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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.

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.

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.

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.

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.

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.

Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of microsecond–millisecond motions opens the opportunity for regulating protein–protein interactions (PPIs) by modulating the dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active-site-directed inhibitors on the dynamics. We used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. We found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively abolished this equilibrium by stabilizing the enzyme in the closed conformation. This work was supported by the Institute for Research in Biomedicine, MINECO-FEDER (Bio2013-40716-R, CTQ2013-48287 and CTQ2012-32183/BQU), and the Generalitat de Catalunya (XRB and Grup Consolidat 2014SGR521). AL has received funding from the Instituto de Salud Carlos III. PB acknowledges the Agence Nationale de la Recherche (SPINHD-ANR-CHEX-2011) and the ATIP-Avenir program for financial support. FHT’s fellowship is co-funded by the INSERM and the University of Copenhagen. Technical assistance from staff at the P12 beam line (EMBL/DESY) is acknowledged.

Peptides are destined to play a major role as therapeutic agents. My laboratory is contributing to speeding up this process. On the one hand, we devote efforts to studying the molecular details and dynamics of the events that occur during molecular recognition at protein surfaces. We succeeded to design and synthesize peptides able to modulate these recognition events either permanently or in response to light. On the other hand, we are discovering and designing peptides able to cross biological barriers. Our aim is to use these peptides as shuttles for targeting therapeutic agents to organs, tissues, or cells, with a special emphasis on drug delivery to the brain. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Noninvasive methods for efficient drug delivery to the brain is an unmet need. Molecular access to the brain is regulated by the blood-brain barrier (BBB) established by the endothelial cells of brain vessels. Passive diffusion is one of the main mechanisms that organic compounds use to travel through these endothelial cells. This passage across the BBB is determined mainly by certain physicochemical properties of the molecule such as lipophilicity, size, and the presence of hydrogen bond donors and acceptors. One emerging strategy to facilitate the passage of organic compounds across the BBB is the use of peptide shuttles.1 In using this approach the permeability in front the BBB is, clearly, determined by the combined physicochemical properties of both the cargo and the shuttle. Herein we report the synthesis of a series of variations of one of the more efficient peptide shuttles, (N-MePhe)n . These include diverse structural features such as various backbone stereochemistries or the presence of non-natural amino acids, including halogenated residues. In several cases, we assessed the BBB permeability of both the shuttles alone and linked to a few cargos. Our results show how factors such as stereochemistry or halogen content influences the passage across the BBB and, more importantly, opens the way to a strategy of peptide shuttles 'à la carte', in which a particular fine-tuned shuttle is used for each specific cargo.

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.

Venoms are currently the focus of many drug discovery programs because they contain highly bioactive and selective components. Among them, apamin, a peptide found in bee venom, has received considerable attention because of its affinity for certain potassium channels and also because of its interesting structure and high stability to extreme pH and temperatures. Although apamin has long been claimed to cross the blood-brain barrier (BBB), only a few studies have been performed producing controversial results. In this article, it is shown that not only apamin is indeed able to penetrate the BBB in a cell-based model but also that an analog reported to be nontoxic passes through this barrier. Furthermore, the permeability values obtained, together with some evidence of an active transport mechanism and an amazing stability to serum proteases, make these peptides promising candidates for BBB shuttles.

Less than 2% of all potential neurotherapeutics cross the blood-brain barrier (BBB). Here, we sought to build a construct with the capacity to cross this barrier, to behave as a chemical delivery system, and, once inside the central nervous system, to be transformed and then act as an enzyme inhibitor. With all this in mind, here, we describe the entire process to obtain such a compound, from the initial candidate selection to preparation of the compound library and posterior evaluation and final selection of the most promising candidates in terms of selectivity, serum stability, and BBB-transport.

Control of membrane traffic: Photoswitchable inhibitors of protein-protein interactions were applied to photoregulate clathrin-mediated endocytosis (CME) in living cells. Traffic light (TL) peptides acting as "stop" and "go" signals for membrane traffic can be used to dissect the role of CME in receptor internalization and in cell growth, division, and differentiation.

