Cucurbit[7]uril Macrocyclic Sensors for Optical Fingerprinting: Predicting Protein Structural Changes to Identifying Disease-Specific Amyloid Assemblies.

In a three-dimensional (3D) representation, each protein molecule displays a specific pattern of chemical and topological features, which are altered during its misfolding and aggregation pathway. Generating a recognizable fingerprint from such features could provide an enticing approach not only to identify these biomolecules but also to gain clues regarding their folding state and the occurrence of pathologically lethal misfolded aggregates. We report here a universal strategy to generate a fluorescent fingerprint from biomolecules by employing the pan-selective molecular recognition feature of a cucurbit[7]uril (CB[7]) macrocyclic receptor. We implemented a direct sensing strategy by covalently tethering CB[7] with a library of fluorescent reporters. When CB[7] recognizes the chemical and geometrical features of a biomolecule, it brings the tethered fluorophore into the vicinity, concomitantly reporting the nature of its binding microenvironment through a change in their optical signature. The photophysical properties of the fluorophores allow a multitude of probing modes, while their structural features provide additional binding diversity, generating a distinct fluorescence fingerprint from the biomolecule. We first used this strategy to rapidly discriminate a diverse range of protein analytes. The macrocyclic sensor was then applied to probe conformational changes in the protein structure and identify the formation of oligomeric and fibrillar species from misfolded proteins. Notably, the sensor system allowed us to differentiate between different self-assembled forms of the disease-specific amyloid-ß (Aß) aggregates and segregated them from other generic amyloid structures with a 100% identification accuracy. Ultimately, this sensor system predicted clinically relevant changes by fingerprinting serum samples from a cohort of pregnant women.

Controlling Ligand Surface Density on Streptavidin-Magnetic Particles by a Simple, Rapid, and Reliable Chemiluminescent Test.

The use of functionalized magnetic particles is increasing because they simplify the analytical process and yield promising results in a wide range of applications. Particularly, streptavidin-coated magnetic beads offer the possibility of rapid and very efficient grafting of biomolecules. Unfortunately, current methods to monitor and compute this grafting process are cumbersome and scarce. We describe herein a simple, rapid, and reliable chemiluminescent assay we have developed to check the grafting rate of functionalized magnetic beads. The power of the assay also relies on its ability to predict the amount of ligands required to obtain a precise grafting rate. In addition, results were correlated with a more general parameter in material functionalization characterization like surface ligand density. Finally, the assay was validated for a wide variety of biotinylated biomolecule sizes, ranging from small molecules (around 200 Da) to antibodies (around 150 kDa). This approach will allow a precise quantification and prediction of the functionalization of magnetic particles that is of enormous importance for quality control in many applications.

Colorimetric immunoassays for the screening and specificity evaluation of molecules disturbing VEGFs/VEGFRs interactions.

Angiogenesis and its involved proteins, particularly Vascular Endothelial Growth Factor family (VEGFs) and VEGF receptors (VEGFRs), have been considered as a target of therapeutic interest for numerous inflammatory and vascular diseases. Acting on this biological process through interaction with VEGFs or VEGFRs has received considerable attention. Indeed, VEGFs and VEGFRs are currently targeted by drugs such as monoclonal antibodies. The feasibility of a therapeutic strategy based on blocking the VEGF/VEGFR interaction by using ligands "other-than-biologics" is also explored. To help to the discovery of new molecules, screening assays have been developed, particularly to evaluate the VEGFA/VEGFR1 interaction. Despite the therapeutic importance of VEGFB and PlGF (Placental Growth Factor), no assays have been developed to evaluate molecules against their interactions with VEGFR1. Here, we present new versatile colorimetric immunoassays to screen and evaluate the specific interaction of discovered molecules with different growth factors (VEGFA, VEGFB, PlGF) and receptors (VEGFR1, VEGFR2). These tests, based on competitive immunoassay format, will provide essential information on specificity and selectivity of molecules for their targets and will help to work on the pharmaco-modulation of molecules for targeting one specific interaction.

Disrupting VEGF-VEGFR1 Interaction: De Novo Designed Linear Helical Peptides to Mimic the VEGF13-25 Fragment.

The interaction between vascular endothelial growth factor (VEGF) and its receptors (VEGFR) has important implications in angiogenesis and cancer, which moved us to search for peptide derivatives able to block this protein-protein interaction. In a previous work we had described a collection of linear 13-mer peptides specially designed to adopt helical conformations (Ac-SSEEX5ARNX8AAX12N-NH2), as well as the evaluation of seven library components for the inhibition of the interaction of VEGF with its Receptor 1 (VEGFR1). This study led to the discovery of some new, quite potent inhibitors of this protein-protein system. The results we found prompted us to extend the study to other peptides of the library. We describe here the evaluation of a new selection of peptides from the initial library that allow us to identify new VEGF-VEGFR1 inhibitors. Among them, the peptide sequence containing F, W, and I residues at the 5, 9, and 12 positions, show a very significant nanomolar IC50 value, competing with VEGF for its receptor 1, VEGFR1 (Flt-1), which could represent a new tool within the therapeutic arsenal for cancer detection and therapy.

