A high throughput screen identifies benzoquinoline compounds as inhibitors of Ebola virus replication

Luthra, Priya; Liang, Jue; Pietzsch, Colette; Khadka, Sudip; Edwards, Megan R.; Wei, Shuguang; De, Sampriti; Posner, Bruce A.; Bukreyev, Alexander; Ready, Joseph M.; Basler, Christopher F.

doi:10.1016/j.antiviral.2017.12.019

Cited by 36 publications

(28 citation statements)

References 48 publications

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“…A screen of 200,000 small molecules identified several compounds sharing an amino-tetrahydrocarbazole scaffold as inhibitors of EBOV RNA synthesis machinery as measured by the EBOV minigenome (MG) assay (Luthra et al, 2017b). In this assay, the plasmids encoding for EBOV polymerase complex proteins, L, NP, VP35 and VP30, are transfected along with a MG plasmid that consists of a Renilla reporter flanked by viral cis-acting regulatory sequences that drive the transcription and translation of the reporter gene.…”

Section: Resultsmentioning

confidence: 99%

“…A single ISRE-FF clone was selected for its optimal signal to background ratio when stimulated with recombinant Universal type I interferon-alpha (uIFN) (PBL Assay Science). The stable HEK 293T IFNβ promoter-reporter cell line has been previously described (Luthra et al, 2017b). …”

Section: Methodsmentioning

confidence: 99%

“…The minigenome assay in 384 well format has been previously described (Edwards et al, 2015; Luthra et al, 2017b). Briefly, HEK293T cells (7.5*10 6 cells) were transfected in T75 flasks using Lipofectamine 2000 (Invitrogen) with plasmids encoding NP (3.7 μg), VP30 (1.5 μg), VP35 (1.9 μg), L (7.5 μg), reporter minigenome plasmid (3 μg) along with T7 polymerase plasmid (3 μg).…”

Section: Methodsmentioning

confidence: 99%

“…A previous report described small molecule inhibitors of EBOV RNA synthesis identified by high throughput screening (HTS) of an EBOV minigenome assay (Luthra et al, 2017b). This assay measures the function of an EBOV RNA synthesis complex that is reconstituted by transfection into mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

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Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses

et al. 2018

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Specific host pathways that may be targeted therapeutically to inhibit the replication of Ebola virus (EBOV) and other emerging viruses remain incompletely defined. A screen of 200,000 compounds for inhibition of an EBOV minigenome (MG) assay that measures the function of the viral polymerase complex identified as hits several compounds with an amino-tetrahydrocarbazole scaffold. This scaffold was structurally similar to GSK983, a compound previously described as having broad-spectrum antiviral activity due to its impairing de novo pyrimidine biosynthesis through inhibition of dihydroorotate dehydrogenase (DHODH). We generated compound SW835, the racemic version of GSK983 and demonstrated that SW835 and brequinar, another DHODH inhibitor, potently inhibit the MG assay and the replication of EBOV, vesicular stomatitis virus (VSV) and Zika (ZIKV) in vitro. Nucleoside and deoxynucleoside supplementation studies demonstrated that depletion of pyrimidine pools contributes to antiviral activity of these compounds. As reported for other DHODH inhibitors, SW835 and brequinar also induced expression of interferon stimulated genes (ISGs). ISG induction was demonstrated to occur without production of IFNα/β and independently of the IFNα receptor and was not blocked by EBOV-encoded suppressors of IFN signaling pathways. Furthermore, we demonstrated that transcription factor IRF1 is required for this ISG induction, and that IRF1 induction requires the DNA damage response kinase ATM. Therefore, de novo pyrimidine biosynthesis is critical for the replication of EBOV and other RNA viruses and inhibition of this pathway activates an ATM and IRF1-dependent innate immune response that subverts EBOV immune evasion functions.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses

et al. 2018

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“…Although the MG system has been used recently to identify small molecule inhibitors of EBOV replication (Edwards et al, 2015; Luthra et al, 2018; Nelson et al, 2017; Welch et al, 2016), these studies have involved high throughput screens of pre-existing libraries of known bioactive compounds. We sought to refine this approach by combining it with a virtual screening cascade to identify compounds - available within our in-house chemical libraries - predicted to bind to the NP pocket.…”

Section: Introductionmentioning

confidence: 99%

Identification of a small molecule inhibitor of Ebola virus genome replication and transcription using in silico screening

et al. 2018

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Ebola virus (EBOV) causes a severe haemorrhagic fever in humans and has a mortality rate over 50%. With no licensed drug treatments available, EBOV poses a significant threat. Investigations into possible therapeutics have been severely hampered by the classification of EBOV as a BSL4 pathogen. Here, we describe a drug discovery pathway combining in silico screening of compounds predicted to bind to a hydrophobic pocket on the nucleoprotein (NP); with a robust and rapid EBOV minigenome assay for inhibitor validation at BSL2. One compound (MCCB4) was efficacious (EC 4.8 μM), exhibited low cytotoxicity (CC > 100 μM) and was specific, with no effect on either a T7 RNA polymerase driven firefly luciferase or a Bunyamwera virus minigenome. Further investigations revealed that this small molecule inhibitor was able to outcompete established replication complexes, an essential aspect for a potential EBOV treatment.

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Phenotypic Prioritization of Diphyllin Derivatives That Block Filoviral Cell Entry by Vacuolar (H⁺)‐ATPase Inhibition

et al. 2018

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Many viruses utilize endosomal pathways to gain entry to cells and propagate infection. Sensing of endosomal acidification is a trigger for release of many virus cores into the cell cytosol. Previous efforts with inhibitors of vacuolar-ATPase have been shown to block endosomal acidification and affect viral entry albeit with limited potential for therapeutic selectivity. Herein, four novel series of derivatives of the vacuolar-ATPase inhibitor diphyllin were synthesized to assess their potential for enhancing potency and anti-filoviral activity over cytotoxicity. Derivatives that suitably blocked cellular entry of Ebola pseudo-typed virus were further evaluated as inhibitors of endosomal acidification and isolated human Vacuolar-ATPase activity. Several compounds with significant increases in potency over diphyllin in these assays also separated from cytotoxic doses in human cell models by >100 fold. Finally, three derivatives were shown to be inhibitors of replication-competent Ebola viral entry into primary macrophages with comparable potencies and enhanced selectivity toward anti-viral activity.

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A high throughput screen identifies benzoquinoline compounds as inhibitors of Ebola virus replication

Cited by 36 publications

References 48 publications

Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses

Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses

Identification of a small molecule inhibitor of Ebola virus genome replication and transcription using in silico screening

Phenotypic Prioritization of Diphyllin Derivatives That Block Filoviral Cell Entry by Vacuolar (H⁺)‐ATPase Inhibition

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A high throughput screen identifies benzoquinoline compounds as inhibitors of Ebola virus replication

Cited by 36 publications

References 48 publications

Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses

Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses

Identification of a small molecule inhibitor of Ebola virus genome replication and transcription using in silico screening

Phenotypic Prioritization of Diphyllin Derivatives That Block Filoviral Cell Entry by Vacuolar (H+)‐ATPase Inhibition

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Product

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Phenotypic Prioritization of Diphyllin Derivatives That Block Filoviral Cell Entry by Vacuolar (H⁺)‐ATPase Inhibition