Pixantrone for COVID-19

Abstract Virus entry is thought to involve binding a unique receptor for cell attachment and cytosolic entry. For SARS-CoV-2 underlying the COVID-19 pandemic, angiotensin- converting enzyme 2 (ACE2) is widely assumed as the receptor. Using advanced light microscopy to resolve individual virions and receptors, we found instead that heparan sulfate (HS), not ACE2, mediates SARS-CoV-2 cell-surface attachment and subsequent endocytosis. ACE2 functions only downstream of HS to enable viral genome expression. Instead of binding single HS molecules that electrostatically interact with viral surface proteins weakly, SARS-CoV-2 binds clusters of ∼6–137 HS molecules projecting 60–410 nm above the plasma membrane. These tall, HS-rich clusters, present at about one per 6 μm², act as docking sites for viral attachment. Blocking HS binding with the clinically used HS- binding agent pixantrone strongly inhibited the clinically relevant SARS-CoV-2 Omicron JN.1 subvariant from attaching to and infecting human airway cells. This work establishes a revised entry paradigm in which HS clusters mediate SARS-CoV-2 attachment and endocytosis, with ACE2 acting downstream, thereby identifying HS interactions as a key anti-COVID-19 strategy. This paradigm and its therapeutic implications may apply broadly beyond COVID-19 because, analogous to SARS-CoV-2, HS binds many other viruses but is only considered an attachment regulator. Statement of Significance Viral entry, a crucial antiviral target, is typically thought to involve binding its unique receptor for the cell surface attachment and subsequent entry. We examined this concept with advanced microscopies to resolve individual receptors and SARS-CoV-2 virions responsible for the COVID-19 pandemic. We discovered two receptors for viral entry: heparan sulfate, a polysaccharide that may bind many viruses, mediates viral attachment and subsequent endocytosis, whereas angiotensin-converting enzyme 2 (ACE2), the generally assumed SARS-CoV-2 receptor, acts only downstream to facilitate viral infection. This new model suggests perturbation of HS binding as a more effective anti-COVID-19 strategy than previously recognized. It may apply broadly beyond COVID-19 because, analogous to SARS-CoV-2, HS binds many other viruses but is only considered an attachment regulator.

AbstractSince the emergence of SARS‐CoV‐2 at the end of 2019, the virus has caused significant global health and economic disruptions. Despite the rapid development of antiviral vaccines and some approved treatments such as remdesivir and paxlovid, effective antiviral pharmacological treatments for COVID‐19 patients remain limited. This study explores Nsp15, a 3′‐uridylate‐specific RNA endonuclease, which has a critical role in immune system evasion and hence in escaping the innate immune sensors. We conducted a comprehensive drug repurposing screen and identified 44 compounds that showed more than 55% inhibition of Nsp15 activity in a real‐time fluorescence assay. A validation pipeline was employed to exclude unspecific interactions, and dose–response assays confirmed 29 compounds with an IC50 below 10 μM. Structural studies, including molecular docking and x‐ray crystallography, revealed key interactions of identified inhibitors, such as TAS‐103 and YM‐155, with the Nsp15 active site and other critical regions. Our findings show that the identified compounds, particularly those retaining potency under different assay conditions, could serve as promising hits for developing Nsp15 inhibitors. Additionally, the study emphasizes the potential of combination therapies targeting multiple viral processes to enhance treatment efficacy and reduce the risk of drug resistance. This research contributes to the ongoing efforts to develop effective antiviral therapies for SARS‐CoV‐2 and possibly other coronaviruses.