scholarly journals Targeting of Non-Structural Protein 9 as a Novel Therapeutic Target for the Treatment of SARS-CoV-2

2020 ◽  
Vol 3 ◽  
Author(s):  
Matthew Anderson ◽  
John Turchi
Keyword(s):  
Viral Replication ◽  
Therapeutic Target ◽  
Rna Binding ◽  
Future Research ◽  
Great Promise ◽  
Dependent Manner ◽  
Nucleic Acid Binding ◽  
Structural Protein ◽  
Concentration Dependent Manner ◽  
Novel Therapeutic

Background/Hypothesis:  The 2019 novel coronavirus (SARS-CoV-2) is a human coronavirus responsible for a global pandemic with over 13 million confirmed cases. Currently, there are no treatments to block viral infection or replication. Exploring novel therapeutic targets for SARS-CoV-2 and future coronaviruses holds great promise for treating the current and future outbreaks. One such target is the non-structural protein 9 (nsp9), which has been shown to be highly conserved and unique to the coronavirus family as well as playing a role in viral replication. We hypothesize nsp9 is a viable target for therapeutic development.     Methods:  Towards determining the utility of targeting nsp9, a series of databases were queried for articles pertaining to nsp9 in SARS-CoV-2 and other coronaviruses and coronaviruses in general. We assessed structural, biochemical and cellular features of nsp9.      Results:  Nsp9 forms a homodimer via a conserved a-helix containing a glycine-rich interaction motif (GxxxG). Dimerization at the GxxxG interface is required for efficient viral replication. Nsp9’s core is an open, six-stranded b-barrel whose fold gives it a structure similar to nucleic acid binding OB-fold proteins. This OB-like fold has not been detected in replicative complexes of other RNA viruses and may reflect the unique and complex CoV replication machinery. Nsp9 is an indispensable component of the replication complex that binds single-stranded RNA in a concentration-dependent manner. A recent bioinformatic approach also found that nsp9 interacts with NF-kappa-B-repressing factor and may play a role in the IL-8/IL-6 mediated chemotaxis of neutrophils and inflammatory response observed in Covid-19 patients.     Conclusion/Potential Impact:   Based on this research, we conclude nsp9 represents a novel therapeutic target whose OB-like-fold may provide a targetable structure for interrupting RNA binding and impairing viral replication. This study will help inform current and future research that seeks to target nsp9’s structure and biochemical interactions as treatment for coronavirus infection. 

scholarly journals The SARS-CoV-2 conserved macrodomain is a mono-ADP-ribosylhydrolase

2020 ◽  
Author(s):  
Yousef M.O. Alhammad ◽  
Maithri M. Kashipathy ◽  
Anuradha Roy ◽  
Jean-Philippe Gagné ◽  
Peter McDonald ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Therapeutic Target ◽  
Viral Infections ◽  
Protein Domain ◽  
Structural Protein ◽  
Potential Therapeutic Target ◽  
Novel Therapeutic Strategies ◽  
Design And Testing ◽  
Translational Process ◽  
Novel Therapeutic

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity.IMPORTANCESARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused greater than 900 thousand deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

scholarly journals The Anti-Aging Potential of Neohesperidin and Its Synergistic Effects with Other Citrus Flavonoids in Extending Chronological Lifespan of Saccharomyces Cerevisiae BY4742

Molecules ◽  
2019 ◽  
Vol 24 (22) ◽  
pp. 4093 ◽  
Author(s):  
Chunxia Guo ◽  
Hua Zhang ◽  
Xin Guan ◽  
Zhiqin Zhou
Keyword(s):  
Synergistic Effects ◽  
Future Research ◽  
Dependent Manner ◽  
Ros Scavenging ◽  
Concentration Dependent Manner ◽  
Chronological Lifespan ◽  
Flavonoid Compounds ◽  
Citrus Flavonoids ◽  
High Throughput Assay

