Helicase Enzymes Research Articles

Drug repurposing screen to identify inhibitors of the RNA polymerase (nsp12) and helicase (nsp13) from SARS-CoV-2 replication and transcription complex

Coronaviruses contain one of the largest genomes among the RNA viruses, coding for 14–16 non-structural proteins (nsp) that are involved in proteolytic processing, genome replication and transcription, and four structural proteins that build the core of the mature virion. Due to conservation across coronaviruses, nsps form a group of promising drug targets as their inhibition directly affects viral replication and, therefore, progression of infection. A minimal but fully functional replication and transcription complex was shown to be formed by one RNA-dependent RNA polymerase (nsp12), one nsp7, two nsp8 accessory subunits, and two helicase (nsp13) enzymes. Our approach involved, targeting nsp12 and nsp13 to allow multiple starting point to interfere with virus infection progression. Here we report a combined in-vitro repurposing screening approach, identifying new and confirming reported SARS-CoV-2 nsp12 and nsp13 inhibitors.

DEAD Box RNA Helicases: Biochemical Properties, Role in RNA Processing and Ribosome Biogenesis.

DEAD box RNA helicases are a versatile group of ATP dependent enzymes that play an essential role in cellular processes like transcription, RNA processing, ribosome biogenesis and translation. These enzymes perform structural rearrangement of complex RNA molecules and enhance the productive folding of RNA and organization of macromolecular complexes. In this review article besides providing the outline about structural organization of helicases, an in-depth discussion will be done on the biochemical properties of RNA helicases like their substrate binding, binding and hydrolysis of ATP and related conformational changes that are important for functioning of the RNA helicase enzymes. I will extensively discuss the physiological role of RNA helicases in RNA processing and ribosome biogenesis.

Exploring the Mangrove Based Phytochemicals as Potential Viral RNA Helicase Inhibitors by in silico Docking and Molecular Dynamics Simulation Methods

A variety of plant-derived molecular compounds from mangrove plants have attracted attention due to the discovery of their antiviral activity. It has been proven that herbal medicines based on them provide good protection against a number of pathogenic viruses. However, it is necessary to screen these effective antiviral compounds to select those that have fewer harmful side effects. This study aimed to screen several bioactive compounds from mangrove plants that could be used as a viral RNA helicase inhibitor. Fifty-nine compounds were selected from the literature and databases for initial study and screening according to Lipinski's rule of five. The resulting selected compounds were subjected to another round of screening through molecular docking studies with five different pathogenic virus RNA helicase enzymes using the Autodock Vina tool followed by ADMET (absorption, distribution, metabolism, excretion and toxicity) analysis. In addition, the best compound-bound helicase-RNA complexes were included in 50 ns molecular dynamics simulations using Gromacs 5.1.1 software followed by molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis. This comparative study predicts that the phytochemical gedunin is an excellent inhibitor of the RNA helicase enzyme of SARS-CoV-2, followed by Japanese encephalitis virus and hepatitis C virus. The results of the study may lead to the development of antiviral compounds against the RNA helicase enzymes of pathogenic viruses.

Determining translocation orientations of nucleic acid helicases.

Helicase enzymes translocate along an RNA or DNA template with a defined polarity to unwind, separate, or remodel duplex strands for a variety of genome maintenance processes. Helicase mutations are commonly associated with a variety of diseases including aging, cancer, and neurodegeneration. Biochemical characterization of these enzymes has provided a wealth of information on the kinetics of unwinding and substrate preferences, and several high-resolution structures of helicases alone and bound to oligonucleotides have been solved. Together, they provide mechanistic insights into the structural translocation and unwinding orientations of helicases. However, these insights rely on structural inferences derived from static snapshots. Instead, continued efforts should be made to combine structure and kinetics to better define active translocation orientations of helicases. This review explores many of the biochemical and biophysical methods utilized to map helicase binding orientation to DNA or RNA substrates and includes several time-dependent methods to unequivocally map the active translocation orientation of these enzymes to better define the active leading and trailing faces.

