Michele Saviano

Acylpeptide Hydrolase Inhibition as Targeted Strategy to Induce Proteasomal Down-Regulation

Acylpeptide hydrolase (APEH), one of the four members of the prolyl oligopeptidase class, catalyses the removal of N-acylated amino acids from acetylated peptides and it has been postulated to play a key role in protein degradation machinery. Disruption of protein turnover has been established as an effective strategy to down-regulate the ubiquitin-proteasome system (UPS) and as a promising approach in anticancer therapy. Here, we illustrate a new pathway modulating UPS and proteasome activity through inhibition of APEH. To find novel molecules able to down-regulate APEH activity, we screened a set of synthetic peptides, reproducing the reactive-site loop of a known archaeal inhibitor of APEH (SsCEI), and the conjugated linoleic acid (CLA) isomers. A 12-mer SsCEI peptide and the trans10-cis12 isomer of CLA, were identified as specific APEH inhibitors and their effects on cell-based assays were paralleled by a dose-dependent reduction of proteasome activity and the activation of the pro-apoptotic caspase cascade. Moreover, cell treatment with the individual compounds increased the cytoplasm levels of several classic hallmarks of proteasome inhibition, such as NFkappaB, p21, and misfolded or polyubiquitinylated proteins, and additive effects were observed in cells exposed to a combination of both inhibitors without any cytotoxicity. Remarkably, transfection of human bronchial epithelial cells with APEH siRNA, promoted a marked accumulation of a mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), herein used as a model of misfolded protein typically degraded by UPS. Finally, molecular modeling studies, to gain insights into the APEH inhibition by the trans10-cis12 CLA isomer, were performed. Our study supports a previously unrecognized role of APEH as a negative effector of proteasome activity by an unknown mechanism and opens new perspectives for the development of strategies aimed at modulation of cancer progression.

Insights into the mechanism of interaction between trehalose-conjugated beta-sheet breaker peptides and Aβ(1–42) fibrils by molecular dynamics simulations

An attractive strategy to contrast the Alzheimer disease (AD) is represented by the development of β-sheet breaker peptides (BSB). β-sheet breakers constitute a class of compounds which have shown a good efficacy in preventing the Aβ fibrillogenesis; however, their mechanism of action has not been precisely understood. In this context, we have studied the structural basis underlying the inhibitory effect of Aβ(1-42) fibrillogenesis explicated by two promising trehalose-conjugated BSB peptides using an all-atom molecular dynamics (MD) approach. Our simulations suggest that the binding on the two protofibril ends occurs through different binding modes. In particular, binding on the odd edge (chain A) is guided by a well defined hydrophobic cleft, which is common to both ligands. Moreover, targeting chain A entails a significant structure destabilization leading to a partial loss of β structure and is an energetically favoured process. A significant contribution of the trehalose moiety to the stability of the complexes emerged from our results. The energetically favoured hydrophobic cleft detected on chain A could represent a good starting point for the design of new molecules with improved anti-aggregating features.

Conformational studies of chiral d-Lys-PNA and achiral PNA system in binding with DNA or RNA through a molecular dynamics approach

The growing interest in peptide nucleic acid (PNA) oligomers has led to the development of a very wide variety of PNA derivatives. Among others, the introduction of charged chiral groups on a PNA oligomer has proven effective in improving DNA binding ability, complexation direction and cellular uptake. In particular, the introduction of three adjacent chiral monomers based on D-Lys in the middle of the PNA sequence (D-Lys-PNA) has produced noteworthy results in modulating the directionality of the binding with the DNA complementary strand and in mismatch detection. Here, through a molecular dynamics approach, a comparative study has been carried out to investigate the structural properties that drive the interaction of the chiral D-Lys-PNA and the corresponding achiral PNA system with DNA as well as RNA complementary strands, starting from the crystal structure of D-Lys-PNA in complex with DNA. The results obtained complement experimental data and indicate that the binding with the RNA molecule, compared to DNA, is differently affected by the addition of three D-Lys groups on the PNA backbone, suggesting that this modification could be taken into account for the development of new PNA-based molecules able to discriminate between DNA and RNA.

Probing the Peptidylglycine α-Hydroxylating Monooxygenase Active Site with Novel 4-Phenyl-3-butenoic Acid Based Inhibitors

Specific inhibition of the copper‐containing peptidylglycine α‐hydroxylating monooxygenase (PHM), which catalyzes the post‐translational modification of peptides involved in carcinogenesis and tumor progression, constitutes a new approach for combating cancer. We carried out a structure–activity study of new compounds derived from a well‐known PHM substrate analogue, the olefinic compound 4‐phenyl‐3‐butenoic acid (PBA). We designed, synthesized, and tested various PBA derivatives both in vitro and in silico. We show that it is possible to increase PBA affinity for PHM by appropriate functionalization of its aromatic nucleus. Compound 2 d, for example, bears a meta‐benzyloxy substituent, and exhibits better inhibition features (Ki=3.9 μM, kinact/Ki=427 M−1 s−1) than the parent PBA (Ki=19 μM, kinact/Ki=82 M−1 s−1). Docking calculations also suggest two different binding modes for PBA derivatives; these results will aid in the development of further PHM inhibitors with improved features.

