Identification and characterization of human interferon alpha inhibitors through a WISH cell line-based reporter gene assay.

Interferons (IFNs) are important glycoproteins which can stimulate or inhibit up to three hundred different genes encoding proteins involved in antiviral defense mechanisms, inflammation, adaptive immunity, angiogenesis and among other processes. Nevertheless, different genetic alterations may lead to interferon alpha (IFN-α) overproduction in human autoimmune diseases like systemic lupus erythematosus. As a consequence, IFN-α is a central molecule whose activity must be regulated to block their harmful effect on those disorders where the endogenous cytokine production constitutes the etiology of the illnesses. In this work, we evaluate the biological activity of eighty-eight compounds, from our own chemo-library, to find potential IFN-α inhibitors by using a reporter gene assay (RGA) WISH-Mx2/EGFP. We identified some compounds able to modulate negatively the IFN-α activity. The most active IFN-α inhibitors were further studied achieving promising results. In addition, some combinations of the most active compounds were analyzed accomplishing a stronger effect to decrease the IFN-α activity than each compound alone. Furthermore, the complete inhibition of the cytokine activity was reached with some combinations of compounds.

Novel erythropoietin-based therapeutic candidates with extra N-glycan sites that block hematopoiesis but preserve neuroplasticity.

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. hEPO-derivatives 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.

Artichoke extracts with potential application in chemoprevention and inflammatory processes

Abstract The aim of this work is to study three cultivars of artichoke (Cynara cardunculus var. scolymus): Gauchito, Guri and Oro Verde in terms of their in vitro chemoprevention and anti-inflammatory properties. These cultivars show good productive performance. The phenolic composition of their fresh leaves and edible bracts was analyzed by high performance liquid chromatography and high resolution mass spectrometry (HPLC-HRMS), showing mainly caffeoylquinic acids and flavonoids. Caffeoylquinic acids were quantified and the highest content was found in Gauchito cultivar. In this cultivar, the content of dicaffeoylquinic acids in fresh bracts was six times higher than that in fresh leaves (10064.5 ± 378.3 mg/kg versus 1451.0 ± 209.3 mg/kg respectively). Luteolin flavonoids were detected in leaves. The extracts from fresh bracts and leaves were assessed in their in vitro bioactivity against human neuroblastoma cells (SH-SY5Y). Inhibition of SH-SY5Y cells proliferation by Gauchito and Guri leaf extracts (8 µg/mL) was higher than 50 %. The leaf extracts of the same cultivars showed an inhibitory effect on human interferon IFN-I, decreasing its activity 50% at 40 µg/mL. Interestingly, the bract extracts did not show in vitro bioactivity at these concentrations, nor did the pure compounds chlorogenic acid, cynarin, apigenin and luteolin (at 2 µg/mL). These results suggest that Gauchito and Guri leaf extracts have potential for human neuroblastoma chemoprevention and treatment of inflammatory processes.

Screening and characterization of molecules that modulate the biological activity of IFNs-I.

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