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Altres ajuts: CERCA Programme/Generalitat de Catalunya, Fundaluce and "la Caixa" foundations (ID 100010434, agreement LCF/PR/HR19/52160010), Co-financed by the European Union Regional Development Fund within the framework of the ERDF Operational Program of Catalonia 2014-2020 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.

Brain metastasis is emerging as a unique entity in oncology based on its particular biology and, consequently, the pharmacological approaches that should be considered. We discuss the current state of modelling this specific progression of cancer and how these experimental models have been used to test multiple pharmacologic strategies over the years. In spite of pre-clinical evidences demonstrating brain metastasis vulnerabilities, many clinical trials have excluded patients with brain metastasis. Fortunately, this trend is getting to an end given the increasing importance of secondary brain tumors in the clinic and a better knowledge of the underlying biology. We discuss emerging trends and unsolved issues that will shape how we will study experimental brain metastasis in the years to come.

Diffuse intrinsic pontine glioma (DIPG) is a chemo-resistant, incurable pediatric tumor of the central nervous system (CNS). The blood–brain barrier (BBB) remains intact in the course of the diseas...

Peptides are a rapidly growing class of therapeutics with various advantages over traditional small molecules, especially for targeting difficult protein¿protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing bioactive cyclic topologies that go beyond natural L-amino acids. Here, we report a generalizable framework that exploits the computational power of Rosetta, in terms of large-scale backbone sampling, side-chain composition and energy scoring, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we developed two new inhibitors (PD-i3 and PD-i6) of programmed cell death 1 (PD-1), a key immune checkpoint in oncology. A comprehensive biophysical evaluation was performed to assess their binding to PD-1 as well as their blocking effect on the endogenous PD-1/PD-L1 interaction. Finally, NMR elucidation of their in-solution structures confirmed our de novo design approach., This study was funded by MINECO-FEDER (BIO 2016-75327-R) and the Generalitat de Catalunya (XRB and 2017SGR-998). We thank the NMR Facility, Dr M. Taulés (CCiT-UB) and the MS Core Facility (IRB Barcelona) for their technical support. We also acknowledge the computer resources at Mare Nostrum and the technical support provided by the Barcelona Supercomputing Center (BCV-2018-1-0005). We are grateful to Prof Tad Holak for providing the PD-1 plasmid and to Dr E. Marcos for useful discussions. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain).

The development and use of nanosystems is an emerging strategy for the diagnosis and treatment of a broad number of diseases, such as Alzheimer's disease (AD). Here, we developed a neurotheranostic nanosystem based on gold nanorods (GNRs) that works as a therapeutic peptide delivery system and can be detected in vivo for microcomputed tomography (micro-CT), being a diagnostic tool. GNRs functionalized with the peptides Ang2 (a shuttle to the Central Nervous System) and D1 (that binds to the Aβ peptide, also inhibiting its aggregation) allowed detecting differences in vivo between wild type and AD mice (APPswe/PSEN1dE9) 15 minutes after a single dose by micro-CT. Moreover, after a recurrent treatment for one month with GNRs-D1/Ang2, we observed a diminution of amyloid load and inflammatory markers in the brain. Thus, this new designed nanosystem exhibits promising properties for neurotheranostics of AD.

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 β-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.

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.

Gold nanoparticles (AuNPs) have been shown to be outstanding tools for drug delivery and biomedical applications, mainly owing to their colloidal stability, surface chemistry, and photothermal properties. The biocompatibility and stability of nanoparticles can be improved by capping the nanoparticles with endogenous proteins, such as albumin. Notably, protein coating of nanoparticles can interfere with and decrease their cell penetration. Therefore, in the present study, we functionalized albumin with the r8 peptide (All-D, octaarginine) and used it for coating NIR-plasmonic anisotropic gold nanoparticles. Gold nanoprisms (AuNPrs) and gold nanorods (AuNRs) were coated with bovine serum albumin (BSA) previously functionalized using a cell penetrating peptide (CPP) with the r8 sequence (BSA-r8). The effect of the coated and r8-functionalized AuNPs on HeLa cell viability was assessed by the MTS assay, showing a low effect on cell viability after BSA coating. Moreover, the internalization of the nanostructures into HeLa cells was assessed by confocal microscopy and transmission electron microscopy (TEM). As a result, both nanoconstructs showed an improved internalization level after being capped with BSA-r8, in contrast to the BSA-functionalized control, suggesting the predominant role of CPP functionalization in cell internalization. Thus, our results validate both novel nanoconstructs as potential candidates to be coated by endogenous proteins and functionalized with a CPP to optimize cell internalization. In a further approach, coating AuNPs with CPP-functionalized BSA can broaden the possibilities for biomedical applications by combining their optical properties, biocompatibility, and cell-penetration abilities.

