Tissue factor derived β-hairpin peptides that bind and inhibit FVII activity.

The formation of the Tissue Factor(TF):Factor VII(FVII) complex is a pivotal event that initiates coagulation; targeting this early step allows for the prevention of the subsequent cascade amplification driven by positive feedback loops. For this reason, the TF:FVII complex is attracting increasing interest as a potential therapeutic target for regulating the coagulation cascade in a specific and timely manner. In order to generate TF-mimics capable of inhibiting this protein-protein interaction, we have designed four small cyclic peptides that simulate a TF region containing the two antiparallel β-strands: 106-110 (RVFSY) and 123-128 (EPLYEN).

Solvent-Free procedure of an A9 Peptide Dimer Exhibiting Specific HER2 Receptor Binding: Fluorescence Spectroscopy Evaluation of the Enhanced Binding Affinity.

HER2-expressing cancers currently benefit from targeted therapies, including monoclonal antibodies and antibody-drug conjugates that specifically bind to the extracellular domain of the receptor. Peptides targeting HER2 represent promising candidates for the development of alternative molecular drugs. In this study, we report a dimeric version of the previously validated A9 peptide as a ligand specifically targeting HER2.

CD38 restrains the activity of extracellular cGAMP in a model of multiple myeloma.

2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) is the endogenous agonist of STING; as such, cGAMP has powerful immunostimulatory activity, due to its capacity to stimulate type I interferon-mediated immunity. Recent evidence indicates that cancer cells, under certain conditions, can release cGAMP extracellularly, a phenomenon currently considered important for therapeutic responses and tumor rejection. Nonetheless, the mechanisms that regulate cGAMP activity in the extracellular environment are still largely unexplored.

Recombinant humanized Fab fragments targeting the CFC domain of human Cripto-1.

In the era of immunotherapy, the targeting of disease-specific biomarkers goes hand in hand with the development of highly selective antibody-based reagents having optimal pharmacological/toxicological profiles. One interesting and debated biomaker for several types of cancers is the onco-fetal protein Cripto-1 that is selectively expressed in many solid tumours and has been actively investigated as potential theranostic target. Starting from previously described anti-CFC/Cripto-1 murine monoclonal antibodies, we have moved forward to prepare the humanized recombinant Fabs which have been engineered so as to bear an MTGase site useful for a one-step site-specific labelling.

Production in Bacteria and Characterization of Engineered Humanized Fab Fragment against the Nodal Protein.

Drug development in recent years is increasingly focused on developing personalized treatments based on blocking molecules selective for therapeutic targets specifically present in individual patients. In this perspective, the specificity of therapeutic targets and blocking agents plays a crucial role. Monoclonal antibodies (mAbs) and their surrogates are increasingly used in this context thanks to their ability to bind therapeutic targets and to inhibit their activity or to transport bioactive molecules into the compartments in which the targets are expressed.

Unveiling CD59-Antibody Interactions to Design Paratope-Mimicking Peptides for Complement Modulation.

CD59 is an abundant immuno-regulatory human protein that protects cells from damage by inhibiting the complement system. CD59 inhibits the assembly of the Membrane Attack Complex (MAC), the bactericidal pore-forming toxin of the innate immune system. In addition, several pathogenic viruses, including HIV-1, escape complement-mediated virolysis by incorporating this complement inhibitor in their own viral envelope.

Oxidized Substrates of APEH as a Tool to Study the Endoprotease Activity of the Enzyme.

APEH is a ubiquitous and cytosolic serine protease belonging to the prolyl oligopeptidase (POP) family, playing a critical role in the processes of degradation of proteins through both exo- and endopeptidase events. Endopeptidase activity has been associated with protein oxidation; however, the actual mechanisms have yet to be elucidated. We show that a synthetic fragment of GDF11 spanning the region 48-64 acquires sensitivity to the endopeptidase activity of APEH only when the methionines are transformed into the corresponding sulphoxide derivatives.

Identification and validation of viral antigens sharing sequence and structural homology with tumor-associated antigens (TAAs).

Background: The host's immune system develops in equilibrium with both cellular self-antigens and non-self-antigens derived from microorganisms which enter the body during lifetime. In addition, during the years, a tumor may arise presenting to the immune system an additional pool of non-self-antigens, namely tumor antigens (tumor-associated antigens, TAAs; tumor-specific antigens, TSAs).

Methods: In the present study, we looked for homology between published TAAs and non-self-viral-derived epitopes.

A structure-based approach for the development of a bicyclic peptide acting as a miniaturized anti-CD55 antibody.

CD55 is a major regulator of the complement system, a complex network of proteins that cooperate to clear tissue and blood pathogens from the organism. Indeed, overexpression of CD55 is associated with many diseases and is connected to the resistance mechanisms exhibited by several cancers towards immunotherapy approaches. High level of CD55 expression on tumour cells renders it a good target for both imaging and immunotherapy.

Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry.

Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb's epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents.

Selective inhibition of acylpeptide hydrolase in SAOS-2 osteosarcoma cells: is this enzyme a viable anticancer target?

Serine hydrolases play crucial roles in many physiological and pathophysiological processes and a panel of these enzymes are targets of approved drugs. Despite this, most of the human serine hydrolases remain poorly characterized with respect to their biological functions and substrates and only a limited number of in vivo active inhibitors have been so far identified. Acylpeptide hydrolase (APEH) is a member of the prolyl-oligopeptidase class, with a unique substrate specificity, that has been suggested to have a potential oncogenic role.

Insights into the Interaction Mechanism of DTP3 with MKK7 by Using STD-NMR and Computational Approaches.