Development of a database of RNA helicase inhibitors (VHIMDB) of pathogenic viruses and in silico screening for the potential drug molecules

Abstract The pathogenic RNA virus that infects human beings contains the RNA helicase enzyme, responsible for the replication of the viral genome. The enzyme is used as a suitable target against which the drug molecule acts. Therefore, the identification and proposal the novel compounds that can be targeted toward the helicase enzymes to stop the functioning of the enzyme is desirable. Although many viral helicase inhibitor molecules have been identified, still yet no unique database is available for these compounds. This research work envisages developing a curated database of RNA helicase inhibitors. The database contains in total of 353 entries that are computationally predicted and experimentally verified RNA helicase inhibitors. The database contains information like compound name, chemical properties, chemical format, and name of the target virus to which it acts against it with a user-friendly menu-driven search engine. Presently, the database is freely available at: https://vhimdb.rsatpathy.in/. Further, in silico screening of the whole database by drug-likeness and toxicity resulted in 14 potential drug molecules. The selected molecules were analyzed for their effectiveness in binding by using molecular docking score and interaction with the helicase enzymes of three categories of pathogenic viruses (SARS-CoV-2, SARS-CoV, and MERS-CoV).

The Regulation of Homologous Recombination by Helicases.

Homologous recombination is essential for DNA repair, replication and the exchange of genetic material between parental chromosomes during meiosis. The stages of recombination involve complex reorganization of DNA structures, and the successful completion of these steps is dependent on the activities of multiple helicase enzymes. Helicases of many different families coordinate the processing of broken DNA ends, and the subsequent formation and disassembly of the recombination intermediates that are necessary for template-based DNA repair. Loss of recombination-associated helicase activities can therefore lead to genomic instability, cell death and increased risk of tumor formation. The efficiency of recombination is also influenced by the ‘anti-recombinase’ effect of certain helicases, which can direct DNA breaks toward repair by other pathways. Other helicases regulate the crossover versus non-crossover outcomes of repair. The use of recombination is increased when replication forks and the transcription machinery collide, or encounter lesions in the DNA template. Successful completion of recombination in these situations is also regulated by helicases, allowing normal cell growth, and the maintenance of genomic integrity.

Hepatitis C Virus NS3 Protease and Helicase Inhibitors from Red Sea Sponge (Amphimedon) Species in Green Synthesized Silver Nanoparticles Assisted by in Silico Modeling and Metabolic Profiling.

BackgroundHepatitis C virus (HCV) infection is a major cause of hepatic diseases all over the world. This necessitates the need to discover novel anti-HCV drugs to overcome emerging drug resistance and liver complications.PurposeTotal extract and petroleum ether fraction of the marine sponge (Amphimedon spp.) were used for silver nanoparticle (SNP) synthesis to explore their HCV NS3 helicase- and protease-inhibitory potential.MethodsCharacterization of the prepared SNPs was carried out with ultraviolet-visible spectroscopy, transmission electron microscopy, and Fourier-transform infrared spectroscopy. The metabolomic profile of different Amphimedon fractions was assessed using liquid chromatography coupled with high-resolution mass spectrometry. Fourteen known compounds were isolated and their HCV helicase and protease activities assessed using in silico modeling of their interaction with both HCV protease and helicase enzymes to reveal their anti-HCV mechanism of action. In vitro anti-HCV activity against HCV NS3 helicase and protease was then conducted to validate the computation results and compared to that of the SNPs.ResultsTransmission electron–microscopy analysis of NPs prepared from Amphimedon total extract and petroleum ether revealed particle sizes of 8.22–14.30 nm and 8.22–9.97 nm, and absorption bands at λmax of 450 and 415 nm, respectively. Metabolomic profiling revealed the richness of Amphimedon spp. with different phytochemical classes. Bioassay-guided isolation resulted in the isolation of 14 known compounds with anti-HCV activity, initially revealed by docking studies. In vitro anti–HCV NS3 helicase and protease assays of both isolated compounds and NPs further confirmed the computational results.ConclusionOur findings indicate that Amphimedon, total extract, petroleum ether fraction, and derived NPs are promising biosources for providing anti-HCV drug candidates, with nakinadine B and 3,4-dihydro-6-hydroxymanzamine A the most potent anti-HCV agents, possessing good oral bioavailability and penetration power.

Interactome Analysis and Docking Sites of MutS Homologs Reveal New Physiological Roles in Arabidopsis thaliana.

Due to their sedentary lifestyle, plants are constantly exposed to different stress stimuli. Stress comes in variety of forms where factors like radiation, free radicals, “replication errors, polymerase slippage”, and chemical mutagens result in genotoxic or cytotoxic damage. In order to face “the base oxidation or DNA replication stress”, plants have developed many sophisticated mechanisms. One of them is the DNA mismatch repair (MMR) pathway. The main part of the MMR is the MutS homologue (MSH) protein family. The genome of Arabidopsis thaliana encodes at least seven homologues of the MSH family: AtMSH1, AtMSH2, AtMSH3, AtMSH4, AtMSH5, AtMSH6, and AtMSH7. Despite their importance, the functions of AtMSH homologs have not been investigated. In this work, bioinformatics tools were used to obtain a better understanding of MSH-mediated DNA repair mechanisms in Arabidopsis thaliana and to understand the additional biological roles of AtMSH family members. In silico analysis, including phylogeny tracking, prediction of 3D structure, interactome analysis, and docking site prediction, suggested interactions with proteins were important for physiological development of A. thaliana. The MSH homologs extensively interacted with both TIL1 and TIL2 (DNA polymerase epsilon catalytic subunit), proteins involved in cell fate determination during plant embryogenesis and involved in flowering time repression. Additionally, interactions with the RECQ protein family (helicase enzymes) and proteins of nucleotide excision repair pathway were detected. Taken together, the results presented here confirm the important role of AtMSH proteins in mismatch repair and suggest important new physiological roles.