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.

The blood–brain barrier (BBB) limits the delivery of therapeutics to the brain but also represents the main gate for nutrient entrance. Targeting the natural transport mechanisms of the BBB offers an attractive route for brain drug delivery. Peptide shuttles are able to use these mechanisms to increase the transport of compounds that cannot cross the BBB unaided. As peptides are a group of biomolecules with unique physicochemical and structural properties, the field of peptide shuttles has substantially evolved in the last few years. In this review, we analyze the main classifications of BBB–peptide shuttles and the leading sources used to discover them.

Peptides are a rapidly growing class of therapeutics with various advantages over traditional small molecules, especially for targeting difficult protein–protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing bioactive cyclic topologies that go beyond natural l-amino acids. Here, we report a generalizable framework that exploits the computational power of Rosetta, in terms of large-scale backbone sampling, side-chain composition and energy scoring, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we developed two new inhibitors (PD-i3 and PD-i6) of programmed cell death 1 (PD-1), a key immune checkpoint in oncology. A comprehensive biophysical evaluation was performed to assess their binding to PD-1 as well as their blocking effect on the endogenous PD-1/PD-L1 interaction. Finally, NMR elucidation of their in-solution structures confirmed our de novo design approach., In silico design of heterochiral cyclic peptides that bind to a specific surface patch on the target protein (PD-1, in this case) and disrupt protein–protein interactions.

Clathrin-mediated endocytosis (CME) is an essential cellular process, which is evolutionarily conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of pharmacological agents that could complement genetics in selectively and reversibly interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor that targets the AP2/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP and demonstrate that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts, thus providing a unique tool for acute and reversible CME modulation in yeast.

Previously, we identified that a cyclic hexapeptide AOA-2 inhibited the interaction of Gram-negative bacilli (GNB) like Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli to host cells thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. In this work, we aimed to evaluate in vitro a library of AOA-2 derivatives in order to improve the effect of AOA-2 against GNB infections. Ten AOA-2 derivatives were synthetized for the in vitro assays. Their toxicities to human lung epithelial cells (A549 cells) for 24 h were evaluated by determining the A549 cells viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of these peptide derivatives and AOA-2 at 250, 125, 62.5, and 31.25 μg/mL on the attachment of A. baumannii ATCC 17978, P. aeruginosa PAO1 and E. coli ATCC 25922 strains to A549 cells was characterized by adherence and viability assays. None of the 10 derivatives showed toxicity to A549 cells. RW01 and RW06 have reduced more the adherence of ATCC 17978, PAO1 and ATCC 2599 strains to A549 cells when compared with the original compound AOA-2. Moreover, both peptides have increased slightly the viability of infected A549 cells by PAO1 and ATCC 25922 than those observed with AOA-2. Finally, RW01 and RW06 have potentiated the activity of colistin against ATCC 17978 strain in the same level with AOA-2. The optimization program of AOA-2 has generated two derivatives (RW01 and RW06) with best effect against interaction of GNB with host cells, specifically against P. aeruginosa and E. coli., This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (Grants PI15/01358 and PI16/01378) and by Plan Nacional de I + D + i 2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Inno-vación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009)—co-financed by the European Development Regional Fund “A way to achieve Europe,” Operative program Intelligent Growth 2014–2020. YS was supported by the Subprograma Miguel Servet Tipo I from the Ministerio de Economía y Competitividad of Spain (CP15/00132).

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., A novel photoswitchable amino acid has provided access to tyrocidine A analogues fully operated under visible light. This has led to linear analogues that have shown good photoswitchability and strain selectivity against A. baumannii and S. pyogenes clinical strains by using different analogues. Activated cis compounds turn back to their deactivated trans form upon short exposure times to sunlight, paving the way to self‐deactivating antibacterials with minor evolutionary pressure on the environment.

