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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.

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.

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.

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.

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.