Helicases g4-dna and drug design software

A helicase interacting compound may disrupt protein oligomerization, binding to dna substrate, or compete with atp binding. Hale institute of fundamental sciences, massey university, private bag 11 222, palmerston north new zealand, fax. Jul 26, 2016 gquadruplex gq is a four stranded dna secondary structure that arises from a guanine rich sequence. Pdf mycobacterium tuberculosis ding is a structurespecific. Singlemolecule imaging reveals a common mechanism shared by.

Indeed, mutations in recq helicases blm and wrn as well as fancj, chlr1, pif1, and rtel1 helicases that have been. A conserved g4 dna binding domain in recq family helicases. The classical swine fever virus csfv is a major pathogen of livestock and belongs to the flaviviridae viral family. Apr 23, 2019 dhx36 also called rhau and g4r1 is an rna helicase that can resolve gquadruplex g4 dna and g4rna with high specificity and affinity 1,2,3,4,5,6. Further structure refinement was carried out through docking a series of focused virtual libraries. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid.

Their main function is to unpackage an organisms genes. Understanding how cfss are protected to prevent genome instability is thus extremely important for. In this direction using the modeled structure of csvf helicase, a 3d pharmacophore was designed. Singlemolecule imaging reveals a common mechanism shared. Nonstructural protein 3 ns3 helicase encoded by the dengue virus denv is considered as a potential drug target to design new and effective drugs against dengue. The nonstructural protein3 ns3 helicase of zikv play an important role in viral rna replication. We have quantified the potential for g4 dna formation g4p of the 16 654 genes in the human refseq database, and then correlated gene function with g4p.

Figure 3a compares recq helicase activity on g4 dna and the h1k1 duplex. Stably formed gq structures can act as a physical blockade to disrupt genomic processes. G4 dna or other similar structures may therefore be a natural target of blm helicase in vivo, and failure to displace interacting grich dna sequences may explain the increase in genetic exchange characteristic of blooms syndrome. This program will start from a seed compound that must be manually positioned in the 3d conformational space of the molecular system fig. Helicase dependent amplification point of care testing. Dna unwinding mechanism of helicases according to the structures, helicases fall broadly into two groupsnonhexameric and hexameric helicases. Dengue fever is an emerging public health concern, with several million viral infections occur annually, for which no effective therapy currently exist. Author summary common fragile sites cfss are large chromosomal regions which are more prone to breakage than other places in the genome. However, the ability of these drugs to pass the bloodbrain barrier to treat infected neurons poses a challenge to antizika virus drug discovery.

Blm prevents instability of structureforming dna sequences. We report the structure of a bacterial recq dna helicase bound to resolved g4 dna. Request pdf helicases, g4dnas, and drug design new helicase assays that recognise therapeutically important g4dna structures will lead to the discovery of. Moreover, we examine g4 ligand design and the application of g4.

Dna helicases involved in dna repair and their roles in cancer. G4 motifs confer the ability to form a g4 dna structure, also known as a quadruplex by analogy with the bform dna duplex. Pdf singlemolecule imaging reveals a common mechanism. Dna unwinding mechanism of helicases atlas of science. Mms1 is an assistant for regulating gquadruplex dna structures. Linking these two activities telomerase and g4 will certainly be an interesting development of. Ddx3 rna helicase has also remarkable oncogenic properties. Considerable circumstantial evidence suggests that these structures can exist in vivo in specific regions of the genome including the telomeric ends of chromosomes and. Jul 21, 2018 braco19 is a 3,6,9trisubstituted acridine derivative, designed by neidle et al.

