Design of biodegradable bi-compartmental microneedles for the stabilization and the controlled release of the labile molecule collagenase for skin healthcare.

Polymeric microneedles (MNs) have emerged as a novel class of drug delivery system thanks to their ability in penetrating the skin with no pain, encapsulate active proteins and in particular, proposed bicompartimental MNs can tune protein release

Destabilisation, aggregation, toxicity and cytosolic mislocalisation of nucleophosmin regions associated with acute myeloid leukemia

Nucleophosmin (NPM1) is a multifunctional protein that is implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments of NPM1 in its biological activities, we dissected the C-terminal domain (CTD) into its helical fragments. Here we focus the attention on the third helix of the NPM1-CTD in its wild-type (H3 wt) and AML-mutated (H3 mutA and H3 mutE) sequences. Conformational studies, by means of CD and NMR spectroscopies, showed that the H3 wt peptide was partially endowed with an a-helical structure, but the AML-sequences exhibited a lower content of this conformation, particularly the H3 mutA peptide. Thioflavin T assays showed that the H3 mutE and the H3 mutA peptides displayed a significant aggregation propensity that was confirmed by CD and DLS assays. In addition, we found that the H3 mutE and H3 mutA peptides, unlike the H3 wt, were moderately and highly toxic, respectively, when exposed to human neuroblastoma cells. Cellular localization experiments confirmed that the mutated sequences hamper their nucleolar accumulation, and more importantly, that the helical conformation of the H3 region is crucial for such a localization

Destabilisation, aggregation, toxicity and cytosolic mislocalisation of nucleophosmin regions associated with acute myeloid leukemia

Nucleophosmin (NPM1) is a multifunctional protein that is implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments of NPM1 in its biological activities, we dissected the C-terminal domain (CTD) into its helical fragments. Here we focus the attention on the third helix of the NPM1-CTD in its wild-type (H3 wt) and AML-mutated (H3 mutA and H3 mutE) sequences. Conformational studies, by means of CD and NMR spectroscopies, showed that the H3 wt peptide was partially endowed with an a-helical structure, but the AML-sequences exhibited a lower content of this conformation, particularly the H3 mutA peptide. Thioflavin T assays showed that the H3 mutE and the H3 mutA peptides displayed a significant aggregation propensity that was confirmed by CD and DLS assays. In addition, we found that the H3 mutE and H3 mutA peptides, unlike the H3 wt, were moderately and highly toxic, respectively, when exposed to human neuroblastoma cells. Cellular localization experiments confirmed that the mutated sequences hamper their nucleolar accumulation, and more importantly, that the helical conformation of the H3 region is crucial for such a localization

Identification of peptide inhibitors of protein-protein interactions as new potential drugs