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 internalised, 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.

NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above.------------------------------------------------------------------------Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-i3 and PD-i6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our de novo design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.

Despite the widespread use of antibodies in clinical applications, the precise molecular mechanisms underlying antibody&ndash, antigen (Ab&ndash, Ag) interactions are often poorly understood. In this study, we exploit the technical features of a typical surface plasmon resonance (SPR) biosensor to dissect the kinetic and thermodynamic components that govern the binding of single-domain Ab or nanobodies to their target antigen, epidermal growth factor (EGF), a key oncogenic protein that is involved in tumour progression. By carefully tuning the experimental conditions and transforming the kinetic data into equilibrium constants, we reveal the complete picture of binding thermodynamics, including the energetics of the complex-formation transition state. This approach, performed using an experimentally simple and high-throughput setup, is expected to facilitate mechanistic studies of Ab-based therapies and, importantly, promote the rational development of new biological drugs with suitable properties.

Platelets have a well-established role in atherosclerosis and related diseases. Lebetins from the venom of Vipera lebetina, lacking the RGD sequence, emerged as a new family of platelet aggregation inhibitors. However, the interaction sites and precise mechanism between lebetin and its substrate remain unclear. Here, we successfully synthesized two peptide analogs, which differ only by one glycine residue at the N-terminus: lebetin 2α (sL2α residues) and lebetin 2β (sL2ββ residues) were produced in sufficient quantities for structural and functional studies. NMR structure determination showed that the sL2α peptide adopts a compact ring conformation stabilized by a disulfide bond, from which emerge one loop and two extended regions, the C- and N-termini. Interestingly, two RGD-like motifs were identified in the structure of the peptides, suggesting an anti-platelet aggregation effect of the two isoforms. Indeed, activity was demonstrated on human and rabbit platelet-rich plasma where sL2α and sL2β showed more potent inhibitory effect on platelet aggregation compared to the previously described native lebetin 1. Synthetic lebetin 2 peptides constitute promising candidates for drug design toward chimeric compounds with high anti-platelet and natriuretic effects. These findings contribute to a novel field of research triggering platelet activation and natriuretic action.

The manipulation of an individual’s genetic information to treat a disease has revolutionized the biomedicine field. Despite the promise of gene therapy, this treatment can have long-term sideeffects. Efforts in the field and recent discoveries have already led to several improvements, including efficient gene delivery and transfer, as well as inpatient safety. Several studies to treat a wide range of pathologies-such as cancer or monogenic diseases- are currently being conducted. Here we provide a broad overview of methodologies available for gene therapy, placing a strong emphasis on treatments for central nervous system diseases. Finally, we give a perspective on current delivery strategies to treat such diseases, with a special focus on systems that use peptides as delivery vectors.

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.

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.

The brain is a complex, regulated organ with a highly controlled access mechanism: The Blood-Brain Barrier (BBB). The selectivity of this barrier is a double-edged sword, being both its greatest strength and weakness. This weakness is evident when trying to target therapeutics against diseases within the brain. Diseases such as metastatic brain cancer have extremely poor prognosis due to the poor permeability of many therapeutics across the BBB. Peptides can be designed to target BBB receptors and gain access to the brain by transcytosis. These peptides (known as BBB-shuttles) can carry compounds, usually excluded from the brain, across the BBB. BBB-shuttles are limited by poor loading of therapeutics and degradation of the peptide and cargo. Likewise, nano- submicro- and microparticles can be fine-tuned to limit their degradation and with high loading of therapeutics. However, most nano- and microparticles’ core materials completely lack efficient targeting, with a few selected materials able to cross the BBB passively. Combining the selectivity of peptides with the high loading potential of nano-, microparticles offers an exciting strategy to develop novel, targeted therapeutics towards many brain disorders and diseases. Nevertheless, at present the field is diverse, in both scope and nomenclature, often with competing or contradictory names. In this review, we will try to address some of these issues and evaluate the current state of peptide mediated nano,-microparticle transport to the brain, analyzing delivery vehicle type and peptide design, the two key components that must act synergistically for optimal therapeutic impact.

