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

Antiviral Res. 2019 Sep:169:104544. doi: 10.1016/j.antiviral.2019.104544. Epub 2019 Jun 27.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding, Competitive*
  • Capsid / drug effects
  • Capsid Proteins / drug effects*
  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism
  • Cell Line
  • Drug Evaluation, Preclinical / methods*
  • Drug Liberation
  • Fluorescence*
  • HIV-1 / drug effects*
  • High-Throughput Screening Assays / methods*
  • Recombinant Proteins
  • T-Lymphocytes
  • Virus Assembly / drug effects*
  • Virus Replication / drug effects


  • Capsid Proteins
  • Recombinant Proteins