How Do Cancer-Related Mutations Affect the Oligomerisation State of the p53 Tetramerisation Domain?

Tumour suppressor p53 plays a key role in the development of cancer and has therefore been widely studied in recent decades. While it is well known that p53 is biologically active as a tetramer, the tetramerisation mechanism is still not completely understood. p53 is mutated in nearly 50% of cancers, and mutations can alter the oligomeric state of the protein, having an impact on the biological function of the protein and on cell fate decisions. Here, we describe the effects of a number of representative cancer-related mutations on tetramerisation domain (TD) oligomerisation defining a peptide length that permits having a folded and structured domain, thus avoiding the effect of the flanking regions and the net charges at the N- and C-terminus. These peptides have been studied under different experimental conditions. We have applied a variety of techniques, including circular dichroism (CD), native mass spectrometry (MS) and high-field solution NMR. Native MS allows us to detect the native state of complexes maintaining the peptide complexes intact in the gas phase; the secondary and quaternary structures were analysed in solution by NMR, and the oligomeric forms were assigned by diffusion NMR experiments. A significant destabilising effect and a variable monomer population were observed for all the mutants studied.

Proteomic Tools for the Quantitative Analysis of Artificial Peptide Libraries: Detection and Characterization of Target-Amplified PD-1 Inhibitors.

We report a quantitative proteomics data analysis pipeline, which coupled to protein-directed dynamic combinatorial chemistry (DDC) experiments, enables the rapid discovery and direct characterization of protein-protein interaction (PPI) modulators. A low-affinity PD-1 binder was incubated with a library of >100 D-peptides under thiol-exchange favoring conditions, in the presence of the target protein PD-1, and we determined the S-linked dimeric species that resulted, amplified in the protein samples versus the controls. We chemically synthesized the target dimer candidates and validated them by thermophoresis binding and protein-protein interaction assays. The results provide a proof-of-concept for using this strategy in the high-throughput search of improved drug-like peptide binders that block therapeutically relevant protein-protein interactions.

A new synthetic protegrin as a promising peptide with antibacterial activity against MDR Gram-negative pathogens.

OBJECTIVES: Protegrins are a family of natural peptides from the innate immune system of vertebrates, with broad-spectrum antimicrobial activity. However, the toxicity and haemolysis of protegrin-1 (PG-1) at low concentrations renders it useless for therapeutic application. We rationally designed PLP-3, a novel synthetic PG-1-like peptide, comprising key activity features of protegrins in a constrained bicyclic structure. Our main objective was to investigate PLP-3's activity against MDR strains of Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae and to analyse its haemolysis and cytotoxicity. METHODS: Peptide synthesis was performed via solid phase and intramolecular ligation in solution, and the correct folding of the peptide was verified by circular dichroism. Antimicrobial activity was performed through broth microdilution. The test panel contained 45 bacterial strains belonging to A. baumannii, P. aeruginosa and K. pneumoniae (15 strains per species) comprising colistin-resistant and MDR strains. Cytotoxicity was assessed by XTT cell viability assays using HeLa and A549 cells and haemolysis of human erythrocytes. RESULTS: PLP-3 was successfully synthesized, and its antiparallel ß-sheet conformation was confirmed. Antimicrobial activity screening showed MIC90 values of 2 mg/L for A. baumannii, 16 mg/L for K. pneumoniae and 8 mg/L for P. aeruginosa. The haemolysis IC50 value was 48.53 mg/L. Cytotoxicity against human HeLa and A549 cells showed values of ca. 200 mg/L in both cell lines resulting in a 100-fold selectivity window for bacterial over human cells. CONCLUSIONS: PLP-3 has potent antimicrobial activity, especially against A. baumannii, while maintaining low haemolysis and toxicity against human cell lines at antimicrobial concentrations. These characteristics make PLP-3 a promising peptide with an interesting therapeutic window.

Controlling Antibacterial Activity Exclusively with Visible Light: Introducing a Tetra-ortho-Chloro-Azobenzene Amino Acid.