Peptides are experiencing a new era in medical research, finding applications ranging from therapeutics to vaccines. In spite of the promising properties of peptide pharmaceuticals, their development continues to be hindered by three weaknesses intrinsic to their structure, namely protease sensitivity, clearance through the kidneys, and immune system activation. Here we report on two retro-D-peptides (H2N-hrpyiah-CONH2 and H2N-pwvpswmpprht-CONH2), which are protease-resistant and retain the original BBB shuttle activity of the parent peptide but are much less immunogenic than the parent peptide. Hence, we envisage that retro-D-peptides, which display a similar topological arrangement as their parent peptides, will expand drug design and help to overcome factors that lead to the failure of peptide pharmaceuticals in pre- and clinical trials. Furthermore, we reveal requirements to avoid or elicit specific humoral responses to therapeutic peptides, which might have a strong impact in both vaccine design and peptide therapeutic agents.

The administration and controlled release of drugs over time remains one of the greatest challenges of science today. In the nanomaterials field, anisotropic gold nanoparticles (AuNPs) with plasmon bands centered at the near-infrared region (NIR), such as gold nanorods (AuNRs) and gold nanoprisms (AuNPrs), under laser irradiation, locally increase the temperature, allowing the release of drugs. In this sense, temporally controlled drug delivery could be promoted by external stimuli using thermo-reversible chemical reactions, such as Diels-Alder cycloadditions from a diene and a dienophile fragment (compound a). In this study, an antitumor drug (methotrexate, MTX) was linked to plasmonic AuNPs by a Diels-Alder adduct (compound c), which after NIR suffers a retro-Diels-Alder reaction, producing release of the drug (compound b). We obtained two nanosystems based on AuNRs and AuNPrs. Both nanoconstructs were coated with BSA-r8 (Bovine Serum Albumin functionalized with Arg8, all-D octa arginine) in order to increase the colloidal stability and promote internalization of the nanosystems on HeLa and SK-BR-3 cells. In addition, the presence of BSA allows protecting the cargo from being released on the extracellular environment and promotes the photothermal release of the drug in the presence of glutathione (GSH). The nanosystems' drug release profile was evaluated after NIR irradiation in the presence and absence of glutathione (GSH), showing a considerable increase of drug release when NIR light and glutathione were combined. This work broadens the range of possibilities of using two complementary strategies for the controlled release of an antitumor drug from AuNRs and AuNPrs: the photothermal cleavage of a thermolabile adduct controlled by an external stimulus (laser irradiation), complemented with the use of the intracellular metabolite GSH.

The present study aims to develop chlorotoxin (CTX), from Giant Yellow Israeli scorpion venom, as a new BBB-shuttle. Minimised versions of CTX were prepared to reduce its complexity while enhancing its BBB-shuttle capacity and preserving its protease-resistance. MiniCTX3, a monocyclic lactam-bridge peptidomimetic, was capable of transporting nanoparticles across endothelial cell monolayers. Our results reveal animal venoms as an outstanding source of new families of BBB-shuttles.

THRre is a protease resistant BBB-shuttle. A branched version of THRre displaying two copies of the peptide increases the transport of a model protein in BBB cell-based models., The blood–brain barrier (BBB) hampers the delivery of therapeutic proteins into the brain. BBB-shuttle peptides have been conjugated to therapeutic payloads to increase the permeability of these molecules. However, most BBB-shuttles have several limitations, such as a lack of resistance to proteases and low effectiveness in transporting large biomolecules. We have previously reported on the THRre peptide as a protease-resistant BBB-shuttle that is able to increase the transport of fluorophores and quantum dots in vivo. In this work, we have evaluated the capacity of linear and branched THRre to increase the permeability of proteins in cellular models of the BBB. With this purpose, we have covalently attached peptides with one or two copies of the BBB-shuttle to proteins in order to develop chemically well-defined peptide–protein conjugates. While THRre does not enhance the uptake and transport of a model protein in BBB cellular models, branched THRre peptides displaying two copies of the BBB-shuttle result in a 2.6-fold increase.

