-KO clone.

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.

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

A C-2-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.

Presented herein is our study of the conformation and reactivity of highly reactive thioglycoside donors. The structural studies have been conducted using NMR spectroscopic and computational methods. The reactivity of these donors has been investigated in bromine-promoted glycosylations of aliphatic and sugar alcohols. Swift reaction times, high yields, and respectable 1,2-cis stereoselectivity were observed in a majority of these glycosylations.

This work reports the synthesis of a series of small‐molecule–drug conjugates containing the αVβ3‐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 αvβ3 receptor and were shown to retain nanomolar IC50 values, in the same range as those of the free ligands. The cytotoxic activity of the conjugates was evaluated in cell viability assays with αvβ3 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 IC50 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.

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

This work reports the synthesis of a series of small-molecule-drug conjugates containing the αV β3 -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 αv β3 receptor and were shown to retain nanomolar IC50 values, in the same range as those of the free ligands. The cytotoxic activity of the conjugates was evaluated in cell viability assays with αv β3 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 IC50 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.

A novel series of β-lactam derivatives that was designed and synthesized to target RGD-binding and leukocyte integrins is reported. The compound library was evaluated by investigating the effects on integrin-mediated cell adhesion and cell signaling in cell lines expressing αvβ3, αvβ5, αvβ6, α5β1, αIIbβ3, α4β1, and αLβ2 integrins. SAR analysis of the new series of azetidinones enabled the recognition of structural elements associated with integrin selectivity. We obtained selective and potent agonists that could induce cell adhesion and promote cell signaling mediated by αvβ3, αvβ5, α5β1, or α4β1 integrin, and antagonists for the integrins αvβ3 and α5β1, as well as α4β1 and αLβ2, preventing the effects elicited by the respective endogenous agonists.

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

Hybrid cyclic α/β-peptides, in which one or more β-amino acids are incorporated into the backbone, are gaining increasing interest as potential therapeutics, thanks to their ability to achieve enhanced binding affinities for a biological target through pre-organization in solution. The in silico prediction of their three dimensional structure through strategies such as MD simulations would substantially advance the rational design process. However, whether the molecular mechanics force fields are accurate in sampling highly constrained cyclopeptides containing β-amino acids remains to be verified. Here, we present a systematic assessment of the ability of 8 widely used force fields to reproduce 79 NMR observables (including chemical shifts and 3J scalar couplings) on five cyclic α/β-peptides that contain the integrin recognition motif isoDGR. Most of the investigated force fields, which include force fields from AMBER, OPLS, CHARMM and GROMOS families, display very good agreement with experimental 3J(HN,Hα), suggesting that MD simulations could be an appropriate tool in the rational design of therapeutic cyclic α-peptides. However, for NMR observables directly related to β-amino acids, we observed a poor agreement with experiments and a remarkable dependence of our evaluation on the choice of Karplus parameters. The force field weaknesses herein unveiled might constitute a source of inspiration for further force field optimization.

In this paper we report the synthesis of new azabicycloalkane scaffolds, which could be exploited to obtain cRGD-based bioconjugates that may find promising application for targeted drug delivery, theranostic, and general cancer-cell labeling. By exploiting a Hosomi–Sakurai intramolecular allylation reaction we efficiently converted a silylated aldehyde precursor into 7,5-fused lactam scaffolds endowed with an exocyclic double bond. The presence of the vinyl function should make it possible to conjugate bioactive compounds to selectively carry them to tumor sites. The optimized synthetic sequence allows the gram-scale preparation of the target scaffolds in a few steps and good 39 % overall yield from readily accessible materials. The high reactivity of the exocyclic olefin moiety was ascertained by performing a Heck coupling reaction with 1-bromo-4-nitrobenzene, which gave the corresponding functionalized derivatives in good (80–91 %) yields.

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.

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

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.

The interaction of a small library of cyclic RGD (Arg-Gly-Asp) peptidomimetics with alphaVbeta6 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 alphaVbeta6 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 alphaVbeta6 integrin. Although the RGD interaction with alphaVbeta6 recapitulates the RGD binding mode observed in alphaVbeta3, differences between the integrin binding pockets can strongly affect the ligand binding ability. In general, the peptidomimetics exhibited IC50 values for integrin alphaVbeta6 (i.e., the concentration of compound required for 50% inhibition of biotinylated fibronectin binding to isolated alphaVbeta6 integrin) in the nanomolar range (77-345 nM), about 10-100 times higher than those for the related alphaVbeta3 receptor, with a single notable ligand displaying a low nanomolar IC50 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.

