Synthesis and Antiviral Evaluation of Bisnoradamantane Sulfites and Related Compounds

The reaction of a series of 1,2-diols with thionyl chloride led to bisnoradamantane sulfites in very good yields. The reaction has also been applied to related polycyclic scaffolds. The compounds have been tested for antiviral activity but none of them showed to be active. Several attempts to generate and trap SO from these polycyclic sulfites have been unsuccessful

Probing the Kinetic and Thermodynamic Fingerprints of Anti-EGF Nanobodies by Surface Plasmon Resonance

Despite the widespread use of antibodies in clinical applications, the precise molecular mechanisms underlying antibody-antigen (Ab-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

Toward a novel drug to target the EGF-EGFR interaction: design of metabolically stable bicyclic peptides

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

Targeted Covalent Inhibition of Prolyl Oligopeptidase (POP): Discovery of Sulfonylfluoride Peptidomimetics

Prolyl oligopeptidase (POP), a serine protease highly expressed in the brain, has recently emerged as an enticing therapeutic target for the treatment of cognitive and neurodegenerative disorders. However, most reported inhibitors suffer from short duration of action, poor protease selectivity, and low blood-brain barrier (BBB) permeability, which altogether limit their potential as drugs. Here, we describe the structure-based design of the first irreversible, selective, and brain-permeable POP inhibitors. At low-nanomolar concentrations, these covalent peptidomimetics produce a fast, specific, and sustained inactivation of POP, both in vitro and in human cells. More importantly, they are >1,000-fold selective against two family-related proteases (DPPIV and FAP) and display high BBB permeability, as shown in both lipid membranes and MDCK cells

Design, synthesis and biophysical evaluation of peptides targeting pharmacologically relevant proteins

[eng] Protein-protein interactions (PPIs) and protein surfaces are considered challenging targets for drug discovery. In this field, conventional medicinal chemistry (i.e. small molecules) has largely failed to provide effective hits. On the other hand, peptides are endowed with a higher degree of structural flexibility (which allows them to better adapt to irregular targets) and are able to display a variety of tailor-made topologies, emerging as an alternative to target proteins that were considered undruggable. In this thesis, we have explored the potential of designed peptides to modulate the function of two therapeutically relevant protein targets involved in cancer (epidermal growth factor, EGF) and cognitive disorders (prolyloligopeptidase, POP). Regarding the discovery of peptide ligands against EGF, docking tools have allowed the de novo design of a family of small peptides, which have shown a reproducible, albeit weak, binding to EGF. In order to obtain more active candidates, the relevant interacting regions of EGFR have been identified and mimicked with a diversity of cycle-constrained peptides. These are larger and structurally richer scaffolds that have proved more efficient in targeting a small and featureless protein, such as EGF. The best peptide hit, a 28-mer cyclic miniprotein (cp28), has served as the starting point in a computer-guided optimization process that strived for more active and structurally constrained analogues. Our design effort has resulted in a series of bicyclic peptides that mimic the mode of binding of cp28 to EGF, albeit with reduced size, increased hydrophilicity and a more restrained topology. The chemical synthesis of these complex bicyclic molecules was enabled by state-of-the-art native chemical ligation techniques. In order to assess the binding of our peptides with EGF, an array of suitable biophysical techniques was explored, and the most suitable ones were implemented to our discovery process. In particular, NMR spectroscopy (combined with expression of recombinant 15N-EGF) has allowed the monitoring of ligand-induced changes on the protein NMR spectra. In parallel, a recently developed acoustic biosensor (SAW) was set up as a low-cost, label-free, and highly sensitive technique to quantify the interactions with EGF. Moreover, our best peptide candidates (cp28 and cp23G) were able to disrupt the EGF-EGFR interaction, an effect that has been tested in several cell-like and living cell assays. Indeed, these peptides were able to halt the proliferation of EGFR(+) human carcinoma cells, an effect that underlines their biological efficacy. Moreover, the bicycle-constrained analogues display an exceptional level of biological stability, especially in serum and hepatic media. In the last part of this thesis, a series of bioactive peptides has been designed with a fundamentally different mechanism of action. Peptides typically possess fast dissociation rates from the protein target, a feature that represents an obstacle when competing with endogenous ligands for the binding to cavities, such as catalytic sites in enzymes. As a proof of concept, a novel class of covalent-acting peptidomimetics were developed to supress the activity of POP, a protease involved in neurodegenerative disorders. In these bifunctional molecules, the peptide backbone and side chains formed a template that selectively binds to the POP active site, whereas a novel sulfonyl fluoride electrophile was optimally positioned to react with the catalytic Ser residue. These compounds showed a high potency in vitro, being able to inactivate POP at low nM concentrations, and their mechanism of action as irreversible inhibitors was confirmed by kinetic assays. Moreover, they displayed a remarkable selectivity against closely related proteases, and they were able to permeate through a lipid bilayer that mimics the composition of the blood- brain barrier. In summary, our findings show how two completely different classes of peptides, bicycle-constrained miniproteins and covalent-acting peptidomimetics, with binding affinities several orders of magnitude apart, can be efficiently designed to target specific protein surfaces. With PPIs and challenging binding sites becoming the focus of current drug discovery projects, these type of ligands are ideally positioned to deliver new drugs for the treatment of disease.[cat] Les interaccions proteïna-proteïna representen un dels principals reptes pel desenvolupament de nous fàrmacs, donat el fracàs de la química terapèutica convencional per adreçar aquest tipus de dianes. En aquesta tesi, hem modelitzat (mitjançant tècniques de docking) una sèrie de pèptids petits capaços d’unir-se a EGF, una proteïna que juga un paper crític pel desenvolupament dels tumors. Per tal d’estudiar les interaccions dels lligands, s’han explorat diverses tècniques biofísiques de les quals la RMN i el biosensor SAW han sigut les més adients. Per tal d’aconseguir candidats més actius que els identificats per docking, s’han dissenyat pèptids cíclics i bicíclics que mimetitzen epítops d’EGFR rellevants pel reconeixement molecular d’EGF. Aquests pèptids, que incorporen aminoàcids no naturals, tenen una gran estabilitat biològica i han estat capaços de disminuir la proliferació de cèl·lules tumorals fins a un 80%. D’altra banda, per tal de modular l’activitat d’una proteasa (la prolil- oligopeptidasa, o POP) que està involucrada en trastorns neurològics, s’han dissenyat diferents peptidomimètics amb acció covalent. Aquests composts són capaços d’inactivar la POP a concentracions nanomolars, a la vegada que són selectius per aquest enzim. A més, aconsegueixen valors elevats de permeabilitat en un assaig de difusió passiva a través de lípids cerebrals, el que posa de manifest el seu potencial com a fàrmacs. Els nostres resultats mostren com diferents classes de pèptids, amb afinitats i mecanismes d’acció dispars, són capaços de modular l’activitat de proteïnes amb interès terapèutic, com ara l’EGF i la POP. Aquest tipus de molècules expandeix les fronteres de la química mèdica convencional per al descobriment de nous fàrmacs

