Here, we present the study of this MLN4924 scaffold including its putative mode of action at the molecular level. Here we report a screening method based on a cellular phenotypic assay utilizing image analysis. Using a SAR131675 clinical trial well-defined experimental setup, this procedure was suitable to characterize active molecules against any steps of the HIV-1 life cycle. When comparing our developed technology to others, based on viral cytopathogenicity, this high-content approach allows visualization of compound activity while simultaneously assessing cellular toxicity in a single assay. Furthermore, due to the microscopy readout, additional biologically relevant markers could be incorporated toward high-content screening. This strategy may be successfully extended to develop screening assays for inhibitors of other viruses. Starting from a relatively small compound library of both diverse and focused scaffolds, we were able to identify hits and find a unique potent compound, IPK1, that does not exhibit cellular toxicity. Further analysis of this biologically active chemical was performed, and we identified its properties. The antiviral activity of the IPK1 compound was in the nanomolar range, which is comparable with known antiviral molecules available on the market. In addition, clinical isolated reverse transcriptase mutants were also inhibited in in vitro replicative assays. Finally, we tried to elucidate the mode of action of this compound by testing classical resistance mutations against NNRTI molecules and biochemical properties. According to the resistance profile obtained with point mutations, a relevant computer modeling was performed. The docking of IPK1 and analogs in the NNRTI pocket correlated with the in vitro assay data. The interaction model corroborates the binding of TMC125 and additional reference compounds such as nevirapine and MK4965. One of the main differences between those co-crystal structures is the involvement of the K103 residue that interacts with nevirapine. In both cases, modeled interactions of IPK1 had a similar binding mode in the pocket of the wild-type reverse transcriptase. Based on the TMC125/K103N mutant co-crystal structure, we were able to model the interaction of IPK1 with the K103N mutant that correlates with the observed resistance level in vitro. We did not observe resistance induced by the mutation V106A, which also corroborates the in silico IPK1 binding mode. In addition, we were able to examine the effect of some chemical modifications that resulted in the loss of biological activity in agreement with the predictions from the docking model.