Light-dependent inhibition of clathrin-mediated endocytosis in yeast unveils conserved functions of the AP2 complex.

Clathrin-mediated endocytosis (CME) is an essential cellular process, conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of specific pharmacological agents to interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor targeting the AP2-ß-adaptin/ß-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, demonstrating that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts. This is despite the ß-adaptin/ß-arrestin interaction not being conserved in yeast. Our data indicate that the AP2 α-adaptin is the functional target of activated TL2. We identified as interacting partners for the α-appendage, the Eps15 and epsin homologues Ede1 and Ent1. This demonstrates that endocytic cargo loading and sensing can be executed by conserved molecular interfaces, regardless of the proteins involved.

IDP-410: a Novel Therapeutic Peptide that Alters N-MYC Stability and Reduces Angiogenesis and Tumor Progression in Glioblastomas.

Glioblastomas (GBMs) are the most frequent and highly aggressive brain tumors, being resistant to all cytotoxic and molecularly targeted agents tested so far. There is, therefore, an urgent need to find novel therapeutic approaches and/or alternative targets to bring treatment options to patients. Here, we first show that GBMs express high levels of N-MYC protein, a transcription factor involved in normal brain development. A novel stapled peptide designed to specifically target N-MYC protein monomer, IDP-410, is able to impair the formation of N-MYC/MAX complex and reduce the stability of N-MYC itself. As a result, the viability of GBM cells is compromised. Moreover, the efficacy is found dependent on the levels of expression of N-MYC. Finally, we demonstrate that IDP-410 reduces GBM growth in vivo when administered systemically, both in subcutaneous and intracranial xenografts, reducing the vascularization of the tumors, highlighting a potential relationship between the function of N-MYC and the expression of mesenchymal/angiogenic genes. Overall, our results strengthen the view of N-MYC as a therapeutic target in GBM and strongly suggest that IDP-410 could be further developed to become a first-in-class inhibitor of N-MYC protein, affecting not only tumor cell proliferation and survival, but also the interplay between GBM cells and their microenvironment.

Correction: Photoswitchable dynasore analogs to control endocytosis with light.

[This corrects the article DOI: 10.1039/D0SC03820B.].

Targeted Nanoswitchable Inhibitors of Protein-Protein Interactions Involved in Apoptosis.

Progress in drug delivery is hampered by a lack of efficient strategies to target drugs with high specificity and precise spatiotemporal regulation. The remote control of nanoparticles and drugs with light allows regulation of their action site and dosage. Peptide-based drugs are highly specific, non-immunogenic, and can be designed to cross the plasma membrane. In order to combine target specificity and remote control of drug action, here we describe a versatile strategy based on a generalized template to design nanoswitchable peptides that modulate protein-protein interactions upon light activation. This approach is demonstrated to promote photomodulation of two important targets involved in apoptosis (the interactions Bcl-xL-Bak and MDM2-p53), but can be also applied to a large pool of therapeutically relevant protein-protein interactions mediated by α-helical motifs. The template can be adjusted using readily available information about hot spots (residues contributing most to the binding energy) at the protein-protein interface of interest.

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.

Synthesis of Bromoundecyl Resorc[4]arenes and Applications of the Cone Stereoisomer as Selector for Liquid Chromatography.

As an extension of our studies on the multifaceted properties of C-alkylated resorc[4]arenes, we planned to immobilize on a solid support resorc[4]arenes with C11-long side chains in the lower rim. To this purpose, we synthesized two conformationally diverse resorc[4]arenes containing a bromoundecyl moiety in the four axial pendants. The cone stereoisomer 6a (30% yield) was selected for the reaction with an aminopropylated silica gel (APSG) obtained from spherical Kromasil Si 100, 5 µm particles, to give the corresponding immobilized SP-C11-resorc[4]arene system. The resulting polar-embedded stationary phase was fully characterized and investigated in the HPLC discrimination of the E/ Z stereoisomers of naturally occurring and semisynthetic combretastatins, a family of ( Z)-stilbene anticancer drugs. The chair stereoisomer 6b (20% yield), when submitted to X-ray diffraction analysis, showed a noteworthy self-assembly in the crystal lattice, with intercalated hydrophobic and polar layers as a result of intermolecular Br···O halogen bond interactions, according to a unique stacking motif. The potential and versatility of the SP-C11-resorc[4]arene stationary phase were shown as well in the separation of highly polar natural products (namely, flavonoids), under reversed-phase (RP) conditions, and of fullerenes C60 and C70, by using apolar solvents as mobile phases.

