Identification and characterization of human interferon-alpha antagonist compounds through a wish cell line-based reporter gene assay

BURGI, MARÍA DE LOS MILAGROS; PRIETO, CLAUDIO; CABRERA, N; HERNÁNDEZ, P; CERECETTO, H; GONZÁLEZ, M; ETCHEVERRIGARAY, MARINA; KRATJE, RICARDO; BOLLATI-FOGOLÍN, MARIELA; OGGERO, M.

Barcelona

Congreso; 24th Meeting of the European Society for Animal Cell Technology (ESACT); 2015

European Society for Animal Cell Technology (ESACT)

Interferons (IFNs) are essential glycoproteins in the immune defense. Nevertheless, different genetic alterations may lead to IFN-α overproduction in human autoimmune diseases, where the cytokine represents the etiology of these illnesses. Thus, blocking the IFN-α increment becomes noteworthy to control them. Consequently, this work proposes the identification and characterization of new antagonists of IFN‑α. The screening of compounds modulating the IFNs biological activity requires versatile, easy and robust methods. Then, a reporter gene assay (RGA), developed in our lab, was used to identify IFN-α activity modulator compounds using a WISH‑Mx2/Egfp reporter cell line where the eGFP gene is driven by the specific human type I IFN (hIFN-I) inducible Mx2 promoter. Consequently, the eGFP percentage, induced by hIFN-I addition, is directly correlated with the cytokine. This RGA allowed the analysis of a complete synthetic chemical library, in a low throughput screening mode. Successfully, some compounds capable to decrease the hIFN-α activity were identified. Finally, twelve leads were selected and their specific properties were analyzed.Experimental approachThe identification of new molecules that antagonize the IFN-α activity was carried out using the WISH cell-based RGA. Their characterization in terms of toxicity, effective dose, action on antiviral and antiproliferative activity (AVA and APA) of IFN, effect on cell cycle and evaluation of their combined action was accomplished.Results and discussionInitially, Z´ factor was measured to evaluate the RGA quality. This factor was estimated in 0.82 ± 0.07, assuring an excellent assay.The non toxic minimum concentration for each compound was calculated and further used for the RGA. Accordingly, twenty-seven compounds demonstrated an inhibitory action on hIFN-α2a activity and twelve out of them, considering their higher responses, were selected to be characterized. Therefore, their effect on AVA and APA was accomplished. These responses were properly correlated with those from the RGA with the exception of the APA where few compounds did not show effects. This result could be attributed to the selectivity of each compound to particularly modulate any of the IFN activities or, also, it depended on the sensitivity of each assay. Furthermore, their effect on cell cycle was studied. Two compounds stopped the cell cycle in G1 phase impeding the DNA synthesis whereas other two increased the S phase demonstrating an increase in DNA synthesis and cell proliferation. Sixteen compounds combinations were also evaluated. All combinations showed higher inhibition of IFN activity than each compound by itself, demonstrating a cooperative effect. The inhibition of AVA and APA of IFN by the compound combinations correlated with that obtained by RGA. Therefore, the reliability of RGA was confirmed. Additional investigations showed their residual effects keeping their inhibitory action on IFN activity after the combinations were removed. Besides, all of them were able to reverse the activation pathway of the IFN activity. Taken as a whole, this search for compounds which can block IFN-α activity shows a big potential in view of their therapeutic implications.