Dna helicase breaks the hydrogen bonds between the paired nitrogen bases in the dna molecule. Jul 09, 2019 genes in red indicate those found in the dgidb 2. Binding of yeast dna2 to yeast and oxytrichia g4 dnathe protein preparations used in this study are shown in fig. Strikingly, g4 dna is a preferred substrate of the blm helicase, as measured both by efficiency of unwinding and by competition. We identify the rqc domain, found only in recq family enzymes, as an independent, high affinity and conserved g4 dna binding domain. Selective interactions of cationic porphyrins with g. Each g4 dna 5 l was combined with a control compound dilution 5 l for a 10 l experimental sample containing 5 m g4 dna and 10 m ligand. Roshan singh thakur from the department of biochemistry, indian institute of science, bangalore 560012, india. What factors drive cells to adopt one cell fate over another. Longloop gquadruplexes are misfolded population minorities with fast transition kinetics in human telomeric sequences j. Mutation or loss in any one of three human recq helicases give rise to genetic instability syndromes. Telomeres are the specialized ends of linear chromosomes comprising a long stretch of a doublestranded tandem repeat sequence and a short singlestranded grich 3. Flavivirus helicases have both atp hydrolysis and rna unwinding activities. First health pharmaceuticals has set its rna research priorities on rna helicase inhibiting lead compounds development focus on rna rna molecules are a small and versatile tool to transport information within the cell and the organism, thus rna represents an optimal way for viruses to store all the necessary information for their survival and.

In humans, five recq family members have been identified. Structurebased design of selective and potent g quadruplexmediated telomerase inhibitors. G4 dna, transcription, rloops, top1, supercoiling, genome stability. The formation of g4 dna requires a cluster of guanineruns within a strand of dna. Of these processes, atp hydrolysis represents the most basic event. Therefore, gquadruplexes serve as potential targets for drug design 12. Our ddx3 rna helicase inhibiting class of compounds do not target viral proteins but temporarily inhibit an enzyme of our own rna cell biology that is of vital importance for the virus during its reproduction cycle. These results suggest that g4 dna may be a natural substrate of blm in vivo and that the failure to unwind g4 dna may cause the genomic instability and increased frequency of sister chromatid exchange characteristic. The present study aims at investigating the mechanisms involved in this specific drug interaction. These molecules are visualized, downloaded, and analyzed by users who range from students. These computerbased methodologies are now becoming an integral part of the drug discovery process and, although the principles of molecular recognition are far.

In this article, we performed hundred nanosecond molecular dynamics simulation and molecular docking of the ns3 helicase. Dna quadruplexes, also known as gquadruplex structures, comprise stacked tetrads, each of which arises from the planar association of four guanines in a cyclic hoogsteen hydrogenbonding arrangement. A lower catalytic threshold of fancj helicase activity is required for the cellular response to dna polymerase inhibition or gquadruplex accumulation compared to repair of interstrand crosslinks. Helicases are a group of nucleic acidbinding atpases that can unwind dna andor rna and function in almost every aspect of nucleic acid metabolism. The present invention relates to a crystal structure of gquadruplexes and its use. For instance, fancj, blm, and wrn, which are dna helicases that can help maintain genomic integrity by resolving guaninequadruplex g4 dna structures, are implicated in genetic disorders of fanconi anemia, blooms syndrome, and werners syndrome, respectively. Among these g4 helicases, pif1 is one of the best characterized dna g4 helicases. We have found that very low and very high g4p correlates with specific functional classes of genes.

Frontiers targeting dengue virus ns3 helicase by ligand. Helicases are a class of enzymes vital to all living organisms. Laboratory of molecular gerontology, national institute on aging, national institutes of health, nih biomedical research center, 251 bayview blvd baltimore, md 21224, laboratory of. Humans possess five distinct recq helicases see figure 1, all of which possess a hallmark recq helicase domain. The identification and characterization of a g4 dna resolvase activity j. The zika virus zikv has gained attention for the last few years due to the congenital microcephaly and guillainbarre syndrome that resulted in humans. Wrn mutation gives werners syndrome ws, blm loss results in bloom syndrome bs, and rothmundthomson syndrome rts is caused. A small molecule helicase inhibitors may interfere with the catalytic activities of dna helicase proteins and their molecular and cellular functions by a variety of mechanisms. Helicases as antiviral drug targets pubmed central pmc. Helicases are a class of enzymes vital to all organisms. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands i. Chd3 helicase domain mutations cause a neurodevelopmental. All five members of the human recq helicase family, helicase pif1, mitochondrial helicase twinkle, and helicasenuclease. Structural basis of zika virus helicase in recognizing its.