The properties of nanometric materials make nanotechnology a promising platform for tackling problems of contemporary medicine. In this work, gold nanorods were synthetized and stabilized with polyethylene glycols and modified with two kinds of peptides. The D1 peptide that recognizes toxic aggregates of Aβ, a peptide involved in Alzheimer's disease (AD); and the Angiopep 2 that can be used to deliver nanorods to the mammalian central nervous system. The nanoconjugates were characterized using absorption spectrophotometry, dynamic light scattering, and transmission electron microscopy, among other techniques. We determined that the nanoconjugate does not affect neuronal viability; it penetrates the cells, and decreases aggregation of Aβ peptide in vitro. We also showed that when we apply our nanosystem to a Caenorhabditis elegans AD model, the toxicity of aggregated Aβ peptide is decreased. This work may contribute to the development of therapies for AD based on metallic nanoparticles.

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.

Cognitive deficits are considered a key feature of schizophrenia, and they usually precede the onset of the illness and continue after psychotic symptoms appear. Current antipsychotic drugs have little or no effect on the cognitive deficits of this disorder. Prolyl oligopeptidase (POP) is an 81-kDa monomeric serine protease that is expressed in brain and other tissues. POP inhibitors have shown neuroprotective, anti-amnesic and cognition-enhancing properties. Here we studied the potential of IPR19, a new POP inhibitor, for the treatment of the cognitive symptoms related to schizophrenia. The efficacy of the inhibitor was evaluated in mouse models based on subchronic phencyclidine and acute dizocilpine administration, and in adult offspring from mothers with immune reaction induced by polyinosinic:polycytidylic acid administration during pregnancy. Acute IPR19 administration (5 mg/kg, i.p.) reversed the cognitive performance deficits of the three mouse models in the novel object recognition test, T-maze, and eight-arm radial maze. The compound also ameliorates deficits of the prepulse inhibition response. The in vitro inhibitory efficacy and selectivity, brain penetration and exposure time after injection of IPR19 were also addressed. Our results indicate that the inhibition of POP using IPR19 may offer a promising strategy to develop drugs to ameliorate the cognitive deficits of schizophrenia.

NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above.------------------------------------------------------------------------Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-i3 and PD-i6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our de novo design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.

The blood–brain barrier (BBB) is a challenge for the treatment of diseases of the central nervous system (CNS) from the systemic circulation. The design of novel strategies to increase drug bioavailability in the CNS is called for. In this work, we synthesized amphiphilic polymeric nanoparticles by the self-assembly of graft copolymers of chitosan (CS, hydrophilic copolymer backbone) hydrophobized in the side-chain with poly­(methyl methacrylate) (PMMA)/poly­(acrylic acid) (PAAc) blocks and surface-decorated with a biologically stable retro-enantio peptide shuttle that improves brain transport. Nanoparticles showed one size population in the 190–210 nm range (intensity distribution) and a relatively small polydispersity index, as measured by dynamic light scattering. The surface charge estimated by the zeta-potential decreased from +35 mV in the unmodified nanoparticles to +14 mV in the modified ones, confirming the exposure of the peptide shuttle at the nanoparticle surface. The cell compatibility and uptake were assayed in hCMEC/D3 cells, a model of BBB endothelium, by a metabolic assay, confocal laser scanning fluorescence microscopy, and imaging flow cytometry in the absence and the presence of endocytosis inhibitors. Results indicated that the peptide shuttle modification improves their cell compatibility and that they are internalized by a clathrin-mediated pathway. In vitro permeability studies conducted in hCMEC/D3 cell monolayers showed that peptide shuttle-modified nanoparticles increase the apparent permeability with respect to the unmodified ones by 3.4 times. Finally, the brain accumulation was investigated upon i.v. administration to Hsd:ICR mice by using fluorescently labeled nanoparticles in an in vivo imaging system and light sheet fluorescence microscopy. Unmodified nanoparticles could be hardly detected in the brain blood vessels and parenchyma. Conversely, nanoparticles modified with the peptide shuttle could be detected after 10 min, with a maximum accumulation at 30 min and a slow concentration decline later on. Calculation of the area under the curve confirmed a 4-fold statistically significant increase in the accumulation of the modified nanoparticles with respect to the unmodified counterparts. These findings demonstrate the promise of this strategy to improve the delivery of nanoencapsulated cargos to the CNS.

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.

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.

