Evaluation of potency and metabolic stability of diphyllin-derived Vacuolar-ATPase inhibitors.

Diphyllin is a naturally occurring lignan comprised of an aryl naphthalene lactone scaffold that demonstrates beneficial biological activities in disease models of cancer, obesity, and viral infection. A target of diphyllin and naturally occurring derivatives is the vacuolar ATPase (V-ATPase) complex. Although diphyllin-related natural products are active with in vitro models for viral entry, the potencies and unknown pharmacokinetic properties limit well-designed in vivo evaluations. Previous studies demonstrated that diphyllin derivatives have the utility of blocking the Ebola virus cell entry pathway. However, diphyllin shows limited potency and poor oral bioavailability in mice. An avenue to improve the potency was used in a new library of synthetic derivatives of diphyllin. Diphyllin derivatives exploiting ether linkages at the 4-position with one-to-three carbon spacers to an oxygen or nitrogen atom provided compounds with EC50 values ranging from 7 to 600 nM potency and selectivity up to >500 against Ebola virus in infection assays. These relative potencies are reflected in the Ebola virus infection of primary macrophages, a cell type involved in early pathogenesis. A target engagement study reveals that reducing the ATPV0a2 protein expression enhanced the potency of diphyllin derivatives to block EBOV entry, consistent with effects on the endosomal V-ATPase function. Despite the substantial enhancement of antiviral potencies, limitations were identified, including rapid clearance predicted by in vitro microsome stability assays. However, compounds with similar or improved half-lives relative to diphyllin demonstrated improved pharmacokinetic profiles in vivo. Importantly, these derivatives displayed suitable plasma levels using oral administration, establishing the feasibility of in vivo antiviral testing.

Identification of potent inhibitors of SARS-CoV-2 infection by combined pharmacological evaluation and cellular network prioritization.

Pharmacologically active compounds with known biological targets were evaluated for inhibition of SARS-CoV-2 infection in cell and tissue models to help identify potent classes of active small molecules and to better understand host-virus interactions. We evaluated 6,710 clinical and preclinical compounds targeting 2,183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target, and cell interactome produced cellular networks important for infection. This analysis revealed 389 small molecules with micromolar to low nanomolar activities, representing >12 scaffold classes and 813 host targets. Representatives were evaluated for mechanism of action in stable and primary human cell models with SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of host factor dependencies and treatments for viral diseases.

Evaluation of Phenol-Substituted Diphyllin Derivatives as Selective Antagonists for Ebola Virus Entry.

Ebola virus (EBOV) is an aggressive filoviral pathogen that can induce severe hemorrhagic fever in humans with up to 90% fatality rate. To date, there are no clinically effective small-molecule drugs for postexposure therapies to treat filoviral infections. EBOV cellular entry and infection involve uptake via macropinocytosis, navigation through the endocytic pathway, and pH-dependent escape into the cytoplasm. We report the inhibition of EBOV cell entry via selective inhibition of vacuolar (V)-ATPase by a new series of phenol-substituted derivatives of the natural product scaffold diphyllin. In cells challenged with Ebola virus, the diphyllin derivatives inhibit viral entry dependent upon structural variations to low nanomolar potencies. Mechanistically, the diphyllin derivatives had no effect on uptake and colocalization of viral particles with endocytic marker LAMP1 but directly modulated endosomal pH. The most potent effects were reversible exhibiting higher selectivity than bafilomycin or the parent diphyllin. Unlike general lysosomotrophic agents, the diphyllin derivatives showed no major disruptions of endocytic populations or morphology when examined with Rab5 and LAMP1 markers. The dilated vacuole phenotype induced by apilimod treatment or in constitutively active Rab5 mutant Q79L-expressing cells was both blocked and reversed by the diphyllin derivatives. The results are consistent with the action of the diphyllin scaffold as a selective pH-dependent viral entry block in late endosomes. Overall, the compounds show improved selectivity and minimal cytotoxicity relative to classical endosomal acidification blocking agents.

Identification of druggable host targets needed for SARS-CoV-2 infection by combined pharmacological evaluation and cellular network directed prioritization both in vitro and in vivo.

Identification of host factors contributing to replication of viruses and resulting disease progression remains a promising approach for development of new therapeutics. Here, we evaluated 6710 clinical and preclinical compounds targeting 2183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target and cell interactome networking produced cellular networks important for infection. This analysis revealed 389 small molecules, >12 scaffold classes and 813 host targets with micromolar to low nanomolar activities. From these classes, representatives were extensively evaluated for mechanism of action in stable and primary human cell models, and additionally against Beta and Delta SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of novel host factor dependencies and treatments for viral diseases.

Inactivation of Ebola Virus and SARS-CoV-2 in Cell Culture Supernatants and Cell Pellets by Gamma Irradiation.