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.

Peptides Targeting EGF Block the EGF-EGFR Interaction.

Epidermal growth factor receptor (EGFR) is a key target in chemotherapy. Some drugs acting on the receptor are currently in use; however, drug resistance, which causes tumour relapse, calls for the discovery of alternative inhibitors. Using docking and receptor hotspot mimicry, we have designed novel peptides directed at EGF, the main growth factor ligand of EGFR. An array of biophysical techniques was used to characterise the structure and interaction of these ligands with the target protein. Both design methods identified peptides able to bind EGF, and the capacity of these peptides to inhibit the interaction between EGF and EGFR was demonstrated in two in vitro systems. Based on targeting the smaller companion of a protein-protein interaction, the new approach described herein can be envisaged as a parallel drug design strategy, and our compounds represent the first in a new class of binders that could serve as complementary compounds in potential multidrug cancer therapy.

Absence of a stable secondary structure is not a limitation for photoswitchable inhibitors of ß-arrestin/ß-Adaptin 2 protein-protein interaction.

Many protein-protein interactions (PPIs) are mediated by short, often helical, linear peptides. Molecules mimicking these peptides have been used to inhibit their PPIs. Recently, photoswitchable peptides with little secondary structure have been developed as modulators of clathrin-mediated endocytosis. Here we perform a systematic analysis of a series of azobenzene-crosslinked peptides based on a ß-arrestin P-long 20-mer peptide (BAP-long) sequence to assess the relevance of secondary structure in their interaction with ß-adaptin 2 and to identify the design requirements for photoswitchable inhibitors of PPI (PIPPIs). We observe that flexible structures show a greater inhibitory capacity and enhanced photoswitching ability and that the absence of helical structures in free inhibitor peptide is not a limitation for PIPPI candidates. Therefore, our PIPPIs expand the field of potential inhibitors of PPIs to the wide group of flexible peptides, and we argue against using a stable secondary structure as a sole criterion when designing PIPPI candidates.

Modulating protein-protein interactions: the potential of peptides.

Protein-protein interactions (PPIs) have emerged as important and challenging targets in chemical biology and medicinal chemistry. The main difficulty encountered in the discovery of small molecule modulators derives from the large contact surfaces involved in PPIs when compared with those that participate in protein-small molecule interactions. Because of their intrinsic features, peptides can explore larger surfaces and therefore represent a useful alternative to modulate PPIs. The use of peptides as therapeutics has been held back by their instability in vivo and poor cell internalization. However, more than 200 peptide drugs and homologous compounds (proteins or antibodies) containing peptide bonds are (or have been) on the market, and many alternatives are now available to tackle these limitations. This review will focus on the latest progress in the field, spanning from "lead" identification methods to binding evaluation techniques, through an update of the most successful examples described in the literature.

Light-regulated stapled peptides to inhibit protein-protein interactions involved in clathrin-mediated endocytosis.

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.

Reaction of nitrosonium cation with resorc[4]arenes activated by supramolecular control: covalent bond formation.

Resorc[4]arenes 1 and 2, which previously proved to entrap NO(+) cation within their cavities under conditions of host-to-guest excess, were treated with a 10-fold excess of NOBF4 salt in chloroform. Kinetic and spectral UV-visible analyses revealed the formation of isomeric 1:2 complexes as a direct evolution of the previously observed event. Accordingly, three-body 1-(NO(+))2 and 2-(NO(+))2 adducts were built by MM and fully optimized by DFT calculations at the B3LYP/6-31G(d) level of theory. Notably, covalent nitration products 4, 5 and 6, 7 were obtained by reaction of NOBF4 salt with host 1 and 2, respectively, involving macrocycle ring-opening and insertion of a nitro group in one of the four aromatic rings. In particular, compounds 4 and 6, both containing a trans-double bond in the place of the methine bridge, were oxidized to aldehydes 5 and 7, respectively, after addition of water to the reaction mixture. Calculation of the charge and frontier orbitals of the aromatic donor (HOMO) and the NO(+) acceptor (LUMO) clearly suggests an ipso electrophilic attack by a first NO(+) unit on the resorcinol ring, mediated by the second NO(+) unit.

Super-secondary structure peptidomimetics: design and synthesis of an α-α hairpin analogue.