Altres ajuts: CERCA Programme/Generalitat de Catalunya Aquest article té una correcció a 10.1039/d0sc90189j The spatiotemporal control of cellular dynamic processes has great fundamental interest but lacks versatile molecular tools. Dynamin is a key protein in endocytosis and an appealing target to manipulate cell trafficking using patterns of light. We have developed the first photoswitchable small-molecule inhibitors of endocytosis (dynazos), by a stepwise design of the photochromic and pharmacological properties of dynasore, a dynamin inhibitor. We have characterized their photochromism with UV-visible and transient absorption spectroscopy and their biological activity using fluorescence microscopies and flow cytometry. Dynazos are water-soluble, cell permeable, and photostable, and enable fast, single-wavelength photoswitchable inhibition of clathrin-mediated endocytosis at micromolar concentration.

Correction for ‘Photoswitchable dynasore analogs to control endocytosis with light’ by Núria Camarero et al., Chem. Sci., 2020, 11, 8981–8988, DOI: 10.1039/d0sc03820b.

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.

Epidermal growth factor receptor (EGFR) inhibitors were among the first type of targeted agents discovered in cancer and currently constitute the standard of care for a wide range of lung and colon malignancies. However, the therapeutic progress achieved with these drugs has been accompanied by the identification of an ever-increasing number of acquired resistance mechanisms that inevitably appear in nearly all patients. Increased knowledge on EGFR biochemistry, cellular crosstalk, and resistance pathways provides an opportunity to establish effective combination therapies and discover novel-acting inhibitors that prevent or overcome therapeutic resistance. One such strategy is the selective blockade of circulating growth factors such as EGF. In this review, we address the uses and limitations of approved EGFR inhibitors and explore the potential of drug combinations and new third avenues to block the activation of the EGFR.

In recent decades, peptide blood-brain barrier shuttles have emerged as a promising solution for brain drugs that are not able to enter this organ. The research and development of these compounds involve the use of in vitro cell-based models of the BBB. Nevertheless, peptide transport quantification implies the use of large amounts of peptide (upper micromolar range for RP-HPLC-PDA) or of derivatives (e.g. fluorophore or quantum-dot attachment, radiolabeling) in the donor compartment in order to enhance the detection of these molecules in the acceptor well, although their structure is highly modified. Therefore, these methodologies either hamper the use of low peptide concentrations, thus hindering mechanistic studies, or do not allow the use of the unmodified peptide. Here we successfully applied a MALDI-TOF MS methodology for transport quantification in an in vitro BBB cell-based model. A light version of the acetylated peptide was evaluated, and the transport was subsequently quantified using a heavy internal standard (isotopically acetylated). We propose that this MALDI-TOF MS approach could also be applied to study the transport across other biological barriers using the appropriate in vitro transport models (e.g. Caco-2, PAMPA).

Background: The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) osimertinib improves therapy for non-small cell lung cancer (NSCLC) patients who possess EGFR mutations. However, acquired resistance appears invariably due to several mechanisms. The strategy of using EGF-targeted nanobodies (Nbs) to block the initial step of the EGFR pathway constitutes a new research area. Nbs also offer several advantages compared to traditional mAbs, such as their reduced size, higher stability and tissue penetration, low production costs and lack of Fc effector function, which provide key advantages for targeting soluble tumoral growth factors. In this study we investigated the efficacy of anti-EGF Nbs to reduce osimertinib-resistance. Experimental procedures: Cell viability and colony formation assays were performed in PC9 and PC9-osimertinib-resistant NSCLC cell lines tested with anti-EGF Nbs alone or in combination with osimertinib. To analyze the mechanism of action of these compounds and its combination, western blot analysis was carried out to study protein expression and activation. Summary of data: Two anti-EGF Nbs, generated in our laboratory, inhibited cell viability and colony formation in PC9 and PC9-derived osimertinib-resistant cell lines. The combination of these Nbs with osimertinib improved the antitumoral efficacy of this EGFR-TKI in cell viability and colony formation experiments. The combination of anti-EGF Nbs with osimertinib clearly decreases the activation of proteins involved in the EGFR pathway, such as, EGFR, Akt and Erk 1/2. In addition, it increases cellular apoptosis, and decreases the expression of Hes1, a marker of cancer stem cells involved in metastasis and osimertinib resistance mechanisms. Conclusions: We conclude that the combination of anti-EGF nanobodies and osimertinib exhibits a good efficacy in inhibiting cell viability and colony formation, while, inhibiting expression and activation of proteins involved in osimertinib resistance. Citation Format: Salvador Guardiola, Macarena Sánchez-Navarro, Ernest Giralt, Rafael Rosell, Jordi Codony-Servat. Nanobodies against EGF enhance the antitumoral effect of osimertinib and overcome resistance in non-small cell lung cancer cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1839.