Brain delivery is one of the major challenges in drug development because of the high number of patients suffering from neural diseases and the low efficiency of the treatments available. Although the blood–brain barrier (BBB) prevents most drugs from reaching their targets, molecular vectors – known as BBB shuttles – offer great promise to safely overcome this formidable obstacle. In recent years, peptide shuttles have received growing attention because of their lower cost, reduced immunogenicity, and higher chemical versatility than traditional Trojan horse antibodies and other proteins.

The phenylpropanoid glycoside verbascoside [2-(3,4-dihydroxyphenylethyl)-1-O-α-l-rhamnopyranosyl-(13)-β-d-(4-O-caffeyl)-glucopyranoside] (1) has been isolated as the main constituent of the crude extract of Buddleja brasiliensis Jacq. ex Spreng from Southern Brazil. The crude extract, main fractions and the compound 1 were evaluated for inhibition of the enzymes acetylcholinesterase (AChE), dipeptidyl peptidase-IV (DPP-IV) and prolyl oligopeptidase (POP). Compound 1 showed weak activity against DPP-IV with an IC50 >> 150 µm and was inactive against AChE, with a pMIQ determined by bioautography of 9.6. In contrast, 1 displayed significant inhibition of POP in a dose-dependent manner with an IC50 value of 1.3 ± 0.2 µm, similar to the positive control, baicalin, with a POP IC50 of 12 ± 3 µm. Copyright © 2012 John Wiley & Sons, Ltd.

Advances in the field of shuttle-mediated drug delivery have been made in the last decade; however, the treatment of brain disorders still remains a great challenge because of the presence of the blood-brain barrier (BBB), a structure that limits the access of drugs to their site of action in the central nervous system. Several strategies have been proposed to enhance the transport of drugs across the BBB. In this Review, we focus on the vector-mediated approach, in which a drug is coupled to a molecule (shuttle) that has the ability to cross the BBB and deliver the drug to the brain.

Wahrend des letzten Jahrzehnts wurden im Bereich der Wirkstofffreisetzung wesentliche Fortschritte erzielt. Die Behandlung von Gehirnerkrankungen bleibt aber wegen der Blut-Hirn-Schranke (BHS; blood–brain barrier) noch immer eine grose Herausforderung. Diese Struktur schrankt den Zugang von Wirkstoffen zu ihrem Wirkort im Zentralnervensystem stark ein. Es wurden verschiedene Strategien vorgeschlagen, um den Transport von Wirkstoffen uber die BHS zu verstarken. In diesem Aufsatz konzentrieren wir uns auf einen vektorvermittelten Ansatz, bei dem der Wirkstoff an ein Schleusermolekul gekuppelt wird, das die Fahigkeit hat, die BHS zu uberqueren und die Substanz ins Gehirn abzugeben.

Cell-penetrating peptides (CPPs) are powerful tools to transport cell-impermeable cargoes into the cytoplasm without damaging the cell membrane. The vast majority of these peptides described to date share several features, among others, they are positively charged at physiological pH. In several cases a clear correlation between an increasing number of positive charges and internalization properties has been reported. Here, we describe what, to the best of our knowledge, is the first anionic CPP. This new compound SAP(E) internalizes into a range of cell lines with good efficiency and it shows low toxicity. We also report on the internalization mechanism. The discovery of this new class of CPP opens the way to the intracellular delivery of new molecular cargoes.

Prolyl oligopeptidase (POP) and dipeptidyl peptidase IV (DPP IV) are serine proteases that belong to the same family of enzymes. These peptidases are relevant because of their association with the pathophysiology of serious illnesses, such as type 2 diabetes (DPP IV), and those related to cognitive disorders (POP). Several NMR-based screening methods are being used to find and validate new hit scaffolds. In particular, (19)F NMR-based screening methods have proven to be powerful tools for the discovery and development of new inhibitors. Here we present an accurate and reliable (19)F NMR-based simultaneous assay that is used to screen for new selective POP and DPP IV inhibitors in compound mixtures. This activity assay consists of the simultaneous performance of POP and DPP-IV (19)F NMR activity assays in the presence of their fluorine-containing substrates. Furthermore, the assays were conducted in the presence of 0.01 % v/v of Triton X-100, which is a detergent that disrupts micelle formation, thereby preventing unspecific aggregate-based inhibition. Finally, this (19)F NMR methodology was applied to screen for ligands in plant extracts. Our results indicate that this method allows the simultaneous and accurate identification of selective POP and DPP IV inhibitors in these compound mixtures.

