scholarly journals Vaccinia Virus Uses Retromer-Independent Cellular Retrograde Transport Pathways To Facilitate the Wrapping of Intracellular Mature Virions during Virus Morphogenesis

2016 ◽  
Vol 90 (22) ◽  
pp. 10120-10132 ◽  
Author(s):  
Kate Harrison ◽  
Ismar R. Haga ◽  
Tali Pechenick Jowers ◽  
Seema Jasim ◽  
Jean-Christophe Cintrat ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Clinical Signs ◽  
Retrograde Transport ◽  
Central Component ◽  
Transport Pathways ◽  
Extracellular Virus ◽  
Garp Complex ◽  
In Cells

ABSTRACTPoxviruses, such as vaccinia virus (VACV), undertake a complex cytoplasmic replication cycle which involves morphogenesis through four distinct virion forms and includes a crucial wrapping step whereby intracellular mature virions (IMVs) are wrapped in two additional membranes to form intracellular enveloped virions (IEVs). To determine if cellular retrograde transport pathways are required for this wrapping step, we examined VACV morphogenesis in cells with reduced expression of the tetrameric tethering factor known as the GARP (Golgi-associated retrograde pathway), a central component of retrograde transport. VACV multistep replication was significantly impaired in cells transfected with small interfering RNA targeting the GARP complex and in cells with a mutated GARP complex. Detailed analysis revealed that depletion of the GARP complex resulted in a reduction in the number of IEVs, thereby linking retrograde transport with the wrapping of IMVs. In addition, foci of viral wrapping membrane proteins without an associated internal core accumulated in cells with a mutated GARP complex, suggesting that impaired retrograde transport uncouples nascent IMVs from the IEV membranes at the site of wrapping. Finally, small-molecule inhibitors of retrograde transport strongly suppressed VACV multistep growthin vitroand reduced weight loss and clinical signs in anin vivomurine model of systemic poxviral disease. This work links cellular retrograde transport pathways with the morphogenesis of poxviruses and identifies a panel of novel inhibitors of poxvirus replication.IMPORTANCECellular retrograde transport pathways traffic cargo from endosomes to thetrans-Golgi network and are a key part of the intracellular membrane network. This work reveals that the prototypic poxvirus vaccinia virus (VACV) exploits cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions and therefore promote the production of extracellular virus. Inhibition of retrograde transport by small-molecule inhibitors reduced the replication of VACV in cell culture and alleviated disease in mice experimentally infected with VACV. This research provides fundamental new knowledge about the wrapping step of poxvirus morphogenesis, furthers our knowledge of the complex cellular retrograde pathways, and identifies a new group of antipoxvirus drugs.

scholarly journals A chemoprobe tracks its target

2019 ◽  
Vol 294 (20) ◽  
pp. 8323-8324
Author(s):  
Aseem Z. Ansari
Keyword(s):  
Small Molecule ◽  
Clinical Utility ◽  
Small Molecule Inhibitors ◽  
Indirect Measure ◽  
Histone Marks ◽  
Histone Lysine ◽  
Histone Lysine Demethylase ◽  
Histone Modifying Enzymes ◽  
Lysine Demethylase ◽  
In Cells

Small-molecule inhibitors of histone-modifying enzymes have significant clinical utility for managing diseases such as cancer. These inhibitors are usually identified and monitored through their effects on the gain or loss of specific histone marks. In cells, multiple related enzymes can place or remove a specific mark; therefore, relying on an indirect measure of inhibitor engagement can be misleading. Mascaró et al. describe a luminescence-based ELISA approach that directly monitors binding of inhibitors to the histone lysine demethylase KDM1A.

scholarly journals A phenolic small molecule inhibitor of RNase L prevents cell death from ADAR1 deficiency

2020 ◽  
Vol 117 (40) ◽  
pp. 24802-24812 ◽  
Author(s):  
Salima Daou ◽  
Manisha Talukdar ◽  
Jinle Tang ◽  
Beihua Dong ◽  
Shuvojit Banerjee ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Therapeutic Potential ◽  
Protein Kinase Inhibitors ◽  
Response To Treatment ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
In Cells

The oligoadenylate synthetase (OAS)–RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.

scholarly journals Inhibitors of the Sec61 Complex and Novel High Throughput Screening Strategies to Target the Protein Translocation Pathway

2021 ◽  
Vol 22 (21) ◽  
pp. 12007
Author(s):  
Eva Pauwels ◽  
Ralf Schülein ◽  
Kurt Vermeire
Keyword(s):  
Small Molecule ◽  
Cell Biology ◽  
High Throughput Screening ◽  
Secretory Pathway ◽  
Small Molecule Inhibitors ◽  
Protein Translocation ◽  
Central Component ◽  
Antibiotic Drugs ◽  
Screening Strategies ◽  
Translocation Pathway

