Mechanistic characterization and rational design of Lassa virus entry inhibitors

PROJECT SUMMARY Lassa virus (LASV), a BSL-4 pathogen and the causative agent of Lassa fever, poses a significant public health threat due to its epidemic potential both within and outside endemic regions and the lack of FDA-approved treatments or vaccines. The LASV glycoprotein complex (GPC), the sole envelope-anchored viral protein, and the entry process it mediates are key targets for developing intervention strategies. Despite extensive efforts, progress on vaccines and immunotherapies has been impeded by glycosylation of GPC, the genetic diversity of LASV, and the cost and time required for these strategies. Small-molecule arenavirus inhibitors have shown promise, but further development has been limited by poor target specificity and a lack of mechanistic insight. These challenges raise a critical question: how can we accelerate the development of effective, accessible LASV therapeutics suitable for broad use, particularly in resource-limited endemic regions? To address this unmet need, we conducted two parallel screens of a 665,000-compound library, controlled by SARS-CoV-2 and Machupo virus, respectively, and identified a family of small-molecule entry inhibitors that are non-toxic, potent, and specific for LASV entry, with pan-lineage activity. To the best of our knowledge, these compounds are the first to inhibit LASV entry without targeting other arenaviruses. Preliminary mechanistic studies suggest their direct interactions with the membrane-proximal region of LASV GPC, indicating a potential mechanism for disrupting conformational changes required for secondary receptor engagement or subsequent membrane fusion. Additionally, these inhibitors exhibit encouraging drug-like properties in both in vitro and in vivo drug metabolism and pharmacokinetics (DMPK) studies. Our initial structure-activity relationship (SAR) analyses support their potential for further optimization towards clinical development. In this proposal, we will develop these LASV-specific entry inhibitors through two independent and synergistic approaches. In Aim 1, we will investigate how these compounds interact with LASV GPC and inhibit viral entry. This effort will reveal the antiviral mechanisms of our lead compounds and inform targets for future LASV-specific antiviral design. In Aim 2, we will further optimize their antiviral efficacy and drug-likeness through detailed SAR and DMPK studies. Together, these studies will advance first-in-class LASV-specific small-molecule therapeutics and establish a robust platform for antiviral discovery against high-priority emerging pathogens.