Protein-protein interactions comprise of collection of molecular recognition events that take place at protein surfaces. A better understanding of the mechanism behind these interactions would provide deeper insight into the nature of many diseases, caused by the malfunction of protein networks, and contribute to design of molecules for efficient modulating of these interactions. One major factor in molecular recognition mechanism is interaction of reacting species with aqueous media. Thus, comparative study of noncovalent complex behavior in solution and gas phase can provide valuable information about the role of the solvent. Here examined interactions of vascular endothelial growth factor (VEGF) protein with five peptidic ligands of the same molecular weight but with different affinities. Interactions of VEGF with ligands in solution were studied by ITC and NMR, and K(D)s were determined. Gas phase stability was addressed using CID-MS approach. The energy transfer model was taken and adapted for the calculation of binding energy. Peptides were ranked on the basis of both solution and gas phase affinity to VEGF. The results indicate that the ranking of peptides in terms of affinity in solution is reversed compared with the gas phase ranking. This observation opens up a vast field for the future study of the system, and the determination and characterization of factors, responsible for the change of stability of noncovalent protein-ligand complexes upon complete or partial removal of the solvent.

Prolyl oligopeptidase (POP) has gained importance as a target for the treatment of neuropsychiatric diseases and cognitive disturbances. Therefore, a variety of strategies are currently used to identify POP inhibitors. Here we performed electron microscopy (EM) studies of human POP. Our data reveal for the first time the presence of a new side opening in POP that was not observed in any of the crystallographic structures described to date. Finally, molecular dynamics, the relevant normal modes that contribute to the fluctuation of the catalytic triad residues and the algorithm CAVERN also support the existence of a new large side opening on POP.

An emerging and attractive target for the treatment of Alzheimer's disease is to inhibit the aggregation of beta-amyloid protein (Abeta). We applied the retro-enantio concept to design an N-methylated peptidic inhibitor of the Abeta42 aggregation process. This inhibitor, inrD, as well as the corresponding all-L (inL) and all-D (inD) analogues were assayed for inhibition of Abeta42 aggregation. They were also screened in neuroblastoma cell cultures to assess their capacity to inhibit Abeta42 cytotoxicity and evaluated for proteolytic stability. The results reveal that inrD and inD inhibit Abeta42 aggregation more effectively than inL, that inrD decreases Abeta42 cytotoxicity to a greater extent than inL and inD, and that as expected, both inD and inrD are stable to proteases. Based on these results, we propose that the retro-enantio approach should be considered in future designs of peptide inhibitors of protein aggregation.

The aim of this study was to design synthetic peptides with D-amino acid substitutions that mimic the human immunodeficiency virus (HIV) gp41 HR2 region. The objective was to develop new and active C34 analogue peptides by introducing D-amino acid point substitutions at nonessential sites for HR1-HR2 interaction without disrupting the structure of the peptide. Herein we report a study with C34L peptide analogues, including the enantiomer peptide C34D, the retro-inverso analogue (RI), and two peptides with D-amino acid point substitutions (C34M2 and C34M3). Our results show that, with the exception of RI, these peptides adopt an alpha-helical structure and are, like C34L, able to interact with HR1, mimicked by the N36 peptide. Furthermore, we show that modifications introduced in C34M2, but not in C34M3, enhance its resistance to trypsin-mediated hydrolysis and increase the stability of C34M2 in physiological medium. Interestingly, our results show that C34 peptide analogues C34M2 and C34M3, but not C34D and its RI analogue, retain their ability to inhibit HIV-1 replication with an efficiency similar to that of the C34L peptide. These data underscore the interest in using D-amino acids at specific sites in the C34 peptide sequence and may lead to a new strategy for the development of more stable and active anti-HIV-1 peptidic drugs.