Proteins targeted to the secretory pathway start their intracellular journey by being transported across biological membranes such as the endoplasmic reticulum (ER). A central component in this protein translocation process across the ER is the Sec61 translocon complex, which is only intracellularly expressed and does not have any enzymatic activity. In addition, Sec61 translocon complexes are difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its function has thus been notoriously difficult. However, such translocation inhibitors may not only be valuable tools for cell biology, but may also represent novel anticancer drugs, given that cancer cells heavily depend on efficient protein translocation into the ER to support their fast growth. In this review, different inhibitors of protein translocation will be discussed, and their specific mode of action will be compared. In addition, recently published screening strategies for small molecule inhibitors targeting the whole SRP-Sec61 targeting/translocation pathway will be summarized. Of note, slightly modified assays may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex, in order to identify novel antibiotic drugs.

scholarly journals Author's overview: identifying SARS-CoV-2 antiviral compounds

2021 ◽  
Vol 478 (13) ◽  
pp. 2533-2535
Author(s):  
John F.X. Diffley
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Chemical Compounds ◽  
Antiviral Compounds ◽  
In Cells

In response to the COVID-19 pandemic, we began a project in March 2020 to identify small molecule inhibitors of SARS-CoV-2 enzymes from a library of chemical compounds containing many established pharmaceuticals. Our hope was that inhibitors we found might slow the replication of the SARS-CoV-2 virus in cells and ultimately be useful in the treatment of COVID-19. The seven accompanying manuscripts describe the results of these chemical screens. This overview summarises the main highlights from these screens and discusses the implications of our results and how our results might be exploited in future.

scholarly journals Small molecule inhibitors of HDM2 ubiquitin ligase activity stabilize and activate p53 in cells

Cancer Cell ◽  
2005 ◽  
Vol 7 (6) ◽  
pp. 547-559 ◽  
Author(s):  
Yili Yang ◽  
Robert L. Ludwig ◽  
Jane P. Jensen ◽  
Shervon A. Pierre ◽  
Maxine V. Medaglia ◽  
...  
Keyword(s):  
Small Molecule ◽  
Ubiquitin Ligase ◽  
Small Molecule Inhibitors ◽  
Ubiquitin Ligase Activity ◽  
In Cells

scholarly journals Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Behnam Nabet ◽  
Fleur M. Ferguson ◽  
Bo Kyung A. Seong ◽  
Miljan Kuljanin ◽  
Alan L. Leggett ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protein Function ◽  
Chemical Biology ◽  
Small Molecule Inhibitors ◽  
Target Protein ◽  
Protein Homeostasis ◽  
Direct Control ◽  
Protein Levels ◽  
In Cells

Abstract Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAGV-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12F36V-tagged proteins. dTAGV-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice.

scholarly journals Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

2020 ◽  
Author(s):  
Behnam Nabet ◽  
Fleur M. Ferguson ◽  
Bo Kyung A. Seong ◽  
Miljan Kuljanin ◽  
Alan L. Leggett ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protein Function ◽  
Chemical Biology ◽  
Small Molecule Inhibitors ◽  
Target Protein ◽  
Protein Homeostasis ◽  
Direct Control ◽  
Protein Levels ◽  
In Cells

ABSTRACTChemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAGV-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12F36V-tagged proteins. dTAGV-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice.

High-throughput screening for the identification of small-molecule inhibitors of retinoblastoma protein phosphorylation in cells

2003 ◽  
Vol 320 (1) ◽  
pp. 66-74 ◽  
Author(s):  
S Elaine Barrie ◽  
Ebun Eno-Amooquaye ◽  
Anthea Hardcastle ◽  
Georgina Platt ◽  
Juliet Richards ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Retinoblastoma Protein ◽  
In Cells

Inhibition of NGLY1 inactivates the transcription factor Nrf1 and potentiates proteasome inhibitor cytotoxicity

2017 ◽  
Author(s):  
Carolyn Bertozzi ◽  
Fred Tomlin ◽  
Ulla Gerling-Driessen ◽  
Yi-Chang Liu ◽  
Ryan Flynn ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
Proteasome Inhibitor ◽  
Activation Mechanism ◽  
Cancer Drugs ◽  
Small Molecule Library ◽  
Library Screen ◽  
In Cells

We discovered that the proteostasis modulating transcription factor Nrf1 requires cytosolic de-N-glycosylation by the N-glycanase NGly1 as part of its activation mechanism. Through a covalent small molecule library screen, we discovered an inhibitor of NGly1 that blocks Nrf1 activation in cells and potentiates the activity of proteasome inhibitor cancer drugs. The requirement of NGly1 for Nrf1 activity likely underlies several pathologies associated with a rare hereditary deficiency in NGly1.

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