Vascular Endothelial Growth Factor Peptide Ligands Explored by Competition Assay and Isothermal Titration Calorimetry.

The v114* cyclic peptide has been identified as a tight vascular endothelial growth factor (VEGF) ligand. Here we report on the use of isothermal titration calorimetry (ITC), 96-well plate competition assay, and circular dichroism (CD) to explore the binding determinants of a new set of related peptides. Anti-VEGF antibodies are currently used in the clinic for regulating angiogenesis in cancer and age-related macular degeneration treatment. In this context, our aim is to develop smaller molecular entities with high affinity for the growth factor by a structure activity relationship approach. The cyclic disulfide peptide v114* was modified in several ways, including truncation, substitution, and variation of the size and nature of the cycle. The results indicated that truncation or substitution of the four N-terminal amino acids did not cause severe loss in affinity, allowing potential peptide labeling. Increase of the cycle size or substitution of the disulfide bridge with a thioether linkage drastically decreased the affinity, due to an enthalpy penalty. The leucine C-terminal residue positively contributed to affinity. Cysteine N-terminal acetylation induced favorable ΔΔG° and ΔΔH° of binding, which correlated with free peptide CD spectra changes. We also propose a biochemical model to extrapolate Ki from IC50 values measured in the displacement assay. These calculated Ki correlate well with the Kd values determined by extensive direct and reverse ITC measurements.

Design and synthesis of C-terminal modified cyclic peptides as VEGFR1 antagonists.

Previously designed cyclic peptide antagonist c[YYDEGLEE]-NH2 disrupts the interaction between vascular endothelial growth factor (VEGF) and its receptors (VEGFRs). It represents a promising tool in the fight against cancer and age-related macular degeneration. We described in this paper the optimization of the lead peptide by C-terminal modification. A new strategy for the synthesis of cyclic peptides is developed, improving the cyclisation efficiency. At 100 µM, several new peptides with an aromatic group flexibly linked at C-terminal end showed significantly increased receptor binding affinities in competition ELISA test. The most active peptide carrying a coumarin group may be a useful tool in anti-angiogenic biological studies.

Dual-Probe SERS Nanosensor: A Promising Approach for Sensitive and Ratiometric Detection of Glucose in Clinical Settings.

Diabetes is a major global health concern, with millions of annual deaths. Monitoring glucose levels is vital for clinical management, and urine samples offer a noninvasive alternative to blood samples. Optical techniques for urine glucose sensing have gained notable traction due to their cost-effectiveness and portability. Among these methods, surface-enhanced Raman spectroscopy (SERS) has attracted considerable attention thanks to its remarkable sensitivity and multiplexing capabilities. However, challenges remain in achieving reliable quantification through SERS. In this study, an alternative approach is proposed to enhance quantification involving the use of dual probes. Each probe is encoded with unique SERS signatures strategically positioned in the biologically silent region. One probe indicates the glucose presence, while the other acts as an internal reference for calibration. This setup enables ratiometric analysis of the SERS signal, directly correlating it with the glucose concentration. The fabrication of the sensor relies on the prefunctionalization of Fe sheets using an aryl diazonium salt bearing a -C≡CH group (internal reference), followed by the immobilization of Ag nanoparticles modified with an aryl diazonium salt bearing a -B(OH)2 group (for glucose capture). A secondary probe bearing a -B(OH)2 group on one side and a -C≡N group on the other side enables the ratiometric analysis by forming a sandwich-like structure in the presence of glucose (glucose indicator). Validation studies in aqueous solutions and artificial urine demonstrated the high spectral stability and the potential of this dual-probe nanosensor for sensitive glucose monitoring in clinical settings.

Helical peptides from VEGF and Vammin hotspots for modulating the VEGF-VEGFR interaction.

The design, synthesis, conformational studies and binding affinity for VEGF receptors of a collection of linear and cyclic peptide analogues of the N-terminal α-helix fragments 13-25 of VEGF and 1-13 of Vammin are described. Linear 13(14)-mer peptides were designed with the help of an AGADIR algorithm and prepared following peptide solid-phase synthetic protocols. Cyclic peptide derivatives were prepared on-resin from linear precursors with conveniently located Glu and Lys residues, by the formation of amide linkages. Conformational analysis, CD and NMR, showed that most synthesized peptides have a clear tendency to be structured as α-helices in solution. Some of the peptides were able to bind a VEGFR-1 receptor with moderate affinity. In addition to the described key residues (Phe17, Tyr21 and Tyr25), Val14 and Val20 seem to be relevant for affinity.