Cell-penetrating peptides (CPPs) are a potential tool for intracellular delivery of different kinds of cargoes. Because of their growing use in nanobiomedicine, both for diagnostics and for treatment, metal nanoparticles are an interesting cargo for CPPs. Here, gold nanoparticles (AuNps) and the amphipathic proline-rich peptide SAP have been used. Conjugation of the peptide onto the AuNps was achieved by addition of a cysteine to the SAP sequence for thiol chemisorption on gold, and the attachment was confirmed by visible spectroscopy, dynamic light scattering (DLS), zeta-potential (ZP), stability towards ionic strength (as high as 1 M NaCl), X-ray photoelectron spectroscopy (XPS) and high-resolution transmission electron microscopy (HR-TEM) coupled to electron energy loss spectroscopy (EELS). AuNp-C-SAP internalization in HeLa cells was observed by three different microscopy techniques-TEM, confocal laser scanning microscopy (CLSM) and transmission X-ray microscopy (TXM)-and all of them have confirmed the effective intracellular delivery of AuNps by SAP.

The purification of a range of protected peptide segments has been carried out using modified reversed-phase chromatographic techniques in which DMF was added to the water and acetonitrile mixtures used as eluents. The purity of the recovered peptides was excellent and recoveries were high in all cases, even for longer hydrophobic segments. In several cases purifications were carried out on the hundreds of milligrams scale. For protected peptide segments containing Met, protection as the sulfoxide avoids its unwanted alkylation and oxidation, and the increased overall polarity can be useful in the purification of protected peptides incorporating this residue. © Munksgaard 1995.

Peptides representing the sequence of the immunodominant loop of foot-and-mouth disease virus strain C-S8 (YTASARGDLAHLTTTHARHLP, residues 136-156 of VP1) and of several variant viruses have been prepared by solid phase methods. In addition, five peptides with single-residue replacements at Leu147 (Ile, Nle, Val, Ala, Gly) have been synthesized. Tosyl and dinitrophenyl protections for histidine have been compared, the latter being found to give better synthetic products. The peptides have been tested in an immunodot assay against a panel of monoclonal antibodies directed towards the VP1 loop. Immunochemical results are discussed on the basis of the mobility of the region reproduced by the peptides and the nature of the side chain of residue 147.

A library of peptides required for a project investigating the factors relevant for blood-brain barrier transport was synthesized on solid phase. As a result of the high N-methylamino acid content in the peptides, their syntheses were challenging and form the basis of the work presented here. The coupling of protected N-methylamino acids with N-methylamino acids generally occurs in low yield. (7-azabenzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP) or PyBOP/1-hydroxy-7-azabenzotriazole (HOAt), are the most promising coupling reagents for these couplings. When a peptide contains an acetylated N-methylamino acid at the N-terminal position, loss of Ac-N-methylamino acid occurs during trifluoroacetic acid (TFA) cleavage of the peptide from the resin. Other side reactions resulting from acidic cleavage are described here, including fragmentation between consecutive N-methylamino acids and formation of diketopiperazines (DKPs). The time of cleavage is shown to greatly influence synthetic results. Finally, high-performance liquid chromatography (HPLC) profiles of N-methyl-rich peptides show multiple peaks because of slow conversion between conformers.

