A HTRF based competitive binding assay for screening specific inhibitors of HIV-1 capsid assembly targeting the C-Terminal domain of capsid

Abstract

Due to its multifaceted essential roles in virus replication and extreme genetic fragility, the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein is a valued therapeutic target. However, CA is as yet unexploited clinically, as there are no antiviral agents that target it currently on the market. To facilitate the identification of potential HIV-1 CA inhibitors, we established a homogeneous time-resolved fluorescence (HTRF) assay to screen for small molecules that target a biologically active and specific binding pocket in the C-terminal domain of HIV-1 CA (CA CTD). The assay, which is based on competition of small molecules for the binding of a known CA inhibitor (CAI) to the CA CTD, exhibited a signal-to-background ratio (S/B) > 10 and a Z' value > 0.9. In a pilot screen of three kinase inhibitor libraries containing 464 compounds, we identified one compound, TX-1918, as a low micromolecular inhibitor of the HIV-1 CA CTD-CAI interaction (IC₅₀ = 3.81 μM) that also inhibited viral replication at moderate micromolar concentration (EC₅₀ = 15.16 μM) and inhibited CA assembly in vitro. Based on the structure of TX-1918, an additional compound with an antiviral EC₅₀ of 6.57 μM and cellular cytotoxicity CC₅₀ of 102.55 μM was obtained from a compound similarity search. Thus, the HTRF-based assay has properties that are suitable for screening large compound libraries to identify novel anti-HIV-1 inhibitors targeting the CA CTD.

Keywords: Capsid C-Terminal domain; Drug discovery; HIV-1 capsid; HTRF; TX-1918.