αvβ3‐ or α5β1‐Integrin‐Selective Peptidomimetics for Surface Coating

Engineering biomaterials with integrin‐binding activity is a very powerful approach to promote cell adhesion, modulate cell behavior, and induce specific biological responses at the surface level. The aim of this Review is to illustrate the evolution of surface‐coating molecules in this field: from peptides and proteins with relatively low integrin‐binding activity and receptor selectivity to highly active and selective peptidomimetic ligands. In particular, we will bring into focus the difficult challenge of achieving selectivity between the two closely related integrin subtypes αvβ3 and α5β1. The functionalization of surfaces with such peptidomimetics opens the way for a new generation of highly specific cell‐instructive surfaces to dissect the biological role of integrin subtypes and for application in tissue engineering and regenerative medicine.     

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.     

Selective Integrin α5β1 Targeting through Spatially Constrained Multivalent DNA-Based Nanoparticles

Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor density across healthy and diseased cells. Engaging receptors above a threshold concentration would be desirable in devising selective diagnostics. Integrins are prime target candidates as they are readily available on the cell surface and have been reported to be overexpressed in diseases. Insights into their spatial organization would therefore be advantageous to design selective targeting agents. Here, we investigated the effect of activation method on integrin α₅β₁ clustering by immunofluorescence and modeled the global neighbor distances with input from an immuno-staining assay and image processing of microscopy images. This data was used to engineer spatially-controlled DNA-scaffolded bivalent ligands, which we used to compare trends in spatial-selective binding observed across HUVEC, CHO and HeLa in resting versus activated conditions in confocal microscopy images. For HUVEC and CHO, the data demonstrated an improved selectivity and localisation of binding for smaller spacings ~7 nm and ~24 nm, in good agreement with the model. A deviation from the mode predictions for HeLa was observed, indicative of a clustered, instead of homogeneous, integrin organization. Our findings demonstrate how low-technology imaging methods can guide the design of spatially controlled ligands to selectively differentiate between cell type and integrin activation state.     

PET Diagnostic Molecules Utilizing Multimeric Cyclic RGD Peptide Analogs for Imaging Integrin αvβ3 Receptors

Multimeric ligands consisting of multiple pharmacophores connected to a single backbone have been widely investigated for diagnostic and therapeutic applications. In this review, we summarize recent developments regarding multimeric radioligands targeting integrin α_vβ₃ receptors on cancer cells for molecular imaging and diagnostic applications using positron emission tomography (PET). Integrin α_vβ₃ receptors are glycoproteins expressed on the cell surface, which have a significant role in tumor angiogenesis. They act as receptors for several extracellular matrix proteins exposing the tripeptide sequence arginine-glycine-aspartic (RGD). Cyclic RDG peptidic ligands c(RGD) have been developed for integrin α_vβ₃ tumor-targeting positron emission tomography (PET) diagnosis. Several c(RGD) pharmacophores, connected with the linker and conjugated to a chelator or precursor for radiolabeling with different PET radionuclides (¹⁸F, ⁶⁴Cu, and ⁶⁸Ga), have resulted in multimeric ligands superior to c(RGD) monomers. The binding avidity, pharmacodynamic, and PET imaging properties of these multimeric c(RGD) radioligands, in relation to their structural characteristics are analyzed and discussed. Furthermore, specific examples from preclinical studies and clinical investigations are included.     

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.     

An αv‐RGD Integrin Inhibitor Toolbox: Drug Discovery Insight,Challenges and Opportunities

There is a requirement for efficacious and safe medicines to treat diseases with high unmet need. The resurgence in αv‐RGD integrin inhibitor drug discovery is poised to contribute to this requirement. However, drug discovery in the αv integrin space is notoriously difficult due to the receptors being structurally very similar as well as the polar zwitterionic nature of the pharmacophore. This Review aims to guide drug discovery research in this field through an αv inhibitor toolbox, consisting of small molecules and antibodies. Small‐molecule αv tool compounds with extended profiles in αvβ1, 3, 5, 6 and 8 cell adhesion assays, with key physicochemical properties, have been collated to assist in the selection of the right tool for the right experiment. This should also facilitate an understanding of partial selectivity profiles of compounds generated in different assays across research institutions. Prospects for further αv integrin research and the critical importance of target validation are discussed, where increased knowledge of the selectivity for individual RGD αv integrins is key. Insights into the design of small‐molecule RGD chemotypes for topical or oral administration are provided and clinical findings on advanced molecules are examined.     

Intrinsically Photoswitchable α/β Peptides toward Two‐State Foldamers

A simple, unsaturated, E–Z photoisomerizable β‐amino acid, (Z)‐3‐aminoprop‐2‐enoic acid, has been introduced into peptide foldamers through a one‐pot chemical coupling, based on Pd/Cu‐catalyzed olefin oxidative amidation, between two peptide segments carrying, respectively, a ‐Gly‐NH₂ residue at the C‐terminus and an acryloyl group at the N‐terminus. Reversible conversion between the Z and E configurations of the 3‐aminoprop‐2‐enoic linkage was achieved photochemically. A crystallographic analysis on two model compounds shed light on the consequences, in terms of 3D structure and self‐association properties, brought about by the different configuration of the unsaturated linkage. As a proof of concept, E–Z photoisomerization of a 3‐aminoprop‐2‐enoic acid residue, inserted as the junction between two conformationally distinct peptide domains (one helical while the other β‐sheet promoter), allowed supramolecular self‐association to be reversibly turned on/off.     

