scholarly journals Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase

2011 ◽  
Vol 92 (7) ◽  
pp. 1607-1616 ◽  
Author(s):  
Ji-Hye Lee ◽  
Intekhab Alam ◽  
Kang Rok Han ◽  
Sunyoung Cho ◽  
Sungho Shin ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Crystal Structures ◽  
Active Site ◽  
Metal Ion ◽  
Health Concern ◽  
Murine Norovirus ◽  
Active Site Residues ◽  
Rna Dependent Rna Polymerase ◽  
Close Proximity ◽  
Functional Features

Norovirus is one of the leading agents of gastroenteritis and is a major public health concern. In this study, the crystal structures of recombinant RNA-dependent RNA polymerase (RdRp) from murine norovirus-1 (MNV-1) and its complex with 5-fluorouracil (5FU) were determined at 2.5 Å resolution. Crystals with C2 symmetry revealed a dimer with half a dimer in the asymmetrical unit, and the protein exists predominantly as a monomer in solution, in equilibrium with a smaller population of dimers, trimers and hexamers. MNV-1 RdRp exhibited polymerization activity with a right-hand fold typical of polynucleotide polymerases. The metal ion modelled in close proximity to the active site was found to be coordinated tetrahedrally to the carboxyl groups of aspartate clusters. The orientation of 5FU observed in three molecules in the asymmetrical unit was found to be slightly different, but it was stabilized by a network of favourable interactions with the conserved active-site residues Arg185, Asp245, Asp346, Asp347 and Arg395. The information gained on the structural and functional features of MNV-1 RdRp will be helpful in understanding replication of norovirus and in designing novel therapeutic agents against this important pathogen.

scholarly journals Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase

2015 ◽  
Vol 60 (1) ◽  
pp. 600-608 ◽  
Author(s):  
Hong-Tao Xu ◽  
Susan P. Colby-Germinario ◽  
Said Hassounah ◽  
Peter K. Quashie ◽  
Yingshan Han ◽  
...  
Keyword(s):  
Drug Design ◽  
Dengue Virus ◽  
Rna Polymerase ◽  
Small Molecule ◽  
Active Site ◽  
Metal Ion ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Molecule Inhibitor ◽  
Hiv 1

ABSTRACTThe viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50sof 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 μM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor.

scholarly journals Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase in complex with 2-thiouridine or ribavirin

Virology ◽  
2012 ◽  
Vol 426 (2) ◽  
pp. 143-151 ◽  
Author(s):  
Intekhab Alam ◽  
Ji-Hye Lee ◽  
Ki Joon Cho ◽  
Kang Rok Han ◽  
Jai Myung Yang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Crystal Structures ◽  
Murine Norovirus ◽  
Rna Dependent Rna Polymerase

scholarly journals Using bioinformatics tools for the discovery of Dengue RNA-dependent RNA polymerase inhibitors

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5068 ◽  
Author(s):  
Nomagugu B. Nncube ◽  
Pritika Ramharack ◽  
Mahmoud E.S. Soliman
Keyword(s):  
Drug Design ◽  
Rna Polymerase ◽  
Active Site ◽  
Mammalian Cells ◽  
Pharmacophore Model ◽  
Pharmacophore Modeling ◽  
Health Concern ◽  
Innovative Drug ◽  
Rna Dependent Rna Polymerase ◽  
Bioinformatics Tools

