Discovery of a Potent Tat-Binding Antiretroviral Compound Using a Two-Step Screening Approach

The transcription of the HIV-1 long-terminal repeat (LTR), driven by the viral transactivator Tat, represents a distinct and druggable step in HIV-1 replication. Targeting Tat-mediated transcription is a promising antiretroviral strategy due to its mechanistic distinction from host cell transcription, although specificity remains a challenge due to potential off-target effects. To identify selective inhibitors, a two-step screening approach was employed. A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was first used to identify compounds that disrupt Tat-trans-activation response (TAR) RNA interactions, yielding 655 initial hits, including five 2-phenyl acrylate derivatives with >50% inhibition. A secondary library of 194 structurally related compounds was then screened using a dual-luciferase reporter assay to evaluate transcriptional inhibition and cytotoxicity. From this, 46 compounds met selection criteria (>50% F-Luc inhibition, > 90% R-Luc activity, > 90% viral inhibition, and >70% cell viability). Among them, methyl (E)-2-(2-((4-(decyloxy)phenoxy)methyl)phenyl)-3-methoxyacrylate showed the highest potency, with IC₅₀ and EC₅₀ values of 1.44 and 0.83 μM, respectively. Time-of-addition (TOA) assays indicated inhibition of the Tat-dependent transcription phase. Surface plasmon resonance analysis revealed binding to the Tat peptide but not to TAR RNA, suggesting the binding target for 019854-B06. Immunoblotting and coimmunoprecipitation showed that 019854-B06 neither promotes Tat degradation nor disrupts the Tat/CycT1 complex, supporting a Tat-centric, nondegradative mechanism. Therefore, this study identifies a novel 2-phenyl acrylate-based inhibitor of Tat-mediated HIV-1 transcription through an integrated biophysical and functional screening strategy.