Ryan Bott

Background/Objectives: We have established a robust, cell-based high-throughput screening platform capable of identifying SARS-CoV-2 entry inhibitors within a BSL-2 facility. Methods: Using a curated compound library, we conducted a screening campaign that led to the discovery of potent viral entry inhibitors active in both pseudoviral and infectious SARS-CoV-2 inhibition assays. Results: Among those, Hit-1 exhibited submicromolar antiviral activity across all tested SARS-CoV-2 strains, including the highly transmissible Omicron subvariants. Biophysical binding assays confirmed that Hit-1 and related compounds directly engage the prefusion-stabilized SARS-CoV-2 spike proteins of both authentic WA1/2020 and Omicron viral strains. To elucidate potential binding orientations and interactions of the hit compounds with the SARS-CoV-2 spike protein, molecular docking studies were performed targeting two putative binding sites. Conclusions: Preliminary structure–activity relationship studies identified a promising subset of drug-like 7,8-dihydropyrido[4,3-d]pyrimidine-based inhibitors with potential for further development as novel therapeutic agents aimed at blocking viral entry and thereby preventing or mitigating SARS-CoV-2 infection. Among these, compound 13 stands out due to its superior in vitro potency and favorable pharmacokinetic properties, positioning it as a strong candidate for in vivo efficacy evaluation.

Several arenaviruses, such as the Old World (OW) Lassa virus (LASV) and the New World (NW) Junin virus (JUNV), can cause severe and lethal viral hemorrhagic fevers in humans. Currently, no vaccines or specific antiviral therapies are FDA-approved for treating arenavirus infections. One major challenge for the development of new therapeutic candidates against these highly pathogenic viruses is that they are BSL-3/4 pathogens that need to be handled in high biocontainment laboratories. In this work, a recombinant non-pathogenic New World arenavirus, Pichinde virus (rPICV), was used for the development of a high-throughput screening (HTS) assay in the BSL-2 laboratory for the screening and identification of small molecule inhibitors against arenaviruses. The rPICV is a replication-competent virus expressing the firefly luciferase reporter gene in the infected cells proportionally to the infection rate. rPICV infection was optimized for an automated HTS in 384-well format with robust Z′ scores, high signal-to-background ratios, and low intrinsic variance. Screening an established library allowed for the identification of five top hit compounds, which included ribavirin, a known inhibitor of arenaviral RNA synthesis, showing good potency and selectivity in inhibiting rPICV replication. The antiviral activity of the top hit compounds was further validated against another recombinant arenavirus, the OW lymphocytic choriomeningitis virus (rLCMV) and against laboratory strains of LASV (Josiah) and JUNV (Romero). The use of rPICV in the HTS-based antiviral assay under BSL-2 condition has proven to be safe and suitable for the identification of broad-spectrum small molecule inhibitors against highly pathogenic arenaviruses.