BackgroundDengue fever has rapidly manifested into a serious global health concern. The emergence of various viral serotypes has prompted the urgent need for innovative drug design techniques. Of the viral non-structural enzymes, the NS5 RNA-dependent RNA polymerase has been established as a promising target due to its lack of an enzymatic counterpart in mammalian cells and its conserved structure amongst all serotypes. The onus is now on scientists to probe further into understanding this enzyme and its mechanism of action. The field of bioinformatics has evolved greatly over recent decades, with updated drug design tools now being publically available.MethodsIn this study, bioinformatics tools were used to provide a comprehensive sequence and structural analysis of the two most prominent serotypes of Dengue RNA-dependent RNA polymerase. A list of popularflavivirusinhibitors were also chosen to dock to the active site of the enzyme. The best docked compound was then used as a template to generate a pharmacophore model that may assist in the design of target-specific Dengue virus inhibitors.ResultsComparative sequence alignment exhibited similarity between all three domains of serotype 2 and 3.Sequence analysis revealed highly conserved regions at residues Meth530, Thr543 Asp597, Glu616, Arg659 and Pro671. Mapping of the active site demonstrated two highly conserved residues: Ser710 and Arg729. Of the active site interacting residues, Ser796 was common amongst all ten docked compounds, indicating its importance in the drug design process. Of the ten dockedflavivirusinhibitors, NITD-203 showed the best binding affinity to the active site. Further pharmacophore modeling of NITD-203 depicted significant pharmacophoric elements that are necessary for stable binding to the active site.DiscussionThis study utilized publically available bioinformatics tools to provide a comprehensive framework on Dengue RNA-dependent RNA polymerase. Based on docking studies, a pharmacophore model was also designed to unveil the crucial pharmacophoric elements that are required when constructing an efficacious DENV inhibitor. We believe that this study will be a cornerstone in paving the road toward the design of target-specific inhibitors against DENV RdRp.

scholarly journals How cyanophage S-2L rejects adenine and incorporates 2-aminoadenine to saturate hydrogen bonding in its DNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dariusz Czernecki ◽  
Pierre Legrand ◽  
Mustafa Tekpinar ◽  
Sandrine Rosario ◽  
Pierre-Alexandre Kaminski ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonding ◽  
Crystal Structures ◽  
Active Site ◽  
Metal Ion ◽  
Restriction Endonucleases ◽  
Structural Element

AbstractBacteriophages have long been known to use modified bases in their DNA to prevent cleavage by the host’s restriction endonucleases. Among them, cyanophage S-2L is unique because its genome has all its adenines (A) systematically replaced by 2-aminoadenines (Z). Here, we identify a member of the PrimPol family as the sole possible polymerase of S-2L and we find it can incorporate both A and Z in front of a T. Its crystal structure at 1.5 Å resolution confirms that there is no structural element in the active site that could lead to the rejection of A in front of T. To resolve this contradiction, we show that a nearby gene is a triphosphohydolase specific of dATP (DatZ), that leaves intact all other dNTPs, including dZTP. This explains the absence of A in S-2L genome. Crystal structures of DatZ with various ligands, including one at sub-angstrom resolution, allow to describe its mechanism as a typical two-metal-ion mechanism and to set the stage for its engineering.

Cordycepin as a Promising Inhibitor of SARS-CoV-2 RNA dependent RNA polymerase (RdRp)

2021 ◽  
Vol 28 ◽  
Author(s):  
Shabana Bibi ◽  
Mohammad Mehedi Hasan ◽  
Yuan-Bing Wang ◽  
Stavros P. Papadakos ◽  
Hong Yu
Keyword(s):  
Rna Polymerase ◽  
Drug Repositioning ◽  
Human Life ◽  
Drug Candidate ◽  
Active Site Residues ◽  
Rna Dependent Rna Polymerase ◽  
Economic Downturns ◽  
Inhibitory Potential ◽  
Dynamics Simulations ◽  
Global Threat

Background: SARS-CoV-2, which emerged in Wuhan, China, is a new global threat that has killed millions of people and continues to do so. This pandemic has not only threatened human life but has also triggered economic downturns across the world. Researchers have made significant strides in discovering molecular insights into SARS-CoV-2 pathogenesis and developing vaccines, but there is still no successful cure for SARS-CoV-2 infected patients. Objective: The present study has proposed a drug-repositioning pipeline for the design and discovery of an effective fungal-derived bioactive metabolite as a drug candidate against SARS-CoV-2. Methods: Fungal derivative “Cordycepin” was selected for this study to investigate the inhibitory properties against RNA-dependent RNA polymerase (RdRp) (PDB ID: 6M71) of SARS-CoV-2. The pharmacological profile, intermolecular interactions, binding energy, and stability of the compound were determined utilizing cheminformatic approaches. Subsequently, molecular dynamic simulation was performed to better understand the binding mechanism of cordycepin to RdRp. Results: The pharmacological data and retrieved molecular dynamics simulations trajectories suggest excellent drug-likeliness and greater structural stability of cordycepin, while the catalytic residues (Asp760, Asp761), as well as other active site residues (Trp617, Asp618, Tyr619, Trp800, Glu811) of RdRp, showed better stability during the overall simulation span. Conclusion: Promising results of pharmacological investigation along with molecular simulations revealed that cordycepin exhibited strong inhibitory potential against SARS-CoV-2 polymerase enzyme (RdRp). Hence, cordycepin should be highly recommended to test in a laboratory to confirm its inhibitory potential against the SARS-CoV-2 polymerase enzyme (RdRp).