Viral pathogens with the potential to cause widespread disruption to human health and society continue to emerge or re-emerge around the world. Research on such viruses often involves high biocontainment laboratories (BSL3 or BSL4), but the development of diagnostics, vaccines and therapeutics often uses assays that are best performed at lower biocontainment. Reliable inactivation is necessary to allow removal of materials to these spaces and to ensure personnel safety. Here, we validate the use of gamma irradiation to inactivate culture supernatants and pellets of cells infected with a representative member of the Filovirus and Coronavirus families. We show that supernatants and cell pellets containing SARS-CoV-2 are readily inactivated with 1.9 MRad, while Ebola virus requires higher doses of 2.6 MRad for supernatants and 3.8 MRad for pellets. While these doses of radiation inactivate viruses, proinflammatory cytokines that are common markers of virus infection are still detected with low losses. The doses required for virus inactivation of supernatants are in line with previously reported values, but the inactivation of cell pellets has not been previously reported and enables new approaches for analysis of protein-based host responses to infection.

Synthesis and antiviral activity of fatty acyl conjugates of remdesivir against severe acute respiratory syndrome coronavirus 2 and Ebola virus.

We report here the synthesis, purification, and characterization of mono- and di-fatty acyl conjugates of remdesivir (RDV) and their in vitro antiviral activity against SAR-CoV-2, an Ebola virus transcription- and replication-competent virus-like particle (trVLP) system, and infectious Ebola virus. The most potent monofatty acyl conjugate was 4b, containing a 4-oxatetradecanolyl at the 3' position. Monofatty acyl conjugates, 3'-O-tetradecanoyl (4a) (IC50(VeroE6) = 2.3 µM; IC50(Calu3) = 0.24 µM), 3'-O-4-oxatetradodecanoyl (4b) (IC50(VeroE6) = 2.0 µM; IC50(Calu3) = 0.18 µM), and 3'-O-(12-ethylthiododecanoyl) (4e) (IC50(VeroE6) = 2.4 µM; IC50(Calu3) = 0.25 µM) derivatives exhibited less activity than RDV (IC50(VeroE6) = 0.85 µM; IC50(Calu3) = 0.06 µM) in both VeroE6 and Calu3 cells. Difatty acylation led to a significant reduction in the antiviral activity of RDV (as shown in conjugates 5a and 5b) against SARS-CoV-2 when compared with monofatty acylation (3a-e and 4a-e). About 77.9% of 4c remained intact after 4 h incubation with human plasma while only 47% of parent RDV was observed at the 2 h time point. The results clearly indicate the effectiveness of fatty acylation to improve the half-life of RDV. The antiviral activities of a number of monofatty acyl conjugates of RDV, such as 3b, 3e, and 4b, were comparable with RDV against the Ebola trVLP system. Meanwhile, the corresponding physical mixtures of RDV and fatty acids 6a and 6b showed 1.6 to 2.2 times less antiviral activity than the corresponding conjugates, 4a and 4c, respectively, against SARS-CoV-2 in VeroE6 cells. A significant reduction in viral RNA synthesis was observed for selected compounds 3a and 4b consistent with the IC50 results. These studies indicate the potential of these compounds as long-acting antiviral agents or prodrugs of RDV.

Development of a Well-Characterized Rhesus Macaque Model of Ebola Virus Disease for Support of Product Development.

Ebola virus (EBOV) is a negative-sense RNA virus that can infect humans and nonhuman primates with severe health consequences. Development of countermeasures requires a thorough understanding of the interaction between host and pathogen, and the course of disease. The goal of this study was to further characterize EBOV disease in a uniformly lethal rhesus macaque model, in order to support development of a well-characterized model following rigorous quality standards. Rhesus macaques were intramuscularly exposed to EBOV and one group was euthanized at predetermined time points to characterize progression of disease. A second group was not scheduled for euthanasia in order to analyze survival, changes in physiology, clinical pathology, terminal pathology, and telemetry kinetics. On day 3, sporadic viremia was observed and pathological evidence was noted in lymph nodes. By day 5, viremia was detected in all EBOV exposed animals and pathological evidence was noted in the liver, spleen, and gastrointestinal tissues. These data support the notion that EBOV infection in rhesus macaques is a rapid systemic disease similar to infection in humans, under a compressed time scale. Biomarkers that correlated with disease progression at the earliest stages of infection were observed thereby identifying potential "trigger-to-treat" for use in therapeutic studies.

Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID50 Assays.

Ongoing efforts to develop effective therapies against filoviruses rely, to different extents, on quantifying the amount of viable virus in samples by plaque, TCID50, and focus assays. Unfortunately, these techniques have inherent variance, and laboratory-specific preferences make direct comparison of data difficult. Additionally, human errors such as operator errors and subjective bias can further compound the differences in outcomes. To overcome these biases, we developed a computer-based automated image-processing method for a focus assay based on the open-source CellProfiler software platform, which enables high-throughput screening of many treatment samples at one time. We compared virus titers calculated using this platform to plaque and TCID50 assays using common stocks of virus for 3 major Filovirus species, Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus with each assay performed by multiple operators on multiple days. We show that plaque assays give comparable findings that differ by less than 3-fold. Focus-forming unit (FFU) and TCID50 assays differ by 10-fold or less from the plaque assays due a higher (FFU) and lower (TCID50) sensitivity. However, reproducibility and accuracy of each assay differs significantly with Neutral Red Agarose Overlay plaque assays and TCID50 with the lowest reproducibility due to subjective analysis and operator error. Both crystal violet methylcellulose overlay plaque assay and focus assays perform best for accuracy and the focus assay performs best for speed and throughput.