The oral route is the preferred for the administration of drugs; however, it has some serious limitations. One of the main disadvantages is poor permeability across the intestinal barrier. Various approaches are currently being adopted to overcome this issue. In this review, we describe the alternatives that use peptides to enhance intestinal absorption. First, we define the various sources of peptide enhancers followed by the analysis of the absorption mechanism used. We then comment on the possible toxic effects derived from their use as permeation enhancers, as well as potential formulation strategies. Finally, the advantages and drawbacks of peptides as intestinal enhancers are examined.

The blood-brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma-targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high-throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d-amino acids, this approach screens only protease-resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state-of-the-art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix-and-split technique to generate a library based on the following: Ac-d-Arg-XXXXX-NH2 , where X were: d-Ala (a), d-Arg (r), d-Ile (i), d-Glu (e), d-Ser (s), d-Trp (w) or d-Pro (p). The assays used comprised the in vitro cell-based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two-step mass spectrometry approach combining LTQ-Orbitrap and Q-trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease-resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Background The genus Hypericum (family Clusiaceae) comprises various species that are used in traditional medicine, such as wound healing, antidepressant, and anticancer agents. Objective The aim of this study was to evaluate the inhibitory capacity of extracts and fractions from two Hypericum species used in the Brazilian folk medicine (H. brasiliense and H. connatum) against the enzymes prolyl oligopeptidase (POP), dipeptidyl peptidase-IV (DPP-IV), and acetylcholinesterase (AChE), as well as to identify their main active constituents. Methods Dried aerial parts of H. connatum and H. brasiliense were subjected to extraction with 8:2 methanol-H2O. Each hydroalcoholic extract was fractioned resulting in ethyl acetate and aqueous fractions. The activity of POP, DPP-IV and AChE was determined in vitro in 96-well microplates. Results The main components identified in the plant extracts were chlorogenic acid (1), quercitrin (2), rutin (3), quercetin (4), and isoquercitrin (5). Hydroalcoholic extracts, ethyl acetate and aqueous fractions showed high POP inhibitory activity with IC50 values of 2.6 to 3.7 µg/mL. AChE and DPP-IV inhibitory effects were very low for all extracts and substances. Conclusion Chlorogenic acid (1) and quercetin (4) were the main constituent responsible for the activity observed against POP. Parallel artificial membrane permeability assay of ethyl acetate fractions of both species showed that the metabolite that can effectively pass through the lipid membrane is 4, the aglycone form of 2, 3 and 5.

Ion mobility mass spectrometry (IMMS) is a biophysical technique that allows the separation of isobaric species on the basis of their size and shape. The high separation capacity, sensitivity and relatively fast time scale measurements confer IMMS great potential for the study of proteins in slow (µs-ms) conformational equilibrium in solution. However, the use of this technique for examining dynamic proteins is still not generalized. One of the major limitations is the instability of protein ions in the gas phase, which raises the question as to what extent the structures detected reflect those in solution. Here, we addressed this issue by analyzing the conformational landscape of prolyl oligopeptidase (POP) - a model of a large dynamic enzyme in the µs-ms range - by native IMMS and compared the results obtained in the gas phase with those obtained in solution. In order to interpret the experimental results, we used theoretical simulations. In addition, the stability of POP gaseous ions was explored by charge reduction and collision-induced unfolding experiments. Our experiments disclosed two species of POP in the gas phase, which correlated well with the open and closed conformations in equilibrium in solution; moreover, a gas-phase collapsed form of POP was also detected. Therefore, our findings not only support the potential of IMMS for the study of multiple co-existing conformations of large proteins in slow dynamic equilibrium in solution but also stress the need for careful data analysis to avoid artifacts. Copyright © 2016 John Wiley & Sons, Ltd.