The α-α helix motif presents key recognition domains in protein-protein and protein-oligonucleotide binding, and is one of the most common super-secondary structures. Herein we describe the design, synthesis and structural characterization of an α-α hairpin analogue based on a tetra-coordinated Pd(II) bis-(iminoisoquinoline) complex as a template for the display of two α-helix mimics. This approach is exemplified by the attachment of two biphenyl peptidomimetics to reproduce the side-chains of the i and i+4 residues of two helices.

Modelling amphetamine/receptor interactions: a gas-phase study of complexes formed between amphetamine and Some chiral amido[4]resorcinarenes.

Diastereomeric proton-bound complexes formed between (R)- and (S)-amphetamine and some chiral amido[4]resorcinarene receptors display significant enantioselectivities when reacting with the enantiomers of 2-aminobutane in the gas phase. The origins of the measured enantioselectivities are discussed in the light of molecular mechanics calculations and molecular dynamics simulations and are ascribed to a combination of structural and dynamic factors, including the lengths and the isomeric structures of the host asymmetric pendants and the frequencies and amplitudes of their motion, as well as those of the proton-bonded amphetamine guests. The emerging picture may represent a starting point for deeper comprehension of the factors determining the different affinities of (R)- and (S)-amphetamine towards various chiral receptors, their selective binding to the monoamine transporters, and their sensitivity to specific inorganic ions.

Nitrosonium complexes of resorc[4]arenes: spectral, kinetic, and theoretical studies.

Resorc[4]arene octamethyl ethers 1-3, when treated with NOBF4 salt in chloroform, form very stable 1:1 nitrosonium (NO+) complexes, which are deeply colored. The complexation process is reversible, and the complexes dissociate and bleach upon addition of methanol or water, to give the starting macrocycles. Resorc[4]arenes 1 and 2 are in the same cone conformation, but with different side-chains, whereas 3 possesses a different conformation (chair), while bearing the same side-chain as 2. Kinetic and spectral UV-visible analysis revealed that NO+ interacts with resorc[4]arenes 1 and 2 both outside and inside their basket, leading to complexes with two absorption patterns growing at different rates, one featuring high-energy bands (HEB) within the near-UV zone, and the other one low-energy bands (LEB), attributed to charge-transfer interactions, within the visible range. The presence of ester carbonyl groups in 2 strongly drives the NO+ cation outside the resorcarene. Resorc[4]arene 3 showed a spectral pattern pointing out a clear involvement of the ester moieties in the NO+ entrapping, beside the formation of significant charge-transfer interactions. 1H NMR spectroscopy and molecular modeling clearly supported these findings.

The contribution of oxazolidinone frame to the biological activity of pharmaceutical drugs and natural products.

The development of resistance by the antibiotics in the Gram-positive pathogenic bacteria over the last twenty years and continuing today has created a need for new antibiotic classes, which may be unaffected by existing bacterial resistance. The oxazolidin-2-ones represent not only a new class with a novel mechanism of action, but also satisfy the requirement for overcoming the resistance mechanisms. Both linezolid and eperozolid, the first chemical candidates, arose from the piperazine subclass, with the first one being chosen further development because of its enhanced pharmacokinetic properties. The main attractive traits of the oxazolidinone series has encouraged further work in the area, and the patent literature reveals that extensive chemical investigation is currently being made. The unexpected early resistance development emphasizes the need for further exploration of features of the oxazolidinone to eliminate these deficiencies. Recently, several changes, involving the C5 side chain as well the N-phenyl heterocyclic ring, give promise for such improvement. Oxazolidinone antibacterial agents comprise also ketolides, derivatives of macrolides, such as erythromycin A, with a newly formed carbamate cycle, with a largely unexplored potential. The oxazolidinone nucleus does not appear only in the structures of antimicrobial drugs, but a number of biological activities are connected with frameworks including the oxazolidinone ring. A partial list of these activities comprises enzyme inhibitors, agonists and antagonists, with a particular citation for a new generation of selective monoamino oxidase inhibitors (befloxatone). The oxazolidinone moiety was found in the structure of few biologically active natural products, such as (-)-cytoxazone and streptazolin. Moreover, in some cases the oxazolidinone ring has been chosen for the preparation of isosteric aza analogues of natural compounds (podophyllotoxin, pilocarpine) that can be more easily synthesised and more hardly inactivated. Finally, the participation of oxazolidinone chiral auxiliaries to several syntheses of natural products must be acknowledged.