Andrea Caporale

Benzoxaborole as a new chemotype for carbonic anhydrase inhibition.

In this paper we report the synthesis of a series of benzoxaborole derivatives, their inhibition properties against some carbonic anhydrases (CAs), recognized as important drug targets, and the characterization of the binding mode of these molecules to the CA active site. Our data provide the first experimental evidence that benzoxaboroles can be efficiently used as CA inhibitors.

Conformational features and binding affinities to Cripto, ALK7 and ALK4 of Nodal synthetic fragments

Nodal, a member of the TGF‐β superfamily, is a potent embryonic morphogen also implicated in tumor progression. As for other TGF‐βs, it triggers the signaling functions through the interaction with the extracellular domains of type I and type II serine/threonine kinase receptors and with the co‐receptor Cripto. Recently, we reported the molecular models of Nodal in complex with its type I receptors (ALK4 and ALK7) as well as with Cripto, as obtained by homology modeling and docking simulations. From such models, potential binding epitopes have been identified. To validate such hypotheses, a series of mutated Nodal fragments have been synthesized. These peptide analogs encompass residues 44–67 of the Nodal protein, corresponding to the pre‐helix loop and the H3 helix, and reproduce the wild‐type sequence or bear some modifications to evaluate the hot‐spot role of modified residues in the receptor binding.

Structural and biochemical insights of CypA and AIF interaction

The Cyclophilin A (CypA)/Apoptosis Inducing Factor (AIF) complex is implicated in the DNA degradation in response to various cellular stress conditions, such as oxidative stress, cerebral hypoxia-ischemia and traumatic brain injury. The pro-apoptotic form of AIF (AIF(Δ1-121)) mainly interacts with CypA through the amino acid region 370–394. The AIF(370-394) synthetic peptide inhibits complex formation in vitro by binding to CypA and exerts neuroprotection in a model of glutamate-mediated oxidative stress. Here, the binding site of AIF(Δ1-121) and AIF(370-394) on CypA has been mapped by NMR spectroscopy and biochemical studies, and a molecular model of the complex has been proposed. We show that AIF(370-394) interacts with CypA on the same surface recognized by AIF(Δ1-121) protein and that the region is very close to the CypA catalytic pocket. Such region partially overlaps with the binding site of cyclosporin A (CsA), the strongest catalytic inhibitor of CypA. Our data point toward distinct CypA structural determinants governing the inhibitor selectivity and the differential biological effects of AIF and CsA, and provide new structural insights for designing CypA/AIF selective inhibitors with therapeutic relevance in neurodegenerative diseases.

The LQSP tetrapeptide is a new highly efficient substrate of microbial transglutaminase for the site‐specific derivatization of peptides and proteins

Transglutaminases catalyze transglutamination reactions on glutamines. Transglutaminases are largely exploited for modifying proteins in pharmaceutical, food, and other biotechnological applications. A library of synthetic peptides has been designed, prepared, and screened to identify new peptide substrates. The new substrates are then used in TGAse‐mediated conjugation reactions to engraft synthons onto biomolecules. These peptide substrates confer the bioactive peptides and proteins with new properties. We have identified an optimized substrate named LQSP, which is recognized and processed by microbial TGAse with a strikingly higher efficiency compared to the well‐known TQGA sequence. The new substrate has been used to selectively modify prototypical bioactive peptides and proteins with fluoresceine or recognition motifs. We show that, where a reactive lysine is available, proteins and peptides of relevant therapeutic interest, can be selectively and smoothly modified in order to incorporate new functions such as fluorescent labels, recognition units, or reactive groups.