The anti-aging activity of many plant flavonoids, as well as their mechanisms of action, have been explored in the current literature. However, the studies on the synergistic effects between the different flavonoid compounds were quite limited in previous reports. In this study, by using a high throughput assay, we tested the synergistic effects between different citrus flavonoids throughout the yeast’s chronological lifespan (CLS). We studied the effect of four flavonoid compounds including naringin, hesperedin, hesperitin, neohesperidin, as well as their different combinations on the CLS of the yeast strain BY4742. Their ROS scavenging ability, in vitro antioxidant activity and the influence on the extracellular pH were also tested. The results showed that neohesperidin extended the yeast’s CLS in a concentration-dependent manner. Especially, we found that neohesperidin showed great potential in extending CLS of budding yeast individually or synergistically with hesperetin. The neohesperidin exhibited the strongest function in decreasing the reactive oxygen species (ROS) accumulation in yeast. These findings clearly indicated that neohesperidin is potentially an anti-aging citrus flavonoid, and its synergistic effect with other flavonoids on yeast’s CLS will be an interesting subject for future research of the anti-aging function of citrus fruits.

scholarly journals The Role of ATP in the RNA Translocation Mechanism of SARS-CoV-2 NSP13 Helicase

2021 ◽  
Author(s):  
Ryan Weber ◽  
Martin McCullagh
Keyword(s):  
Rational Design ◽  
Rna Binding ◽  
Gaussian Mixture ◽  
Dynamics Simulation ◽  
Dependent Manner ◽  
Structural Protein ◽  
Linear Discriminant ◽  
Translocation Mechanism ◽  
Key Aspects ◽  
Antiviral Targets

The COVID-19 pandemic has demonstrated the need to develop potent and transferable therapeutics to treat coronavirus infections. Numerous antiviral targets are being investigated, but non-structural protein 13 (nsp13) stands out as a highly conserved and yet under studied target. Nsp13 is a superfamily 1 (SF1) helicase that translocates along and unwinds viral RNA in an ATP dependent manner. Currently, there are no available structures of nsp13 from SARS-CoV-1 or SARS-CoV-2 with either ATP or RNA bound presenting a significant hurdle to the rational design of therapeutics. To address this knowledge gap, we have built models of SARS-CoV-2 nsp13 in Apo, ATP, ssRNA and ssRNA+ATP substrate states. Using 30 microseconds of Gaussian accelerated molecular dynamics simulation (at least 6 microseconds per substrate state), these models were confirmed to maintain substrate binding poses that are similar to other SF1 helicases. A gaussian mixture model and linear discriminant analysis structural clustering protocol was used to identify key aspects of the ATP-dependent RNA translocation mechanism. Namely, four RNA-nsp13 structures are identified that exhibit ATP-dependent populations and support the inch-worm mechanism for translocation. These four states are characterized by different RNA-binding poses for motifs Ia, IV, and V and suggest a powerstroke--like motion of domain 2A relative to domain 1A. This structural and mechanistic insight of nsp13 RNA translocation presents novel targets for the further development of antivirals.

scholarly journals Interplay between RNA-binding protein HuR and Nox4 as a novel therapeutic target in diabetic kidney disease

2020 ◽  
Vol 36 ◽  
pp. 100968 ◽  
Author(s):  
Qian Shi ◽  
Doug-Yoon Lee ◽  
Denis Féliers ◽  
Hanna E. Abboud ◽  
Manzoor A. Bhat ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Therapeutic Target ◽  
Diabetic Kidney Disease ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Diabetic Kidney ◽  
Novel Therapeutic

scholarly journals Sirt1 Is a Novel Therapeutic Target in T-ALL

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2221-2221
Author(s):  
Olga Lancho ◽  
Amartya Singh ◽  
Victoria da Silva-Diz ◽  
Patricia Renck Nunes ◽  
Luca Tottone ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Gene Expression Profiling ◽  
Therapeutic Target ◽  
Expression Profiling ◽  
Conditional Knockout ◽  
Dependent Manner ◽  
Notch1 Signaling ◽  
Novel Therapeutic