Even though g4 dna is not a substrate for all helicases, both yeast sgs1p and its close human homolog, blm, unwind g4 dna with at least 15. We previously reported that the gquadruplex g4 ligand rhps4 potentiates the antitumor activity of camptothecins both in vitro and in tumor xenografts. The 90kda heat shock protein hsp90 is a molecular chaperone that maintains the proper folding of its client proteins including protein kinases and steroid hormone receptors. The deah aspglualahis box rna helicase associated with aurich element rhau also named dhx36 or g4r1 specifically binds and unwinds g4 structures. The ns3h structures in ns3hssdnada 6 and ns3hssdnadt 6 were superimposable to each other, and also to the polypeptide chains. Dna helicases use the energy of atp hydrolysis to disrupt dna base pairing and displace proteins from dna in order to. Most of the studies in this area are focused on the genes that are expressed in different cell types and proteins that regulate the expression of those genes. The most promising compound was synthesised and it exhibited a submicromolar inhibition of the hcv helicase. For structural studies, we have made two constructs helicase 172617 and helicase 180617 to express the zikv helicase. Instrumental music, calming music, soft music, sleep music duration. Author summary severe acute respiratory syndrome coronavirus sarscov and middle east respiratory syndrome coronavirus merscov caused global pandemics in 2003 and 2012 with the fatality rates of 1035%. Quadruplex g4 dna structures are one of the most stable non.

Our results show that the ability to unwind g4 dna is not a universal property of dna helicases. The invention provides a crystal of an intramolecular gquadruplex structure having a hexagonal space group p6, and unit cell dimensions ab56. The unimolecular forms often occur naturally near the ends of the chromosomes, better known as the telomeric regions, and in transcriptional regulatory. China has solved the structure of the zika virus helicase, which is a key target for. Outbreak of merscov in the republic of korea in 2015 highlighted that the newly emerged covs remain a concern for the public health.

Mergny jl 2015 gquadruplex dna and ligand interaction in living cells. Dhx36 also called rhau and g4r1 is an rna helicase that can resolve gquadruplex g4 dna and g4rna with high specificity and affinity. Structurebased drug design methods utilize knowledge of a threedimensional structure of an enzymereceptor to develop small molecules able to bind to the desired target, generating a specific biological response. Mechanisms of dna helicase inhibitors and therapeutic strategies. Combination index test was used to evaluate the interaction between g4 ligands and standard or novel topo i. The essential unit of g4 dna is the gquartet, a planar array of guanines stabilized by hoogsteen base pairing between the n7 group of one guanine and. Identification of helicase proteins as clients for hsp90. It is the first rationally designed telomerase inhibitor 30 that exhibited strong antitumor activity. What does dna helicase do during replication answers. Gquadruplex dna presents a potential target for the design and development of novel anticancer drugs. If g4 structures are a regulatory tool in the cell, fast and efficient. The dna2 protein, which is involved in the maintenance of genomic stability, is a multifaceted enzyme, with 5. Dhx36 activity in targeting and unfolding g4rna is implicated in many mrna and long noncoding rna processes.

The role of wrn helicaseexonuclease in dna replication. In particular, g4 dna can accommodate a large diversity of small molecules due to the presence of 3 loops and 4 grooves in the vicinity of the guanines quartets. Telomere targeting with a new g4 ligand enhances radiation. Helicase dependent amplification hda is an isothermal dna amplification method that is similar to conventional pcr, but utilizes the action of a thermostable helicase enzyme, rather than heat to separate the nucleic acids and enable labeled primers to anneal to the dna template and elongate under the action of the polymerase. They are a part of normal chromosome structure and are present in all human beings, but are also hotspots for chromosomal rearrangement during oncogenesis. When found in gene promoter regions, gquadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Intramural research program of the national institutes of health, nia.