scholarly journals Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Nidhi Kaushik ◽  
Chandru Subramani ◽  
Saumya Anang ◽  
Rajagopalan Muthumohan ◽  
Shalimar ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Hepatitis E Virus ◽  
Zinc Supplementation ◽  
Hepatitis E ◽  
Viral Rna ◽  
Health Concern ◽  
Healthy Individuals ◽  
Genomic Rna ◽  
Rna Dependent Rna Polymerase ◽  
Zinc Salts

ABSTRACT Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection. IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used to treat HEV cases, there are known side effects and limitations of such therapy. Our discovery of the ability of zinc salts to block HEV replication by virtue of their ability to inhibit the activity of viral RdRp is important because these findings pave the way to test the efficacy of zinc supplementation therapy in HEV-infected patients. Since zinc supplementation therapy is known to be safe in healthy individuals and since high-dose zinc is used in the treatment of Wilson's disease, it may be possible to control HEV-associated health problems following a similar treatment regimen.

scholarly journals Structural basis for VPg-induced formation of RNA-dependent RNA polymerase multimeric complexes

2014 ◽  
Vol 70 (a1) ◽  
pp. C1601-C1601
Author(s):  
Ji-Hye Lee ◽  
Yeon Bin Chung ◽  
Jong Hyeon Seok ◽  
Kang Rok Han ◽  
Sella Kim ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
De Novo ◽  
Helical Structure ◽  
Science Research ◽  
Binding Mode ◽  
Structural Basis ◽  
Murine Norovirus ◽  
Virion Protein ◽  
Electron Microscopic ◽  
Rna Dependent Rna Polymerase

Norovirus is the leading cause of epidemic acute, nonbacterial gastroenteritis, and adopts de novo and VPg (Virion protein genome linked)-primed RNA synthesis by RNA-dependent RNA polymerase (RdRp). To understand the interaction between RdRp and VPg in replication of murine norovirus-1 (MNV-1), we determined the crystal structure of MNV-1 RdRp-VPg(1-73)-RNA complex. VPg was bound to the base of the palm domain and the tip of the fingers domain of RdRp simultaneously, but RNA template could not be modeled. The binding affinity constants (Kd) for RdRp-VPg was 3.7411.57 nM and VPg(1-73) showed approximately 90-fold less affinity than that of full-length VPg. In addition to this multiple binding mode, VPg enhanced the interactions of RdRp hexamers, leading to the formation of high-order multimers or tubular fibrils with significantly increased polymerase activity, confirmed by electron microscopic and biochemical studies. Our data indicated that MNV-1 VPg with helical structure was bound to RdRp at multiple sites and induces RdRp multimerization in viral replication. The multimers of RdRp-VPg-RNA can provide a mechanistic understanding of viral polymerase multimeric arrays and a new tool for development of antivirals to control norovirus outbreaks. This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health, Welfare and Family Affairs (A085119 K.H.K), Basic Science Research Program through the National Research Foundation (NRF-2013R1A1A2064940, L.J-H), Korea University Grant (L.J-H), and the BK21 plus program of the Ministry of Education, Korea.

scholarly journals Structure based protein engineering to confer selectivity of guanine deaminase

2014 ◽  
Vol 70 (a1) ◽  
pp. C437-C437
Author(s):  
Aruna Bitra ◽  
Ruchi Anand
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
X Ray ◽  
Secondary Activity ◽  
Guanine Deaminase ◽  
Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

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