Cysteine co-oxidation process driven by native peptide folding: an example on HER2 receptor model system

Synthetic models of receptors that have relevant biological roles are valuable tools for studying receptors itself and the corresponding ligands. Their properties can be validated at first by their capacity to fold in solution under native-like conditions and to assume conformations structurally and functionally equivalent to those in the native receptor. In this context, a new strategy to prepare the two-fragments synthetic receptor model HER2-DIVMP, an independent structural and functional motif of HER2, has been developed and the folding properties have been investigated. The strategy is based on a one-step cysteine co-oxidation procedure in slightly alkaline aqueous buffers, whereby the two separate peptide chains are allowed to self-assemble in solution. Under these conditions, the two chains spontaneously form the expected heterodimer with the correct pattern of disulfide bridges. To gain insights on the folding mechanism, we investigated the folding of two scrambled variants of the constituent peptide chains.

Eco-friendly microwave-assisted protocol to prepare hyaluronan-fatty acid conjugates and to induce their self-assembly process.

An environmentally sustainable and energy-efficient synthetic process has been developed to prepare hyaluronan-based nano-sized material. It consists in a microwave-promoted acylation of the hydroxyl function of the polysaccharide with natural fatty acids, performed under solvent-free conditions. The efficient interaction of the solid reagents with the MW radiation accounts for the obtained high yielded products. The self-assembly process of the obtained compounds very fast occurred in an aqueous medium under MW-radiation, thus allowing the development of a green protocol for the nano-particles preparation.

Peptide Fragments of Odin-Sam1: Conformational Analysis and Interaction Studies with EphA2-Sam

Odin is a protein belonging to the ANKS family, and has two tandem Sam domains. The first, Odin‐Sam1, binds to the Sam domain of the EphA2 receptor (EphA2‐Sam); this interaction could be crucial for the regulation of receptor endocytosis and might have an impact on cancer. Odin‐Sam1 associates with EphA2‐Sam by adopting a “mid‐loop/end‐helix” model. In this study three peptide sequences, encompassing the mid‐loop interacting portion of Odin‐Sam1 and its C‐terminal α5 helix, were designed. Their conformational properties were analyzed by CD and NMR. In addition, their abilities to interact with EphA2‐Sam were investigated by SPR studies. The peptides adopt a predominantly disordered state in aqueous buffer, but a higher helical content is evident in the presence of the cosolvent trifluoroethanol. Dissociation constants towards EphA2‐Sam were in the high micromolar range. The structural findings suggest further routes for the design of potential anti‐cancer therapeutics as inhibitors of EphA2‐Sam heterotypic interactions.

Silver nanoparticles functionalized with a fluorescent cyclic RGD peptide: a versatile integrin targeting platform for cells and bacteria

The arginine–glycine–aspartic acid (RGD) peptide sequence is known to specifically interact with integrins, which are chief receptors participating at various stages of cancer disease and in bacterial adhesion/invasion processes. In particular, vitronectin receptor (αvβ3) and fibronectin receptor (α5β1) integrins are involved respectively in tumour cell targeting and bacteria internalization inhibition. Silver nanoparticles (AgNPs) have elicited a lot of interest as a theranostic platform, owing to their unique optoelectronic as well as self-therapeutic properties as bactericides. The goal of this work was the comprehensive physicochemical characterization of a hybrid peptide–metal nanoparticle biointerface fabricated by the immobilisation, through thiol chemistry, of a fluorescent cyclic RGD peptide onto AgNPs of 13 nm diameter. RGD peptide-functionalized AgNPs were investigated by a multi-technique approach, including various spectroscopic (XPS, FTIR and UV-visible), spectrometric (ToF-SIMS) and microscopic (SEM, TEM, AFM) methods as well as dynamic light scattering and ζ-potential measurements. Proof-of-work experiments by confocal microscopy imaging of the cellular uptake by human neuroblastoma SH-SY5Y and chronic myelogenous leukaemia K562 cells, overexpressing respectively αvβ3 and α5β1 integrins, demonstrated a receptor-specific activity of the RGD peptide-functionalised AgNPs, which make them very promising as a multifaceted platform in applications with cells and bacteria.

Determination of the secondary structure of peptides in the presence of Gram positive bacterium S. epidermidis cells

A protocol to run CD experiments in the presence of Gram positive bacterial cells was set up. The secondary structure of the antimicrobial peptides temporin L and TB_KKG6A with S. epidermidis cells was determined. CD experiments were also reported for temporin L in the presence of Gram negative bacterium E. coli cells.

A biocompatible process to prepare hyaluronan-based material able to self-assemble into stable nano-particles

New self-assembled nano-particles were developed by chemical conjugation of natural fatty acids to the backbone of hyaluronan (HA). The chemical structure and the self-association behavior of them were studied by FT-IR, NMR, fluorescence and dynamic light scattering. The HA derivatives form stable spherical shape aggregates, as assessed by transmission electron microscopy.