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Marburg virus (MARV) causes sporadic outbreaks of severe disease with high case fatality rates in humans. To date, neither therapeutics nor prophylactic approaches have been approved for MARV disease. The MARV matrix protein VP40 (mVP40) plays central roles in virus assembly and budding. mVP40 also inhibits interferon signaling by inhibiting the function of Janus kinase 1. This suppression of host antiviral defenses likely contributes to MARV virulence and therefore is a potential therapeutic target. We developed and optimized a cell-based high-throughput screening (HTS) assay in 384-well format to measure mVP40 interferon (IFN) antagonist function such that inhibitors could be identified. We performed a pilot screen of 1280 bioactive compounds and identified 3 hits, azaguanine-8, tosufloxacin hydrochloride, and linezolid, with Z scores > 3 and no significant cytotoxicity. Of these, azaguanine-8 inhibited MARV growth at noncytotoxic concentrations. These data demonstrate the suitability of the HTS mVP40 assay for drug discovery and suggest potential directions for anti-MARV therapeutic development.

A series of five benzimidazole-based compounds were identified using a machine learning algorithm as potential inhibitors of the respiratory syncytial virus (RSV) fusion protein. These compounds were synthesized, and compound 2 in particular exhibited excellent in vitro potency with an EC 50 value of 5 nM. This new scaffold was then further refined leading to the identification of compound 44 , which exhibited a 10-fold improvement in activity with an EC 50 value of 0.5 nM.

Marburg virus (MARV) causes severe disease in humans and is known to activate nuclear factor erythroid 2-related factor 2 (Nrf2), the major transcription factor of the antioxidant response. Canonical activation of Nrf2 involves oxidative or electrophilic stress that prevents Kelch-like ECH-associated protein 1 (Keap1) targeted degradation of Nrf2, leading to Nrf2 stabilization and activation of the antioxidant response. MARV activation of Nrf2 is noncanonical with the MARV VP24 protein (mVP24) interacting with Keap1, freeing Nrf2 from degradation. A high-throughput screening (HTS) assay was developed to identify inhibitors of mVP24-induced Nrf2 activity and used to screen more than 55,000 compounds. Hit compounds were further screened against secondary HTS assays for the inhibition of antioxidant activity induced by additional canonical and noncanonical mechanisms. This pipeline identified 14 compounds that suppress the response, dependent on the inducer, with 50% inhibitory concentrations below 5 μM and selectivity index values greater than 10. Notably, several of the identified compounds specifically inhibit mVP24-induced Nrf2 activity.