EGFR phosphorylation and KI-67 accumulation in the nuclei was unaffected by tecovirimat treatment (Figure 5(C4,D4)) and thus was on a level similar to the untreated controls (Figure 5(C1,D1)). host-directed inhibitors afatinib and cetuximab were approx. 100-fold more efficient against CPXV in the 3D infection model, similar to previous results with gefitinib. In summary, inhibition of EGFR-signaling downregulates virus replication comparable to established virus-directed antivirals. However, in contrast to virus-directed inhibitors, in vitro efficacy of host-directed antivirals might be seriously affected by cell cultivation. Results obtained for afatinib and cetuximab suggest that screening of such drugs in standard monolayer culture might underestimate their potential as Nilutamide antivirals. (CPXV) infections is strongly enhanced in 3D cultures of primary normal human epithelial keratinocytes (NHEK) compared to the respective monolayer cultures. Gefitinib intracellularly targets the human epidermal growth factor receptor (EGFR) and thus inhibits EGFR-dependent signaling via viral homologs of the epidermal growth factor (EGF), which is essential for poxvirus replication [10]. For example, (VACV), which encodes the growth factor (VGF), an EGF homologue, hijacks the EGF signaling pathway to spread more efficiently in vivo as well as in vitro [12]. The real potential of gefitinib as an antiviral therapeutic interfering with this pathway became clear only through the use of 3D cell cultures as a first line in vitro identification tool and would have been underestimated and potentially dismissed by screening in conventional monolayer cultures [10]. This finding therefore may be of great relevance because so far there is tecovirimat as the only FDA-approved treatment option for poxvirus infections [13]. Different orthopoxviruses are genetically highly similar. They are of interest for public health because (CPXV) and (MPXV) are zoonotic viruses, while there Nilutamide is also a potential risk of (VARV) being used as a biological weapon [14,15,16]. As another benefit, treatment with gefitinib represents a host-directed antiviral approach which minimizes the probability of viral escape mutations in contrast to the virus-directed tecovirimat where escape mutations have already been shown in cell culture [17,18]. Because gefitinib is already FDA-approved for treatment of specific forms of non-small cell lung cancer (NSCLC), repurposing of this drug would cause significantly fewer costs for clinical trials than approving Nilutamide Nilutamide new compounds [19,20]. Besides gefitinib, which is a first-generation receptor tyrosine kinase inhibitor (RTKI), there are several other FDA-approved EGFR-targeting drugs for treatment of different types of cancer whose antiviral potential still has to be elucidated. Among them, there are RTKIs of the first (erlotinib), second (afatinib), and third (osimertinib) generation which have different binding affinities and specificities for the EGFR. While members of the first generation bind reversibly to the intracellular receptor tyrosine kinase (RTK) domain of wild-type EGFR and receptor forms with activating mutations, substances from the second generation bind the EGFR irreversibly without preference for the mutation state [21,22]. The third-generation members, however, bind preferentially mutated RTK domains in an irreversible manner [23]. Another possibility to inhibit EGFR signaling is represented by approved therapeutic antibodies like cetuximab which bind to the EGFR extracellularly and thus could already prevent the binding of viral EGF homologs and subsequent downstream signaling [24]. In this study, we used different EGFR-targeting drugs which were already FDA-approved for treatment of different types of cancer as potential novel host-directed antiviral substances against poxvirus infections. Studies were performed in 3D cell cultures of NHEK which were, compared to our previous studies, optimized regarding Rabbit Polyclonal to NXPH4 culture format and time to qualify them for high-throughput approaches. To evaluate a possible influence of the culture method on the drug efficacy, as already shown for gefitinib, data from 3D culture were compared to the respective conventional monolayer culture. To analyze whether this effect of cell culture on antiviral activity is a phenomenon specific to just one inhibitor blocking signaling of EFGR or if it is a more general feature, the effect of tradition conditions within the cell-targeted antiviral activity was analyzed with several EGFR inhibitors with different modes of action and compared to virus-directed treatment with tecovirimat and cidofovir [17,25]. 2. Materials and Methods 2.1. Cells and Tradition Conditions Pooled main normal human being epidermal keratinocytes (NHEK; PromoCell, Heidelberg, Germany) from juvenile foreskin were cultivated in keratinocyte growth medium 2 (KGM2 ready-to-use; PromoCell). Cells were cultured at 37 C inside a 5% CO2 humidified atmosphere and regularly screened for the absence of mycoplasma contamination by qPCR [26]. 2.2. Generation of 3D Cell Cultures on Decellularized Biological.