Stable Peptides Instead of Stapled Peptides: Highly Potent αvβ6‐Selective Integrin Ligands

The αvβ6 integrin binds the RGD‐containing peptide of the foot and mouth disease virus with high selectivity. In this study, the long binding helix of this ligand was downsized to an enzymatically stable cyclic peptide endowed with sub‐nanomolar binding affinity toward the αvβ6 receptor and remarkable selectivity against other integrins. Computational studies were performed to disclose the molecular bases underlying the high binding affinity and receptor subtype selectivity of this peptide. Finally, the utility of the ligand for use in biomedical studies was also demonstrated here.     

The homo‐PADAM Protocol: Stereoselective and Operationally Simple Synthesis of α‐Oxo‐ or α‐Hydroxy‐γ‐acylaminoamides and Chromanes

A straightforward and fully stereoselective synthesis of a new class of peptidomimetics, that is α‐oxo‐γ‐acylaminoamides, was achieved starting from various benzaldehydes by a sequence of 1) an asymmetric organocatalytic Mannich reaction, 2) a Passerini multicomponent reaction, 3) an amine deprotection–acyl migration protocol, and 4) a final oxidation. The whole sequence can be performed without purification of the intermediates and represents the first example of a homo‐Passerini–amine deprotection–acyl migration (PADAM) strategy. Highly stereoselective reduction of the α‐oxo‐γ‐acylaminoamides afforded α‐hydroxy‐γ‐acylaminoamides as well. In some cases both diastereomers were obtained by simply changing the reducing agent. Finally, starting from protected salicylaldehyde, the same sequence, followed by a Mitsunobu cyclization, afforded highly substituted chromanes.     

Synthesis and Analysis of the Conformational Preferences of 5‐Aminomethyloxazolidine‐2,4‐dione Scaffolds: First Examples of β2‐ and β2, 2‐Homo‐Freidinger Lactam Analogues

Constrained peptidomimetic scaffolds are of considerable interest for the design of therapeutically useful analogues of bioactive peptides. We present the single‐step cyclization of (S)‐ or (R)‐α‐hydroxy‐β²‐ or α‐substituted‐α‐hydroxy‐β^{2, 2}‐amino acids already incorporated within oligopeptides to 5‐aminomethyl‐oxazolidine‐2,4‐dione (Amo) rings. These scaffolds can be regarded as unprecedented β²‐ or β^{2, 2}‐homo‐Freidinger lactam analogues, and can be equipped with a proteinogenic side chain at each residue. In a biomimetic environment, Amo rings act as inducers of extended, semi‐bent or folded geometries, depending on the relative stereochemistry and the presence of α‐substituents.     

A Minimal β‐Lactone Fragment for Selective β5c or β5i Proteasome Inhibitors

Broad‐spectrum proteasome inhibitors are applied as anticancer drugs, whereas selective blockage of the immunoproteasome represents a promising therapeutic rationale for autoimmune diseases. We here aimed at identifying minimal structural elements that confer β5c or β5i selectivity on proteasome inhibitors. Based on the natural product belactosin C, we synthesized two β‐lactones featuring a dimethoxybenzyl moiety and either a methylpropyl (pseudo‐isoleucin) or an isopropyl (pseudo‐valine) P1 side chain. Although the two compounds differ only by one methyl group, the isoleucine analogue is six times more potent for β5i (IC₅₀=14 nM ) than the valine counterpart. Cell culture experiments demonstrate the cell‐permeability of the compounds and X‐ray crystallography data highlight them as minimal fragments that occupy primed and non‐primed pockets of the active sites of the proteasome. Together, these results qualify β‐lactones as a promising lead‐structure motif for potent nonpeptidic proteasome inhibitors with diverse pharmaceutical applications.     

Incorporation of β‐Silicon‐β3‐Amino Acids in the Antimicrobial Peptide Alamethicin Provides a 20‐Fold Increase in Membrane Permeabilization

Incorporation of silicon‐containing amino acids in peptides is known to endow the peptide with desirable properties such as improved proteolytic stability and increased lipophilicity. In the presented study, we demonstrate that incorporation of β‐silicon‐β3‐amino acids into the antimicrobial peptide alamethicin provides the peptide with improved membrane permeabilizing properties. A robust synthetic procedure for the construction of β‐silicon‐β3‐amino acids was developed and the amino acid analogues were incorporated into alamethicin at different positions of the hydrophobic face of the amphipathic helix by using SPPS. The incorporation was shown to provide up to 20‐fold increase in calcein release as compared with wild‐type alamethicin.