GADD45β/MKK7 complex is a non-redundant, cancer cell-restricted survival module downstream of the NF-kB survival pathway, and it has a pathogenically critical role in multiple myeloma, an incurable malignancy of plasma cells. The first-in-class GADD45β/MKK7 inhibitor DTP3 effectively kills MM cells expressing its molecular target, both in vitro and in vivo, by inducing MKK7/JNK-dependent apoptosis with no apparent toxicity to normal cells. DTP3 combines favorable drug-like properties, with on-target-specific pharmacology, resulting in a safe and cancer-selective therapeutic effect; however, its mode of action is only partially understood.

Generation and testing of engineered multimeric Fabs of trastuzumab.

Recombinant antibodies fragments in several new formats are routinely investigated and used in diagnostic and therapeutic applications as anti-cancers molecules. New antibody formats are generated to compensate the need for multispecificity and site-specific introduction of fluorescent dyes, cytotoxic payloads or for generating semisynthetic multimeric molecules. Fabs of trastuzumab bearing transglutaminase (MTG) reactive sites were generated by periplasmic expression in E.

The role of Nodal and Cripto-1 in human oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a common epithelial malignancy of the oral cavity. Nodal and Cripto-1 (CR-1) are important developmental morphogens expressed in several adult cancers and are associated with disease progression. Whether Nodal and CR-1 are simultaneously expressed in the same tumor and how this affects cancer biology are unclear.

Structure-based design of small bicyclic peptide inhibitors of Cripto-1 activity.

Bicyclic peptides assembled around small organic scaffolds are gaining an increasing interest as new potent, stable and highly selective therapeutics because of their uncommon ability to specifically recognize protein targets, of their small size that favor tissue penetration and of the versatility and easiness of the synthesis. We have here rationally designed bicyclic peptides assembled around a common tri-bromo-methylbenzene moiety in order to mimic the structure of the CFC domain of the oncogene Cripto-1 and, more specifically, to orient in the most fruitful way the hot spot residues H120 and W123. Through the CFC domain, Cripto-1 binds the ALK4 receptor and other protein partners supporting uncontrolled cell growth and proliferation.

Oral Delivery of a Tetrameric Tripeptide Inhibitor of VEGFR1 Suppresses Pathological Choroid Neovascularization.

Age-related macular degeneration (AMD) is the primary cause of blindness in advanced countries. Repeated intravitreal delivery of anti-vascular endothelial growth factor (VEGF) agents has represented an important advancement for the therapy of wet AMD with significative results in terms of blindness prevention and partial vision restore. Nonetheless, some patients are not responsive or do not attain significant visual improvement, intravitreal injection may cause serious complications and important side effects have been reported for the prolonged block of VEGF-A.

Investigating the oxidative refolding mechanism of Cripto-1 CFC domain.

Using a combined approach based on MS, enzyme digestion and advanced MD studies we have determined the sequential order of formation of the three disulfide bridges of the Cripto-1 CFC domain. The domain has a rare pattern of bridges and is involved in the recognition of several receptors. The bridge formation order is C1-C4, C3-C5, C2-C6, however formation of C1-C4 plays no roles for the formation of the others.

Development of conformational antibodies targeting Cripto-1 with neutralizing effects in vitro.

Human Cripto-1 (Cripto-1), the founding member of the EGF-CFC superfamily, is a key regulator of many processes during embryonic development and oncogenesis. Cripto-1 is barely present or even absent in normal adult tissues while it is aberrantly re-expressed in various tumors. Blockade of the CFC domain-mediated Cripto-1 functions is acknowledged as a promising therapeutic intervention point to inhibit the tumorigenic activity of the protein.

Intrinsic structural versatility of the highly conserved 412-423 epitope of the Hepatitis C Virus E2 protein.

HCV infection is a major threaten for human health as it affects hundreds of million people worldwide. Here we investigated the conformational properties of the 412-423 fragment of the envelope E2 protein, one of the most immunogenic regions of the virus proteome whose characterization may provide interesting insights for anti-HCV vaccine development. The spectroscopic characterization of the polypeptide unravels its unexpected tendency to form amyloid-like aggregates.

Probing the interaction interface of the GADD45β/MKK7 and MKK7/DTP3 complexes by chemical cross-linking mass spectrometry.

GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45β physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45β/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45β-overexpressing cancer cells.

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.

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).

A Synthetic MUC1 Glycopeptide Bearing βGalNAc-Thr as a Tn Antigen Isomer Induces the Production of Antibodies against Tumor Cells.

This study presents the synthesis of the novel protected O-glycosylated amino acid derivatives 1 and 2, containing βGalNAc-SerOBn and βGalNAc-ThrOBn units, respectively, as mimetics of the natural Tn antigen (αGalNAc-Ser/Thr), along with the solid-phase assembly of the glycopeptides NHAcSer-Ala-Pro-Asp-Thr[αGalNAc]-Arg-Pro-Ala-Pro-Gly-BSA (3-BSA) and NHAcSer-Ala-Pro-Asp-Thr[βGalNAc]-Arg-Pro-Ala-Pro-Gly-BSA (4-BSA), bearing αGalNAc-Thr or βGalNAc-Thr units, respectively, as mimetics of MUC1 tumor mucin glycoproteins. According to ELISA tests, immunizations of mice with βGalNAc-glycopeptide 4-BSA induced higher sera titers (1:320 000) than immunizations with αGalNAc-glycopeptide 3-BSA (1:40 000). Likewise, flow cytometry assays showed higher capacity of the obtained anti-glycopeptide 4-BSA antibodies to recognize MCF-7 tumor cells.