Fluoroquinolones are an important class of wide‐spectrum antibacterial agents. The first quinolone described was nalidixic acid, which showed a narrow spectrum of activity. The evolution of quinolones to more potent molecules was based on changes at positions 1, 6, 7 and 8 of the chemical structure of nalidixic acid. Quinolones inhibit DNA gyrase and topoisomerase IV activities, two enzymes essential for bacteria viability. The acquisition of quinolone resistance is frequently related to (i) chromosomal mutations such as those in the genes encoding the A and B subunits of the protein targets (gyrA, gyrB, parC and parE), or mutations causing reduced drug accumulation, either by a decreased uptake or by an increased efflux, and (ii) quinolone resistance genes associated with plasmids have been also described, i.e. the qnr gene that encodes a pentapeptide, which blocks the action of quinolones on the DNA gyrase and topoisomerase IV; the aac(6′)‐Ib‐cr gene that encodes an acetylase that modifies the amino group of the piperazin ring of the fluoroquinolones and efflux pump encoded by the qepA gene that decreases intracellular drug levels. These plasmid‐mediated mechanisms of resistance confer low levels of resistance but provide a favourable background in which selection of additional chromosomally encoded quinolone resistance mechanisms can occur.

The blood-brain barrier (BBB) regulates the passage of molecules between the bloodstream and the brain. Overcoming the difficulty of delivery drugs to specific areas of the brain is a major challenge. The BBB exerts a neuroprotective function as it hinders the delivery of diagnostic and therapeutic agents to the brain. Here, we provide an overview of the way in which peptides and nanotechnology are being exploited in tandem to address this problem. Peptides can be used as specialised coatings able to transport nanoparticles with specific properties, such as targeting. The nanoparticle can also carry a peptide drug. Furthermore, peptides can be used in less conventional approaches such as all-peptide nanoparticles. In summary, the combined use of peptides and nanotechnology offers tremendous hope in the treatment of brain disorders.

The present work is a combined structural study, using Nuclear Magnetic Resonance (NMR) and Molecular Dynamics(MD), of the amidated and the free acid forms of substance P in water and methanol. The results obtained using both approaches were compared in order to characterize the structural features of both peptides in solution. From the NMR experiments it was derived that the free acid form adopts an extended conformation at the N-terminus and a helical conformation at the C-terminal segment of the peptide in both water and methanol; these structural features are in qualitative agreement with the results of the MD simulations. No significant differences in behavior were observed between the amidated and the free acid forms of the peptide in the simulations and in the experiments carried out in water, suggesting that the different activities of these analogs are due to their different mode of interaction with the receptor rather than to their structural preferences. Finally, we propose that the structure of substance P can be partially inferred from its sequence due to the presence of a Pro-X-Pro motif on the N-terminus and a Gly–Leu sequence on the C-terminus. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

HIV-1 protease (HIV-1 PR), which is encoded by retroviruses, is required for the processing of gag and pol polyprotein precursors, hence it is essential for the production of infectious viral particles. In vitro inhibition of the enzyme results in the production of progeny virions that are immature and noninfectious, suggesting its potential as a therapeutic target for AIDS. Although a number of potent protease inhibitor drugs are now available, the onset of resistance to these agents due to mutations in HIV-1 PR has created an urgent need for new means of HIV-1 PR inhibition. Whereas enzymes are usually inactivated by blocking of the active site, the structure of dimeric HIV-1 PR allows an alternative inhibitory mechanism. Since the active site is formed by two half-enzymes, which are connected by a four-stranded antiparallel β-sheet involving the N- and C- termini of both monomers, enzyme activity can be abolished by reagents targeting the dimer interface in a region relatively free of mutations would interfere with formation or stability of the functional HIV-1 PR dimer. This strategy has been explored by several groups who targeted the four-stranded antiparallel β-sheet that contributes close to 75% of the dimerization energy. Interface peptides corresponding to native monomer N- or C-termini of several of their mimetics demonstrated, mainly on the basis of kinetic analyses, to act as dimerization inhibitors. However, to the best of our knowledge, neither X-ray crystallography nor NMR structural studies of the enzyme-inhibitor complex have been performed to date. In this article we report a structural study of the dimerization inhibition of HIV-1 PR by NMR using selective Trp side chain labeling. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 164–173, 2007. This article was originally published online as an accepted preprint. The ‘Published Online’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

The applied interaction of synthetic molecules with defined regions of protein surfaces is an emerging strategy for the modulation of protein activity and/or stability. In spite of recent advances, the design of these molecules is not trivial. Among the most challenging aspects in designing these compounds is that they must compete with water molecules for interaction with polar patches of protein surfaces. Herein is reported the preparation of an arginine-rich peptide that interacts in aqueous solution with a very hydrophilic patch at the surface of the tetramerization domain of the tumor suppressor protein p53. The interaction has been studied by several complementary techniques. By using this peptide as a template, a library of peptides has been prepared and evaluated in order to examine the different factors that contribute to the recognition event. The conclusions extracted from this work could be useful for the design of ligands directed at highly hydrophilic protein surface patches.