G-quadruplexes and helicases.

Guanine-rich DNA strands can fold in vitro into non-canonical DNA structures called G-quadruplexes. These structures may be very stable under physiological conditions. Evidence suggests that G-quadruplex structures may act as ‘knots’ within genomic DNA, and it has been hypothesized that proteins may have evolved to remove these structures. The first indication of how G-quadruplex structures could be unfolded enzymatically came in the late 1990s with reports that some well-known duplex DNA helicases resolved these structures in vitro. Since then, the number of studies reporting G-quadruplex DNA unfolding by helicase enzymes has rapidly increased. The present review aims to present a general overview of the helicase/G-quadruplex field.

Cooperative base pair melting by helicase and polymerase positioned one nucleotide from each other.

Leading strand DNA synthesis requires functional coupling between replicative helicase and DNA polymerase (DNAP) enzymes, but the structural and mechanistic basis of coupling is poorly understood. This study defines the precise positions of T7 helicase and T7 DNAP at the replication fork junction with single-base resolution to create a structural model that explains the mutual stimulation of activities. Our 2-aminopurine studies show that helicase and polymerase both participate in DNA melting, but each enzyme melts the junction base pair partially. When combined, the junction base pair is melted cooperatively provided the helicase is located one nucleotide ahead of the primer-end. The synergistic shift in equilibrium of junction base pair melting by combined enzymes explains the cooperativity, wherein helicase stimulates the polymerase by promoting dNTP binding (decreasing dNTP Km), polymerase stimulates the helicase by increasing the unwinding rate-constant (kcat), consequently the combined enzymes unwind DNA with kinetic parameters resembling enzymes translocating on single-stranded DNA.

Hepatitis E virus (HEV) protease: a chymotrypsin-like enzyme that processes both non-structural (pORF1) and capsid (pORF2) protein.

Hepatitis E virus (HEV), a major cause of acute viral hepatitis across the world, is a non-enveloped, plus-strand RNA virus. Its genome codes three proteins, pORF1 (multifunctional polyprotein), pORF2 (capsid protein) and pORF3 (multi-regulatory protein). pORF1 encodes methyltransferase, putative papain-like cysteine protease, helicase and replicase enzymes. Of these, the protease domain has not been characterized. On the basis of sequence analysis, we cloned and expressed a protein covering aa 440-610 of pORF1, expression of which led to cell death in Escherichia coli BL-21 and Huh7 hepatoma cells. Finally, we expressed and purified this protein from E. coli C43 cells (resistant to toxic proteins). The refolded form of this protein showed protease activity in gelatin zymography. Digestion assays showed cleavage of both pORF1 and pORF2 as observed previously. MS revealed digestion of capsid protein at both the N and C termini. N-terminal sequencing of the ~35 kDa methyltransferase, ~35 kDa replicase and ~56 kDa pORF2 proteins released by protease digestion revealed that the cleavage sites were alanine15/isoleucine16, alanine1364/valine1365 in pORF1 and leucine197/valine198 in pORF2. Specificity of these cleavage sites was validated by site-directed mutagenesis. Further characterization of the HEV protease, carried out using twelve inhibitors, showed chymostatin and PMSF to be the most efficient inhibitors, indicating this protein as a chymotrypsin-like protease. The specificity was further confirmed by cleavage of the chymotrypsin-specific fluorogenic peptide N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Mutational analysis of the conserved serine/cysteine/histidine residues suggested that H443 and C472/C481/C483 are possibly the active site residues. To our knowledge, this is the first direct demonstration of HEV protease and its function.