Inhibitors of the M2 channel of influenza A virus

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32392Influenza is a highly contagious, major respiratory tract disease affecting millions of people each year. At present, two classes of antivirals are available: the neuraminidase inhibitors and the M2 proton channel blockers amantadine and rimantadine. However, rapid emergence of M2 blockers resistance makes imperative the development of new anti-influenza drugs. In the last few years several groups have synthesized and evaluated several analogs of amantadine. While several of them are active against wild-type M2 channel only a few are able to inhibit the mutant ion channels that lead to amantadine-resistance

Inhibitors of the M2 channel of influenza A virus

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32392Influenza is a highly contagious, major respiratory tract disease affecting millions of people each year. At present, two classes of antivirals are available: the neuraminidase inhibitors and the M2 proton channel blockers amantadine and rimantadine. However, rapid emergence of M2 blockers resistance makes imperative the development of new anti-influenza drugs. In the last few years several groups have synthesized and evaluated several analogs of amantadine. While several of them are active against wild-type M2 channel only a few are able to inhibit the mutant ion channels that lead to amantadine-resistance

Proteomic tools for the quantitative analysis of artificial peptide libraries: detection and characterization of target-amplified PD-1 inhibitors

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

Toward a novel drug to target the EGF-EGFR interaction: design of metabolically stable bicyclic peptides

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

Combating virulence of Gram-negative bacilli by OmpA inhibition

Preventing the adhesion of pathogens to host cells provides an innovative approach to tackling multidrug-resistant bacteria. In this regard, the identification of outer membrane protein A (OmpA) as a key bacterial virulence factor has been a major breakthrough. The use of virtual screening helped us to identify a cyclic hexapeptide AOA-2 that inhibits the adhesion of Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli to host cells and the formation of biofilm, thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. Inhibition of OmpA offers a strategy as monotherapy to address the urgent need for treatments for infections caused by Gram-negative bacilli