Acinetobacter baumannii is a multiresistant opportunistic nosocomial pathogen. A protein fraction was purified and analyzed by 2-DE. Twenty-nine major protein spots were selected for protein identification using trypsin digestion and MS analysis. As the A. baumannii genome has not yet been described, protein identification was performed by homology with other Acinetobacter species in the NCBi database. We identified ribosomal proteins, chaperones, elongation factors and outer membrane proteins (Omp), such as OmpA and the 33-36-kDa OMP. Proteomic analysis of A. baumannii provides a platform for further studies in antimicrobial resistance.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.

Amino acid sequences and linear or head-to-tail cyclopeptides can be represented conveniently in one-line text formulae using the three-letter symbols. However, other – but nonetheless important – topologies of peptides are ‘side chain-to-head (or tail)’, ‘backbone-to-backbone’, ‘side chain-to-side chain’ cyclopeptides, ‘side chain-to-side chain’ connected peptide strands, and branched peptides (like peptide dendrimers). In general, such structures cannot be described using the three-letter symbols in one-line text: a chemical structure editor is required for symbolic representations according to the IUPAC-IUBMB recommendations. The aim of this contribution is to offer an unambiguous and general nomenclature system that enables researchers to represent all cyclic and branched homo- and hetero-detic peptides in a coherent manner in one-line text – as long as their as constituents can be represented in (three)-letter codes. The application of this new nomenclature would overcome the existing difficulties and provide a way to express complex situations in the shortest way in order to highlight more clearly the salient points in a given scientific communication.

Keywords: bipolar affective disorder; neuropeptides; prolyl oligopeptidase; schizophrenia; structural homology model; proteomics

The discovery of cell-penetrating peptides as gene delivery systems and the interest in the mechanism by which these vectors cross the cell membrane have generated a large number of studies. Among the parameters involved in the translocation process, controversy has arisen about the role of the amphipathicity of the carriers in the interaction and reorganization of the cell membrane. In this review we have summarized the vectors with primary or secondary amphipathicity related to secondary structure. Some of the insights into the relationship between the aggregation state of the peptide at the concentrations used for internalization studies and its interaction with the cell membrane result from our contribution to the field with a new family of amphipathic proline-rich peptides.

Zeins are maize storage proteins that accumulate inside large vesicles called protein bodies. gamma-Zein lines the inner surface of the protein body membrane, and its N-terminal, proline-rich, repetitive domain with the sequence (VHLPPP)(8) appears to be necessary for the accumulation of the protein within the organelle. Synthetic (VHLPPP)(8) adopts an amphipathic polyproline II conformation and forms cylindrical micelles in aqueous solution. Here we explore the interaction of (VHLPPP)(8) with soybean phosphatidylcholine unilamellar lipid vesicles and examine its effect on the stability and permeability of the liposome membrane. The amphipathic N-terminal domain of gamma-zein interacts with the membrane and assembles to form extended domains over the phospholipid membrane. The interaction between the peptide and the membrane increases the stability and permeability of the liposome membrane. The spontaneous amphipathic aggregation of (VHLPPP)(8) on the membrane suggests a mechanism of gamma-zein deposition inside maize protein bodies.