A novel light, oxygen, or voltage fluorescent tag for structural and biophysical analysis of DNA repair enzymes (989.1)

DNA is constantly threatened by internal and external pressures. Xeroderma Pigmentosum (XP) complementation groups D (XPD) and B (XPB) are helicases that unwind helix‐distorting lesions in damaged DNA allowing for repair to occur. These proteins are critical components of the Transcription Factor II H complex, and are essential for nucleotide excision repair. Three rare genetic diseases characterized by cancer and premature aging are caused by mutations in the genes that code for these helicase enzymes. In order to understand how proteins function at the molecular level, it is necessary to investigate the 3‐dimensional structure. To accomplish this, we amplified, cloned, and expressed human XPD and XPB, as well as archeal XPD from Sulfolobus acidocaldarius (SaXPD), and an associated DNA repair protein. Since crystallographic structures will be formed in an anaerobic environment, we employed the flavoprotein improved light, oxygen, or voltage sensing (iLOV) fluorescent tag to investigate target molecules in E. coli. We purified single‐domain llama nanobodies using affinity chromatography. The anti‐iLOV variants were used to optimize large‐scale purification of the DNA repair proteins of interest. We conclude that the novel DNA iLOV fusions will facilitate anaerobic crystallography techniques.Grant Funding Source: NSF ATE DUE 1205059

A holistic evolutionary and structural study offlaviviridaeprovides insights into the function and inhibition of HCV helicase

Viral RNA helicases are involved in duplex unwinding during the RNA replication of the virus. It is suggested that these helicases represent very promising antiviral targets. Viruses of the flaviviridae family are the causative agents of many common and devastating diseases, including hepatitis, yellow fever and dengue fever. As there is currently no available anti-Flaviviridae therapy, there is urgent need for the development of efficient anti-viral pharmaceutical strategies. Herein, we report the complete phylogenetic analysis across flaviviridae alongside a more in-depth evolutionary study that revealed a series of conserved and invariant amino acids that are predicted to be key to the function of the helicase. Structural molecular modelling analysis revealed the strategic significance of these residues based on their relative positioning on the 3D structures of the helicase enzymes, which may be used as pharmacological targets. We previously reported a novel series of highly potent HCV helicase inhibitors, and we now re-assess their antiviral potential using the 3D structural model of the invariant helicase residues. It was found that the most active compound of the series, compound C4, exhibited an IC50 in the submicromolar range, whereas its stereoisomer (compound C12) was completely inactive. Useful insights were obtained from molecular modelling and conformational search studies via molecular dynamics simulations. C12 tends to bend and lock in an almost “U” shape conformation, failing to establish vital interactions with the active site of HCV. On the contrary, C4 spends most of its conformational time in a straight, more rigid formation that allows it to successfully block the passage of the oligonucleotide in the ssRNA channel of the HCV helicase. This study paves the way and provides the necessary framework for the in-depth analysis required to enable the future design of new and potent anti-viral agents.

Sequence-specific electrochemical detection of double-strand PCR amplicons of PML/RARα fusion gene in acute promyelocytic leukemia

A novel electrochemical method for the sequence-specific detection of double-stranded polymerase chain reaction (PCR) products of PML/RARα fusion gene in acute promyelocytic leukemia (APL) was described in detail. Based on a "sandwich" sensing mode involving a pair of locked nucleic acids probes (capture probe and reporter probe), this DNA sensor exhibited excellent selectivity and specificity. The direct and quantitative analysis of double-stranded complementary was firstly performed by our sensor without the use of alkali, helicase enzymes, or denaturants. Finally, combining PCR technique with electrochemical detection scheme, PCR amplicons (191 bp) of the PML/RARα fusion gene were obtained and rapidly identified with a low detection limit of 79 fmol in the 100-μL hybridization system. The results clearly showed the power of sensor as a promising tool for the sensitive, specific, and portable detection of APL and other diseases.

ATP utilization and RNA conformational rearrangement by DEAD-box proteins.

RNA helicase enzymes catalyze the in vivo folding and conformational re-arrangement of RNA. DEAD-box proteins (DBPs) make up the largest family of RNA helicases and are found across all phyla. DBPs are molecular motor proteins that utilize chemical energy in cycles of ATP binding, hydrolysis, and product release to perform mechanical work resulting in reorganization of cellular RNAs. DBPs contain a highly conserved motor domain helicase core. Auxiliary domains, enzymatic adaptations, and regulatory partner proteins contribute to the diversity of DBP function throughout RNA metabolism. In this review we focus on the current understanding of the DBP ATP utilization mechanism in rearranging and unwinding RNA structures. We discuss DBP structural properties, kinetic pathways, and thermodynamic features of nucleotide-dependent interactions with RNA. We highlight recent advances in the DBP field derived from biochemical and molecular biophysical investigations aimed at developing a quantitative mechanistic understanding of DBP molecular motor function.