Trifluoroacetylated tyrosine-rich D-tetrapeptides have potent antioxidant activity

Graphical abstract Figure. No Caption available. HighlightsIdentification of trifluoroacetylated d‐peptides with potent anti‐oxidant activity.Tetrapeptides have the common CF3—CO‐tyr‐tyr moiety on the N‐terminal side.SAR studies identify CF3—tyr‐tyr as the core responsible of antioxidant activity.Structural studies suggest that activity correlates with tridimensional structure.Peptides protect keratinocytes from lipids peroxidation. Abstract The term “oxidative stress” indicates a set of chemical reactions unleashed by a disparate number of events inducing DNA damage, lipid peroxidation, protein modification and other effects, which are responsible of altering the physiological status of cells or tissues. Excessive Reactive Oxygen Species (ROS) levels may accelerate ageing of tissues or induce damage of biomolecules thus promoting cell death or proliferation in dependence of cell status and of targeted molecules. In this context, new antioxidants preventing such effects may have a relevant role as modulators of cell homeostasis and as therapeutic agents. Following an approach of peptide libraries synthesis and screening by an ORACFL assay, we have isolated potent anti‐oxidant compounds with well‐defined structures. Most effective peptides are N‐terminally trifluoroacetylated (CF3) and have the sequence tyr‐tyr‐his‐pro or tyr‐tyr‐pro‐his. Slight changes in the sequence or removal of the CF3 group strongly reduced antioxidant ability, suggesting an active role of both the fluorine atoms and of peptide structure. We have determined the NMR solution structures of the active peptides and found a common structural motif that could underpin the radical scavenging activity. The peptides protect keratinocytes from exogenous oxidation, thereby from potential external damaging cues, suggesting their use as skin ageing protectant and as cell surviving agents.

Monoclonal antibodies against pools of mono- and polyacetylated peptides selectively recognize acetylated lysines within the context of the original antigen

ABSTRACT Post-translational modifications (PTMs) strongly influence the structure and function of proteins. Lysine side chain acetylation is one of the most widespread PTMs, and it plays a major role in several physiological and pathological mechanisms. Protein acetylation may be detected by mass spectrometry (MS), but the use of monoclonal antibodies (mAbs) is a useful and cheaper option. Here, we explored the feasibility of generating mAbs against single or multiple acetylations within the context of a specific sequence. As a model, we used the unstructured N-terminal domain of APE1, which is acetylated on Lys27, Lys31, Lys32 and Lys35. As immunogen, we used a peptide mixture containing all combinations of single or multi-acetylated variants encompassing the 24–39 protein region. Targeted screening of the resulting clones yielded mAbs that bind with high affinity to only the acetylated APE1 peptides and the acetylated protein. No binding was seen with the non-acetylated variant or unrelated acetylated peptides and proteins, suggesting a high specificity for the APE1 acetylated molecules. MAbs could not finely discriminate between the differently acetylated variants; however, they specifically bound the acetylated protein in mammalian cell extracts and in intact cells and tissue slices from both breast cancers and from a patient affected by idiopathic dilated cardiomyopathy. The data suggest that our approach is a rapid and cost-effective method to generate mAbs against specific proteins modified by multiple acetylations or other PTMs.

Automatic procedures for the synthesis of difficult peptides using oxyma as activating reagent: A comparative study on the use of bases and on different deprotection and agitation conditions

HIGHLIGHTSA systematic study for optimizing yield and purity of so‐called difficult sequences using oxyma/DIC as first choice cheap coupling agent.Morpholine/DBU are identified as preferred deprotection agents over other reagents commonly used in solid phase peptide synthesis.Collidine is identified as preferred base over other organic bases commonly used in solid phase synthesis of strongly aggregating peptides.Persisted vortexing during the coupling steps has a role in improving yield and purity of difficult peptides. ABSTRACT Solid‐Phase Peptide Synthesis (SPPS) is a rapid and efficient methodology for the chemical synthesis of peptides and small proteins. However, the assembly of peptide sequences classified as “difficult” poses severe synthetic problems in SPPS for the occurrence of extensive aggregation of growing peptide chains which often leads to synthesis failure. In this framework, we have investigated the impact of different synthetic procedures on the yield and final purity of three well‐known “difficult peptides” prepared using oxyma as additive for the coupling steps. In particular, we have comparatively investigated the use of piperidine and morpholine/DBU as deprotection reagents, the addition of DIPEA, collidine and N‐methylmorpholine as bases to the coupling reagent. Moreover, the effect of different agitation modalities during the acylation reactions has been investigated. Data obtained represent a step forward in optimizing strategies for the synthesis of “difficult peptides”.