The design of peptide drugs to treat central nervous system (CNS) diseases is hampered by our ignorance of the factors that determine whether a given peptide can cross the blood-brain-barrier (BBB). We are developing an approach to this problem that combines computer-aided ligand design, parallel synthesis of peptide libraries, and biological evaluation using in vitro BBB models. We present a genetic algorithm (GA) to search for peptides that can cross the BBB. In the design and optimization of this GA we used a genetic meta-algorithm to optimize the GA parameters. The GA is validated in silico by virtual screening of a peptide library of more than 10 1 5 molecules. We used a virtual fitness function dervied from statistical analysis of the few experimental data on peptide-BBB permeability available.

1H and 13C NMR data for 13 nitrohistidine derivatives are reported, providing a diagnostic method for the elucidation of the N1-(2) and the N3-substituted (3) regioisomers. Spectral assignments of constrained surrogates of His (4) and of the His–Gly dipeptide (5 and 6) are also described. The structure of the compounds was confirmed by NOESY and heteronuclear (13C, 1H) short- and long-range correlation experiments. Copyright © 2003 John Wiley & Sons, Ltd.

Preface. List of Contributors. PART I PRINCIPLES. 1 The Discovery and Characterization of Protein-Protein Interactions (C.W. Bertoncini, A. Higueruelo, and X. Salvatella). 1.1 Introduction. 1.2 Techniques to Identify Protein-Protein Interactions. 1.3 Techniques to Characterize Protein-Protein Interactions. 1.4 Structure and Dynamics of Protein Complexes. 1.5 Protein-Protein Complexes as Therapeutic Targets. 1.6 Conclusions. References. 2 Biophysics of Protein-Protein Interactions (Irene Luque). 2.1 Introduction. 2.2 Intermolecular Forces in Protein Recognition. 2.3 Basic Binding Thermodynamics. 2.4 Thermodynamically Driven Drug Design. 2.5 Measurement of Binding Energetics. 2.6 Structure-based Calculation of Protein Binding Energetics. 2.7 Interfacial Water Molecules in Protein Recognition. 2.8 The Linkage Between Binding and Conformational Equilibrium in Proteins. References. PART II APPROACHES. 3 On the Logic of Natural Product Binding in Protein-Protein Interactivity (James J. La Clair). 3.1 Introduction. 3.2 Structural Logic. 3.3 Functional Logic. 3.4 The Need for Programmers. 3.5 Compiling the NPPI Mapper. References. 4 Interface peptide inhibitors of PPIs (Mark W. Peczuh, and Richard T. Desmond). 4.1 Interface Peptides Defined. 4.2 Unmodified Peptides. 4.3 Modified Peptides. 4.4 Summary/Perspective. References. 5 Inhibition of Protein-Protein Interactions by Peptide Mimics (Jorge Becerril, Johanna M. Rodriguez, Pauline N. Wyrembak, and Andrew D. Hamilton). 5.1 Introduction. 5.2 Inhibition of Calmodulin. 5.3 Inhibition of HIV-1 Fusion. 5.4 Inhibition of the Nuclear Estrogen Receptor. 5.5 Inhibition of the Bcl-x /Bak Interaction. 5.6 Inhibition of the p53/MDM2 Interaction. 5.7 Miscellaneous Protein Targets. 5.8 Conclusion. References. 6 Discovery of Inhibitors of Protein-Protein Interactions by Screening Chemical Libraries (Carlos Garcia-Echeverria). 6.1 Introduction. 6.2 Screening Strategies to Identify and Develop Antagonists of Protein-Protein Interactions. 6.3 Mimetics of Common Protein Structure Motifs and Structure-based Design of Peptidomimetics. 6.4 Conclusions and Outlook. References. PART III TECHNIQUES. 7 High-throughput Methods of Chemical Synthesis Applied to the Preparation of Inhibitors of Protein-Protein Interactions (Annaliese K. Franz, Jared T. Shaw, and Yuchen Tang). 7.1 Introduction. 7.2 Survey of High-throughput Organic Synthesis. 7.3 Synthesis of 'Peptide-Inspired' Compounds and Libraries. 7.4 Synthesis of 'Natural Product-Inspired' Compounds and Libraries. 7.5 Diversity Oriented Synthesis (DOS) in the Discovery of PPI Inhibitors. 7.6 Summary and Outlook. References. 8 In Silico screening (F.J. Luque, and Xavier Barril). 8.1 Introduction. 8.2 Methods for Virtual Ligand Screening. 8.3 Binding Site Characterization. 8.4 Case Studies. 8.5 Outlook and Conclusions. References. 9.1 In Vitro Screening: Screening by Nuclear Magnetic Resonance (Ernest Giralt). 9.1.1 Saturation Transfer Difference (STD). 9.1.2 STD in Fragment-based Drug Design. 9.1.3 Chemical Shift Perturbation (CSP). 9.1.4 19F-NMR in Molecular Recognition Studies. References. 9.2 In Vitro Screening: Methods of High-throughput Screening (Wenjiao Song and Qing Lin). 9.2.1 Introduction. 9.2.2 Statistical Evaluation of the HTS Assay Performance. 9.2.3 Biochemical Assays. 9.2.4 Cell-based Assays. 9.2.5 Conclusion. References. PART IV CASE STUDIES. 10 Case Study: Inhibitors of the MDM2-p53 Protein-Protein Interaction (Sanjeev Shangary, Denzil Bernard, and Shaomeng Wang). 10.1 MDM2-p53 Protein-Protein Interaction: A Case Study. 10.2 Regulation of p53 by the MDM2-p53 Protein-Protein Interaction. 10.3 Structural Basis of the MDM2-p53 Interaction. 10.4 Design of p53-based Peptides. 10.5 Design of Nonpeptidic Small-Molecule Inhibitors of the MDM2-p53 Interaction. 10.6 Challenges in the Design of Small Molecule Inhibitors of the MDM2-p53 Interaction. 10.7 Reactivation of p53 by Inhibitors of the MDM2-p53 Interaction. 10.8 Development of MDM2 Inhibitors and New Anticancer Drugs. 10.9 Concluding Remarks. References. 11 Case Study: The Discovery of Potent LFA-1 Antagonists (Tom Gadek). 11.1 Introduction. 11.2 Structural, Molecular and Cellular Biologies of LFA-1. 11.3 The Search for Small Molecule LFA-1 Antagonists. 11.4 Screening Assays. 11.5 Lead Identification and Optimization. 11.6 Protein and Small Molecule Structure Activity Relationships (PSAR) in the LFA-1/ICAM-1 Interaction. 11.7 Summary. References. Index.

