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As a proof of principle of a functional screen for Medfly tsl putative genes, we planned to use a Drosophila tsl mutant strain, elav, encoding a neuronal RNA-binding protein, and perform embryos injections of a BAC genomic tsl+ fragment (100 kb long) to confirm a transient functional rescue. We first confirmed that the elavts1 stock was inviable at 25oC, by placing vials of flies at this non-permissive temperature. Adults did not die immediately and some even lived long enough to lay a few eggs. Some of these survived long enough to hatch as larvae and a small number survived to adulthood. However, no F1 adults lived long enough to propagate a viable stock at 25oC. Adults were also allowed to lay eggs at the permissive temperature of 18oC. After 24 hours of egg-laying, vials were cultured in parallel at 18oC and 25oC. Larvae and a small number of adults were observed following continuous culture at 25oC. A transient heat-shock of 1 hr at 37 oC were also applied to 24 hour embryo collections. Again, larvae and a small number of adults were observed in the vials. Therefore, under these conditions elavts does not offer the proper temperature-sensitivity required to test the ability of a BAC to rescue ts-induced embryonic lethality. We started to explore alternative Drosophila embryonic tsl mutant strains to be used within the next months. Medfly XX-only embryos injections of recombinant Medfly synthetic MOY protein induced phenotypic and molecular intersexuality, confirming the maintenance of function of the synthetic molecule. A first series of Medfly XX-only injections of recombinant BdMOY and BoMOY proteins led to mild transient shift of Cctra splicing toward male-specific one, and in XX/XY embryos not phenotypic effects at adult stages. A first series of embryos injections of a MoY transgenic vector (PiggyBac based) led to a dozen adult flies which were crossed and the subsequent progeny was screened for transgenics. No fluorescent individuals in F1 were detected. We attempted to integrate by Cas9 a dsRED transgene into MoY locus to obtain detectable XY females to be maintained by crossing them with XX reverted males (by Cctra RNAi). We obtained few XY reverted females but lacking integration of the tramsgene.

Background The host’s immune system develops in equilibrium with both cellular self-antigens and non-selfantigens 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 (tumorassociated antigens, TAAs; tumor-specific antigens, TSAs). Methods In the present study, we looked for homology between published TAAs and non-self-viralderived epitopes. Bioinformatics analyses and ex vivo immunological validations have been performed. Results Surprisingly, several of such homologies have been found. Moreover, structural similarities between paired TAAs and viral peptides as well as comparable patterns of contact with HLA and T cell receptor (TCR) α and β chains have been observed. Therefore, the two classes of non-self-antigens (viral antigens and tumor antigens) may converge, eliciting cross-reacting CD8+ T cell responses which possibly drive the fate of cancer development and progression. Conclusions An established antiviral T cell memory may turn out to be an anticancer T cell memory, able to control the growth of a cancer developed during the lifetime if the expressed TAA is similar to the viral epitope. This may ultimately represent a relevant selective advantage for patients with cancer and may lead to a novel preventive anticancer vaccine strategy.

Peptides are emerging as an increasingly dependable class of therapeutics in the treatment of cancer and metabolic and cardiovascular diseases, which are all areas of high interest to the pharmaceutical industry. The global market for peptide therapeutics was valued at about 25 billion USD in 2018 and is estimated to reach 57.2 billion USD by the end of 2027. Here, we describe a method for the screening and deconvolution of combinatorial peptide libraries to discover compounds that target discrete signaling components of the NF-κB pathway. Recently, we used this approach to specifically disrupt the interaction between the JNK-activating kinase, MKK7, and the NF-κB-regulated antiapoptotic factor, GADD45β, in multiple myeloma (MM). We showed that the GADD45β/MKK7 complex is a functionally critical survival module downstream of NF-κB in MM cells and as such provides an attractive therapeutic target to selectively inhibit NF-κB antiapoptotic signaling in cancer cells. By integrating the library screening and deconvolution methods described here with a rational chemical optimization strategy, we developed the first-in-class GADD45β/MKK7 inhibitor, DTP3 (a D-tripeptide), which is now being trialed in MM and diffuse large B-cell lymphoma (DLBCL) patients. The same drug discovery approach may be generally applied to therapeutically target other key components of the NF-κB pathway in cancers beyond MM and DLBCL, as well as in non-malignant NF-κB-driven diseases.