Understanding how binding events modulate functional motions of multidomain proteins is a major issue in chemical biology. We address several aspects of this problem by analyzing the differential dynamics of αvβ3 integrin bound to wild type (wtFN10, agonist) or high affinity (hFN10, antagonist) mutants of fibronectin. We compare the dynamics of complexes from large-scale domain motions to inter-residue coordinated fluctuations to characterize the distinctive traits of conformational evolution and shed light on the determinants of differential αvβ3 activation induced by different FN sequences. We propose an allosteric model for ligand-based integrin modulation: the conserved integrin binding pocket anchors the ligand, while different residues on the two FN10’s act as the drivers that reorganize relevant interaction networks, guiding the shift towards inactive (hFN10-bound) or active states (wtFN10-bound). We discuss the implications of results for the design of integrin inhibitors., Author Summary Interactions between proteins are at the basis of all biological processes in the cell. In this context, the study of conformational responses of protein receptors to the binding of endogenous ligands may be a source of inspiration for the design of small molecule modulators that permit to control such biological processes. To progress towards this goal, we need to unravel the molecular determinants that underlie the correlations between sequences, structures and the onset of functional motions in the receptor, eventually illuminating the links between the fine atomic-scale protein-ligand interactions and the large-scale protein motions. Herein, we have concentrated on the multidomain receptor αvβ3 integrin bound to two different sequences of the endogenous ligand fibronectin: the wild type one, wtFN10, which acts as an agonist activating the receptor, and a high affinity mutant, hFN10, which acts as a true antagonist inhibiting the receptor. Through the comparative analysis of several dynamic descriptors at different levels of resolution, from the residue to domain level, we shed light on the salient conformational dynamics differences determined by fibronectin sequence mutations: we show that it is possible to identify interaction hotspots in the integrin binding site that specifically respond to the fibronectin sequence variations, and allosterically drive conformational changes towards integrin activation (in the case of wtFN10 binding) or inhibition (hFN10 binding). Finally we propose an allosteric model of integrin regulation that can be used in the design of small molecule integrin inhibitors or modulators.

The interaction of a small library of cyclic DKP-RGD peptidomimetics with α5β1 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 α5β1 is highly expressed. The NMR data were compared with the docking calculations of the RGD ligands in the crystal structure of the α5β1 binding site, and were integrated with competitive binding assays to the purified α5β1 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 α5β1 ligand of the series, displaying a nanomolar IC50 value.

The interaction of a small library of cyclic DKP–RGD peptidomimetics with α5β1 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 α5β1 is highly expressed. The NMR data were compared with the docking calculations of the RGD ligands in the crystal structure of the α5β1 binding site, and were integrated with competitive binding assays to the purified α5β1 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 α5β1 ligand of the series, displaying a nanomolar IC 50 value.

In the case of flexible molecules, the standard approach of transforming NOE intensities into spatial restraints and of building conformational models minimizing these restraints greatly neglects the richness of molecular conformations. Making use of NOE intensities measured in triplicate and of an iterative molecular–dynamics scheme, we optimized a force field to generate a set of conformations whose ensemble is compatible with the experimental data, and is weighted according to the Boltzmann distribution. This scheme is applied to two cyclic peptidomimetic ligands of integrins. Their difference in binding affinity is recapitulated in terms of their difference in conformational fluctuations.

We have designed a new synthetic strategy for the preparation of a new class of cyclic RGD integrin ligands in which the azabicycloalkane scaffold can be envisaged as a (γ,α) dipeptide mimic. The synthesis and in vitro biological evaluation of these RGD derivatives, as well as the computational study of their conformational properties and binding modes to αVβ3 integrin are described. Compound 3 has shown to be a promising candidate as αVβ3 integrin antagonist able to interfere with both cell adhesion and movement on vitronectin with no evidence of cytotoxic effects.

The synthesis of eight bifunctional diketopiperazine (DKP) scaffolds is described; these were formally derived from 2,3-diaminopropionic acid and aspartic acid (DKP-1DKP-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 alphavbeta3 and alphavbeta5, which are involved in tumor angiogenesis. Nanomolar IC50 values were obtained for the RGD peptidomimetics derived from trans DKP scaffolds (DKP-2DKP-8). Conformational studies of the cyclic RGD peptidomimetics by 1H 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 [C beta(Arg)-C beta(Asp) average distance >8.8 angstrom]. 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 alphavbeta3 complexed with the cyclic pentapeptide, Cilengitide. The highest affinity ligands produced top-ranked poses conserving all the important interactions of the X-ray complex.

αVβ3 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 αVβ3 integrin-overexpressing tumors. DA364's binding affinity for αVβ3 integrin was first evaluated in vitro. Human αVβ3 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 αVβ3 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 αVβ3 integrin-overexpressing tumors. Copyright © 2011 John Wiley & Sons, Ltd.

Isoxazoline-containing peptidomimetics, designed to be effective α(v)β(3) and α(5)β(1) integrin ligands, were synthesized through an original procedure involving N,O-bis(trimethylsilyl)hydroxyamine conjugate addition to alkylidene acetoacetates, followed by intramolecular hemiketalization. To mimic the RGD recognition sequence, basic and acidic terminal appendages were introduced, and the final products were tested in cell adhesion inhibition assays. All the synthesized compounds proved to be excellent ligands for both integrin receptors, and a strong influence on intracellular signaling and phosphorylation pathways was demonstrated by evaluation of fibronectin-induced phosphorylation of ERK. The molecular basis of the observed inhibitory activity was suggested on the results of docking experiments.