The introduction of a novel tetra-ortho-chloroazobenzene amino acid (CEBA) has enabled photoswitching of the antimicrobial activity of tyrocidine A analogues by using exclusively visible light, granting spatiotemporal control under benign conditions. Compounds bearing this photoswitchable amino acid become active upon irradiation with red light, but quickly turn-off upon exposure to other visible light wavelengths. Critically, sunlight quickly triggers isomerisation of the red light-activated compounds into their original trans form, offering an ideal platform for self-deactivation upon release into the environment. Linear analogues of tyrocidine A were found to provide the best photocontrol of their antimicrobial activity, leading to compounds active against Acinetobacter baumannii upon isomerisation. Exploration of their N- and C-termini has provided insights into key elements of their structure and has allowed obtaining new antimicrobials displaying excellent strain selectivity and photocontrol.

The Combined Use of Gold Nanoparticles and Infrared Radiation Enables Cytosolic Protein Delivery.

Cytosolic protein delivery remains elusive. The inability of most proteins to cross the cellular membrane is a huge hurdle. Here we explore the unique photothermal properties of gold nanorods (AuNRs) to trigger cytosolic delivery of proteins. Both partners, protein and AuNRs, are modified with a protease-resistant cell-penetrating peptide with nuclear targeting properties to induce internalization. Once internalized, spatiotemporal control of protein release is achieved by near-infrared laser irradiation in the safe second biological window. Importantly, catalytic amounts of AuNRs are sufficient to trigger cytosolic protein delivery. To the best of our knowledge, this is the first time that AuNRs with their maximum of absorption in the second biological window are used to deliver proteins into the intracellular space. This strategy represents a powerful tool for the cytosolic delivery of virtually any class of protein.

Small molecule inhibits α-synuclein aggregation, disrupts amyloid fibrils, and prevents degeneration of dopaminergic neurons.

Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons, a process that current therapeutic approaches cannot prevent. In PD, the typical pathological hallmark is the accumulation of intracellular protein inclusions, known as Lewy bodies and Lewy neurites, which are mainly composed of α-synuclein. Here, we exploited a high-throughput screening methodology to identify a small molecule (SynuClean-D) able to inhibit α-synuclein aggregation. SynuClean-D significantly reduces the in vitro aggregation of wild-type α-synuclein and the familiar A30P and H50Q variants in a substoichiometric molar ratio. This compound prevents fibril propagation in protein-misfolding cyclic amplification assays and decreases the number of α-synuclein inclusions in human neuroglioma cells. Computational analysis suggests that SynuClean-D can bind to cavities in mature α-synuclein fibrils and, indeed, it displays a strong fibril disaggregation activity. The treatment with SynuClean-D of two PD Caenorhabditis elegans models, expressing α-synuclein either in muscle or in dopaminergic neurons, significantly reduces the toxicity exerted by α-synuclein. SynuClean-D-treated worms show decreased α-synuclein aggregation in muscle and a concomitant motility recovery. More importantly, this compound is able to rescue dopaminergic neurons from α-synuclein-induced degeneration. Overall, SynuClean-D appears to be a promising molecule for therapeutic intervention in Parkinson's disease.

Adrenergic Modulation With Photochromic Ligands.

Adrenoceptors are ubiquitous and mediate important autonomic functions as well as modulating arousal, cognition, and pain on a central level. Understanding these physiological processes and their underlying neural circuits requires manipulating adrenergic neurotransmission with high spatio-temporal precision. Here we present a first generation of photochromic ligands (adrenoswitches) obtained via azologization of a class of cyclic amidines related to the known ligand clonidine. Their pharmacology, photochromism, bioavailability, and lack of toxicity allow for broad biological applications, as demonstrated by controlling locomotion in zebrafish and pupillary responses in mice.

Self-Assembly of DNA-Peptide Supermolecules: Coiled-Coil Peptide Structures Templated by d-DNA and l-DNA Triplexes Exhibit Chirality-Independent but Orientation-Dependent Stabilizing Cooperativity.

