Human ACE2 Stable Cell Line - HeLa

Background
This study introduces FragmentScout, a fragment-based pharmacophore virtual screening workflow that integrates multiple experimental X-ray fragment poses into a joint pharmacophore query per binding site. Applied to SARS-CoV-2 NSP13 helicase, the approach identified 13 novel micromolar inhibitors validated in both cellular antiviral and ThermoFluor biophysical assays. FragmentScout outperformed conventional Glide docking in hit discovery across nucleotide and 5'-RNA binding pockets, demonstrating its potential for systematic mining of XChem datasets and advancing fragment-based lead discovery towards potent antiviral agents.
Key Findings
Key findings include identification of 13 novel non-cytotoxic inhibitors of SARS-CoV-2 NSP13 with low micromolar EC50 values (1.07–9.31 μM) in cellular assays, most showing consistent thermal stabilization in ThermoFluor assays. Compounds exhibited broad-spectrum activity against SARS-CoV-2, SARS-CoV, and HCoV-229E, with several more potent against HCoV-229E. FragmentScout, which merged pharmacophore queries from 63 XChem fragment poses clustered into 19 binding sites, proved more effective than Glide SP docking for nucleotide and 5'-RNA sites. The best hits targeted conserved 5'-RNA, nucleotide, and Stalk-Zinc pockets, providing new starting points for antiviral development, including six scaffolds distinct from CACHE Challenge #2.
Contribution
Creative Biogene supplied the HeLa-hACE2 cells used in the HCoV-229E antiviral assays, enabling evaluation of broad-spectrum efficacy of the discovered inhibitors.

Background
Researchers identified Z-Tyr-Ala-CHN2, a small-molecule cathepsin L inhibitor, from a cell-based antiviral screen. It showed sub-micromolar activity against SARS-CoV-2, SARS-CoV-1, and HCoV-229E. Time-of-addition and pseudovirus assays indicated early entry inhibition via the endosomal cathepsin L pathway, not TMPRSS2-dependent entry. Activity was cell-specific: potent in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but absent in Caco-2 cells and human nasal epithelial cells. The compound did not disrupt lysosomal trafficking and represents a useful tool for coronavirus entry studies.
Key Findings
Z-Tyr-Ala-CHN2 was discovered in a phenotypic screen and acts as a potent cathepsin L inhibitor. It displayed sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and HCoV-229E, with EC50 values of 0.32 µM in viral RNA yield assays and 0.006–0.05 µM in high-content imaging assays. Time-of-addition studies placed its action at the early entry phase, and pseudotyped virus assays showed loss of activity in TMPRSS2-expressing cells, indicating endosomal entry dependence. Consequently, the compound inhibited SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells but not in Caco-2 or primary human nasal epithelial cells. It did not cause lysosomal accumulation, supporting specific cathepsin L inhibition rather than nonspecific endolysosomal disruption. It is a useful tool compound for coronavirus entry and replication studies.
Contribution
Creative Biogene contributed by supplying HeLa-hACE2 cells used as a human cell model in antiviral assays. These cells enabled evaluation of Z-Tyr-Ala-CHN2 against SARS-CoV-2, SARS-CoV-1, and HCoV-229E, and supported mechanism-of-action studies showing cathepsin L-dependent, cell-specific entry inhibition.