The innovative concept that proteins exert their function by establishing intricate contact networks rather than acting as independent entities is of outstanding importance both in the comprehension of molecular mechanisms underlying biological processes and in modern drug discovery. Protein-protein interactions are the ensemble of fine tuned recognition events that take place at protein surfaces. These contacts frequently involve large protein surface areas, which comprise several contact sites but, alternatively, they can be mediated by “hot spots” represented by few crucial amino acids. Many studies have highlighted the role of peptide molecules as powerful tools for the characterization and the regulation of these interactions. Peptides are particularly suitable as models for proteins that are not fully folded in their isolated states but that achieve conformational stability upon the formation of complexes with other partners. The characterization of the structural determinants of protein-protein recognition represents an important step for the development of molecular entities able to modulate these interactions. The identification of compounds able to modulate protein-protein interactions represents a major goal for modern drug discovery. Both structure-based design and screening of combinatorial collections have been successfully applied, in this instance often small peptides and peptidomimetics have been selected as inhibitors of protein complexes. In many cases, the lack of sufficient structural information on protein interacting regions renders the combinatorial approach the most suitable method, but the use of completely random peptide libraries often produces redundant chemical species that complicate the deconvolution phases of screening and puzzles the way to the identification of active compounds. Thus, in order to avoid the synthesis of too large combinatorial libraries in two case-studies during this doctoral thesis a simplified approach was followed in it libraries of short peptides, made of a small subset of amino acids were prepared and screened. This approach has been applied to the identification of inhibitors of the protein complex between PED/PEA15 and PLD1 that is involved in the molecular mechanisms of insulin resistance occurring in type 2 diabetes. This study led to the identification of several short peptide inhibitors rich in aromatic and H-donor donor features through the employment of ELISA and SPR binding assay. These short sequences will be converted in small organic molecules following medicinal chemistry rules and actually can be considered as interesting starting scaffold for the design of new therapeutic agents in type 2 diabetes. On the other hand, the screening of small but focused libraries is increasingly used to target specific systems. This method has been applied to the identification of mimicking peptides of KIR region of SOCS1 that is involved in JAK2 recognition. This interaction plays a key role in molecular mechanisms of psoriasis. After the identification of a shorter SOCS1-KIR domain, through an Ala-scanning investigation, focused peptide library in which non essential residues were randomized in a simplified approach were built and assayed. This screening led to the selection of new peptides more effective than natural sequences; indeed one decapeptide resulted binds to JAK2 with a dissociation constant in the nanomolar range and was able to block STAT1 phosphorylation. Actually cellular and in vivo experiments are ongoing in order to investigate its therapeutic applicability. Rational approaches for the identification of protein interacting regions provide deeper insights into the recognition molecular mechanism This rational approach has been applied to other two projects in this thesis. The complex between proteins CypA and AIF is involved in apoptotic cell death, but at structural level only a docked theoretical model is available. In this model few molecular contacts between proteins are detectable, thus a set of sequences were designed in order to map crucial residues of AIF domains involved in the interaction with CypA. The selective disruption of the CypA-AIF complex with these peptides mimicking the protein surfaces was analysed by competition and direct binding SPR experiments. These experiments allowed to identify an active sequence that, once properly modified, could be as a potential suppress the pro-apoptotic action of AIF. This minimalistic approach was successfully applied to another protein complex IB-α/Tat, two proteins involved in the replication of HIV-1. Here IB-α-based peptides were designed and tested in SPR assays. Also in this project the interacting region of IB-α was restricted to a shorter region (26 residues) representing a promising starting point for the development of molecules able to block HIV-1 infection. The characterization of IB-α peptides has provided novel insights into the intrinsic properties of IB-α fragments and structural determinants of IB-α/Tat recognition. In the investigation of protein folding determinants and then in the design of specific modulators of proteic folding and biological function, the protein reassembly through host-chemistry strategy could represent a valid approach. During a period of six months spent at Cambridge University, a protein splitting approach was applied to the small a regulator protein Ubiqutin. Two dissected protein regions were obtained by chemical synthesis: at the C-terminal fragment (residues 47-76) was inserted a glycine as a spacer and then a tryptophan indole group, for the binding to the macrocycle CB[8]; instead at the C-terminus of N-terminal fragment (residues 1-46), a cysteine was inserted to allow the connection with "methylviologen", which has an aromatic ring strongly depleted of electrons for the formation of the ternary complex with CB[8]. The preliminary synthetic results obtained resulted promising for the future development of this project that will be structural studies of individual fragments and the structural and functional characterization of the ternary complex

Identification of Inhibitors of Biological Interactions Involving Intrinsically Disordered Proteins

Protein–protein interactions involving disordered partners have unique features and represent prominent targets in drug discovery processes. Intrinsically Disordered Proteins (IDPs) are involved in cellular regulation, signaling and control: they bind to multiple partners and these high-specificity/low-affinity interactions play crucial roles in many human diseases. Disordered regions, terminal tails and flexible linkers are particularly abundant in DNA-binding proteins and play crucial roles in the affinity and specificity of DNA recognizing processes. Protein complexes involving IDPs are short-lived and typically involve short amino acid stretches bearing few “hot spots”, thus the identification of molecules able to modulate them can produce important lead compounds: in this scenario peptides and/or peptidomimetics, deriving from structure-based, combinatorial or protein dissection approaches, can play a key role as hit compounds. Here, we propose a panoramic review of the structural features of IDPs and how they regulate molecular recognition mechanisms focusing attention on recently reported drug-design strategies in the field of IDPs

