Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.

Neuronal Apolipoprotein E4 Expression Results in Proteome-Wide Alterations and Compromises Bioenergetic Capacity by Disrupting Mitochondrial Function.

Apolipoprotein (apo) E4, the major genetic risk factor for Alzheimer's disease (AD), alters mitochondrial function and metabolism early in AD pathogenesis. When injured or stressed, neurons increase apoE synthesis. Because of its structural difference from apoE3, apoE4 undergoes neuron-specific proteolysis, generating fragments that enter the cytosol, interact with mitochondria, and cause neurotoxicity. However, apoE4's effect on mitochondrial respiration and metabolism is not understood in detail. Here we used biochemical assays and proteomic profiling to more completely characterize the effects of apoE4 on mitochondrial function and cellular metabolism in Neuro-2a neuronal cells stably expressing apoE4 or apoE3. Under basal conditions, apoE4 impaired respiration and increased glycolysis, but when challenged or stressed, apoE4-expressing neurons had 50% less reserve capacity to generate ATP to meet energy requirements than apoE3-expressing neurons. ApoE4 expression also decreased the NAD+/NADH ratio and increased the levels of reactive oxygen species and mitochondrial calcium. Global proteomic profiling revealed widespread changes in mitochondrial processes in apoE4 cells, including reduced levels of numerous respiratory complex subunits and major disruptions to all detected subunits in complex V (ATP synthase). Also altered in apoE4 cells were levels of proteins related to mitochondrial endoplasmic reticulum-associated membranes, mitochondrial fusion/fission, mitochondrial protein translocation, proteases, and mitochondrial ribosomal proteins. ApoE4-induced bioenergetic deficits led to extensive metabolic rewiring, but despite numerous cellular adaptations, apoE4-expressing neurons remained vulnerable to metabolic stress. Our results provide insights into potential molecular targets of therapies to correct apoE4-associated mitochondrial dysfunction and altered cellular metabolism.

The association of spiritual care providers' activities with family members' satisfaction with care after a death in the ICU*.

OBJECTIVES: Spiritual distress is common in the ICU, and spiritual care providers are often called upon to provide care for patients and their families. Our goal was to evaluate the activities spiritual care providers' conduct to support patients and families and whether those activities are associated with family satisfaction with ICU care. DESIGN: Prospective cohort study. SETTING: Three hundred fifty-bed tertiary care teaching hospital with 65 ICU beds. SUBJECTS: Spiritual care providers and family members of patients who died in the ICU or within 30 hours of transfer from the ICU. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Spiritual care providers completed surveys reporting their activities. Family members completed validated measures of satisfaction with care and satisfaction with spiritual care. Clustered regression was used to assess the association between activities completed by spiritual care providers and family ratings of care. Of 494 eligible patients, 275 family members completed surveys (response rate, 56%). Fifty-seven spiritual care providers received surveys relating to 268 patients, completing 285 surveys for 244 patients (response rate, 91%). Spiritual care providers commonly reported activities related to supporting religious and spiritual needs (≥ 90%) and providing support for family feelings (90%). Discussions about the patient's wishes for end-of-life care and a greater number of spiritual care activities performed were both associated with increased overall family satisfaction with ICU care (p < 0.05). Discussions about a patient's end-of-life wishes, preparation for a family conference, and total number of activities performed were associated with improved family satisfaction with decision-making in the ICU (p < 0.05). CONCLUSIONS: Spiritual care providers engage in a variety of activities with families of ICU patients; several are associated with increased family satisfaction with ICU care in general and decision-making in the ICU specifically. These findings provide insight into spiritual care provider activities and provide guidance for interventions to improve spiritual care delivered to families of critically ill patients.

Dispersed sites of HIV Vif-dependent polyubiquitination in the DNA deaminase APOBEC3F.

APOBEC3F (A3F) and APOBEC3G (A3G) are DNA cytosine deaminases that potently restrict human immunodeficiency virus type 1 replication when the virus is deprived of its accessory protein Vif (virion infectivity factor). Vif counteracts these restriction factors by recruiting A3F and A3G to an E3 ubiquitin (Ub) ligase complex that mediates their polyubiquitination (polyUb) and proteasomal degradation. While previous efforts have identified single amino acid residues in APOBEC3 proteins required for Vif recognition, less is known about the downstream Ub acceptor sites that are targeted. One prior report identified a cluster of polyubiquitinated residues in A3G and proposed an antiparallel model of A3G interaction with the Vif-E3 Ub ligase complex wherein Vif binding at one terminus of A3G orients the opposite terminus for polyUb [Iwatani et al. (2009). Proc. Natl. Acad. Sci. USA, 106, 19539-19544]. To test the generalizability of this model, we carried out a complete mutagenesis of the lysine residues in A3F and used a complementary, unbiased proteomic approach to identify Ub acceptor sites targeted by Vif. Our data indicate that internal lysines are the dominant Ub acceptor sites in both A3F and A3G. In contrast with the proposed antiparallel model, however, we find that the Vif-dependent polyUb of A3F and A3G can occur at multiple acceptor sites dispersed along predicted lysine-enriched surfaces of both the N- and C-terminal deaminase domains. These data suggest an alternative model for binding of APOBEC3 proteins to the Vif-E3 Ub ligase complex and diminish enthusiasm for the amenability of APOBEC3 Ub acceptor sites to therapeutic intervention.