DNA nanostructures have been designed and used in many different applications. However, the use of nucleic acid scaffolds to promote the self-assembly of artificial protein mimics is only starting to emerge. Herein five coiled-coil peptide structures were templated by the hybridization of a d-DNA triplex or its mirror-image counterpart, an l-DNA triplex. The self-assembly of the desired trimeric structures in solution was confirmed by gel electrophoresis and small-angle X-ray scattering, and the stabilizing synergy between the two domains was found to be chirality-independent but orientation-dependent. This is the first example of using a nucleic acid scaffold of l-DNA to template the formation of artificial protein mimics. The results may advance the emerging POC-based nanotechnology field by adding two extra dimensions, that is, chirality and polarity, to provide innovative molecular tools for rational design and bottom-up construction of artificial protein mimics, programmable materials and responsive nanodevices.

Bottom-Up Design Approach for OBOC Peptide Libraries.

One-bead-one-compound peptide libraries, developed following the top-down experimental approach, have attracted great interest in the identification of potential ligands or active peptides. By exploiting a reverse experimental design approach based on the bottom-up strategy, we aimed to develop simplified, maximally diverse peptide libraries that resulted in the successful characterization of mixture components. We show that libraries of 32 and 48 components can be successfully detected in a single run using chromatography coupled to mass spectrometry (UPLC-MS). The proposed libraries were further theoretically evaluated in terms of their composition and physico-chemical properties. By combining the knowledge obtained on single libraries we can cover larger sequence spaces and provide a controlled exploration of the peptide chemical space both theoretically and experimentally. Designing libraries by using the bottom-up approach opens up the possibility of rationally fine-tuning the library complexity based on the available analytical methods.

Enthalpy- versus Entropy-Driven Molecular Recognition in the Era of Biologics.

Our laboratory has recently identified two nanobodies (small antibodies produced by camelids)-Nb1 and Nb6-that bind efficiently to epithelial growth factor (EGF) and inhibit its ability to activate its receptor (EGFR). Because of the relevance of the EGF/EGFR axis as a target in oncology, these new nanobodies have promising therapeutic potential. This article, however, is focused on another feature of these nanobodies: their distinct thermodynamic signatures. Nb1 binds to EGF through an entropy-driven mechanism whereas Nb6 binds to this factor under enthalpic control. We discuss the advantages and disadvantages of each mechanism in the contexts of traditional medical chemistry (small-molecule drugs) and also of biological drugs. In this latter case, the implications in terms of selectivity are far from being clearly established and further experimental data are required. Their monomeric natures, high stability, and ease of recombinant production make nanobodies ideally suited for thermodynamic studies. Moreover, nanobodies, thanks to their simpler structures in comparison with conventional antibodies, might provide better understanding of the structural basis of the thermodynamic parameters of antigen recognition.

Protein Chemical Synthesis Combined with Mirror-Image Phage Display Yields d-Peptide EGF Ligands that Block the EGF-EGFR Interaction.

The epidermal growth factor (EGF) pathway, being overactive in a number of cancers, is a good target for clinical therapy. Although several drugs targeting the EGF receptor (EGFR) are on the market, tumours acquire resistance very rapidly. As an alternative, small molecules and peptides targeting EGF have been developed, although with moderate success. Herein, we report the use of mirror-image phage display technology to discover protease-resistant peptides with the capacity to inhibit the EGF-EGFR interaction. After the chemical synthesis of the enantiomeric protein d-EGF, two phage-display peptide libraries were used to select binding sequences. The d versions of these peptides bound to natural EGF, as confirmed by surface acoustic waves (SAWs). High-field NMR spectroscopy showed that the best EGF binder, d-PI_4, interacts preferentially with an EGF region that partially overlaps with the receptor binding interface. Importantly, we also show that d-PI_4 efficiently disrupts the EGF-EGFR interaction. This methodology represents a straightforward approach to find new protease-resistant peptides with potential applications in cancer therapy.

Expanding the MiniAp-4 BBB-shuttle family: Evaluation of proline cis-trans ratio as tool to fine-tune transport.

Venoms have recently emerged as a promising field in drug discovery due to their good selectivity and affinity for a wide range of biological targets. Among their multiple potential applications, venoms are a rich source of blood-brain barrier (BBB) peptide shuttles. We previously described a short nontoxic derivative of apamin, MiniAp-4, which can transport a wide range of cargoes across the BBB. Here, we have studied the conformation of the proline residue of a range of MiniAp-4 analogues by high-field NMR techniques, with the aim to identify whether there is a direct relation between the cis/trans population and a range of features, such as the capacity to transport molecules across a human-based cellular model and stability in various media. The most promising candidate showed improved transport properties for a relevant small fluorophore.