Graphical abstract, Highlights • DTP3 eliminates any viable MM cells in mice upon i.v. bolus administration. • DTP3 exhibits highly favourable PK and ADME profiles, with long plasma half life. • DTP3 had no adverse effect on vital organ systems in GLP safety pharmacology studies. • DTP3 was tolerated in repeat-dose 28-day toxicity studies with wide exposure margins., Aberrant NF-κB activity drives oncogenesis and cell survival in multiple myeloma (MM) and many other cancers. However, despite an aggressive effort by the pharmaceutical industry over the past 30 years, no specific IκBα kinase (IKK)β/NF-κB inhibitor has been clinically approved, due to the multiple dose-limiting toxicities of conventional NF-κB-targeting drugs. To overcome this barrier to therapeutic NF-κB inhibition, we developed the first-in-class growth arrest and DNA-damage-inducible (GADD45)β/mitogen-activated protein kinase kinase (MKK)7 inhibitor, DTP3, which targets an essential, cancer-selective cell-survival module downstream of the NF-κB pathway. As a result, DTP3 specifically kills MM cells, ex vivo and in vivo, ablating MM xenografts in mice, with no apparent adverse effects, nor evident toxicity to healthy cells. Here, we report the results from the preclinical regulatory pharmacodynamic (PD), safety pharmacology, pharmacokinetic (PK), and toxicology programmes of DTP3, leading to the approval for clinical trials in oncology. These results demonstrate that DTP3 combines on-target-selective pharmacology, therapeutic anticancer efficacy, favourable drug-like properties, long plasma half-life and good bioavailability, with no target-organs of toxicity and no adverse effects preclusive of its clinical development in oncology, upon daily repeat-dose administration in both rodent and non-rodent species. Our study underscores the clinical potential of DTP3 as a conceptually novel candidate therapeutic selectively blocking NF-κB survival signalling in MM and potentially other NF-κB-driven cancers.

The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a ?-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape.

Most biological processes involve permanent and temporary interactions between different proteins: protein complexes often play key roles in human diseases and, as a consequence, molecules that prevent protein-protein interactions can be potential new therapeutic agents to treat diseases. Here, we describe a simplified approach by which small synthetic peptide libraries were screened to identify the inhibitors of the complex between phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes15 (PED/PEA15) and D4??, a functional domain of the phospholipase D1, that is involved in the molecular mechanisms of insulin resistance occurring in type 2 diabetes. By using an enzyme-linked immunosorbent assay (ELISA)-based screening, performed on a fully automated platform, we analyzed two simplified peptide libraries in a positional scanning format. This screening led to the identification of small peptides able to inhibit PED/PEA15-D4?? interaction. The selection of inhibitors was carried out employing combined competitive and direct experiments, through ELISA and surface plasmon resonance techniques, providing peptides with IC 50 values in the micromolar range. Our results showed that the protein complex PED/PEA15-D4?? is susceptible to peptides having H-donor groups and aromatic rings on specific positions. These small sequences can be considered as promising scaffolds that could be converted into higher-affinity inhibitor compounds. By a simplified approach small synthetic peptide libraries were screened to identify inhibitors of the complex between PED/PEA15 and D4?? that is involved in molecular mechanisms of insulin resistance in type 2 diabetes. Small peptide sequences having H-donor groups and aromatic rings on specific positions are able to inhibit the complex and are promising scaffolds that could be converted into higher-affinity inhibitor compounds