Dnas, and drug design hale, tracy k norris, gillian e jameson, geoffrey b filichev, vyacheslav v. Not all helicases can unwind g4 dnaspecific helicases are associated with replication and recombination, but these helicases could mitigate against formation of gdna only if they can unwind this substrate efficiently. Therefore, to achieve therapeutic selectivity using gquadruplexes as targets for drug design, it will be necessary to differentiate. Most strikingly, by comparing the unwinding activity of blm on g4 dna and on synthetic duplex substrates, we have found that g4 dna is a preferred substrate of the blm helicase. In contrast, depletion of the fancjbrip1 helicase causes persistent replication. A g4 motif consists of four runs of at least three guanines per run, separated by other bases n. Although g4 dna and potential involvement of dna helicases will. Stable formation of gq in genomic dna can be counteracted by the resolving activity of specialized helicases including rna helicase au associated with au rich elements rhau g4 resolvase 1, bloom helicase blm, and werner helicase wrn. Aug 04, 2015 gquadruplexes g4 are fourstranded nucleic acid structures implicated in important biological processes. To counteract such an effect, cells possess special helicases dedicated to unfolding gq structures. Here, we show that g4 dna is a preferred target for blm helicase within the context of long dna molecules. In an effort to shed light to the molecular mechanisms underlying the structural and drug design potential of the viral helicase. Because gquadruplex dna exhibits structural polymorphism, different gquadruplex typologies may be associated with different cellular processes.

Although the first generations of g4 ligands were mainly restricted to planar aromatic molecules 2426, several recent studies reported the design of secondgeneration quadruplex. The rcsb pdb also provides a variety of tools and resources. Gquadruplexes significantly stimulate pif1 helicase. Multiple helicases have been identified to regulate g4 structures and by this preserve. G4 dna is a noncanonical dna structure consisting of a stacked array of gquartets held together by base pairing between guanine bases. Dhx36 also called rhau and g4r1 is an rna helicase that can resolve gquadruplex g4 dna and g4rna with high specificity and affinity 1,2,3,4,5,6. New insights into dna helicases as druggable targets for. Identification of gquadruplexbinding inhibitors of myc. In addition to the familiar duplex dna, certain dna sequences can fold into secondary structures that are fourstranded. Molecular modeling and pharmacophore elucidation study of. Dna damage persistence as determinant of tumor sensitivity to. In molecular biology, gquadruplex secondary structures g4 are formed in nucleic acids by sequences that are rich in guanine.

Mechanism of atp hydrolysis by the zika virus helicase. Genetic interactions of gquadruplexes in humans elife. As a member of the wwpdb, the rcsb pdb curates and annotates pdb data according to agreed upon standards. We identify the rqc domain, found only in recq family enzymes, as an independent, high af. Maintaining the integrity of the singlestrand overhang is essential for cell survival because telomere endend fusion and erosion could cause genomic. These include overexpression of three different g4 dna binding proteins, loss of the g4 dna unwinding helicase sgs1, or treatment with small molecule g4 dna ligands. Crystal structure of parallel quadruplexes from human. Crystal structure of middle east respiratory syndrome. New helicase assays that recognise therapeutically important g4dna structures will lead to the discovery of novel molecular entities that bind not only to g4tetrads, but also to grooves and. Researchers solve the structure of the zika virus helicase. This causes the molecule to separate into two individual strands. G4 dna structures are resistant to the many helicases and nucleases that process intermediates arising in the course of dna replication and repair.

Herein we report a successful application of a computeraided design approach to identify a novel hcv helicase inhibitor. Oct 29, 2012 the recq family of dna helicases is highly conserved throughout evolution, and is important for the maintenance of genome stability. G4s were initially considered a structural curiosity, but recent evidence suggests their involvement in key genome functions such as transcription, replication, genome stability, and epigenetic regulation, together with numerous connections to cancer. They are helical in shape and contain guanine tetrads that can form from one, two or four strands. Computational methods to study gquadruplexligand complexes. Mycobacterium tuberculosis ding is a structurespecific. Stabilization of a gquadruplex from unfolding by replication. Rna gquadruplex is resolved by repetitive and atpdependent. The crystal structure of the ns3 helicase of zikv was obtained from protein data bank with the pdb id 5jrz 35.

G4 structures are typically present in gcrich regions. Jul 26, 2016 gquadruplex gq is a fourstranded dna structure that forms in the human genome and influences gene expression and dna replication. The prepared samples were annealed by heating to 95 c for 5 min and then cooling to 25 c at 3 cmin in a life eco thermocycler bioer. A guanineflipping and sequestration mechanism for gquadruplex. Here we used singlemolecule fret to define the mechanism of interaction of blm helicase with intrastranded g4 structures. Three conformational snapshots of the hepatitis c virus. Fancj promotes dna synthesis through gquadruplex structures. Putative dna gquadruplex formation within the promoters of. Guaninerich nucleic acid sequences are capable of folding into an intramolecular fourstranded structure called a gquadruplex. Us7312082b2 crystal structure of gquadruplex containing a. Users can perform simple and advanced searches based on annotations relating to sequence, structure and function. Dna structures will lead to the discovery of novel molecular entities that bind not only to g4.

