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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

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

Zhang Da-Wei DW Luo Rong-Hua RH Xu Lei L Yang Liu-Meng LM Xu Xiao-Shuang XS Bedwell Gregory J GJ Engelman Alan N AN Zheng Yong-Tang YT Chang Shan S

Antiviral research 20190627

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 biologica ...[more]

PMID: 31254557

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Project description:Polyomaviruses are a family of ubiquitous double-stranded DNA viruses many of which are human pathogens. These include BK polyomavirus which causes severe urinary tract infection in immunocompromised patients and Merkel cell polyomavirus associated with aggressive cancers. The small genome of polyomaviruses lacks conventional drug targets, and no specific drugs are available at present. Here we focus on the main structural protein VP1 of BK polyomavirus which is responsible for icosahedral capsid formation. To provide a foundation towards rational drug design, we crystallized truncated VP1 pentamers and subjected them to a high-throughput screening for binding drug-like fragments through a direct X-ray analysis. To enable a highly performant screening, rigorous optimization of the crystallographic pipeline and processing with the latest generation PanDDA2 software were necessary. As a result, a total of 144 binding hits were established. Importantly, the hits are well clustered in six surface pockets. Three pockets are located on the outside of the pentamer and map on the regions where the 'invading' C-terminal arm of another pentamer is attached upon capsid assembly. Another set of three pockets is situated within the wide pore along the five-fold axis of the VP1 pentamer. These pockets are situated at the interaction interface with the minor capsid protein VP2 which is indispensable for normal functioning of the virus. Here we systematically analyse the three outside pockets which are highly conserved across various polyomaviruses, while point mutations in these pockets are detrimental for viral replication. We show that one of the pockets can accommodate antipsychotic drug trifluoperazine. For each pocket, we derive pharmacophore features which enable the design of small molecules preventing the interaction between VP1 pentamers and therefore inhibiting capsid assembly. Our data lay a foundation towards a rational development of first-in-class drugs targeting polyomavirus capsid.

Dataset Information

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

Publications

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

Similar Datasets

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