Toward a Novel Drug To Target the EGF-EGFR Interaction: Design of Metabolically Stable Bicyclic Peptides.

In cancer, proliferation of malignant cells is driven by overactivation of growth-signalling mechanisms, such as the epidermal growth factor receptor (EGFR) pathway. Despite its therapeutic relevance, the EGF-EGFR interaction has remained elusive to inhibition by synthetic molecules, mostly as a result of its large size and lack of binding pockets and cavities. Designed peptides, featuring cyclic motifs and other structural constraints, have the potential to modulate such challenging protein-protein interactions (PPIs). Herein, we present the structure-based design of a series of bicyclic constrained peptides that mimic an interface domain of EGFR and inhibit the EGF-EGFR interaction by targeting the smaller partner (i.e., EGF). This design process was guided by the integrated use of in silico methods and biophysical techniques, such as NMR spectroscopy and surface acoustic wave. The best analogues were able to reduce selectively the viability of EGFR+ human cancer cells. In addition to their efficacy, these bicyclic peptides are endowed with exceptional stability and metabolic resistance-two features that make them suitable candidates for in vivo applications.

Synergistic activity of an OmpA inhibitor and colistin against colistin-resistant Acinetobacter baumannii: mechanistic analysis and in vivo efficacy.

Objectives: Preventing bacterial contact with host cells can provide an additional approach to tackling MDR Acinetobacter baumannii. Recently, we identified AOA-2 as a potential blocker of A. baumannii outer membrane protein A without presenting bactericidal activity. Here, we aimed to study whether AOA-2 can increase the activity of colistin against colistin-resistant A. baumannii in vitro and in vivo. Methods: Reference and clinical A. baumannii strains susceptible and resistant to colistin (CST-S and CST-R) were used. Microdilution and time-kill curve assays were performed to determine the synergy between AOA-2 and colistin. SDS-PAGE assays with CST-S and CST-R outer membrane proteins and MALDI-TOF-TOF (MS-MS/MS) analysis were performed to determine the AOA-2 and colistin synergy mechanism. In a murine peritoneal sepsis model, the therapeutic efficacy of AOA-2 (10 mg/kg/24 h) in combination with a sub-optimal dose of colistin (10 mg/kg/24 h) against CST-R was evaluated by determining the bacterial load in tissues and blood, and mouse survival. Results: We showed that AOA-2 increased the in vitro colistin susceptibility of reference and clinical CST-S and CST-R strains. This combination also enhanced their killing activity after 24 h of drug exposure. This synergy is mediated by the overexpression of Omp25. In vivo, the combination of AOA-2 with colistin significantly reduced the bacterial load in tissues and blood, and increased mouse survival, compared with colistin monotherapy. Conclusions: We identified a novel class of antimicrobial agents that has proven to be effective in combination with colistin in an experimental model of severe infection by CST-R A. baumannii.

Jumping Hurdles: Peptides Able To Overcome Biological Barriers.

The cell membrane, the gastrointestinal tract, and the blood-brain barrier (BBB) are good examples of biological barriers that define and protect cells and organs. They impose different levels of restriction, but they also share common features. For instance, they all display a high lipophilic character. For this reason, hydrophilic compounds, like peptides, proteins, or nucleic acids have long been considered as unable to bypass them. However, the discovery of cell-penetrating peptides (CPPs) opened a vast field of research. Nowadays, CPPs, homing peptides, and blood-brain barrier peptide shuttles (BBB-shuttles) are good examples of peptides able to target and to cross various biological barriers. CPPs are a group of peptides able to interact with the plasma membrane and enter the cell. They display some common characteristics like positively charged residues, mainly arginines, and amphipathicity. In this field, our group has been focused on the development of proline rich CPPs and in the analysis of the importance of secondary amphipathicity in the internalization process. Proline has a privileged structure being the only amino acid with a secondary amine and a cyclic side chain. These features constrain its structure and hamper the formation of H-bonds. Taking advantage of this privileged structure, three different families of proline-rich peptides have been developed, namely, a proline-rich dendrimer, the sweet arrow peptide (SAP), and a group of foldamers based on γ-peptides. The structure and the mechanism of internalization of all of them has been evaluated and analyzed. BBB-shuttles are peptides able to cross the BBB and to carry with them compounds that cannot reach the brain parenchyma unaided. These peptides take advantage of the natural transport mechanisms present at the BBB, which are divided in active and passive transport mechanisms. On the one hand, we have developed BBB-shuttles that cross the BBB by a passive transport mechanism, like diketoperazines (DKPs), (N-MePhe)n, or (PhPro)n. On the other hand, we have investigated BBB-shuttles that utilize active transport mechanisms such as SGV, THRre, or MiniAp-4. For the development of both groups, we have explored several approaches, such as the use of peptide libraries, both chemical and phage display, or hit-to-lead optimization processes. In this Account, we describe, in chronologic order, our contribution to the development of peptides able to overcome various biological barriers and our efforts to understand the mechanisms that they display. In addition, the potential use of both CPPs and BBB-shuttles to improve the transport of promising therapeutic compounds is described.