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematological malignancy that affects both children and adults. Still, 20%-50% of patients show primary resistance or relapse after treatment, and ultimately die from their disease. Aberrant NOTCH1 signaling has a major role in the pathogenesis of T-ALL, as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. In this context, small-molecule γ-secretase inhibitors (GSIs), which effectively block NOTCH1 activation via inhibition of a critical intramembrane proteolytic cleavage required for NOTCH1 signaling, are being tested in clinical trials for the treatment of relapsed and refractory T-ALL. However, the clinical development of anti-NOTCH1 therapies in T-ALL has been hampered by limited and delayed therapeutic response to these drugs, underscoring the need to identify novel therapeutic targets and to develop more effective drugs for the treatment of this disease. We previously demonstratedthe importance of NOTCH1-driven metabolic pathways in the response to anti-NOTCH1 therapies (gamma-secretase inhibitors, GSIs). Moreover, epigenetic plasticity has also been proposed to mediate resistance to GSIs. Thus, we postulated that central regulators that control both the metabolic and epigenetic status of cells could act as master regulators of NOTCH1-induced transformation. Indeed, our results have identified the SIRT1 histone deacetylase, a central epigenetic and metabolic regulator, as a key player in T-ALL. Analyses of gene expression profiling data from T-ALL patients revealed a significant upregulation of SIRT1 in T-ALL. Consistently, SIRT1 protein levels are significantly upregulated in T-ALL cells as compared to normal human thymus. Moreover, through the integration of GSI-washout experiments, epigenetic profiling and CRISPR/Cas9-induced experiments, we have identified a distal enhancer of Sirt1 that is bound and controlled by NOTCH1, which might help explain the broad upregulation of Sirt1 observed in T-ALL patients. Next, and to formally test the effects of Sirt1 on T-cell transformation, we generated NOTCH1-driven primary T-ALLs from different Sirt1 genetic backgrounds. In this context, our results demonstrate that Sirt1 genetic overexpression leads to accelerated kinetics of NOTCH1-induced T-ALL and promotes resistance to GSI treatment in T-ALL in vivo in a deacetylase-dependent manner. Conversely, germinal loss of Sirt1 leads to delayed T-ALL development and reduced disease penetrance. Moreover, pharmacological inhibition of SIRT1 with EX-527 shows anti-leukemic and synergistic effects with NOTCH1 inhibition in T-ALL cell lines in vitro. Finally, genetic deletion of Sirt1 in already established primary isogenic Sirt1 conditional knockout leukemiasleads to significant and highly synergistic anti-leukemic effects with GSI treatment in vivo. Mechanistically, acetyl-proteomics analyses revealed that acute deletion of Sirt1 consistently leads to hyperacetylation of Kat7 and Brd1, which are both part of a histone acetyltransferase complex. Indeed, Sirt1 loss results in global epigenetic changes including decreased levels of H4K12ac, which is a Kat7-target mark. Moreover, gene expression profiling analyses upon Sirt1 loss in leukemia in vivo revealed broad transcriptional changes. Gene-set enrichment analyses revealed that the transcriptional signature upon Sirt1 loss significantly correlates with the one obtained upon Kat7 loss, overall suggesting that Sirt1 loss leads to hyperacetylation of Kat7, which might be less active. Finally, our gene expression analyses also revealed a marked block in mTOR signaling, suggesting leukemia cells suffer a metabolic crisis upon Sirt1 loss. Consistently, acute deletion of Sirt1 results in prominent global metabolic changes in glycolysis, glutaminolysis and TCA, with concomitant activation of AMPK, resulting in markedly cytotoxic effects. Overall, our results reveal an oncogenic role for Sirt1 in T-ALL generation and progression, demonstrate that Sirt1 contributes to mediate resistance to anti-NOTCH1 therapies, identify a novel Notch1-Sirt1-Kat7 link and uncover Sirt1 as a novel therapeutic target for the treatment of T-ALL. Disclosures No relevant conflicts of interest to declare.

Genetics of novel therapeutic targets in schizophrenia

1999 ◽  
Vol 174 (S38) ◽  
pp. 1-4 ◽  
Author(s):  
R. Kerwin ◽  
M. Owen
Keyword(s):  
Adverse Effects ◽  
Therapeutic Target ◽  
Atypical Antipsychotics ◽  
Therapeutic Targets ◽  
New Drugs ◽  
Future Research ◽  
Receptor Blockade ◽  
Targeted Agents ◽  
Novel Therapeutic ◽  
Made In

For many years, following the introduction of chlorpromazine in the 1950s, little progress was made in the discovery of new drugs for schizophrenia (Reynolds, 1992). Dopamine D2 receptor blockade was recognised as the only therapeutic target for antipsychotics (Creese et al, 1976) and the inevitable consequences of striatal blockade remained problematic. However, the strategies and stimuli for discovery of new drugs changed with the introduction of new, atypical antipsychotics in the 1990s. These include clozapine, remoxipride (now withdrawn), olanzapine, risperidone and sertindole (Kerwin & Taylor, 1996). The goal of antipsychotic drug development has always been to widen the therapeutic ratio between efficacy and adverse effects. These new drugs have in the main achieved this. However, which therapeutic targets these drugs employ remains a mystery, and this information is clearly important for future research into more selectively targeted agents.

scholarly journals Norovirus RNA Synthesis Is Modulated by an Interaction between the Viral RNA-Dependent RNA Polymerase and the Major Capsid Protein, VP1

2012 ◽  
Vol 86 (18) ◽  
pp. 10138-10149 ◽  
Author(s):  
Chennareddy V. Subba-Reddy ◽  
Muhammad Amir Yunus ◽  
Ian G. Goodfellow ◽  
C. Cheng Kao
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Murine Norovirus ◽  
Structural Protein ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Concentration Dependent Manner

Using a cell-based assay for RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of noroviruses, we previously observed that VP1, the major structural protein of the human GII.4 norovirus, enhanced the GII.4 RdRp activity but not that of the related murine norovirus (MNV) or other unrelated RNA viruses (C. V. Subba-Reddy, I. Goodfellow, and C. C. Kao, J. Virol. 85:13027–13037, 2011). Here, we examine the mechanism of VP1 enhancement of RdRp activity and the mechanism of mouse norovirus replication. We determined that the GII.4 and MNV VP1 proteins can enhance cognate RdRp activities in a concentration-dependent manner. The VP1 proteins coimmunoprecipitated with their cognate RdRps. Coexpression of individual domains of VP1 with the viral RdRps showed that the VP1 shell domain (SD) was sufficient to enhance polymerase activity. Using SD chimeras from GII.4 and MNV, three loops connecting the central β-barrel structure were found to be responsible for the species-specific enhancement of RdRp activity. A differential scanning fluorimetry assay showed that recombinant SDs can bind to the purified RdRpsin vitro. An MNV replicon with a frameshift mutation in open reading frame 2 (ORF2) that disrupts VP1 expression was defective for RNA replication, as quantified by luciferase reporter assay and real-time quantitative reverse transcription-PCR (qRT-PCR).Trans-complementation of VP1 or its SD significantly recovered the VP1 knockout MNV replicon replication, and the presence or absence of VP1 affected the kinetics of viral RNA synthesis. The results document a regulatory role for VP1 in the norovirus replication cycle, further highlighting the paradigm of viral structural proteins playing additional functional roles in the virus life cycle.

scholarly journals The SARS-CoV-2 conserved macrodomain is a mono-ADP-ribosylhydrolase

2020 ◽  
Author(s):  
Yousef M.O. Alhammad ◽  
Maithri M. Kashipathy ◽  
Anuradha Roy ◽  
Jean-Philippe Gagné ◽  
Peter McDonald ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Therapeutic Target ◽  
Viral Infections ◽  
Protein Domain ◽  
Structural Protein ◽  
Potential Therapeutic Target ◽  
Novel Therapeutic Strategies ◽  
Design And Testing ◽  
Translational Process ◽  
Novel Therapeutic

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. IMPORTANCE SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused greater than 1.2 million deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

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