The Characterization of Multifaceted PREP1 Peptides Provides Insights into Correlations between Spectroscopic and Structural Properties of Amyloid‐like Assemblies

: The widespread ability of proteins and peptides to self-assemble by forming cross-β structure is one of the most significant discoveries in structural biology. Intriguingly, the cross-β association of proteins/peptides may generate intricate supramolecular architectures with uncommon spectroscopic properties. We have recently characterized self-assembled peptides extracted from the PREP1 protein that are endowed with interesting structural/spectroscopic properties. We here demonstrate that the green fluorescence emission of the peptide PREP1[117-132] (λem ~ 520 nm), can be induced by excitation with UV radiation. The associated unusually large Stokes shift (Δλ ~ 150 nm) represents, to the best of our knowledge, the first evidence of an internal resonance energy transfer in amyloid-like structures, where the blue emission of some assemblies becomes the excitation radiation for others. Moreover, the characterization of PREP1[117-132] variants provides insights into the sequence/structure and structure/spectroscopic properties relationships. Our data suggests that the green fluorescence is plausibly associated with antiparallel β-sheet states of the peptide whereas parallel β-sheet assemblies are only endowed with blue fluorescence. Notably, the different PREP1[117-132] variants also form assemblies characterized by distinct morphologies. Indeed, the parent peptide and single mutants form compact but structured aggregates whereas most of the double mutants exhibit elongated and highly extended fibers

Hydrogel particles-on-chip (HyPoC): a fluorescence micro-sensor array for IgG immunoassay

Novel microparticles have generated growing interest in diagnostics for potential sensitivity and specificity in biomolecule detection and for the possibility to be integrated in a micro-system array as a lab-on-chip. Indeed, bead-based technologies integrated in microfluidics could speed up incubation steps, reduce reagent consumption and improve accessibility of diagnostic devices to non-expert users. To limit non-specific interactions with interfering molecules and to exploit the whole particle volume for bioconjugation, hydrogel microparticles, particularly polyethylene glycol-based, have emerged as promising materials to develop high-performing biosensors since their network can be functionalized to concentrate the target and improve detection. However, the limitations in positioning, trapping and mainly fine manipulation of a precise number of particles in microfluidics have largely impaired point-of-care applications. Herein, we developed an on-chip sandwich immunoassay for the detection of human immunoglobulin G in biological fluids. The detection system is based on finely engineered cleavable PEG-based microparticles, functionalized with specific monoclonal antibodies. By changing the particle number, we demonstrated tuneable specificity and sensitivity (down to 3 pM) in serum and urine. Therefore, a controlled number of hydrogel particles have been integrated in a microfluidic device for on-chip detection (HyPoC) allowing for their precise positioning and fluid exchange for incubation, washing and target detection. HyPoC dramatically decreases incubation time from 180 minutes to one minute and reduces washing volumes from 3.5 ml to 90 mu L, achieving a limit of detection of 0.07 nM (with a dynamic range of 0.07-1 nM). Thus, the developed approach represents a versatile, fast and easy point-of-care testing platform for immunoassays

Cell mechanosensory recognizes ligand compliance at biomaterial interface

Cells activate signalling through ligand-receptor bonds by sensing the mechanical properties of the surrounding extracellular matrix (ECM). Ligands, indeed, have to withstand the pulling force elicited by cell receptors through focal adhesions (FAs). On this basis, we developed functional ligands to be simply adsorbed on surfaces and constituted by a two-domain peptide: one derived from ECM proteins and available to receptors to offer biochemical cues, and another adsorbed on material to withstand the tension upon receptor engagement. Tuneable compliance of the anchoring domain of the peptide ligand was verified by single peptide analysis through molecular dynamics and adsorption measurements. We showed that the highest adsorbed peptides combined with integrin cell-binding motifs allow for the cell recognition and polarization with larger mature FA areas. On the contrary, the lowest adsorbed sequences did not provide mechanical resistance to the integrin pulling action, leading to more rounded cells with smaller FA areas. This evidence demonstrates that cell mechanosensory can discriminate ligands on surfaces and should be considered as a criterion in ligand design for material bioactivation