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??M) that also inhibited viral replication at moderate micromolar concentration (EC50?=?15.16??M) and inhibited CA assembly in vitro. Based on the structure of TX-1918, an additional compound with an antiviral EC50 of 6.57??M and cellular cytotoxicity CC50 of 102.55??M 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.

SUBMITTER: Zhang DW 

PROVIDER: S-EPMC6901019 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature