A cellular screening platform, stably expressing DENV2 NS5, defines a novel anti-DENV mechanism of action of Apigenin based on STAT2 activation

Abstract

Type I interferon (IFN–I) evasion by Dengue virus (DENV) is key in DENV pathogenesis. The non-structural protein 5 (NS5) antagonizes IFN-I response through the degradation of the signal transducer and activator of transcription 2 (STAT2). We developed a K562 cell-based platform, for high throughput screening of compounds potentially counteracting the NS5-mediated antagonism of IFN-I signaling. Upon a screening with a library of 1220 approved drugs, 3 compounds previously linked to DENV inhibition (Apigenin, Chrysin, and Luteolin) were identified. Luteolin and Apigenin determined a significant inhibition of DENV2 replication in Huh7 cells and the restoration of STAT2 phosphorylation in both cell systems. Apigenin and Luteolin were able to stimulate STAT2 even in the absence of infection. Despite the “promiscuous” and “pan-assay-interfering” nature of Luteolin, Apigenin promotes STAT2 Tyr 689 phosphorylation and activation, highlighting the importance of screening for compounds able to interact with host factors, to counteract viral proteins capable of dampening innate immune responses. A) K562 cells were engineered by means of a double retroviral transduction followed by cell sorting to express the luciferase reporter gene under the control of a ISRE sequence-containing promoter, non targeting shRNA to mimic PRR stimulation elicited by a RNA virus infection and either GFP or GFP-NS5. After TPA-mediated megakaryocytes differentiation, compounds are administered before IFN-α stimulation. A day later, the luciferase assay is performed. Signals of different intensities are obtained in the luminescence assay, lower for cells containing the GFP-NS5 fusion protein compared to cells expressing GFP alone. The addition of a compound X to cells expressing GFP-NS5 fusion protein and potentially capable of inhibiting the anti-IFN function of the viral protein, should, at least partially, restore the signal obtained in control cells in response to IFN-α treatment. B) Schematic workflow of the 1220 compound library screening in a miniaturized format. At day 1, cells are seeded at a specific density, differetiated with TPA, and then seeded again in 384 cell plates. After 72 h of differentiation (day 4), cells were treated with the 1220 compound library and after 2 h (day 4 + 2 h) were stimulated with IFN-α for further 24 h. At day 5, cell were analyzed for light emission upon lysis, using a microplate luminometer, C) Possible mechanism of action of the identified anti-NS5 compound Apigenin as DENV NS5 inhibitor. Apigenin can determine the phosphorylation of STAT2 even in the absence of DENV infection or virus-induced IFN-I stimulation. Pre-activated STAT-2 may be more resistant to Dengue NS5 inhibition/degradation and, therefore, more prone to interact with activated STAT1 and interferon regulatory factor 9 (IRF-9) to promote interferon stimulated genes (ISGs) and therefore the antiviral state, All images of this graphical abstract were created with BioRender.com . • We developed a luciferase-based K562 cell platform to identify inhibitors of anti–IFN–I activity of DENV NS5 protein. • Apigenin, Chrysin, and Luteolin were identified screening a library of approved drugs, with Luteolin behaving as PAINS. • Apigenin determines STAT2 Tyr 689 phosphorylation in Huh7 cells not infected and infected with DENV2. • The host factor STAT2 can be a valuable targets for therapeutic interventions aimed at inhibiting NS5 anti-IFN-I activity. • Our reporter K562 cellular platform, due to the successful miniaturization, can be exploited for future HTS campaigns.

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