Novel Small-Molecule Inhibitors of Transmissible Gastroenteritis Virus

Glutaminase, which converts glutamine to glutamate, is involved in Warburg effect in cancer cells. Two human glutaminase genes have been identified, GLS (GLS1) and GLS2. Two alternative transcripts arise from each glutaminase gene: first, the kidney isoform (KGA) and glutaminase C (GAC) for GLS; and, second, the liver isoform (LGA) and glutaminase B (GAB) for GLS2. While GLS1 is considered as a cancer therapeutic target, the potential role of GLS2 in cancer remains unclear. Here, we discovered a series of alkyl benzoquinones that preferentially inhibit glutaminase B isoform (GAB, GLS2) rather than the kidney isoform of glutaminase (KGA, GLS1). We identified amino acid residues in an allosteric binding pocket responsible for the selectivity. Treatment with the alkyl benzoquinones decreased intracellular glutaminase activity and glutamate levels. GLS2 inhibition by either alkyl benzoquinones or GLS2 siRNA reduced carcinoma cell proliferation and anchorage-independent colony formation, and induced autophagy via AMPK mediated mTORC1 inhibition. Our findings demonstrate amino acid sequences for selective inhibition of glutaminase isozymes and validate GLS2 as a potential anti-cancer target.

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“…Compound C was obtained from Sigma–Aldrich (St. Louis, MO). Western blotting was performed as described [40]. …”

Section: Methodsmentioning

confidence: 99%

Discovery of selective inhibitors of Glutaminase-2, which inhibit mTORC1, activate autophagy and inhibit proliferation in cancer cells

et al. 2014

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“…Cells, viruses, immunofluorescent assay (IFA), and cytopathic effect (CPE) Swine testicular (ST) epithelial cells and the Taiwan fieldisolated virulent strain of TGEV were grown and propagated as described (Yang et al, 2007b). IFA and CPE and cytotoxicity assays were also as described (Yang et al, 2007b). Briefly, ST cells in 96-well plates, with or without a 2 h pretreatment with test compounds, were infected with TGEV at a multiplicity of infection (MOI) of 10 for IFA.…”

Section: Methodsmentioning

confidence: 99%

“…These assays were performed as described (Yang et al, 2007b); antibody against human GAPDH was purchased from Cell Signaling Technology Inc. (MA, USA). DMSO, PEG400 and DMA were purchased from Sigma Aldrich Inc. (MO, USA).…”

Section: Chemicals Western Blot Analysis Viral Rna Isolation and Rementioning

confidence: 99%

Identification of phenanthroindolizines and phenanthroquinolizidines as novel potent anti-coronaviral agents for porcine enteropathogenic coronavirus transmissible gastroenteritis virus and human severe acute respiratory syndrome coronavirus

et al. 2010

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The discovery and development of new, highly potent anti-coronavirus agents and effective approaches for controlling the potential emergence of epidemic coronaviruses still remains an important mission. Here, we identified tylophorine compounds, including naturally occurring and synthetic phenanthroindolizidines and phenanthroquinolizidines, as potent in vitro inhibitors of enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV). The potent compounds showed 50% maximal effective concentration (EC₅₀) values ranging from 8 to 1468 nM as determined by immunofluorescent assay of the expression of TGEV N and S proteins and by real time-quantitative PCR analysis of viral yields. Furthermore, the potent tylophorine compounds exerted profound anti-TGEV replication activity and thereby blocked the TGEV-induced apoptosis and subsequent cytopathic effect in ST cells. Analysis of the structure-activity relations indicated that the most active tylophorine analogues were compounds with a hydroxyl group at the C14 position of the indolizidine moiety or at the C3 position of the phenanthrene moiety and that the quinolizidine counterparts were more potent than indolizidines. In addition, tylophorine compounds strongly reduced cytopathic effect in Vero 76 cells induced by human severe acute respiratory syndrome coronavirus (SARS CoV), with EC₅₀ values ranging from less than 5 to 340 nM. Moreover, a pharmacokinetic study demonstrated high and comparable oral bioavailabilities of 7-methoxycryptopleurine (52.7%) and the naturally occurring tylophorine (65.7%) in rats. Thus, our results suggest that tylophorine compounds are novel and potent anti-coronavirus agents that may be developed into therapeutic agents for treating TGEV or SARS CoV infection.

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“…They have demonstrated these results by the correlation of in vivo and in vitro activities using mouse paw edema (Tseng et al 2005). 143 Yang et al (2007) synthesized a series of benzothiazolium compounds (144 and 145) which showed inhibitory activity against gastroenteritis virus (TGEV). They used swine testicle (ST) cells infected with transmissible gastroenteritis virus (TGEV) and an indirect immunofluorescent assay with antibodies against TGEV spike and nucleocapsid proteins to screen the benzothiazolium compounds that inhibit TGEV replication.…”

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confidence: 99%

“…They used swine testicle (ST) cells infected with transmissible gastroenteritis virus (TGEV) and an indirect immunofluorescent assay with antibodies against TGEV spike and nucleocapsid proteins to screen the benzothiazolium compounds that inhibit TGEV replication. The benzothiazolium compounds were found to have inhibitory activity against TGEV 3CL(pro) (Yang et al 2007). …”

mentioning

confidence: 99%