Raman reporters derived from aryl diazonium salts for SERS encoded-nanoparticles.

Surface-enhanced Raman scattering (SERS) tags are usually prepared by immobilizing Raman reporters on plasmonic nanoparticles (NPs) via thiol-based self-assembled monolayers. We describe here the first example of SERS tags obtained by combining gold NPs and aryl diazonium salts. This strategy results in robust Au-C covalent bonds between the Raman reporter and the NPs, thus ensuring a high stability of the nanohybrid interface.

Multichannel DNA Sensor Array Fingerprints Cell States and Identifies Pharmacological Effectors of Catabolic Processes.

Cells at disease onset are often associated with subtle changes in the expression level of a single or few molecular components, making traditionally used biomarker-driven clinical diagnosis a challenging task. We demonstrate here the design of a DNA nanosensor array with multichannel output that identifies the normal or pathological state of a cell based on the alteration of its global proteomic signature. Fluorophore-encoded single-stranded DNA (ssDNA) strands were coupled via supramolecular interaction with a surface-functionalized gold nanoparticle quencher to generate this integrated sensor array. In this design, ssDNA sequences exhibit dual roles, where they provide differential affinities with the receptor gold nanoparticle as well as act as transducer elements. The unique interaction mode of the analyte molecules disrupts the noncovalent supramolecular complexation, generating simultaneous multichannel fluorescence output to enable signature-based analyte identification via a linear discriminant analysis-based machine learning algorithm. Different cell types, particularly normal and cancerous cells, were effectively distinguished using their fluorescent fingerprints. Additionally, this DNA sensor array displayed excellent sensitivity to identify cellular alterations associated with chemical modulation of catabolic processes. Importantly, pharmacological effectors, which could modulate autophagic flux, have been effectively distinguished by generating responses from their global protein signatures. Taken together, these studies demonstrate that our multichannel DNA nanosensor is well suited for rapid identification of subtle changes in a complex mixture and thus can be readily expanded for point-of-care clinical diagnosis, high-throughput drug screening, or predicting the therapeutic outcome from a limited sample volume.

VEGF (Vascular Endothelial Growth Factor) Functionalized Magnetic Beads in a Microfluidic Device to Improve the Angiogenic Balance in Preeclampsia.

Preeclampsia is a hypertensive pregnancy disease associated with a massive increase in sFlt-1 (soluble form of the vascular endothelial growth factor 1) in the maternal circulation, responsible for angiogenic imbalance and endothelial dysfunction. Pilot studies suggest that extracorporeal apheresis may reduce circulating sFlt-1 and prolong pregnancy. Nonspecific apheresis systems have potential adverse effects because of the capture of many other molecules. Our concept is based on a specific and competitive apheresis approach using VEGF (vascular endothelial growth factor) functionalized magnetic beads to capture sFlt-1 while releasing endogenous PlGF (placental growth factor) to restore a physiological angiogenic balance. Magnetic beads were functionalized with VEGF to capture sFlt-1. Experiments were performed using PBS, conditioned media from human trophoblastic cells, and human plasma. The proof of concept was validated in dynamic conditions in a microfluidic device as an approach mimicking real apheresis. Magnetic beads were functionalized with VEGF and characterized to evaluate their surface ligand density and recognition capabilities. VEGF-coated magnetic beads proved to be an efficient support in capturing sFlt-1 and releasing PlGF. In static conditions, sFlt-1 concentration decreased by 33±13%, whereas PlGF concentration increased by 27±10%. In dynamic conditions, the performances were improved, with 40% reduction of sFlt-1 and up to 2-fold increase of free PlGF. The sFlt-1/PlGF ratio was reduced by 63% in the plasma of preeclamptic patients. Apheresis was also associated with VEGF release. A ligand-based approach using VEGF-coated beads is an effective approach to the capture of sFlt-1 and the release of endogenous PlGF. It offers new perspectives for the treatment of preeclampsia.

Identification of Peptidic Antagonists of Vascular Endothelial Growth Factor Receptor 1 by Scanning the Binding Epitopes of Its Ligands.

Cancer angiogenesis is mainly initiated by vascular endothelial growth factors (VEGFs). On the basis of the reported crystal structures of three natural ligands (VEGF-A, -B, and PlGF) with the major receptors VEGFR-1 and VEGFR-2, we scanned receptor-binding epitopes of these ligands by designing linear and cyclic peptides with the aim to disrupt the VEGF-A/VEGFR-1 interaction, which is implicated in cancer development. The ability of peptides to inhibit this interaction was evaluated by an ELISA-based assay. Several peptides, especially those mimicking loop 1 (L1) of these ligands that binds primarily to domain D3 of VEGFRs, have demonstrated higher inhibition for VEGF-A/VEGFR-1 binding. They have also shown inhibitory effects on VEGF-induced tube formation in HUVECs (human umbilical vein endothelial cells). These results validate the domain D3 of VEGFRs as an efficient target for the design of VEGFR antagonists.