Antigen-antibody binding is regarded as one of the most representative examples of specific molecular recognition in nature. The simplistic view of antigenic recognition in terms of a lock-and-key mechanism is obsolete, as it is evident that both antigens and antibodies are flexible and can undergo substantial mutual adaptation. This flexibility is the source of complexities such as degeneracy and nonadditivity in antigenic recognition. We have used surface plasmon resonance to study the effects of combining multiple amino acid replacements within the sequence of the antigenic GH loop of foot-and-mouth disease virus. Our aim was 2-fold: to explore the extent to which antigenic degeneracy can be extended in this particular case, and to search for potential nonadditive effects in introducing multiple amino acid replacements. Combined analysis of one such multiply substituted peptide by SPR, solution NMR and X-ray diffraction shows that antigenic degeneracy can be expected as long as residues directly interacting with the paratope are conserved and the peptide bioactive folding is unaltered.

A simplified strategy is described for the assignment of disulfide bonds in proteins of medium to high molecular mass (10-30 kDa). The method combines the use of high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) and HPLC with UV diode-array detection (HPLC diode array). The denatured protein is subjected to proteolysis and the peptide mixture is divided into three fractions: (i) underivatized peptides, (ii) ethylpyridylated peptides, and (iii) reduced and ethylpyridylated peptides. The three peptide ensembles are then subjected to chromatographic and spectroscopic analysis. A systematic methodology is described to analyze the large amount of data obtained. The method was applied to the localization of disulfide bonds in alpha-L-fucosidase from pea. The two disulfide bonds were located between residues Cys64 and Cys109 and between Cys162 and Cys169, while Cys127 was free.