Unique true predicted neoantigens (TPNAs) correlates with anti-tumor immune control in HCC patients

BackgroundA novel prediction algorithm is needed for the identification of effective tumor associated mutated neoantigens. Only those with no homology to self wild type antigens are true predicted neoantigens (TPNAs) and can elicit an antitumor T cell response, not attenuated by central tolerance. To this aim, the mutational landscape was evaluated in HCV-associated hepatocellular carcinoma.MethodsLiver tumor biopsies and adjacent non-tumor liver tissues were obtained from 9 HCV-chronically infected subjects and subjected to RNA-Seq analysis. Mutant peptides were derived from single nucleotide variations and TPNAs were predicted using two prediction servers (e.g. NetTepi and NetMHCstabpan) by comparison with corresponding wild-type sequences, non-related self and pathogen-related antigens. Immunological confirmation was obtained in preclinical as well as clinical setting.ResultsThe development of such an improved algorithm resulted in a handful of TPNAs despite the large number of predicted neoantigens. Furthermore, TPNAs may share homology to pathogen’s antigens and be targeted by a pre-existing T cell immunity. Cross-reactivity between such antigens was confirmed in an experimental pre-clinical setting. Finally, TPNAs homologous to pathogen’s antigens were found in the only HCC long-term survival patient, suggesting a correlation between the pre-existing T cell immunity specific for these TPNAs and the favourable clinical outcome.ConclusionsThe new algorithm allowed the identification of the very few TPNAs in cancer cells, and those targeted by a pre-existing immunity strongly correlated with long-term survival. Only such TPNAs represent the optimal candidates for immunotherapy strategies.

Targeting VEGF receptors with non-neutralizing cyclopeptides for imaging applications

Pharmacological strategies aimed at preventing cancer growth are in most cases paralleled by diagnostic investigations for monitoring and prognosticating therapeutic efficacy. A relevant approach in cancer is the suppression of pathological angiogenesis, which is principally driven by vascular endothelial growth factor (VEGF) or closely related factors and by activation of specific receptors, prevailingly VEGFR1 and VEGFR2, set on the surface of endothelial cells. Monitoring the presence of these receptors in vivo is henceforth a way to predict therapy outcome. We have designed small peptides able to bind and possibly antagonize VEGF ligands by targeting VEGF receptors. Peptide systems have been designed to be small, cyclic and to host triplets of residues known to be essential for VEGF receptors recognition and we named them ‘mini-factors’. They have been structurally characterized by CD, NMR and molecular dynamics (MD) simulations. Mini-factors do bind with different specificity and affinity VEGF receptors but none blocks receptor activity. Following derivatization with suitable tracers they have been employed as molecular probes for sensing receptors on cell surface without affecting their activity as is usually observed with other binders having neutralizing activity.

Structural insights into the interaction of a monoclonal antibody and Nodal peptides by STD-NMR spectroscopy

Nodal is a growth factor expressed during early embryonic development, but reactivated in several advanced-stage cancers. Targeting of Nodal signaling, which occurs via the binding to Cripto-1 co-receptor, results in inhibition of cell aggressiveness and reduced tumor growth. The Nodal binding region to Cripto-1 was identified and targeted with a high affinity monoclonal antibody (3D1). By STD-NMR technique, we investigated the interaction of Nodal fragments with 3D1 with the aim to elucidate at atomic level the interaction surface. Data indicate with high accuracy the antibody-antigen contact atoms and confirm the information previously obtained by immune-enzymatic methods. Main residues contacted by 3D1 are P46, V47, E49 and E50, which belong to the Nodal loop involved in the interaction with the co-receptor.