Fc epsilon RI alpha found on the surface of mast cells and basophiles mediates allergic diseases, anaphylaxis and asthma through binding of IgE. Disrupting this interaction with anti-IgE mAbs has proven an efficient approach to control these diseases. The crystallographic structure of the complex formed between the IgE-Fc and Fc epsilon RI alpha extracellular domain has shown that recognition is mediated by residues in the second Ig-like domain of the receptor (132) and in the loop connecting the D1 and D2 domains. In an attempt to obtain specific IgE antagonists, we have designed and prepared a polypeptide named IgE-Trap that partially reproduces the IgE receptor-binding sites and binds with micromolar affinity to soluble IgE. The polypepticle contains loops C'-E [residues 129-134] and F-G [residues 151-1611 from the D2 domain joined by a linker, and loop B-C [residues 110-113]. Peptide binding to IgE has been assessed by SPR analyses and the data fit with a biphasic model of interaction, in agreement with the two-site mechanism reported for the native receptor. The polypeptide binds to immobilized IgE in a dose-dependent manner with a K(D) estimated to be around 6 mu M, while it does not recognize IgG nor IgA. Polypeptide sub-domains involved in IgE binding have also been defined, showing that loop C-E connected to loop B-C, but also the isolated loop B-C alone suffice to bind immunoglobulins E with high selectively though with reduced affinity compared to IgE-Trap. ELISA and cytometric assays on RBL2H3 cells demonstrate that the interacting peptides are able to displace the binding of IgE to receptor, confirming affinity and specificity of these ligands and suggesting a potential application as modulators of disorders associated with inappropriate IgE production. (C) 2009 Elsevier Ltd. All rights reserved.

Cripto is one of the key regulators of embryonic stem cells (ESCs) differentiation into cardiomyocites vs neuronal fate. Cripto-/- murine ESCs have been utilized to investigate the molecular mechanisms underlying early events of mammalian lineage differentiation. 2D/LC-MS/MS and a label-free LC-MS approaches were used to qualitatively and quantitatively profile the cripto-/- ESC proteome, providing an integral view of the alterations induced in stem cell functions by deleting the cripto gene. Cripto is one of the key regulators of embryonic stem cells (ESCs) differentiation into cardiomyocites vs neuronal fate. Cripto-/- murine ESCs have been utilized to investigate the molecular mechanisms underlying early events of mammalian lineage differentiation. 2D/LC-MS/MS and a label-free LC-MS approaches were used to qualitatively and quantitatively profile the cripto-/- ESC proteome, providing an integral view of the alterations induced in stem cell functions by deleting the cripto gene. © 2009 American Chemical Society.

Using a commercially available time-resolved fluorescence resonance energy transfer (TR-FRET)—based assay for IKKβ, the authors have automated the assay procedure on a high-throughput screening station to carry out screening campaigns on multiwell plates. They have determined the Z' factor and optimized volumes, times, and time-resolved fluorescence parameters. They have also compared 2 kinases with different fusion tags, the influence of different enzyme/substrate ratios and of DMSO presence at different concentration. The authors found that glutathione S-transferase (GST)—fused IKKβ shows better signal-to-noise (S/N) ratios over the poly-histidine-tagged variant. The substrate can be used at 50 nM with optimal performances when the enzyme is used at 2 nM. DMSO at 0.2% and 1% only slightly affects the S/N ratio, whereas when used at 2%, the final concentration deriving from a 50-fold dilution from a 5-mM stock solution in pure solvent, S/N undergoes a decrease of about 15%. Under the optimized conditions, the assay Z' factor calculated over 192 data points has an optimized value of 0.881 and allows the testing of 94 molecules in quadruplicate in 140 min.

Secondary structure motifs and small protein domains can act as building blocks that are isolated and investigated to gain insights into protein global structure but can also modulate interactions with external partners. Most progress has been made in this field using synthetic peptides. Fragmentation of folded proteins by proteolytic enzymes that act preferentially on exposed and less structured sites can help to isolate shorter polypeptides with preserved secondary and tertiary structures that mimic the original protein architecture. Such mols. can be used as probes for structural studies and as tools for in vitro assays to select active fragments useful as agonists or antagonists of the original protein or as scaffolds for the design of more potent and selective ligands. This simple but effective proteolytic methodol. has been successfully applied to det. antagonists of protein-protein interactions, allowing the identification of inhibitors with high efficacy and specificity. Here, several studies are presented, including the complex between phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15)and phospholipase 1 (PLD1), believed to play a relevant role in the insulin resistance mechanism in phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-over-expressing tissues, the self-assocn. of BCL10 caspase recruitment domain that mediates a protein oligomerization process responsible for NF-.kappa.B activation, and the self-assocn. of growth arrest and DNA damage-inducible factor 45.beta. (GADD45.beta.), a major player of the endogenous NF-.kappa.B-mediated resistance to apoptosis.