High throughput screening identifies inhibitors for parvovirus B19 infection of human erythroid progenitor cells.

Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or the prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high throughput screening assay, which is based on an in vitro nicking assay using recombinant N-terminal 1-176 amino acids of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe (OriQ) that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using ex vivo expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds demonstrated >50% in vivo inhibition of B19V infection at 10 μM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index [half maximal cytotoxic concentration (CC50)/half maximal effective concentration (EC50)] in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC90). A purine derivative (P7), demonstrated an antiviral effect (EC50=1.46 μM) without prominent cytotoxicity (CC50=71.8 μM) in EPCs, exhibited 92% inhibition of B19V infection in EPCs at 3.32 μM, which can be used as the lead compound in future studies for the treatment of B19V infection caused hematological disorders. Importance B19V encodes a large non-structural protein NS1. Its N-terminal domain (NS1N) consisting of 1-176 amino acids binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling hairpin-dependent B19V DNA replication. For high throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based in vitro nicking assay, which employs a fluorophore-labeled probe spanning the trs and the NS1N protein, into a 384-well plate format. The HTS assay showed a high reliability and capability in screening 17,040 compounds. Based on the therapeutic index [half maximal cytotoxic concentration (CC50)/half maximal effective concentration (EC50)] in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 μM (two times EC90). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.