Development of High-Throughput Screening Assay for Antihantaviral Therapeutics

Abstract

Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. There is no treatment for hantavirus diseases at present. Therapeutic intervention during early stages of viral infection can improve the outcome of this zoonotic viral illness. The interaction between an evolutionary conserved sequence at the 5′ terminus of hantaviral genomic RNA and hantavirus nucleocapsid protein plays a critical role in the hantavirus replication cycle. This unique interaction is a novel target for therapeutic intervention of hantavirus disease. We developed a very sensitive, tractable, and cost-effective fluorescence-based assay to monitor the interaction between the nucleocapsid protein and the target RNA sequence. The assay was optimized for high-throughput screening of chemical libraries to identify molecules that interrupt this RNA–protein interaction. The assay was validated using a library of 6880 chemical compounds. This validation screen demonstrated the reproducibility and validity of required statistical criteria for high-throughput screening. The assay is ready to use for high-throughput screening of large chemical libraries to identify antihantaviral therapeutic molecules and can be amenable to similar targets in other viruses.

Hantavirus induced cardiopulmonary syndrome: A public health concern

“…There was no statistical difference in the number of deaths, P/F ratio, drop in cardiac index, incidence of ventricular tachycardia, and ventricular fibrillation in both groups . Recently, a high throughput screening approach identified few novel compounds that selectively inhibit hantavirus replication in cell culture . These compounds appear promising for the development of anti‐hantaviral therapeutics.…”

“…69 Recently, a high throughput screening approach identified few novel compounds that selectively inhibit hantavirus replication in cell culture. 70,71 These compounds appear promising for the development of anti-hantaviral therapeutics.…”

Integration of a miniaturized DMMB assay with high-throughput screening for identifying regulators of proteoglycan metabolism

“…A proof-of-concept chemical screening was conducted using the miniaturized DMMB assay, and both positive and negative regulatory candidates were identified from the library. The majority of the 960 compounds (79.69%) showed readouts within 3 × S.D., which were considered insignificant 36 . Notably, the screening results showed a skewed distribution, and a larger panel of compounds with < 3 × S.D was identified (15.10% vs 5.21%).…”

“…Plate uniformity was evaluated as previously described to investigate the signal variability across different plates 36 . A library containing 960 small molecules (ChemBridge Co., US) with molecular weights ranging between 160 and 6600 Da was arrayed on three plates (320 compounds/plate) in triplicate to assess the assay for screening in high-throughput mode.…”

SARS-CoV-2 Nucleocapsid Protein Is a Potential Therapeutic Target for Anticoronavirus Drug Discovery

“…While the majority of anticoronavirus drug development efforts target RNA-dependent RNA polymerase (RdRp) ( 8 , 9 ), spike protein ( 10 ), envelope protein ( 11 ), 3CL pro ( 12 , 13 ), and M pro ( 14 , 15 ), the N protein as an important therapeutic target has not been much explored. Using a high-throughput screening approach ( 16 , 17 ), we previously identified a novel molecule [4-(3-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-6-carboxylic acid], referred to here as K31 ( Fig. 1E , inset), that targets the hantavirus nucleocapsid protein and inhibits hantavirus replication in cells with a selective index (SI) of >18 ( 18 – 20 ).…”