Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2 (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 80 million cases and 1.7 million deaths to date while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.

Characterization of cell-cell communication in COVID-19 patients (preprint)

COVID-19 patients display a wide range of disease severity, ranging from asymptomatic to critical symptoms with high mortality risk. Our ability to understand the interaction of SARS-CoV-2 infected cells within the lung, and of protective or dysfunctional immune responses to the virus, is critical to effectively treat these patients. Currently, our understanding of cell-cell interactions across different disease states, and how such interactions may drive pathogenic outcomes, is incomplete. Here, we developed a generalizable workflow for identifying cells that are differentially interacting across COVID-19 patients with distinct disease outcomes and use it to examine five public single-cell RNA-seq datasets with a total of 85 individual samples. By characterizing the cell-cell interaction patterns across epithelial and immune cells in lung tissues for patients with varying disease severity, we illustrate diverse communication patterns across individuals, and discover heterogeneous communication patterns among moderate and severe patients. We further illustrate patterns derived from cell-cell interactions are potential signatures for discriminating between moderate and severe patients.

Discovery of SARS-CoV-2 antiviral synergy between remdesivir and approved drugs in human lung cells (preprint)

The SARS coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic with currently 29 million confirmed cases and close to a million deaths. At this time, there are no FDA-approved vaccines or therapeutics for COVID-19, but Emergency Use Authorization has been granted for remdesivir, a broad-spectrum antiviral nucleoside analog. However, remdesivir is only moderately efficacious against SARS-CoV-2 in the clinic, and improved treatment strategies are urgently needed. To accomplish this goal, we devised a strategy to identify compounds that act synergistically with remdesivir in preventing SARS-CoV-2 replication. We conducted combinatorial high-throughput screening in the presence of submaximal remdesivir concentrations, using a human lung epithelial cell line infected with a clinical isolate of SARS-CoV-2. We identified 20 approved drugs that act synergistically with remdesivir, many with favorable pharmacokinetic and safety profiles. Strongest effects were observed with established antivirals, Hepatitis C virus nonstructural protein 5 A (HCV NS5A) inhibitors velpatasvir and elbasvir. Combination with their partner drugs sofosbuvir and grazoprevir further increased efficacy, increasing remdesivir's apparent potency 25-fold. We therefore suggest that the FDA-approved Hepatitis C therapeutics Epclusa (velpatasvir/sofosbuvir) and Zepatier (elbasvir/grazoprevir) should be fast-tracked for clinical evaluation in combination with remdesivir to improve treatment of acute SARS-CoV-2 infections.

Single-cell RNA Expression of SARS-CoV-2 Cell Entry Factors in Human Endometrium during Preconception (preprint)

We investigated potential SARS-CoV-2 tropism in human endometrium by single-cell RNA-sequencing of viral entry-associated genes in healthy women. Percentages of endometrial cells expressing ACE2, TMPRSS2, CTSB, or CTSL were <2%, 12%, 80%, and 80%, respectively, with 0.7% of cells expressing all four genes. Our findings imply low efficiency of SARS-CoV-2 infection in the endometrium before embryo implantation, providing information to assess preconception risk in asymptomatic carriers.

Ambroxol Hydrochloride Inhibits the Interaction between Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein's Receptor Binding Domain and Recombinant Human ACE2. (preprint)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), enters the host cells through two main pathways, both involving key interactions between viral envelope-anchored spike glycoprotein of the novel coronavirus and the host receptor, angiotensin-converting enzyme 2 (ACE2). To date, SARS-CoV-2 has infected up to 26 million people worldwide; yet, there is no clinically approved drug or vaccine available. Therefore, a rapid and coordinated effort to re-purpose clinically approved drugs that prevent or disrupt these critical entry pathways of SARS-CoV-2 spike glycoprotein interaction with human ACE2, could potentially accelerate the identification and clinical advancement of prophylactic and/or treatment options against COVID-19, thus providing possible countermeasures against viral entry, pathogenesis and survival. Herein, we discovered that Ambroxol hydrochloride (AMB), and its progenitor, Bromhexine hydrochloride (BHH), both clinically approved drugs are potent effective modulators of the key interaction between the receptor binding domain (RBD) of SARS-CoV-2 spike protein and human ACE2. We also found that both compounds inhibited SARS-CoV-2 infection-induced cytopathic effect at micromolar concentrations. Therefore, in addition to the known TMPRSS2 activity of BHH; we report for the first time that the BHH and AMB pharmacophore has the capacity to target and modulate yet another key protein-protein interaction essential for the two known SARS-CoV-2 entry pathways into host cells. Altogether, the potent efficacy, excellent safety and pharmacologic profile of both drugs along with their affordability and availability, makes them promising candidates for drug repurposing as possible prophylactic and/or treatment options against SARS-CoV-2 infection.

Non-permissive SARS-CoV-2 infection of neural cells in the developing human brain and neurospheres (preprint)

Coronavirus disease 2019 (COVID-19) was initially described as a viral infection of the respiratory tract. It is now known, however, that many other biological systems are affected, including the central nervous system (CNS). Neurological manifestations such as stroke, encephalitis, and psychiatric conditions have been reported in COVID-19 patients, but its neurotropic potential is still debated. Here, we investigate the presence of SARS-CoV-2 in the brain from an infant patient deceased from COVID-19. The susceptibility to virus infection was compatible with the expression levels of viral receptor ACE2, which is increased in the ChP in comparison to other brain areas. To better comprehend the dynamics of the viral infection in neural cells, we exposed human neurospheres to SARS-CoV-2. Similarly to the human tissue, we found viral RNA in neurospheres, although viral particles in the culture supernatant were not infective. Based on our observations in vivo and in vitro, we hypothesize that SARS-CoV-2 does not generate productive infection in developing neural cells and that infection of ChP weakens the blood-cerebrospinal fluid barrier allowing viruses, immune cells, and cytokines to access the CNS, causing neural damage in the young brain.

SARS CoV-2 nucleocapsid protein forms condensates with viral genomic RNA (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes COVID-19, a pandemic that seriously threatens global health. SARS CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with unstructured RNA, it forms mesh like-structures with viral RNA strands that contain secondary structure elements. Cross-linking mass spectrometry identified an intrinsically-disordered region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. By screening 1,200 FDA approved drugs in vitro, we identified a kinase inhibitor nilotinib, which affects the morphology of N condensates in vitro and disrupts phase separation of the N protein in vivo. These results indicate that the N protein compartmentalizes viral RNA in infected cells through liquid-liquid phase separation, and this process can be disrupted by a possible drug candidate.