Mitochondria-targeted Triphenylamine Derivatives Activatable by Two-Photon Excitation for Triggering and Imaging Cell Apoptosis.

Photodynamic therapy (PDT) leads to cell death by using a combination of a photosensitizer and an external light source for the production of lethal doses of reactive oxygen species (ROS). Since a major limitation of PDT is the poor penetration of UV-visible light in tissues, there is a strong need for organic compounds whose activation is compatible with near-infrared excitation. Triphenylamines (TPAs) are fluorescent compounds, recently shown to efficiently trigger cell death upon visible light irradiation (458 nm), however outside the so-called optical/therapeutic window. Here, we report that TPAs target cytosolic organelles of living cells, mainly mitochondria, triggering a fast apoptosis upon two-photon excitation, thanks to their large two-photon absorption cross-sections in the 760-860 nm range. Direct ROS imaging in the cell context upon multiphoton excitation of TPA and three-color flow cytometric analysis showing phosphatidylserine externalization indicate that TPA photoactivation is primarily related to the mitochondrial apoptotic pathway via ROS production, although significant differences in the time courses of cell death-related events were observed, depending on the compound. TPAs represent a new class of water-soluble organic photosensitizers compatible with direct two-photon excitation, enabling simultaneous multiphoton fluorescence imaging of cell death since a concomitant subcellular TPA re-distribution occurs in apoptotic cells.

Biophysical Studies of the Induced Dimerization of Human VEGF Receptor 1 Binding Domain by Divalent Metals Competing with VEGF-A.

Angiogenesis is tightly regulated through the binding of vascular endothelial growth factors (VEGFs) to their receptors (VEGFRs). In this context, we showed that human VEGFR1 domain 2 crystallizes in the presence of Zn2+, Co2+ or Cu2+ as a dimer that forms via metal-ion interactions and interlocked hydrophobic surfaces. SAXS, NMR and size exclusion chromatography analyses confirm the formation of this dimer in solution in the presence of Co2+, Cd2+ or Cu2+. Since the metal-induced dimerization masks the VEGFs binding surface, we investigated the ability of metal ions to displace the VEGF-A binding to hVEGFR1: using a competition assay, we evidenced that the metals displaced the VEGF-A binding to hVEGFR1 extracellular domain binding at micromolar level.

De novo designed library of linear helical peptides: an exploratory tool in the discovery of protein-protein interaction modulators.

Protein-protein interactions (PPIs) have emerged as important targets for pharmaceutical intervention because of their essential role in numerous physiological and pathological processes, but screening efforts using small-molecules have led to very low hit rates. Linear peptides could represent a quick and effective approach to discover initial PPI hits, particularly if they have inherent ability to adopt specific peptide secondary structures. Here, we address this hypothesis through a linear helical peptide library, composed of four sublibraries, which was designed by theoretical predictions of helicity (Agadir software). The 13-mer peptides of this collection fixes either a combination of three aromatic or two aromatic and one aliphatic residues on one face of the helix (Ac-SSEEX(5)ARNX(9)AAX(12)N-NH2), since these are structural features quite common at PPIs interfaces. The 81 designed peptides were conveniently synthesized by parallel solid-phase methodologies, and the tendency of some representative library components to adopt the intended secondary structure was corroborated through CD and NMR experiments. As proof of concept in the search for PPI modulators, the usefulness of this library was verified on the widely studied p53-MDM2 interaction and on the communication between VEGF and its receptor Flt-1, two PPIs for which a hydrophobic α-helix is essential for the interaction. We have demonstrated here that, in both cases, selected peptides from the library, containing the right hydrophobic sequence of the hot-spot in one of the protein partners, are able to interact with the complementary protein. Moreover, we have discover some new, quite potent inhibitors of the VEGF-Flt-1 interaction, just by replacing one of the aromatic residues of the initial F(5)Y(9)Y(12) peptide by W, in agreement with previous results on related antiangiogenic peptides. Finally, the HTS evaluation of the full collection on thermoTRPs has led to a few antagonists of TRPV1 and TRPA1 channels, which open new avenues on the way to innovative modulators of these channels.

A blue-absorbing photolabile protecting group for in vivo chromatically orthogonal photoactivation.

The small and synthetically easily accessible 7-diethylamino-4-thiocoumarinylmethyl photolabile protecting group has been validated for uncaging with blue light. It exhibits a significant action cross-section for uncaging in the 470-500 nm wavelength range and a low light absorption between 350 and 400 nm. These attractive features have been implemented in living zebrafish embryos to perform chromatic orthogonal photoactivation of two biologically active species controlling biological development with UV and blue-cyan light sources, respectively.