Helicases are enzymes that use atpdriven motor force to unwind doublestranded dna or rna. Dna, including several human helicases wrn, blm, fancj, and pif1. The kinetic parameters of atp hydrolysis for the long form of the zikv helicase 172617 were determined using the. The essential unit of g4 dna is the gquartet, a planar array of guanines stabilized by hoogsteen base pairing between the n7 group of one guanine and the extracyclic amino group of its neighbor. Interaction between g4 and proteins is essential for the cellular functions of g4. This study proposes innovative strategies to design drugs that are capable of passing the bloodbrain barrier, and to be able to use drugs that are impermeable via drug delivery mechanisms. Blm helicase has a high specificity for noncanonical gquadruplex g4 dna structures, which are formed by grich dna strands and play an important role in the maintenance of genomic integrity. Furthermore, dna damage induced by a strong g4 stabilizing ligand. New helicase assays that recognise therapeutically important g4.

Ddx3 is part of the dead box proteins involved in rna metabolism. Recq helicase is comparably active on duplex and g4 dna substrates, as measured by direct comparison of protein activity and by competition assays. The crystal structure pdb 1a1v that was cocrystallized. Gene function correlates with potential for g4 dna formation. For the former group, a lot of in vitro experimental data showed that some are inactive in dna unwinding in the form of monomer while others are active in the form of monomer.

Accordingly, dhx36 is found enriched in g4 loci in cells. Understanding the roles of g4resolving dna helicases may lead to new insights for the design of g4 ligands that block g4 helicases or related proteins, thereby interfering with telomere semiconservative replication or impeding telomerasecatalysed elongation in cancer cells and causing them to senesce fig. How recq family helicases recognize this substrate has not been established. One critical aspect of drug design and discovery is identification of compounds that inhibit or stimulate specific.

They are helical structures containing guanine tetrads that can form from one, two or four strands. The interplay between gquadruplex and transcription. G, h overlap of the all 290 robust g4 sensitisers g and the 40 g4 sensitisers common to both ligands h with the drug genome interaction database. The ligands were searched from the literature and newly reported compounds were selected for molecular docking 37. Since the discovery of gquadruplex dna in the early 1960s, a number.

The methyltransferase and helicase enzymes as therapeutic. In our study, we examined three such gqresolving helicases, using single. Grich genomic regions can form g4 dna upon transcription or replication. A team led by researchers from tianjin university p. Insights into gquadruplex specific recognition by the deah. There are two main categories of gquadruplex ligands 1 telomestatin and amidebased macrocyclic ligands, and 2 porphyrin and related pyrrole and isoindolebased macrocyclic ligands 8. Molecular docking and simulation of zika virus ns3 helicase. Even though there arent any verified zoonosis cases yet, the outcomes of csfv epidemics have been devastating to local communities. Jul 24, 2014 mycobacterium tuberculosis ding is a structurespecific helicase that unwinds g4 dna. Structurebased drug design the design of the new series of molecules via structurebased drug design was conducted using ligbuilder version 1. Here we have used a genomewide bioinformatic approach to identify putative gquadruplex sequences pqs in the plasmodium. Gquadruplex gq is a four stranded dna secondary structure that arises from a guanine rich sequence. Helicases catalytically unwind duplex dna or rna using energy derived from the hydrolysis of nucleoside triphosphates and are attractive drug targets because they are required for viral replication.

We discovered that the bap1 promoter has a high gc content, which is 69. Guaninerich dna sequences can fold into fourstranded, noncanonical secondary structures called gquadruplexes g4s. The tool assigns each of these windows a score, and selects windows. Dna helicase, dna replication, gquadruplex, genomic. Gquadruplexes in the bap1 promoter positively regulate its. Dna helicases have been found that preferentially bind and unwind g4. The druggable genome denotes genes with known or predicted drug interactions.