Bromotryptophans and their incorporation in cyclic and bicyclic privileged peptides.

While revisiting biologically active natural peptides, the importance of the tryptophan residue became clear. In this article, the incorporation of this amino acid, brominated at different positions of the indole ring, into cyclic peptides was successfully achieved. These products demonstrated improved properties in terms of passive diffusion, permeability across membranes, biostability in human serum and cytotoxicity. Moreover, these brominated tryptophans at positions 5, 6, or 7 proved to be compatible as building blocks to prepare bicyclic stapled peptides by performing on-resin Suzuki-Miyaura cross-coupling reactions.

Blocking EGFR Activation with Anti-EGF Nanobodies via Two Distinct Molecular Recognition Mechanisms.

One of the hallmarks of cancer is the overproduction of growth factors such as EGF. Despite the clinical success achieved by EGFR-targeted therapies, their long-term efficacy is compromised by the onset of drug-resistant mutations. To address this issue, a family of camelid-derived single-domain antibodies (Nbs) were generated, obtaining the first direct EGF inhibitors that prevent EGFR phosphorylation and pathway activation through this new mechanism of action. The two best Nbs were subjected to a detailed investigation of their interaction mechanism that revealed important differences in their binding kinetics and equilibrium thermodynamics. These distinct behaviors at the biophysical level translate into an equally efficient inhibition of the cellular EGFR phosphorylation, thus proving the efficacy of these Nbs to turn off the initiation of this key oncogenic pathway in cancer cells.

Measuring the Spin-Polarization Power of a Single Chiral Molecule.

The electronic spin filtering capability of a single chiral helical peptide is measured. A ferromagnetic electrode source is employed to inject spin-polarized electrons in an asymmetric single-molecule junction bridging an α-helical peptide sequence of known chirality. The conductance comparison between both isomers allows the direct determination of the polarization power of an individual chiral molecule.

Phage display as a tool to discover blood-brain barrier (BBB)-shuttle peptides: panning against a human BBB cellular model.

Most potential drugs for the treatment of central nervous system disorders do not cross the blood-brain barrier (BBB). Much research effort has been devoted to the discovery of new BBB-shuttle peptides-most of which have been identified by phage display. Here we report for the first time on the use of phage display against a human BBB cellular model which mimics the characteristics of the BBB. From the panning experiment of a 12-mer library, the SGVYKVAYDWQH (SGV) peptide sequence was selected and its permeability validated in the aforementioned model. Furthermore, internalization studies suggested that SGV internalizes through a clathrin-mediated mechanism and that it increases the uptake of a cargo in endothelial cells. These results highlight the usefulness of in vitro BBB models for the discovery of BBB-shuttle peptides through phage display libraries.

Bike peptides: a ride through the membrane.

Several natural peptides have a biaryl or biaryl ether motif in their biologically active structures. A model bicyclic pentapeptide containing a biaryl bridge has been synthesized by solid-phase peptide synthesis combining on-resin Suzuki and Miyaura cross-coupling reactions. Its biological properties in terms of permeability, stability and cytotoxicity have been studied, demonstrating the positive contribution of the biaryl bridge, excellent membrane penetration and serum stability Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.