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Keywords 22Cell-based RGA, hIFN-I, Natural and synthetic compounds, Low Throughput 23Screening, Mx2/EGFP 24 2 Abstract 25Type I Interferons (IFNs-I) are species-specific glycoproteins which play an 26 important role as primary defence against viral infections and that can also 27 modulate the adaptive immune system. In some autoimmune diseases, 28 interferons (IFNs) are over-produced. IFNs are widely used as 29 biopharmaceuticals for a variety of cancer indications, chronic viral diseases, 30and for their immuno-modulatory action in patients with multiple sclerosis; 31 therefore, increasing their therapeutic efficiency and decreasing their side 32 effects is of high clinical value. In this sense, it is interesting to find molecules 33 that can modulate the activity of IFNs. In order to achieve that, it was necessary 34 to establish a simple, fast and robust assay to analyze numerous compounds 35 simultaneously. We developed four reporter gene assays (RGAs) to identify IFN 36 activity modulator compounds by using WISH-Mx2/EGFP, HeLa-Mx2/EGFP, 37 A549-Mx2/EGFP, and HEp2-Mx2/EGFP reporter cell lines (RCLs). All of them 38 present a Z' factor higher than 0.7. By using these RGAs, natural and synthetic 39 compounds were analyzed simultaneously. A total of 442 compounds were 40 studied by the Low Throughput Screening (LTS) assay using the four RCLs to 41 discriminate between their inhibitory or enhancing effects on IFN activity. Some 42 of them were characterized and 15 leads were identified. Finally, one promising 43 candidate with enhancing effect on IFN-α/-β activity and five compounds with 44 inhibitory effect were described. 45

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Crosslinked casein micelles bound paclitaxel as enzyme activated intracellular drug delivery systems for cancer therapy,

Activated mPEG carbonates are important reagents that have been widely used for the PEGylation of several peptides and proteins by means of stable urethane linkages. In fact, mPEG-N-hydroxysuccinimidyl carbonate and mPEG-p-nitrophenyl carbonate are among the most used reagents in PEGylation technology. However, the synthesis and storage of these reagents are not always easy to resolve. With the aim of surpassing some of the drawbacks associated with the use of activated mPEG-carbonates we have prepared and evaluated a new mPEG-carbonylimidazolium iodide, which can be used for the conjugation of the NH 2 group by means of urethane linkages, as an interesting alternative to the known reagents. It is noteworthy that the novel reagent is prepared by a simple two-step procedure under mild experimental conditions. Moreover, we performed a detailed study of the conjugation reaction of interferon-a2b with the carbonylimidazolium derivative, and evaluated the conjugate in in vitro and in vivo studies.

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Neurological disorders affect millions of people causing behavior-cognitive disabilities.Nowadays they have no effective treatment. Human erythropoietin (hEPO) has been clinically used because of its neurotrophic and cytoprotective properties. However, the erythropoietic activity (EA) should be considered as a side effect. Some analogs like non-sialylated EPO, carbamylated EPO, or EPO peptides have been developed showing different weaknesses: erythropoiesis preservation, low stability, potential immunogenicity, or fast clearance. Herein, we used a novel strategy that blocks the EA but preserves hEPO neurobiological actions. N-glycoengineering was accomplished to add a new glycosylation site within the hEPO sequence responsible for its EA. hEPOderivatives were produced by CHO.K1 cells, affinity-purified and functionally analyzed studying their in vitro and in vivo EA, their in vitro neuronal plasticity in hippocampal neurons and their neuroprotective action by rescuing hippocampal neurons from apoptosis. Muteins Mut 45_47 (K45 > N45 + N47 > T47), Mut 104 (S104 > N104), and Mut 151_153 (G151 > N151 + K153 > T153) lost their EA but preserved their neuroprotection activity and enhanced neuroplasticity more efficiently than hEPO. Interestingly, Mut 45_47 resulted in a promising candidate to explore as neurotherapeutic considering not only its biopotency but also its pharmacokinetic potential due to the hyperglycosylation.

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