DNA helicase and helicase-nuclease enzymes with a conserved iron-sulfur cluster

Conserved Iron–Sulfur (Fe–S) clusters are found in a growing family of metalloproteins that are implicated in prokaryotic and eukaryotic DNA replication and repair. Among these are DNA helicase and helicase–nuclease enzymes that preserve chromosomal stability and are genetically linked to diseases characterized by DNA repair defects and/or a poor response to replication stress. Insight to the structural and functional importance of the conserved Fe–S domain in DNA helicases has been gleaned from structural studies of the purified proteins and characterization of Fe–S cluster site-directed mutants. In this review, we will provide a current perspective of what is known about the Fe–S cluster helicases, with an emphasis on how the conserved redox active domain may facilitate mechanistic aspects of helicase function. We will discuss testable models for how the conserved Fe–S cluster might operate in helicase and helicase–nuclease enzymes to conduct their specialized functions that help to preserve the integrity of the genome.

Isolation and Identification of Helicase from Escherichia coli for Biotechnology Processes

Helicases are able to unwind duplex DNA enzymatically at one temperature without prior heat denaturation. Therefore, the current study was undertaken to isolate and identify E. coli for in house production of helicase enzymes. Bacteria were isolated from patient stool samples from Penang General Hospital, Penang, Malaysia. The light and scanning microscopy technique were used to identify the morphology of the isolate. Chromosomal DNA from the organism was isolated and used to amplify 16S rRNA and uvrD gene fragments. The gene was amplified by a set of universal primers (F_UNI16S and R_UNI16S). The phylogenetic tree, homological analysis, and detailed comparison of the sequences showed that 16S rRNA gene sequence of the isolate had closest similarities with E. coli. Isolates gave PCR fragments of 2163bp which represent the uvrD gene and 1500bp which represent the 16S rRNA respectively of E. coli. E. coli was successfully isolated and identified by using 16S rRNA gene sequence analysis with uvrD gene

Mechanistic insight into Type I restriction endonucleases

Restriction and modification are two opposing activities that are used to protect bacteria from cellular invasion by DNA (e.g. bacteriophage infection). Restriction activity involves cleavage of the DNA; while modification activity is the mechanism used to "mark" host DNA and involves DNA methylation. The study of Type I restriction enzymes has often been seen as an esoteric exercise and this reflects some of their more unusual properties - non-stoichiometric (non-catalytic) cleavage of the DNA substrate, random cleavage of DNA, a massive ATPase activity, and the ability to both cleave DNA and methylate DNA. Yet these enzymes have been found in many bacteria and are very efficient as a means of protecting bacteria against bacteriophage infection, indicating they are successful enzymes. In this review, we summarise recent work on the mechanisms of action, describe switching of function and review their mechanism of action. We also discuss structural rearrangements and cellular localisation, which provide powerful mechanisms for controlling the enzyme activity. Finally, we speculate as to their involvement in recombination and discuss their relationship to helicase enzymes.

Unwinding and Rewinding: Double Faces of Helicase?

Helicases are enzymes that use ATP-driven motor force to unwind double-stranded DNA or RNA. Recently, increasing evidence demonstrates that some helicases also possess rewinding activity—in other words, they can anneal two complementary single-stranded nucleic acids. All five members of the human RecQ helicase family, helicase PIF1, mitochondrial helicase TWINKLE, and helicase/nuclease Dna2 have been shown to possess strand-annealing activity. Moreover, two recently identified helicases—HARP and AH2 have only ATP-dependent rewinding activity. These findings not only enhance our understanding of helicase enzymes but also establish the presence of a new type of protein: annealing helicases. This paper discusses what is known about these helicases, focusing on their biochemical activity to zip and unzip double-stranded DNA and/or RNA, their possible regulation mechanisms, and biological functions.

Small Molecule Drug Discovery for Dengue and West Nile Viruses: Applying Experience from Hepatitis C Virus

There are currently no specific treatments for infection with Dengue virus (DENV) and West Nile Virus (WNV). Drug-discovery programs are underway for both viruses, but as yet no small molecules have advanced to clinical trials. Hepatitis C virus (HCV) is a related flavivirus that has been the focus of intense drug discovery efforts for the last two decades. Many approaches currently being pursued for DENV and WNV have been previously attempted for HCV with varying degrees of success. The experience with HCV may direct DENV and WNV efforts towards approaches with the best chance of success. Based on experience with HCV, the viral polymerase and protease are attractive targets to focus on since these have been most successful to date. Cell-based phenotypic screening may also yield attractive inhibitors. The helicase and methyltransferase enzymes are likely to prove difficult targets and host target approaches are fraught with safety concerns.